Publications

Weiner S., Seligman J., Nadav-Ziv L., Haddad E., Asscher Y., Ovechkina M., Regev L., Mintz E. & Boaretto E. 2024. The mosaic tesserae in the industrial Byzantine wine press, Yavne, Israel: A natural unusually hard chalk or a chemically transformed chalk? Journal of Archaeological Science. 2024 Jan , 161 .

[Abstract]

Many of the surfaces of a large industrial scale wine press from the Byzantine period in Yavne, Israel, are covered in mosaic tesserae. These surfaces are part of the grape presses, storage pools, fermentation pools and also the walkways between these structures. The calcitic tesserae resemble limestone in color, texture and fracture properties. We were therefore surprised to discover that they are more disordered at the atomic level than consolidated limestone and in fact they are as disordered as porous and friable chalk. In addition SEM examination of fracture surfaces reveals abundant nannofossils, mostly coccoliths and foraminifera; properties that are also reminiscent of chalk. The nanofossil identifications shows that the tesserae are from the Middle Eocene, and this includes the chalky Maresha Formation that outcrops about 15kms from the site. SEM examination of polished surfaces shows that the larger pores of tesserae are lined with euhedral calcite crystals. Vickers hardness measurements show that the tesserae are much harder than chalk, and are comparable to limestone. There are two scenarios for explaining these seemingly contradictory observations. One scenario is that a chalky limestone geological formation with all these properties exists somewhere in the Mediterranean region and is the source of the raw material for the tesserae. The second scenario is that the Byzantine constructors of the wine press transformed a chalk into a harder and more consolidated material. Chalk transformation processes akin to plaster formation can be excluded based on the analyses of the stable carbon isotopic compositions of the tesserae, as well as their ¹⁴C concentrations. A chemical transformation using a weak acid that would only affect the calcite in the vicinity of the accessible pores, and leave the bulk of the chalk and its microfossils in a good state of preservation, is conceivable. In fact a by-product of producing wine is vinegar and the acetic acid of the vinegar could conceivably have been used for this purpose. If indeed a chemical transformation of soft chalk into a hard durable material was used, this would represent a “lost” technology that might even be useful in present times.
Milgram J., Rehav K., Ibrahim J., Shahar R. & Weiner S. 2023. The 3D organization of the mineralized scales of the sturgeon has structures reminiscent of dentin and bone: A FIB-SEM study Journal of Structural Biology. 2023 Dec , 215 (4).

[Abstract]

Scales are structures composed of mineralized collagen fibrils embedded in the skin of fish. Here we investigate structures contributing to the bulk of the scale material of the sturgeon (Acipencer guldenstatii) at the millimeter, micrometer and nanometer length scales. Polished and fracture surfaces were prepared in each of the three anatomic planes for imaging with light and electron microscopy, as well as focused ion beam – scanning electron microscopy (FIB-SEM). The scale is composed of three layers, upper and lower layers forming the bulk of the scale, as well as a thin surface layer. FTIR shows that the scale is composed mainly of collagen and carbonated hydroxyapatite. Lacunae are present throughout the structure. Fracture surfaces of all three layers are characterized by large diameter collagen fibril bundles (CFBs) emanating from a plane comprising smaller diameter CFBs orientated in different directions. Fine lineations seen in polished surfaces of both major layers are used to define planes called here the striation planes. FIB-SEM image stacks of the upper and lower layers acquired in planes aligned with the striation planes, show that CFBs are oriented in various directions within the striation plane, with larger CFBs emanating from the striation plane. Fibril bundles oriented in different directions in the same plane is reminiscent of a similar organization in orthodentin. The large collagen fibril bundles emanating out of this plane are analogous to von Korff fibrils found in developing dentin with respect to size and orientation. Scales of the sturgeon are unusual in that their mineralized collagen fibril organization contains structural elements of both dentin and bone. The sturgeon scale may be an example of an early evolved mineralized material which is neither bone nor dentin but contains characteristics of both materials, however, the fossil data required to confirm this is missing.
Shaked N., Addadi S., Goliand I., Fox S., Barinova S., Lia Addadi A. & Weiner S. 2023. Intra- to extracellular crystallization of calcite in the freshwater green algae Phacotus lenticularis Acta Biomaterialia. 2023 Jun , .

[Abstract]

Phacotus lenticularis is a freshwater unicellular green alga that forms lens-shaped calcitic shells around the cell. We documented P. lenticularis biomineralization pathways in live daughter cells while still within the reproductive complex, using scanning confocal microscopy and after vitrification using cryo-scanning electron microscopy (cryo-SEM). We show that some or all of the calcium ions required for mineral formation enter the cell through endocytosis, as inferred from the uptake of calcein fluorescent dye. Ions first concentrate inside intracellular vesicles to form small crystals that were detected by birefringence, reflectance, and cryo-SEM of cells in near-native, hydrated state. The crystals later exit the cell and build up the lens-shaped shell. The small crystals first cover the outer lorica surface and later fuse to form a thin continuous shell. This is most likely followed by a second shell maturation phase in which the shell undergoes thickening and crystal reorganization. Crystal assembly within the confined protected volume of the reproduction complex allows controlled shell formation outside the daughter cell. Only two other unicellular marine calcifiers, coccolithophores and miliolid foraminifera, are known to perform intracellular crystal formation. Statement of significance: Calcium carbonate (CaCO₃) deposition in aquatic environments is a major component of the global carbon cycle, which determines the CO₂ content of the atmosphere. In freshwater ecosystems, the green alga Phacotus lenticularis is considered the main contributor of autochthonous calcite precipitation and the only algal species known to form its shell through a controlled process. The chemical and ecological effects of P. lenticularis are intensively investigated, but our understanding of its shell formation is limited. We used advanced confocal laser scanning microscopy and cryo-scanning electron microscopy (cryo-SEM) to provide new insights into mineral formation and trafficking in the calcifying P. lenticularis cells.
Ibrahim J., Rechav K., Boaretto E. & Weiner S. 2023. Three dimensional structures of the inner and outer pig petrous bone using FIB-SEM: Implications for development and ancient DNA preservation Journal of Structural Biology. 2023 Sept , 215 (3).

[Abstract]

We report on the 3D ultrastructure of the mineralized petrous bone of mature pig using focused ion beam – scanning electron microscopy (FIB-SEM). We divide the petrous bone into two zones based on the degree of mineralization; one zone close to the otic chamber has higher mineral density than the second zone further away from the otic chamber. The hypermineralization of the petrous bone results in the collagen D-banding being poorly revealed in the lower mineral density zone (LMD), and absent in the high mineral density zone (HMD). We therefore could not use D-banding to decipher the 3D structure of the collagen assembly. Instead we exploited the anisotropy option in the Dragonfly image processing software to visualize the less mineralized collagen fibrils and/or nanopores that surround the more mineralized zones known as tesselles. This approach therefore indirectly tracks the orientations of the collagen fibrils in the matrix itself. We show that the HMD bone has a structure similar to that of woven bone, and the LMD is composed of lamellar bone with a plywood-like structural motif. This agrees with the fact that the bone close to the otic chamber is fetal bone and is not remodeled. The lamellar structure of the bone further away from the otic chamber is consistent with modeling/remodeling. The absence of the less mineralized collagen fibrils and nanopores resulting from the confluence of the mineral tesselles may contribute to shielding DNA during diagenesis. We show that anisotropy evaluation of the less mineralized collagen fibrils could be a useful tool to analyze bone ultrastructures and in particular the directionality of collagen fibril bundles that make up the bone matrix.
Garralda M. D., Weiner S., Arensburg B., Maureille B. & Vandermeersch B. 2022. Dental Paleobiology in a Juvenile Neanderthal (Combe-Grenal, Southwestern France) Biology. 2022 Sept , 11 (9).

[Abstract]

Combe-Grenal site (Southwest France) was excavated by F. Bordes between 1953 and 1965. He found several human remains in Mousterian levels 60, 39, 35 and especially 25, corresponding to MIS 4 (~75–70/60 ky BP) and with Quina Mousterian lithics. One of the fossils found in level 25 is Combe-Grenal IV, consisting of a fragment of the left corpus of a juvenile mandible. This fragment displays initial juvenile periodontitis, and the two preserved teeth (LLP4 and LLM1) show moderate attrition and dental calculus. The SEM tartar analysis demonstrates the presence of cocci and filamentous types of bacteria, the former being more prevalent. This result is quite different from those obtained for the two adult Neanderthals Kebara 2 and Subalyuk 1, where more filamentous bacteria appear, especially in the Subalyuk 1 sample from Central Europe. These findings agree with the available biomedical data on periodontitis and tartar development in extant individuals, despite the different environmental conditions and diets documented by numerous archeological, taphonomical and geological data available on Neanderthals and present-day populations. New metagenomic analyses are extending this information, and despite the inherent difficulties, they will open important perspectives in studying this ancient human pathology.
Kahil K., Kaplan-Ashiri I., Wolf S. G., Rechav K., Weiner S. & Addadi L. 2022. Elemental compositions of sea urchin larval cell vesicles evaluated by cryo-STEM-EDS and cryo-SEM-EDS Acta Biomaterialia. 2022 Nov , .

[Abstract]

During spicule formation in sea urchin larvae, calcium ions translocate within the primary mesenchymal cells (PMCs) from endocytosed seawater vacuoles to various organelles and vesicles where they accumulate, and subsequently precipitate. During this process, calcium ions are concentrated by more than three orders of magnitude, while other abundant ions (Na, Mg) must be removed. To obtain information about the overall ion composition in the vesicles, we used quantitative cryo-SEM-EDS and cryo-STEM-EDS analyzes. For cryo-STEM-EDS, thin (500 nm) frozen hydrated lamellae of PMCs were fabricated using cryo-focused ion beam-SEM. The lamellae were then loaded into a cryo-TEM, imaged and the ion composition of electron dense bodies was measured. Analyzes performed on 18 Ca-rich particles/particle clusters from 6 cells contained Ca, Na, Mg, S and P in different ratios. Surprisingly, all the Ca-rich particles contained P in amounts up to almost 1:1 of Ca. These cryo-STEM-EDS results were qualitatively confirmed by cryo-SEM-EDS analyzes of 310 vesicles, performed on high pressure frozen and cryo-planed samples. We discuss the advantages and limitations of the two techniques, and their potential applicability, especially to study ion transport pathways and ion trafficking in cells involved in mineralization.
Ibrahim J., Brumfeld V., Addadi Y., Rubin S., Weiner S. & Boaretto E. 2022. The petrous bone contains high concentrations of osteocytes: One possible reason why ancient DNA is better preserved in this bone PLoS ONE. 2022 Oct , 17 (10).

[Abstract]

The characterization of ancient DNA in fossil bones is providing invaluable information on the genetics of past human and other animal populations. These studies have been aided enormously by the discovery that ancient DNA is relatively well preserved in the petrous bone compared to most other bones. The reasons for this better preservation are however not well understood. Here we examine the hypothesis that one reason for better DNA preservation in the petrous bone is that fresh petrous bone contains more DNA than other bones. We therefore determined the concentrations of osteocyte cells occluded inside lacunae within the petrous bone and compared these concentrations to other bones from the domestic pig using high resolution microCT. We show that the concentrations of osteocyte lacunae in the inner layer of the pig petrous bone adjacent to the otic chamber are about three times higher (around 95,000 lacunae per mm3) than in the mastoid of the temporal bone (around 28,000 lacunae per mm3), as well as the cortical bone of the femur (around 27,000 lacunae per mm3). The sizes and shapes of the lacuna in the inner layer of the petrous bone are similar to those in the femur. We also show that the pig petrous bone lacunae do contain osteocytes using a histological stain for DNA. We therefore confirm and significantly expand upon previous observations of osteocytic lacuna concentrations in the petrous bone, supporting the notion that one possible reason for better preservation of ancient DNA in the petrous bone is that this bone initially contains at least three times more DNA than other bones. Thus during diagenesis more DNA is likely to be preserved in the petrous bone compared to other bones.
Friedman O., Böhm A., Rechav K., Pinkas I., Brumfeld V., Pass G., Weiner S. & Addadi L. 2022. Structural organization of xanthine crystals in the median ocellus of a member of the ancestral insect group Archaeognatha Journal of Structural Biology. 2022 Mar , 214 (1):107834-107834.

[Abstract]

Biogenic purine crystals function in vision as mirrors, multilayer reflectors and light scatterers. We investigated a light sensory organ in a primarily wingless insect, the jumping bristletail Lepismachilis rozsypali (Archaeognatha), an ancestral group. The visual system of this animal comprises two compound eyes, two lateral ocelli, and a median ocellus, which is located on the front of the head, pointing downwards to the ground surface. We determined that the median ocellus contains crystals of xanthine, and we obtained insights into their function. To date, xanthine biocrystals have only been found in the Archaeognatha. We performed a structural analysis, using reflection light microscopy, cryo-FIB-SEM, microCT and cryo-SEM. The xanthine crystals cover the bottom of a bowl-shaped volume in the median ocellus, in analogy to a tapetum, and reflect photons to light-sensitive receptors that are spread in the volume without apparent order or preferential orientation. We infer that the median ocellus operates as an irregular multifocal reflector, which is not capable of forming images. A possible function of this organ is to improve photon capture, and by so doing assess distances from the ground surface when jumping by determining changes in the intensity and contrast of the incident light.
Barzilai O., Oron M., Porat N., White D., Timms R., Blockley S., Zular A., Avni Y., Faershtein G., Weiner S. & Boaretto E. 2022. Expansion of eastern Mediterranean Middle Paleolithic into the desert region in early marine isotopic stage 5 Scientific Reports. 2022 Mar , 12 (1).

[Abstract]

Marine Isotopic Stage 5 is associated with wetter climatic conditions in the Saharo-Arabian deserts. This stage also corresponds to the establishment of Middle Paleolithic hominins and their associated material culture in two geographical provinces in southwest Asia-the Eastern Mediterranean woodland and the Arabian Peninsula desert. The lithic industry of the Eastern Mediterranean is characterized by the centripetal Levallois method, whereas the Nubian Levallois method characterizes the populations of the Arabian desert. The Negev Desert, situated between these regions is a key area to comprehend population movement in correlation to climatic zones. This investigation addresses the nature of the Middle Paleolithic settlement in the Negev Desert during MIS 5 by studying the site of Nahal Aqev. High resolution chronological results based on luminescence dating and cryptotephra show the site was occupied from MIS 5e to MIS 5d. The lithic industries at Nahal Aqev are dominated by centripetal Levallois core method. These data demonstrate that Nahal Aqev is much closer in its cultural attributes to the Eastern Mediterranean Middle Paleolithic than to the Arabian Desert entity. We conclude that Nahal Aqev represents an expansion of Middle Paleolithic groups from the Mediterranean woodland into the desert, triggered by better climatic conditions. These groups possibly interacted with hominin groups bearing the Nubian core tradition from the vast region of Arabia.
Sibony-Nevo O., Rechav K., Farstey V., Shimoni E., Varsano N., Addadi L. & Weiner S. 2022. The shell microstructure of the pteropod Creseis acicula is composed of nested arrays of S-shaped aragonite fibers: A unique biological material MRS Bulletin. 2022 Jan , 47 (1):18-28.

[Abstract]

Abstract: Snails of the superfamily Cavolinioidea, known as pteropods, are very abundant in the surface waters of all the oceans. Their transparent and lightweight shells are composed of densely packed, well-aligned, continuously crystalline curved aragonite fibers. Previous studies of the shell microstructure using mainly scanning electron microscopy, transmission electron microscopy, and x-ray diffraction suggested that the aragonite fibers adopt a helical motif. We mainly used focused ion beam-scanning electron microscopy to obtain three-dimensional information on the shell structure of Creseis acicula. We show that the basic structural motif in the central part of the shell (teleoconch) comprises aragonite fibers that are not helical, but are organized in nested S-shaped arcs arranged in planar arrays. This plane is oblique to the outer shell surface by approximately 20°. The planes stack in the third dimension with local displacements, to form a unique biological material. Impact statement: Of the seven basic materials used by mollusks to build their shells, the structure of one of these materials remains enigmatic, even though the snails that form this structure are by far the most abundant mollusks on earth. These so-called pteropods live in the surface waters of all the oceans and produce a significant amount of all the calcium carbonate formed in the open oceans. Since the first study of the pteropod shell structure in 1972, the basic structural motif of the arrays of highly elongated aragonite crystal fibers was inferred to be helical, although no one actually documented an entire helix. Here we resolved the 3D structure of the shell of one pteropod species using an instrument (FIB-SEM) that produces a high resolution 3D structure. We show that the basic repeating unit is a planar layer of nested S-shaped aragonite crystal fibers. Furthermore this planar layer is oblique to the shell outer surface. Besides resolving a fundamental basic question concerning mollusk shell structures, this unique organization of crystals raises fascinating questions about the mechanical properties of this most unusual curved space filling structure that will hopefully inspire materials scientists to produce superior synthetic materials.
Zhong J., Pierantoni M., Weinkamer R., Brumfeld V., Zheng K., Chen J., Swain M., Weiner S. & Li Q. 2021. Microstructural heterogeneity of the collagenous network in the loaded and unloaded periodontal ligament and its biomechanical implications Journal of Structural Biology. 2021 Sept , 213 (3).

[Abstract]

The periodontal ligament (PDL) is a highly heterogeneous fibrous connective tissue and plays a critical role in distributing occlusal forces and regulating tissue remodeling. Its mechanical properties are largely determined by the extracellular matrix, comprising a collagenous fiber network interacting with the capillary system as well as interstitial fluid containing proteoglycans. While the phase-contrast micro-CT technique has portrayed the 3D microscopic heterogeneity of PDL, the topological parameters of its network, which is crucial to understanding the multiscale constitutive behavior of this tissue, has not been characterized quantitatively. This study aimed to provide new understanding of such microscopic heterogeneity of the PDL with quantifications at both tissue and collagen network levels in a spatial manner, by combining phase-contrast micro-CT imaging and a purpose-built image processing algorithm for fiber analysis. Both variations within a PDL and among the PDL with different shapes, i.e. round-shaped and kidney-shaped PDLs, are described in terms of tissue thickness, fiber distribution, local fiber densities, and fiber orientation (namely azimuthal and elevation angles). Furthermore, the tissue and collagen fiber network responses to mechanical loading were evaluated in a similar manner. A 3D helical alignment pattern was observed in the fiber network, which appears to regulate and adapt a screw-like tooth motion under occlusion. The microstructural heterogeneity quantified here allows development of samplespecific constitutive models to characterize the PDL's functional and pathological loading responses, thereby providing a new multiscale framework for advancing our knowledge of this complex limited mobility soft-hard tissue interface.
Varsano N., Kahil K., Haimov H., Rechav K., Addadi L. & Weiner S. 2021. Characterization of the growth plate-bone interphase region using cryo-FIB SEM 3D volume imaging Journal of Structural Biology. 2021 Dec , 213 (4).

[Abstract]

The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.
Kahil Guterman K., Weiner S., Addadi L. & Gal A. 2021. Ion Pathways in Biomineralization: Perspectives on Uptake, Transport and Deposition of Calcium, Carbonate and Phosphate Journal of the American Chemical Society. 2021 Dec , .

[Abstract]

Minerals are formed by organisms in all of the kingdoms of life. Mineral formation pathways all involve uptake of ions from the environment, transport of ions by cells, sometimes temporary storage, and ultimately deposition in or outside of the cells. Even though the details of how all this is achieved vary enormously, all pathways need to respect both the chemical limitations of ion manipulation, as well as the many “housekeeping” roles of ions in cell functioning. Here we provide a chemical perspective on the biological pathways of biomineralization. Our approach is to compare and contrast the ion pathways involving calcium, phosphate, and carbonate in three very different organisms: the enormously abundant unicellular marine coccolithophores, the well investigated sea urchin larval model for single crystal formation, and the complex pathways used by vertebrates to form their bones. The comparison highlights both common and unique processes. Significantly, phosphate is involved in regulating calcium carbonate deposition and carbonate is involved in regulating calcium phosphate deposition. One often overlooked commonality is that, from uptake to deposition, the solutions involved are usually supersaturated. This therefore requires not only avoiding mineral deposition where it is not needed but also exploiting this saturated state to produce unstable mineral precursors that can be conveniently stored, redissolved, and manipulated into diverse shapes and upon deposition transformed into more ordered and hence often functional final deposits.
Boaretto E., Hernandez M., Goder-Goldberger M., Aldeias V., Regev L., Caracuta V., McPherron S. P., Hublin J., Weiner S. & Barzilai O. 2021. The absolute chronology of Boker Tachtit (Israel) and implications for the Middle to Upper Paleolithic transition in the Levant Proceedings of the National Academy of Sciences - PNAS. 2021 Jun , 118 (25).

[Abstract]

The Initial Upper Paleolithic (IUP) is a crucial lithic assemblage type in the archaeology of southwest Asia because it marks a dramatic shift in hominin populations accompanied by technological changes in material culture. This phase is conventionally divided into two chronocultural phases based on the Boker Tachtit site, central Negev, Israel. While lithic technologies at Boker Tachtit are well defined, showing continuity from one phase to another, the absolute chronology is poorly resolved because the radiocarbon method used had a large uncertainty. Nevertheless, Boker Tachtit is considered to be the origin of the succeeding Early Upper Paleolithic Ahmarian tradition that dates in the Negev to ∼42,000 y ago (42 ka). Here, we provide 14C and optically stimulated luminescence dates obtained from a recent excavation of Boker Tachtit. The new dates show that the early phase at Boker Tachtit, the Emirian, dates to 50 through 49 ka, while the late phase dates to 47.3 ka and ends by 44.3 ka. These results show that the IUP started in the Levant during the final stages of the Late Middle Paleolithic some 50,000 y ago. The later IUP phase in the Negev chronologically overlaps with the Early Upper Paleolithic Ahmarian of the Mediterranean woodland region between 47 and 44 ka. We conclude that Boker Tachtit is the earliest manifestation of the IUP in Eurasia. The study shows that distinguishing the chronology of the IUP from the Late Middle Paleolithic, as well as from the Early Upper Paleolithic, is much more complex than previously thought.
Ullman M., Brailovsky L., Schechter H. C., Weissbrod L., Zuckerman-Cooper R., Toffolo M. B., Caracuta V., Boaretto E., Weiner S., Abramov J., Bar-Yosef Mayer D. E., Avrutis V. W., Kol-Ya'kov S. & Frumkin A. 2021. The early Pre-Pottery Neolithic B site at Nesher-Ramla Quarry, Israel Quaternary International. 2021 Apr , 624 (30):148-167.

[Abstract]

Routine quarrying activity at the Nesher-Ramla Quarry, in the Judean Lowlands, Israel, has recently exposed a new Early Holocene archaeological site located in a small natural sinkhole, one of many dolines scattered in the area, dated to the Early Pre-Pottery Neolithic B (EPPNB). It is the first site of this period to be uncovered in the narrow strip of land between the Judean Mts. and the coastal plain. This site, dubbed NRQN, contains lithic artifacts, groundstone tools, shells and beads as well as botanical and faunal remains. Here we combine data from a series of studies on the site's stratigraphy and radiometric dating, paleoenvironment, sediments and material culture, with the aim of understanding the role of the site in the EPPNB sphere. Various human activities took place in or immediately adjacent to the sinkhole, predominantly domestic in nature, including stone-tool making and food consumption. However, some of the sediments deep within the sinkhole underwent intense in situ combustion, possibly associated with episodes of lime-plaster production. The filling of the sinkhole appears to have occurred rapidly, not exceeding a few hundred years (ca. 10,500–10,300 cal. BP) and was driven by both geogenic and anthropogenic sedimentation processes. Good preservation of microvertebrate, macrovertebrae, short-lived plants and wood remains at the site, provides a unique opportunity to study the environmental characteristics of this geographical area during the Early Holocene, which appears to have been of an open grassy landscape with patchy Mediterranean forest, resembling the current environmental conditions. Studying the characteristics of Early Holocene human activity at the site, its paleoenvironment, and the site formation mechanisms, also provides useful comparisons with the nearby NRQ Middle Paleolithic site (this issue).
Weiner S., Raguin E. & Shahar R. 2021. High resolution 3D structures of mineralized tissues in health and disease Nature reviews. Endocrinology. 2021 Mar , .

[Abstract]

A thorough knowledge of the structures of healthy mineralized tissues, such as bone or cartilage, is key to understanding the pathological changes occurring during disease. Such knowledge enables the underlying mechanisms that are responsible for pathology to be pinpointed. One high-resolution 3D method in particular - focused ion beam-scanning electron microscopy (FIB-SEM) - has fundamentally changed our understanding of healthy vertebrate mineralized tissues. FIB-SEM can be used to study demineralized matrix, the hydrated components of tissue (including cells) using cryo-fixation and even untreated mineralized tissue. The latter requires minimal sample preparation, making it possible to study enough samples to carry out studies capable of detecting statistically significant differences - a pre-requisite for the study of pathological tissues. Here, we present an imaging and characterization strategy for tissue structures at different length scales, describe new insights obtained on healthy mineralized tissues using FIB-SEM, and suggest future research directions for both healthy and diseased mineralized tissues.
Weiner S., Pinkas I., Kossoy A. & Feldman Y. 2021. Calcium sulfate hemihydrate (Bassanite) crystals in the wood of the tamarix tree Minerals. 2021 Mar , 11 (3).

[Abstract]

The most abundant mineral produced in the wood and leaves of trees is calcium oxalate monohydrate (whewellite), and after burning the wood the ash obtained is calcite. In the case of the Tamarix sp. tree, the freshly prepared ash is calcium sulfate (anhydrite). The aim of this study is to determine the calcium sulfate mineral phase in the fresh wood of Tamarix aphylla prior to burning. SEM images of the crystals show that they express smooth faces, are about 5–15 microns in their longest dimensions and are located in the ray cells. Fourier transform infrared spectroscopy (FTIR) and Raman microspectroscopy of the crystals in the wood and after extraction, both showed that the crystals are composed of calcium sulfate hemihydrate (bassanite). As elemental analyses of the crystals showed that in addition to calcium and sulfur, around 20 atom percent of the cations are sodium and potassium, we also obtained an X-ray powder diffraction pattern. This pattern excluded the possibility that the crystals are composed of another related mineral, and confirmed that, indeed, the crystals in the T. aphylla wood are composed of calcium sulfate hemihydrate (bassanite).
Shipov A., Zaslansky P., Riesemeier H., Segev G., Atkins A., Kalish-Achrai N., Weiner S. & Shahar R. 2021. The influence of estrogen deficiency on the structural and mechanical properties of rat cortical bone PeerJ. 2021 Jan , 9 .

[Abstract]

Background. Post-menopausal osteoporosis is a common health problem worldwide, most commonly caused by estrogen deficiency. Most of the information regarding the skeletal effects of this disease relates to trabecular bone, while cortical bone is less studied. The purpose of this study was to evaluate the influence of estrogen deficiency on the structure and mechanical properties of cortical bone. Methods. Eight ovariectomized (OVH) and eight intact (control) Sprague Dawley rats were used. Structural features of femoral cortical bone were studied by light microscopy, scanning electron microscopy and synchrotron-based microcomputer-tomography and their mechanical properties determined by nano-indentation. Results. Cortical bone of both study groups contains two distinct regions: organized circumferential lamellae and disordered bone with highly mineralized cartilaginous islands. Lacunar volume was lower in the OVH group both in the lamellar and disorganized regions (182 ± 75 µm³ vs 232 ± 106 µm³, P < 0.001 and 195 ± 86 µm³ vs. 247 ± 106 µm³, P < 0.001, respectively). Lacunar density was also lower in both bone regions of the OVH group (40 ± 18 ×10³ lacunae/mm³ vs. 47 ± 9×10³ lacunae/mm³ in the lamellar region, P = 0.003 and 63 ± 18×10³ lacunae/mm³ vs. 75 ± 13×10³ lacunae/mm³ in the disorganized region, P < 0.001). Vascular canal volume was lower in the disorganized region of the bone in the OVH group compared to the same region in the control group (P < 0.001). Indentation moduli were not different between the study groups in both bone regions. Discussion. Changes to cortical bone associated with estrogen deficiency in rats require high-resolution methods for detection. Caution is required in the application of these results to humans due to major structural differences between human and rat bone.
Raguin E., Rechav K., Shahar R. & Weiner S. 2021. Focused ion beam-SEM 3D analysis of mineralized osteonal bone: lamellae and cement sheath structures Acta Biomaterialia. 2021 Feb , 121 :497-513.

[Abstract]

The mineralized collagen fibril is the basic building block of bone, and hence is the key to understanding bone structure and function. Here we report imaging of mineralized pig bone samples in 3D using the focused ion beam-scanning electron microscope (FIB-SEM) under conditions that reveal the 67 nm D-banding of mineralized collagen fibrils. We show that in adult pig osteons, the lamellar bone comprises alternating layers with either collagen fibrils predominantly aligned in one direction, and layers in which fibrils are predominantly aligned in two directions. The cement sheath contains thin layers of both these motifs, but its dominant structural component comprises a very complex layer of fibrils predominantly aligned in three or more directions. The degree of mineralization of the cement sheath is comparable to that of the osteon interior. The extent of alignment (dispersion) of the collagen fibrils in the osteonal lamellar bone is significantly higher than in the cement sheath. Canaliculi within the cement sheath are mainly aligned parallel to the cement sheath boundary, whereas in the lamellar bone they are mainly aligned perpendicular to the lamellar boundaries. This study further characterizes the presence of two types of collagen fibril arrangements previously identified in demineralized lamellar bone from other species. The simple sample preparation procedure for mineralized bone and the lower risk of introducing artifacts opens the possibility of using FIB-SEM to study more samples, to obtain automatic quantitative information on collagen fibril organization and to evaluate the degrees of mineralization all in relatively large volumes of bone.
Jantschke A., Pinkas I., Schertel A., Addadi L. & Weiner S. 2020. Biomineralization pathways in calcifying dinoflagellates: Uptake, storage in MgCaP-rich bodies and formation of the shell Acta Biomaterialia. 2020 Jan , 102 :427-439.

[Abstract]

Little is known about shell formation of calcareous dinoflagellates, despite the fact that they are one of the major calcifying organisms of the phytoplankton. Here, calcitic cyst formation in two representative members of calcareous dinoflagellates is investigated using cryo-electron microscopy (cryo-SEM and cryo-FIB-SEM) in combination with micro-Raman and infrared spectroscopy. Only calcein-AM and not calcein enters these cells, indicating active uptake of calcium and other divalent cations. Multifunctional vacuoles containing crystalline inclusions are observed, and the crystals are identified as anhydrous guanine in the beta-form. The same vacuolar enclosures contain dense magnesium-, calcium-, and phosphorous-rich mineral bodies. These bodies are presumably secreted into the outer matrix where calcite forms. Calcite formation occurs via multiple independent nucleation events, and the different crystals grow with preferred orientation into a dense reticular network that forms the mature calcitic shell. We suggest a biomineralization pathway for calcareous dinoflagellates that includes (1) active uptake of calcium through the membranes, (2) deposition of Mg2+- and Ca2+-ions inside disordered MgCaP-rich mineral bodies, (3) secretion of these bodies to the inter-membrane space, and (4) Formation and growth of calcite into a dense reticulate network. This study provides new insights into calcium uptake, storage and transport in calcifying dinoflagellates.Statement of significanceLittle is known about the shell formation of calcareous dinoflagellates, despite the fact that they are one of the major calcifying organisms of the phytoplankton. We used state-of-the-art cryo-electron microscopy (cryo-SEM and cryo-FIB-SEM) in combination with micro-Raman spectroscopy to provide new insights into mineral formation in calcifying dinoflagellates.To date, intracellular crystalline calcite was assumed to be involved in calcite shell formation. Surprisingly, we identify these crystalline inclusions as anhydrous guanine suggesting that they are not involved in biomineralization. Instead, a key finding is that MgCaP-rich bodies are probably secreted into the outer matrix where the calcite shell is formed. We suggest that these bodies are an essential part of Ca-uptake, -storage and -transport and propose a new biomineralization model.
Kahil K., Varsano N., Sorrentino A., Pereiro E., Rez P., Weiner S. & Addadi L. 2020. Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae Proceedings of the National Academy of Sciences. 2020 Dec , 117 (49):30957-30965.

[Abstract]

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L2'/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L2'/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.
Raguin E., Rechav K., Brumfeld V., Shahar R. & Weiner S. 2020. Unique three-dimensional structure of a fish pharyngeal jaw subjected to unusually high mechanical loads Journal of Structural Biology. 2020 Aug , 211 (2).

[Abstract]

We examine the structure of the bone of the pharyngeal jaws of a large fish, the black drum (Pogonias cromis), that uses its tooth-jaw complex to crush hard-shelled bivalve mollusks. During mastication huge compressive forces are concentrated in a tiny zone at the tooth-bone interface. We report on the structure of this bone, with emphasis on its contact with the teeth, at different hierarchical levels and in 3D. Micro-CT shows that the molariform teeth do not have roots and are supported by a circular narrow bony rim that surrounds the periphery of the tooth base. The lower pharyngeal jaw is highly porous, as seen by reflected light microscopy and secondary electron microscopy (SE-SEM). Porosity decreases close to the bone-tooth interface and back-scattered electron (BSE-SEM) microscopy shows a slight elevation in mineral density. Focused ion beam - scanning electron microscopy (FIB-SEM) in the serial surface view (SSV) mode reveals a most surprising organization at the nanoscale level: parallel arrays of mineralized collagen fibrils surrounding channels of similar to 100 nm diameter, both with their long axes oriented along the load direction. The channels are filled with organic matter. These fibril-channel arrays are surrounded by a highly disordered mineralized material. This unusual structure clearly functions efficiently under compression, but the precise way by which this unique arrangement achieves this function is unknown.
Palmer B. A., Yallapragada V. J., Schiffmann N., Wormser E. M., Elad N., Aflalo E. D., Sagi A., Weiner S., Addadi L. & Oron D. 2020. A highly reflective biogenic photonic material from core-shell birefringent nanoparticles Nature Nanotechnology. 2020 Feb , 15 (2):138-144.

[Abstract]

The birefringence of isoxanthopterin crystalline spherulites enhances the reflectivity of a biological photonic crystal.Spectacular natural optical phenomena are produced by highly reflective assemblies of organic crystals. Here we show how the tapetum reflector in a shrimp eye is constructed from arrays of spherical isoxanthopterin nanoparticles and relate the particle properties to their optical function. The nanoparticles are composed of single-crystal isoxanthopterin nanoplates arranged in concentric lamellae around a hollow core. The spherulitic birefringence of the nanoparticles, which originates from the radial alignment of the plates, results in a significant enhancement of the back-scattering. This enables the organism to maximize the reflectivity of the ultrathin tapetum, which functions to increase the eye's sensitivity and preserve visual acuity. The particle size, core/shell ratio and packing are also controlled to optimize the intensity and spectral properties of the tapetum back-scattering. This system offers inspiration for the design of photonic crystals constructed from spherically symmetric birefringent particles for use in ultrathin reflectors and as non-iridescent pigments.
Haimov H., Shimoni E., Brumfeld V., Shemesh M., Varsano N., Addadi L. & Weiner S. 2020. Mineralization pathways in the active murine epiphyseal growth plate Bone. 2020 Jan , 130 .

[Abstract]

Endochondral ossification in the growth plate of long bones involves cartilage mineralization, bone formation and the budding vasculature. Many of these processes take place in a complex and dynamic zone, the provisional ossification zone, of the growth plate. Here we investigate aspects of mineralization in 2D and 3D in the provisional ossification zone at different length scales using samples preserved under cryogenic or fully hydrated conditions. We use confocal light microscopy, cryo-SEM and micro-CT in the phase contrast mode. We show in 9 week old BALB/c mice the presence of vesicles containing mineral particles in the blood serum, as well as mineral particles without membranes integrated with the blood vessel walls. We also observe labeled mineral particles within cells associated with bone formation, but not in the hypertrophic cartilage cells that are involved with cartilage mineralization. High resolution micro-CT images of fresh hydrated tibiae, show that there are open continuous pathways between the blood vessel extremities and the hypertrophic chondrocyte zone. As the blood vessel extremities, the mineralizing cartilage and the forming bone are all closely associated within this narrow zone, we raise the possibility that in addition to ion transport, mineral necessary for both cartilage and bone formation is also transported through the vasculature.
Schiffmann N., Wormser E. M., Brumfeld V., Addadi Y., Pinkas I., Yallapragada V. J., Aflalo E. D., Sagi A., Palmer B. A., Weiner S. & Addadi L. 2020. Characterization and possible function of an enigmatic reflector in the eye of the shrimp Litopenaeus vannamei Faraday Discussions. 2020 Oct , 223 :278-294.

[Abstract]

Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 μm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.
Berna F., Boaretto E., Wiebe M. C., Goder-Goldberger M., Abulafia T., Lavi R., Barzilai O., Marder O. & Weiner S. 2020. Site formation processes at Manot Cave, Israel: Interplay between strata accumulation in the occupation area and the talus Journal of Human Evolution. 2020 Oct , .

[Abstract]

Manot Cave contains important human fossils and archaeological assemblages related to the origin and dispersal of anatomically modern humans and the Upper Paleolithic period. This record is divided between an elevated in situ occupation area and a connecting talus. We, thus, investigated the interplay between the accumulation of the sediments and their associated artifacts in the occupation areas and the translocation of part of these sediments and artifacts down the talus. We examined the lithostratigraphy of two excavation locations in the occupation area (areas E and I), and two in the talus (areas C and D). We also assessed the diagenetic processes that have affected all these areas. A linear array of stalagmites and stalactites separates the occupation area from the talus, demarcating a major topographic barrier between the two. We infer that during human occupation, sediment accumulation of soil, wood ash, and bone was rapid and that some sediments with their associated artifacts overflowed the barrier and translocated down the talus. During periods of nonoccupation, the ash in the occupation area partially dissolved owing to the release of acid from the degrading bat and bird guano, and the layer thicknesses decreased. The south side of the talus (area C) has a normally stratified archaeological record, with the older archaeological materials underlying the younger materials. This suggests that the barrier between the occupation area and area C was relatively shallow and allowed a fairly continuous sediment accumulation in the talus. In the central part of the talus (area D), the stratigraphy is complex and shows mixing, presumably owing to the steep underlying bedrock topography and the mixing that occurs when sediments move down a steep slope. Finally, the distribution of secondary phosphates is consistent with the location of a main cave entrance to the south of the Paleolithic occupation area.
Weiner S., Nagorsky A., Feldman Y. (. & Kossoy A. 2020. Archaeological Ceramic Diagenesis: Clay Mineral Recrystallization in Sherds from a Late Byzantine Kiln, Israel Minerals. 2020 May , 10 (5).

[Abstract]

The pseudo-amorphous clay components of some of the pottery sherds that formed a surface in the firing chamber of a Late Byzantine kiln were shown by Fourier Transform Infrared Spectroscopy to have undergone almost complete recrystallization. Powder X-ray diffraction showed that the crystalline montmorillonite component of these sherds increased and kaolinite formed de novo. As this recrystallization process only occurred in the center of the firing chamber, we infer that the recrystallization process was due to repeated exposure of the sherds to high temperatures. The zeolite gonnardite was identified by X-ray diffraction. The chemical compositions of sodium-rich minerals, determined by energy dispersive X-ray spectroscopy (EDS), are consistent with the presence of gonnardite and analcime, and showed that the sodium was partially substituted by calcium and other cations. As these zeolites were also present in sherds from the upper pottery chamber, they did not form only as a result of repeated exposure to high temperatures. The demonstration that the clay mineral component of ceramics can undergo diagenetic recrystallization supports the possibility that provenience studies based on elemental analyses, especially of cooking pots that are repeatedly exposed to high temperatures, may be affected by recrystallization.
Chiou A. E., Hinckley J. A., Khaitan R., Varsano N., Wang J., Malarkey H. F., Hernandez C. J., Williams R. M., Estroff L. A., Weiner S., Addadi L., Wiesner U. B. & Fischbach C. 2020. Fluorescent Silica Nanoparticles to Label Metastatic Tumor Cells in Mineralized Bone Microenvironments Small. 2020 May , .

[Abstract]

During breast cancer bone metastasis, tumor cells interact with bone microenvironment components including inorganic minerals. Bone mineralization is a dynamic process and varies spatiotemporally as a function of cancer-promoting conditions such as age and diet. The functional relationship between skeletal dissemination of tumor cells and bone mineralization, however, is unclear. Standard histological analysis of bone metastasis frequently relies on prior demineralization of bone, while methods that maintain mineral are often harsh and damage fluorophores commonly used to label tumor cells. Here, fluorescent silica nanoparticles (SNPs) are introduced as a robust and versatile labeling strategy to analyze tumor cells within mineralized bone. SNP uptake and labeling efficiency of MDA-MB-231 breast cancer cells is characterized with cryo-scanning electron microscopy and different tissue processing methods. Using a 3D in vitro model of marrow-containing, mineralized bone as well as an in vivo model of bone metastasis, SNPs are demonstrated to allow visualization of labeled tumor cells in mineralized bone using various imaging modalities including widefield, confocal, and light sheet microscopy. This work suggests that SNPs are valuable tools to analyze tumor cells within mineralized bone using a broad range of bone processing and imaging techniques with the potential to increase the understanding of bone metastasis.
Weiner S., Nagorsky A., Taxel I., Asscher Y., Albert R. M., Regev L., Yan X., Natalio F. & Boaretto E. 2020. High temperature pyrotechnology: A macro- and microarchaeology study of a late Byzantine-beginning of Early Islamic period (7th century CE) pottery kiln from Tel Qatra/Gedera, Israel Journal of Archaeological Science: Reports. 2020 Jun , 31 .

[Abstract]

Pottery kilns are usually recognizable in the archaeological record based on their prominent and characteristic architectural features, evidence of exposure to high temperatures and associated waste products. Here we describe how we identified a kiln that has no readily recognizable architectural features, but does have an upper chamber full of broken pottery, and a lower chamber full of ash. The typology of the pottery in the upper chamber and radiocarbon dating show that the kiln was constructed after around 600 CE and was used until the early 7th century. The confining structures around these two chambers are sediments, some of which were deliberately brought to the site. A detailed study of the section through the kiln using on-site and off-site microarchaeological techniques revealed much about the firing conditions. The ash is composed almost entirely of phytoliths with a small amount of calcite. Surprisingly the diversity of phytolith morphotypes identified was low, and many of the phytoliths are unusually large multicells. The phytolith analysis shows that the fuel was mainly the stalks/chaff of domesticated grasses, most probably wheat, and probably not animal dung. Many of the phytolith morphologies are distorted and charred due to exposure to high temperatures, and show birefringence. Infrared spectra show that the phytoliths have partially recrystallized into the high temperature silicate mineral cristobalite. Slags formed close to the inner surfaces of the confining sediments presumably from melted phytoliths, and some of the clay-rich sediment close to these surfaces is clearly heat altered. These observations show that the temperatures in the firing chamber ranged from around 700 to 900 °C. The calcite in this ash is extremely disordered at the atomic level, based on the grinding curve analysis. A partial reconstruction of the kiln structure shows that the pottery chamber is above the firing chamber, and there is no constructed partition that separates the two chambers. The identification and characterization of this unusual kiln depended significantly on the use of the microarchaeological approach.
Hirsch A., Palmer B. A., Ramasubramaniam A., Williams P. A., Harris K. D. M., Pokroy B., Weiner S., Addadi L., Leiserowitz L. & Kronik L. 2019. Structure and Morphology of Light-Reflecting Synthetic and Biogenic Polymorphs of Isoxanthopterin: A Comparison Chemistry of Materials. 2019 Jun , 31 (12):4479-4489.

[Abstract]

Until recently it was thought that the only optical function of pteridines in biology was to act as light-absorbing pigments, but a recent report by some of us revealed that crystalline isoxanthopterin is a reflector in the eyes of decapod crustaceans. Here, we report the formation of crystalline isoxanthopterin synthetically from the polar dimethyl sulfoxide solvent, with X-ray diffraction analysis revealing a crystal structure different from that of biogenic isoxanthopterin. The structure of the new polymorph was determined in two independent ways. In one approach, it was generated and optimized using first-principles calculations, followed by comparison of simulation and experiment for high-resolution powder X-ray diffraction (PXRD) and electron diffraction. In the other approach, the structure was obtained definitively from PXRD data using a direct-space genetic algorithm for structure solution followed by Rietveld refinement. The synthetic structure is different from its biogenic counterpart, especially in having a nonplanar criss-cross H-bonded arrangement. We also rationalized the morphology of the crystals and the effect of the DMSO thereon, via a comparison between observed and theoretical growth morphologies. In addition, we calculated the optical properties of the synthetic structure and found its two dominant refractive indices to be somewhat lower than those of its biogenic counterpart, but still as high as those of reflecting guanine crystals. Synthetic isoxanthopterin therefore emerges as a promising candidate for incorporation in artificial optical systems.
Jantschke A., Pinkas I., Hirsch A., Elad N., Schertel A., Addadi L. & Weiner S. 2019. Anhydrous beta-guanine crystals in a marine dinoflagellate: Structure and suggested function Journal of Structural Biology. 2019 Jul , 207 (1):12-20.

[Abstract]

Guanine crystals are used by certain animals, including vertebrates, to produce structural colors or to enhance vision, because of their distinctive reflective properties. Here we use cryo-SEM, cryo-FIB SEM and Raman spectroscopic imaging to characterize crystalline inclusions in a single celled photosynthesizing marine dinoflagellate species. We demonstrate spectroscopically that these inclusions are blocky crystals of anhydrous guanine in the beta-polymorph. Two-dimensional cryo-SEM and three-dimensional cryo-FIB-SEM serial block face imaging show that the deposits of anhydrous guanine crystals are closely associated with the chloroplasts. We suggest that the crystalline deposits scatter light either to enhance light exploitation by the chloroplasts, or possibly for protection from UV radiation. This is consistent with the crystal locations within the cell, their shapes and their sizes. As the dinoflagellates are extremely abundant in the oceans and are a major group of photosynthesizing marine organisms, the presence of guanine crystals in this marine organism may have broad significance.
Zou Z., Habraken W. J. E. M., Matveeva G., Jensen A. C. S., Bertinetti L., Hood M. A., Sun C., Gilbert P. U. P. A., Polishchuk I., Pokroy B., Mahamid J., Politi Y., Weiner S., Werner P., Bette S., Dinnebier R., Kolb U., Zolotoyabko E. & Fratzl P. 2019. A hydrated crystalline calcium carbonate phase: Calcium carbonate hemihydrate Science. 2019 Jan , 363 (6425): 396-400.

[Abstract]

As one of the most abundant materials in the world, calcium carbonate, CaCO3, is the main constituent of the skeletons and shells of various marine organisms. It is used in the cement industry and plays a crucial role in the global carbon cycle and formation of sedimentary rocks. For more than a century, only three polymorphs of pure CaCO3-calcite, aragonite, and vaterite-were known to exist at ambient conditions, as well as two hydrated crystal phases, monohydrocalcite (CaCO3-1H(2)O) and ikaite (CaCO3-6H(2)O). While investigating the role of magnesium ions in crystallization pathways of amorphous calcium carbonate, we unexpectedly discovered an unknown crystalline phase, hemihydrate CaCO3.1/2H(2)O, with monoclinic structure. This discovery may have important implications in biomineralization, geology, and industrial processes based on hydration of CaCO3.
Ben-Zvi Y., Maria R., Pierantoni M., Brumfeld V., Shahar R. & Weiner S. 2019. Response of the tooth-periodontal ligament-bone complex to load: A microCT study of the minipig molar Journal of Structural Biology. 2019 Feb , 205 (2):155-162.

[Abstract]

One strategy evolved by teeth to avoid irreversible damage is to move and deform under the loads incurred during mastication. A key component in this regard is the periodontal ligament (PDL). The role of the bone underlying the PDL is less well defined. We study the interplay between the PDL and the underlying alveolar bone when loaded in the minipig. Using an Instron loading device we confirmed that the force-displacement curves of the molars and premolars of relatively fresh minipig intact mandibles are similar to those obtained for humans and other animals. We then used this information to obtain 3D images of the teeth before and after loading the tooth in a microCT such that the load applied is in the third linear part of the force displacement curve. We observed that at many locations there is a complimentary topography of the cementum and alveolar bone surface, strongly suggesting an active interplay between the tooth and the bone during mastication. We also observed that the loaded tooth does not come into direct contact with the underlying bone surface. A highly compressed layer of PDL is present between the tooth and the bone. The structure of the bone in the upper furcation region has a unique appearance with little obvious microstructure, abundant pores that have a large size range and at many locations the bone at the PDL interface has a needle -like shape. We conclude that there is a close interaction between the tooth, the PDL and the underlying alveolar bone during mastication. The highly compressed PDL layer that separates the tooth from the bone may fulfill a key shock absorbing function.
Sibony-Nevo O., Pinkas I., Farstey V., Baron H., Addadi L. & Weiner S. 2019. The Pteropod Creseis acicula Forms Its Shell through a Disordered Nascent Aragonite Phase Crystal Growth and Design. 2019 May , 19 (5):2564-2573.

[Abstract]

Shelled pteropods are holoplanktonic mollusks that build lightweight shells containing aragonite crystals. The complex shell architecture is composed of well-aligned, curved aragonitic fibers. Each curved fiber is continuously crystalline. We used in vivo micro-Raman spectroscopy to study the mineral composition of shells of living Creseis acicula pteropods at the larval (veliger) and adult stages. The spectra obtained from the growing edge have weak and broad peaks indicative of a highly disordered nascent aragonite phase. The disordered precursor phase is detected in the newly formed regions both at the shell edge, and during thickening in the internal part of the shell. As the shell grows and thickens throughout the life of the animal, the mineral matures from a disordered transient precursor phase to crystalline aragonite. We conclude that the shell of C. acicula is formed via a disordered nascent form of aragonite, which, being isotropic, facilitates the formation of the convoluted morphology in the continuously crystalline fibers of aragonite.
Suzuki M., Kubota K., Nishimura R., Negishi L., Komatsu K., Kagi H., Rehav K., Cohen S. & Weiner S. 2019. A unique methionine-rich protein-aragonite crystal complex: Structure and mechanical functions of the Pinctada fucata bivalve hinge ligament Acta Biomaterialia. 2019 Dec , 100 :1-9.

[Abstract]

The bivalve hinge ligament holds the two shells together. The ligament functions as a spring to open the shells after they were closed by the adductor muscle. The ligament is a mineralized tissue that bears no resemblance to any other known tissue. About half the ligament is composed of a protein-rich matrix, and half of long and extremely thin segmented aragonite crystals. Here we study the hinge ligament of the pearl oyster Pinctada fucata. FIB SEM shows that the 3D organization is remarkably ordered. The full sequence of the major protein component contains a continuous segment of 30 repeats of MMMLPD. There is no known homologous protein. Knockdown of this protein prevents crystal formation, demonstrating that the integrity of the matrix is necessary for crystals to form. X-ray diffraction shows that the aragonite crystals are more aligned in the compressed ligament, indicating that the crystals may be actively contributing to the elastic properties. The fusion interphase that joins the ligament to the shell nacre is composed of a prismatic mineralized tissue with a thin organic-rich layer at its center. Nanoindentation of the dry interphase shows that the elastic modulus of the nacre adjacent to the interphase gradually decreases until it approximates that of the interphase. The interphase modulus slightly increases until it matches the ligament. All these observations demonstrate that the ligament shell complex is a remarkable biological tissue that has evolved unique properties that enable bivalves to open their shell effectively innumerable times during the lifetime of the animal.Statement of significance - The hinge ligament shell complex is a unique functioning structural tissue whose elastic properties enable the shell to open without expending energy. Methionine-rich proteins are not known elsewhere raising fundamental questions about secondary and tertiary structures contributing to its elastic properties. The segmented and extremely thin aragonite crystals embedded in this matrix may also have unexpected elastic materials properties as they flex during compression. The structure of the interphase comprises a fascinating biological joint that connects two very different materials. The interphase materials, including the nacre, are graded with respect to elastic modulus so as to approximately match the connecting components. The interphase incorporates a thin organic rich layer that presumably functions as a gasket. This study raises many fundamental questions relevant to the diverse fields of protein chemistry, biomineralization and biological materials. (C) 2019 Published by Elsevier Ltd on behalf of Acta Materialia Inc.
Akiva A., Nelkenbaum O., Schertel A., Yaniv K., Weiner S. & Addadi L. 2019. Intercellular pathways from the vasculature to the forming bone in the zebrafish larval caudal fin: Possible role in bone formation Journal of Structural Biology. 2019 May , 206 (2):139-148.

[Abstract]

The pathway of ion supply from the source to the site of bone deposition in vertebrates is thought to involve transport through the vasculature, followed by ion concentration in osteoblasts. The cells deposit a precursor mineral phase in vesicles, which are then exocytosed into the extracellular matrix. We observed that the entire skeleton of zebrafish larvae, is labelled within minutes after injection of calcein or FITC-dextran into the blood. This raised the possibility that there is an additional pathway of solute transport that can account for the rapid labelling. We used cryo-FIB-SEM serial block face imaging to reconstruct at high resolution the 3D ultrastructure of the caudal tail of the zebrafish larva. This reconstruction clearly shows that there is a continuous intercellular pathway from the artery to the forming bone, and from the forming bone to the vein. Fluorescence light microscopy shows that calcein and FITC-dextran form a reticulate network pattern in this tissue, which we attribute to the dye being present in the intercellular space. We conclude that this intercellular continuous space may be a supply route for ions, mineral and other solute or particulate material to the fast forming bone.
Zhang G., Hirsch A., Shmul G., Avram L., Elad N., Brumfeld V., Pinkas I., Feldman Y., Ben Asher R., Palmer B. A., Kronik L., Leiserowitz L., Weiner S. & Addadi L. 2019. Guanine and 7,8-Dihydroxanthopterin Reflecting Crystals in the Zander Fish Eye: Crystal Locations, Compositions, and Structures Journal of the American Chemical Society. 2019 Dec , 141 (50):19736-19745.

[Abstract]

The eyes of many fish contain a reflecting layer of organic crystals partially surrounding the photoreceptors of the retina, which are commonly believed to be composed of guanine. Here we study an unusual fish eye from Stizostedion lucioperca that contains two layers of organic crystals. The crystals in the outer layer are thin plates, whereas the crystals in the inner tapetum layer are block-shaped. We show that the outer layer indeed contains guanine crystals. Analyses of solutions of crystals from the inner layer indicated that the block-shaped crystals are composed of xanthopterin. A model of the structure of the block-shaped crystals was produced using symmetry arguments based on electron diffraction data followed by dispersion-augmented DFT calculations. The resulting crystal structure of xanthopterin included, however, a problematic repulsive interaction between C=O and N of two adjacent molecules. Knowing that dissolved 7,8-dihydroxanthopterin can oxidize to xanthopterin, we replaced xanthopterin with 7,8-dihydroxanthopterin in the model. An excellent fit was obtained with the powder X-ray diffraction pattern of the biogenic crystals. We then analyzed the biogenic block-shaped crystals in their solid state, using MALDI-TOF and Raman spectroscopy. All three methods unequivocally prove that the block-shaped crystals in the eye of S. lucioperca are crystals of 7,8-dihydroxanthopterin. On the basis of the eye anatomy, we deduce that the guanine crystals form a reflective layer producing the silvery color present on part of the eye surface, whereas the block-shaped crystals backscatter light into the retina in order to increase the light sensitivity of the eye.
Maria R., Ben-Zvi Y., Rechav K., Klein E., Shahar R. & Weiner S. 2019. An unusual disordered alveolar bone material in the upper furcation region of minipig mandibles: A 3D hierarchical structural study Journal of Structural Biology. 2019 Apr , 206 (1):128-137.

[Abstract]

Teeth are subjected to compressive loads during mastication. Under small loads the soft tissue periodontal ligament (PDL) deforms most. However when the loads increase and the PDL is highly compressed, the tooth and the alveolar bone supporting the tooth, begin to deform. Here we report on the structure of this alveolar bone in the upper furcation region of the first molars of mature minipigs. Using light microscopy and scanning electron microscopy (SEM) of bone cross-sections, we show that this bone is hypermineralized, containing abundant small pores around 1-5 mu m in diameter, lacunae around 10-20 mu m as well as larger spaces. This bone does not possess the typical lamellar motif or other repeating structures normally found in cortical or trabecular mammalian bone. We also use high resolution focused ion beam scanning electron microscopy (FIB-SEM) in the serial surface mode to image the 3D organization of the demineralized bone matrix. We show that the upper furcation bone matrix has a disordered isotropic structure composed mainly of individual collagen fibrils with no preferred orientation, as well as highly staining material that is probably proteoglycans. Much larger aligned arrays of collagen fibers - presumably Sharpey's fibers - are embedded in this material. This unusual furcation bone material is similar to the disordered material found in human lamellar bone. In the upper furcation region this disordered bone comprises almost all the volume excluding Sharpey's fibers. We surmise that this most unusual bone type functions to resist the repeating compressive loads incurred by molars during mastication.
Pierantoni M., Brumfeld V., Addadi L. & Weiner S. 2019. A 3D study of the relationship between leaf vein structure and mechanical function Acta Biomaterialia. 2019 Apr , 88 :111-119.

[Abstract]

We investigate the structures and mechanical properties of leaf midribs of Ficus microcarpa and Prunus dulcis, which deposit calcium oxalate crystals, and of Olea europaea midribs which contain no mineral deposits, but do contain lignified fibers. The midrib mechanical performance contributes to the leafs ability to maintain a flat conformation for light harvesting and to efficiently reconfigure to reduce wind drag. We use a novel approach involving 3D visualization of the vein structure during mechanical load. This involves the use of customized mechanical loading devices that fit inside a microCT chamber. We show that the elastic, compression and torsional moduli of the midribs of leaves from the 3 species examined vary significantly. We also observed different modes of fracture and buckling of the leaves during compression. We assess the contributions of the calcium oxalate crystals to the mechanical and fracture properties. In F. microcarpa midrib linear arrays of calcium oxalate crystals contribute to resisting the bending, in contrast to P. dulcis leaves, where the calcium oxalate crystals do not resist bending. In both F. microcarpa and P. dulcis isolated calcium oxalate crystals enable high torsional compliance. The integrated microCT- mechanical testing approach could be used to investigate the structure-mechanics relationships in other complex biological samples.Statement of significanceLeaves need to maintain a flat conformation for light harvesting, but they also need to efficiently reconfigure to reduce wind drag. The leaf central vein (midrib) is a key structural component for leaf mechanicss. 3D visualization of the vein structure under mechanical loads showed that veins can be stiffened by reinforcement units composed of calcium oxalates crystals and lignin. The stiffening units can influence the bending and fracture properties of the midribs, and can contribute to determine if buckling will occur during folding. Mineral stiffening elements could be a widespread strategy to reinforce leaf veins and other biological structures. This structural-mechanical approach could be used to study other complex biological samples. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Boaretto E., Asscher Y., Hitchcock L. A., Lehmann G., Maeir A. M. & Weiner S. 2019. The chronology of the late bronze (LB)-Iron Age (IA) Transition in the Southern Levant: A response to finkelstein's critique Radiocarbon. 2019 Feb , 61 (1):1-11.

[Abstract]

The question under discussion is whether the dates of the Late Bronze (LBIIB)-LBIII (Iron IA) transitions in three sites in the southern Levant, namely Megiddo, Tell es-Safi/Gath and Qubur el-Walaydah occur at the same time, as has been proposed by Israel Finkelstein in his article in 2016 in Egypt and Levant. Here we respond to Finkelstein's comments. We add some new data, clarify the issues that were raised, and conclude that the Late Bronze (LBIIB)-LBIII (Iron IA) transitions occurred at different times in northern and southern Israel.
Palmer B. A., Gur D., Weiner S., Addadi L. & Oron D. 2018. The Organic Crystalline Materials of Vision: Structure-Function Considerations from the Nanometer to the Millimeter Scale Advanced Materials. 2018 Oct , 30 (41).

[Abstract]

Vision mechanisms in animals, especially those living in water, are diverse. Many eyes have reflective elements that consist of multilayers of nanometer-sized crystalline plates, composed of organic molecules. The crystal multilayer assemblies owe their enhanced reflectivity to the high refractive indices of the crystals in preferred crystallographic directions. The high refractive indices are due to the molecular arrangements in their crystal structures. Herein, data regarding these difficult-to-characterize crystals are reviewed. This is followed by a discussion on the function of these crystalline assemblies, especially in visual systems whose anatomy has been well characterized under close to in vivo conditions. Three test cases are presented, and then the relations between the reflecting crystalline components and their functions, including the relations between molecular structure, crystal structure, and reflecting properties are discussed. Some of the underlying mechanisms are also discussed, and finally open questions in the field are identified.
Pierantoni M., Tenne R., Rephael B., Brumfeld V., van Casteren A., Kupczik K., Oron D., Addadi L. & Weiner S. 2018. Mineral Deposits in Ficus Leaves: Morphologies and Locations in Relation to Function Plant Physiology. 2018 Feb , 176 (2):1751-1763.

[Abstract]

Ficus trees are adapted to diverse environments and have some of the highest rates of photosynthesis among trees. Ficus leaves can deposit one or more of the three major mineral types found in leaves: amorphous calcium carbonate cystoliths, calcium oxalates, and silica phytoliths. In order to better understand the functions of these minerals and the control that the leaf exerts over mineral deposition, we investigated leaves from 10 Ficus species from vastly different environments (Rehovot, Israel; Bologna, Italy; Issa Valley, Tanzania; and Ngogo, Uganda). We identified the mineral locations in the soft tissues, the relative distributions of the minerals, and mineral volume contents using microcomputed tomography. Each Ficus species is characterized by a unique 3D mineral distribution that is preserved in different environments. The mineral distribution patterns are generally different on the adaxial and abaxial sides of the leaf. All species examined have abundant calcium oxalate deposits around the veins. We used micromodulated fluorimetry to examine the effect of cystoliths on photosynthetic efficiency in two species having cystoliths abaxially and adaxially (Ficus microcarpa) or only abaxially (Ficus carica). In F. microcarpa, both adaxial and abaxial cystoliths efficiently contributed to light redistribution inside the leaf and, hence, increased photosynthetic efficiency, whereas in F. carica, the abaxial cystoliths did not increase photosynthetic efficiency.
Shahar R. & Weiner S. 2018. Open questions on the 3D structures of collagen containing vertebrate mineralized tissues: A perspective Journal of Structural Biology. 2018 Mar , 201 (3):187-198.

[Abstract]

Our current understanding of the structures of vertebrate mineralized tissues is largely based on light microscopy/histology and projections of 3D structures onto 2D planes using electron microscopy. We know little about the fine details of these structures in 3D at the length scales of their basic building blocks, the inherent variations of structure within a tissue and the cell-extracellular tissue interfaces. This limits progress in understanding tissue formation, relating structure to mechanical and metabolic functions, and obtaining deeper insights into pathologies and the evolution of these tissues. In this perspective we identify and discuss a series of open questions pertaining to collagen containing vertebrate mineralized tissues that can be addressed using appropriate 3D structural determination methods. By so doing we hope to encourage more research into the 3D structures of mineralized vertebrate tissues.
Palmer B. A., Hirsch A., Brumfeld V., Aflalo E. D., Pinkas I., Sagi A., Rosenne S., Oron D., Leiserowitz L., Kronik L., Weiner S. & Addadi L. 2018. Optically functional isoxanthopterin crystals in the mirrored eyes of decapod crustaceans Proceedings of the National Academy of Sciences of the United States of America. 2018 Mar , 115 (10):2299-2304.

[Abstract]

The eyes of some aquatic animals form images through reflective optics. Shrimp, lobsters, crayfish, and prawns possess reflecting superposition compound eyes, composed of thousands of square-faceted eye units (ommatidia). Mirrors in the upper part of the eye (the distal mirror) reflect light collected from many ommatidia onto the photosensitive elements of the retina, the rhabdoms. A second reflector, the tapetum, underlying the retina, back-scatters dispersed light onto the rhabdoms. Using microCT and cryo-SEM imaging accompanied by in situ micro-X-ray diffraction and micro-Raman spectroscopy, we investigated the hierarchical organization and materials properties of the reflective systems at high resolution and under close-to-physiological conditions. We show that the distal mirror consists of three or four layers of plate-like nanocrystals. The tapetum is a diffuse reflector composed of hollow nanoparticles constructed from concentric lamellae of crystals. Isoxanthopterin, a pteridine analog of guanine, forms both the reflectors in the distal mirror and in the tapetum. The crystal structure of isoxanthopterin was determined from crystal-structure prediction calculations and verified by comparison with experimental X-ray diffraction. The extended hydrogen-bonded layers of the molecules result in an extremely high calculated refractive index in the H-bonded plane, n = 1.96, which makes isoxanthopterin crystals an ideal reflecting material. The crystal structure of isoxanthopterin, together with a detailed knowledge of the reflector superstructures, provide a rationalization of the reflective optics of the crustacean eye.
Khalifa G. M., Kahil K., Erez J., Ashiri I. K., Shimoni E., Pinkas I., Addadi L. & Weiner S. 2018. Characterization of unusual MgCa particles involved in the formation of foraminifera shells using a novel quantitative cryo SEM/EDS protocol Acta Biomaterialia. 2018 Sept , 77 :342-351.

[Abstract]

Quantifying ion concentrations and mapping their intracellular distributions at high resolution can provide much insight into the formation of biomaterials. The key to achieving this goal is cryo-fixation, where the biological materials, tissues and associated solutions are rapidly frozen and preserved in a vitreous state. We developed a correlative cryo-Scanning Electron Microscopy (SEM)/Energy Dispersive Spectroscopy (EDS) protocol that provides quantitative elemental analysis correlated with spatial imaging of cryo-immobilized specimens. We report the accuracy and sensitivity of the cryo-EDS method, as well as insights we derive on biomineralization pathways in a foraminifer. Foraminifera are marine protozoans that produce Mg-containing calcitic shells and are major calcifying organisms in the oceans. We use the cryo-SEM/EDS correlative method to characterize unusual Mg and Ca-rich particles in the cytoplasm of a benthic foraminifer. The Mg/Ca ratio of these particles is consistently lower than that of seawater, the source solution for these ions. We infer that these particles are involved in Ca ion supply to the shell. We document the internal structure of the MgCa particles, which in some cases include a separate Si rich core phase. This approach to mapping ion distribution in cryo-preserved specimens may have broad applications to other mineralized biomaterials.Statement of significanceIons are an integral part of life, and some ions play fundamental roles in cell metabolism. Determining the concentrations of ions in cells and between cells, as well as their distributions at high resolution can provide valuable insights into ion uptake, storage, functions and the formation of biomaterials. Here we present a new cryo-SEM/EDS protocol that allows the mapping of different ion distributions in solutions and biological samples that have been cryo-preserved. We demonstrate the value of this novel approach by characterizing a novel biogenic mineral phase rich in Mg found in foraminifera, single celled marine organisms. This method has wide applicability in biology, and especially in understanding the formation and function of mineral-containing hard tissues. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Stepka Z., Barzilai O., Weiner S. & Boaretto E. 2018. Microflint in archaeological sediments from Boker Tachtit, Israel: A new method for quantifying concentrations of small flint fragments Journal of Archaeological Science. 2018 Mar , 91 :52-64.

[Abstract]

Flint is one of the most common rock types used for producing stone tools. During flint knapping huge amounts of microscopic sized flint particles are produced. Thus the presence of high concentrations of microflint in a sedimentary layer, could be a good indication that flint was knapped at that location. We have developed and tested a method for quantification of microflint concentrations in sediments. The method involves concentrating the microflints in specific density fractions, and then counting a representative proportion of the flint fragments using a polarized light microscope. We show that the method successfully identifies a knapping layer in an Initial Upper Palaeolithic level at the site of Boker Tachtit, Israel. This level also contains macroscopic flint debitage, including refitted artifacts. Microflint quantification can aid in identifying knapping areas and be useful for better understanding site formation processes. (C) 2018 Elsevier Ltd. All rights reserved.
Hun L., Martin M. A. S., Finkelstein I. & Weiner S. 2018. A powder preparation kit from the Middle Bronze Age at Megiddo, Israel: Tools and raw materials Journal of Archaeological Science: Reports. 2018 Oct , 21 :667-678.

[Abstract]

A unique assemblage of tools dating to the Middle Bronze Age was uncovered at Megiddo (Israel). The assemblage included pestles, pounding stones, many worked stones and various colored materials. We used mainly Fourier Transform Infrared Spectroscopy (FTIR) in order to identify the bulk materials of the artifacts, as well as the materials adhering to the surfaces of the artifacts. An unusual kaolinite, quartz, calcite and hematite containing compound was used for producing red powder, charcoal for black powder and chalk, shells and bone for white powders. We conclude that the assemblage was a kit used to produce relatively small amounts of colored powders. The possible uses of the kit are discussed, including for decoration of intramural burials found in the immediate vicinity.
Weiner S. & Boaretto E. 2018. Microarchaeology at Tell es-Safi/Gath, Area A Near Eastern Archaeology. 2018 Mar , 81 (1):24-27.

[Abstract]

The overall objective of archaeological excavations is to extract as much reliable information as possible from the whole archaeological record: both macroscopic and microscopic. An effective approach to achieve this goal is to integrate observations on the macroscopic and microscopic records as the excavation proceeds by operating an on-site analytical laboratory at the excavation. In this way important primary context locations can be better identified as they are exposed, and the excavation and sampling strategy can be adapted accordingly (Weiner 2010).
Pierantoni M., Tenne R., Brumfeld V., Kiss V., Oron D., Addadi L. & Weiner S. 2017. Plants and Light Manipulation: The Integrated Mineral System in Okra Leaves Advanced Science. 2017 May , 4 (5).

[Abstract]

Calcium oxalate and silica minerals are common components of a variety of plant leaves. These minerals are found at different locations within the leaf, and there is little conclusive evidence about the functions they perform. Here tools are used from the fields of biology, optics, and imaging to investigate the distributions of calcium oxalate, silica minerals, and chloroplasts in okra leaves, in relation to their functions. A correlative approach is developed to simultaneously visualize calcium oxalates, silica minerals, chloroplasts, and leaf soft tissue in 3D without affecting the minerals or the organic components. This method shows that in okra leaves silica and calcium oxalates, together with chloroplasts, form a complex system with a highly regulated relative distribution. This distribution points to a significant role of oxalate and silica minerals to synergistically optimize the light regime in the leaf. The authors also show directly that the light scattered by the calcium oxalate crystals is utilized for photosynthesis, and that the ultraviolet component of light passing through silica bodies, is absorbed. This study thus demonstrates that calcium oxalates increase the illumination level into the underlying tissue by scattering the incoming light, and silica reduces the amount of UV radiation entering the tissue.
Zou Z., Habraken W. J. E. M., Bertinetti L., Politi Y., Gal A., Weiner S., Addadi L. & Fratzl P. 2017. On the Phase Diagram of Calcium Carbonate Solutions Advanced Materials Interfaces. 2017 Jan , 4 (1).

[Abstract]

A liquid–liquid phase boundary is experimentally determined in calcium carbonate solutions obtained by rapid mixing. Nanoparticles of amorphous calcium carbonate form at concentration higher than this boundary and their size diverges close to the boundary, which is interpreted as a spinodal line. At lower concentrations, the solution is metastable against the nucleation of crystalline calcium carbonate phases.
Silvent J., Akiva A., Brumfeld V., Reznikov N., Rechav K., Yaniv K., Addadi L. & Weiner S. 2017. Zebrafish skeleton development: High resolution micro-CT and FIB-SEM block surface serial imaging for phenotype identification PLoS ONE. 2017 Dec , 12 (12):e0177731-e0177731.

[Abstract]

Although bone is one of the most studied living materials, many questions about the manner in which bones form remain unresolved, including fine details of the skeletal structure during development. In this study, we monitored skeleton development of zebrafish larvae, using calcein fluorescence, high-resolution micro-CT 3D images and FIB-SEM in the block surface serial imaging mode. We compared calcein staining of the skeletons of the wild type and nacre mutants, which are transparent zebrafish, with micro-CT for the first 30 days post fertilization embryos, and identified significant differences. We quantified the bone volumes and mineral contents of bones, including otoliths,during development, and showed that such developmental differences, including otolith development, could be helpful in identifying phenotypes. In addition, high-resolution imaging revealed the presence of mineralized aggregates in the notochord, before the formation of the first bone in the axial skeleton. These structures might play a role in the storage of the mineral. Our results highlight the potential of these high-resolution 3D approaches to characterize the zebrafish skeleton, which in turn could prove invaluable information for better understanding the development and the characterization of skeletal phenotypes.
Gur D., Palmer B. A., Weiner S. & Addadi L. 2017. Light Manipulation by Guanine Crystals in Organisms: Biogenic Scatterers, Mirrors, Multilayer Reflectors and Photonic Crystals Advanced Functional Materials. 2017 Feb , 27 (6).

[Abstract]

Guanine crystals are widely used in nature to manipulate light. The first part of this feature article explores how organisms are able to construct an extraordinary array of optical "devices" including diffuse scatterers, broad-band and narrowband reflectors, tunable photonic crystals, and image-forming mirrors by varying the size, morphology, and arrangement of guanine crystals. The second part presents an overview of some of the properties of crystalline guanine to explain why this material is ideally suited for such optical applications. The high reflectivity of many natural optical systems ultimately derives from the fact that guanine crystals have an extremely high refractive index-a product of its anisotropic crystal structure comprised of densely stacked H-bonded layers. In order to optimize their reflectivity, many organisms exert exquisite control over the crystal morphology, forming plate-like single crystals in which the high refractive index face is preferentially expressed. Guanine-based optics are used in a wide range of biological functions such as in camouflage, display, and vision, and exhibit a degree of versatility, tunability, and complexity that is difficult to incorporate into artificial devices using conventional engineering approaches. These biological systems could inspire the next generation of advanced optical materials.
Hirsch A., Palmer B. A., Elad N., Gur D., Weiner S., Addadi L., Kronik L. & Leiserowitz L. 2017. Biologically Controlled Morphology and Twinning in Guanine Crystals ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 2017 Aug , 56 (32):9420-9424.

[Abstract]

Guanine crystals are widely used in nature as components of multilayer reflectors. Guanine-based reflective systems found in the copepod cuticle and in the mirror of the scallop eye are unique in that the multilayered reflectors are tiled to form a contiguous packed array. In the copepod cuticle, hexagonal crystals are closely packed to produce brilliant colors. In the scallop eye, square crystals are tiled to obtain an image-forming reflecting mirror. The tiles are about 1 mm in size and 70 nm thick. According to analysis of their electron diffraction patterns, the hexagon and square tiles are not single crystals. Rather, each tile type is a composite of what appears to be three crystalline domains differently oriented and stacked onto one another, achieved through a twice-repeated twinning about their and crystal axes, respectively. By these means, the monoclinic guanine crystal mimics higher symmetry hexagonal and tetragonal structures to achieve unique morphologies.
Palmer B. A., Taylor G. J., Brumfeld V., Gur D., Shemesh M., Elad N., Osherov A., Oron D., Weiner S. & Addadi L. 2017. The image-forming mirror in the eye of the scallop Science. 2017 Dec , 358 (6367): 1172-1175.

[Abstract]

Scallops possess a visual system comprising up to 200 eyes, each containing a concave mirror rather than a lens to focus light. The hierarchical organization of the multilayered mirror is controlled for image formation, from the component guanine crystals at the nanoscale to the complex three-dimensional morphology at the millimeter level. The layered structure of the mirror is tuned to reflect the wavelengths of light penetrating the scallop's habitat and is tiled with a mosaic of square guanine crystals, which reduces optical aberrations. The mirror forms images on a double-layered retina used for separately imaging the peripheral and central fields of view. The tiled, off-axis mirror of the scallop eye bears a striking resemblance to the segmented mirrors of reflecting telescopes.
Ben-Zvi Y., Reznikov N., Shahar R. & Weiner S. 2017. 3D architecture of Trabecular Bone in the Pig Mandible and Femur: inter-Trabecular angle Distributions Frontiers in Materials. 2017 Sept , 4 .

[Abstract]

Cancellous bone is an intricate network of interconnected trabeculae, to which analysis of network topology can be applied. The inter-trabecular angle (ITA) analysis-an analysis of network topological parameters and regularity of network-forming nodes-was previously carried out on human proximal femora and showed that trabecular bone follows two main principles: sparsity of the network connectedness (prevalence of nodes with low connectivity in the network) and maximal space spanning (angular offset of connected elements is maximal for their number and approximates the values of geometrically symmetric shapes). These observations suggest that 3D organization of trabecular bone, irrespective of size and shape of individual elements, reflects a tradeoff between minimal metabolic cost of maintenance and maximal network stability under conditions of multidirectional loading. In this study, we validate the ITA application using additional 3D structures (cork and 3D-printed metal lattices), analyze the ITA parameters in porcine proximal femora and mandibles, and carry out a spatial analysis of the most common node type in the porcine mandibular condyle. The validation shows that the ITA application reliably detects designed or evolved topological parameters. The ITA parameters of porcine trabecular bones are similar to those of human bones. We demonstrate functional adaptation in the pig mandibular condyle by showing that the planar nodes with three edges are preferentially aligned in relation to the muscle forces that are applied to the condyle. We conclude that the ITA topological parameters are remarkably conserved, but locally do adapt to applied stresses.
Funt N., Palmer B. A., Weiner S. & Addadi L. 2017. Koi fish-scale iridophore cells orient guanine crystals to maximize light reflection ChemPlusChem. 2017 Jun , 82 (6):914-923.

[Abstract]

Fish-scale iridophore cells deposit guanine crystals and assemble them into multilayer reflectors to produce silvery reflectance. The crystal orientation controls the reflective properties of the fish scales, but little is known about the degree of orientation of the guanine crystals and whether this orientation is pre-determined at the level of an individual cell. Koi fish-scaleattached iridophores, iridophores on regenerated scales, and cultured iridophores were examined by using light microscopy and synchrotron micro-X-ray diffraction. More than 95% of the thin {100} guanine crystal plates in the iridophores of the mature and regenerated scales are oriented parallel to the scale surface and perpendicular to the direction of the incoming light. More than 70% of the crystals in cultured iridophore cells are also in this orientation. The crystals are elongated and within each cell on the mature scale and in the cultured cells the long morphological axes are well aligned with the long axis of the iridophore. In contrast to the cultured iridophores, in the mature scale the iridophore cells are co-aligned with each other. Cultured iridophores are flexible and motile, and azimuthal crystal orientations vary as the cells move. We conclude that iridophore cells function as independent units and that the control over crystal orientation is pre-determined at the individual cell level in the direction that is essential for function, namely, exposing the large reflecting crystal surface to light.
Asscher Y., Weiner S. & Boaretto E. 2017. A new method for extracting the insoluble occluded carbon in archaeological and modern phytoliths: Detection of ¹⁴C depleted carbon fraction and implications for radiocarbon dating Journal of Archaeological Science. 2017 Feb , 78 :57-65.

[Abstract]

Phytolith-rich layers in archaeological sites constitute well defined stratigraphic horizons that would be invaluable if absolutely dated. Previous attempts to radiocarbon date phytoliths produced inconsistent results using plants with known ages. In this study a new approach to extract and analyze the silica occluded carbon was tested on well-dated archaeological contexts in Beth Shemesh and Tell es-Safi/Gath, and on modern wheat plants that grew in a controlled environment. Results show that by dissolving the silica using mild conditions, phytolith insoluble fractions can be extracted and their radiocarbon contents analyzed reproducibly. After phytolith dissolution, the remaining insoluble fractions with 10–30%C have radiocarbon concentrations that are statistically similar to associated charred seeds (within 2σ), and insoluble fractions with 40%C show concentrations that are identical to the seeds. These results show that the insoluble fraction of phytoliths is a suitable material for answering chronological questions.
Toffolo M. B., Ullman M., Caracuta V., Weiner S. & Boaretto E. 2017. A 10,400-year-old sunken lime kiln from the Early Pre-Pottery Neolithic B at the Nesher-Ramla quarry (el-Khirbe), Israel Journal of Archaeological Science: Reports. 2017 Aug , 14 :353-364.

[Abstract]

The Pre-Pottery Neolithic B (PPNB) is the first period in human cultural evolution that is characterized by the extensive production of lime plaster for architectural, decorative and ritual purposes. The production of large quantities of lime plaster requires the operation of a lime kiln, a structure where limestone cobbles are heated to high temperatures (> 600 degrees C) to obtain quicklime, which is then mixed with water to form a moldable lime putty. However, little is known about lime kilns and plaster production processes during the PPNB. This may be because the technology used at that time was simple and left few traces that are unique to lime plaster production. These include combustion features rich in lime plaster and heat-altered sediments, which are difficult to identify through only a visual inspection of the archaeological context. Here we report the study of a small sinkhole at the Nesher-Ramla quarry in Israel, which yielded Early PPNB artifacts. Using infrared spectrometry and micromorphology of sediments, we identified in-situ deposits of heat-altered sediments rich in fragments of burnt limestone, lime plaster, and wood ash in the form of charcoal, phytoliths and siliceous aggregates, which are features consistent with the operation of a lime kiln. Charred botanical remains were characterized and used for radiocarbon dating, which determined the age of the site at 10,400 cal BP. We therefore conclude, based on the examination of the microscopic archaeological record, that this sinkhole was used as a lime kiln during the Early PPNB.
Goder-Goldberger M., Weiner S., Barzilai O. & Boaretto E. 2017. Heating of flint artifacts from the site of Boker Tachtit (Israel) was not detected using FTIR peak broadening Journal of Archaeological Science: Reports. 2017 Apr , 12 :173-182.

[Abstract]

Heat treatment of flint changes its mechanical properties and improves its knappability. Here we examine flint artifacts from two occupational levels at the site of Boker Tachtit (Israel). Boker Tachtit is an important site for understanding the Middle to Upper Paleolithic transition in the Levant. The thin and stratified archeological levels together with a well-defined lithic technology make the site suitable for addressing the issue of heat treatment of raw materials for tool production. We use Fourier transform infrared spectroscopy to assess heat treatment of flint artifacts and compare them to geogenic flint nodules collected in Wadi Zin,
Vidavsky N., Akiva A., Kaplan-Ashiri I., Rechav K., Addadi L., Weiner S. & Schertel A. 2016. Cryo-FIB-SEM serial milling and block face imaging: Large volume structural analysis of biological tissues preserved close to their native state Journal of Structural Biology. 2016 Dec , 196 (3):487-495.

[Abstract]

Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000 μm³) at high resolution (5–20 nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20 μm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues.
Gur D., Leshem B., Farstey V., Oron D., Addadi L. & Weiner S. 2016. Light-Induced Color Change in the Sapphirinid Copepods: Tunable Photonic Crystals Advanced Functional Materials. 2016 Mar , 26 (9):1393-1399.

[Abstract]

Light-induced tunable photonic systems are rare in nature, and generally beyond the state-of-the-art in artificial systems. Sapphirinid male copepods produce some of the most spectacular colors in nature. The male coloration, used for communication purposes, is structural and is produced from ordered layers of guanine crystals separated by cytoplasm. It is generally accepted that the colors of the males are related to their location in the epipelagic zone. By combining correlative reflectance and cryoelectron microscopy image analyses, together with optical time lapse recording and transfer matrix modeling, it is shown that male sapphirinids have the remarkable ability to change their reflectance spectrum in response to changes in the light conditions. It is also shown that this color change is achieved by a change in the thickness of the cytoplasm layers that separate the guanine crystals. This change is reversible, and is both intensity and wavelength dependent. This capability provides the male with the ability to efficiently reflect light under certain conditions, while remaining transparent and hence camouflaged under other conditions. These copepods can thus provide inspiration for producing synthetic tunable photonic arrays.
Addadi L., Vidavsky N. A., Addadi S., Schertel A., Ben-Ezra D., Shpigel M. & Weiner S. 2016. Calcium transport into the cells of the sea urchin larva in relation to spicule formation Proceedings of the National Academy of Sciences of the United States of America. 2016 Nov , 113 (45):12637-12642.

[Abstract]

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake.We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.
Akiva A., Kerschnitzki M., Pinkas I., Wagermaier W., Yaniv K., Fratzl P., Addadi L. & Weiner S. 2016. Mineral Formation in the Larval Zebrafish Tail Bone Occurs via an Acidic Disordered Calcium Phosphate Phase Journal of the American Chemical Society. 2016 Nov , 138 (43):14481-14487.

[Abstract]

Both in vivo and ex vivo observations support the hypothesis that bone mineral formation proceeds via disordered precursor phases. The characteristics of the precursor phases are not well defined, but octacalcium phosphate-like, amorphous calcium phosphate-like, and HPO₄^2--enriched phases were detected. Here we use in vivo Raman spectroscopy and high-resolution wide-angle X-ray diffraction (WAXD) to characterize and map at 2 μm resolution the mineral phases in the rapidly forming tail fin bones of living zebrafish larvae and zebrafish larvae immediately after sacrifice, respectively. Raman spectroscopy shows the presence of an acidic disordered calcium phosphate phase with additional characteristic features of HPO₄^2- at the bone-cell interface. The complexity in the position and shape of the ν₁ PO₄ peak viewed by in vivo Raman spectroscopy emphasizes the heterogeneity of the mineral during bone formation. WAXD detects an additional isolated peak, appearing alone or together with the characteristic diffraction pattern of carbonated hydroxyapatite. This unidentified phase is located at the interface between the mature bone and the surrounding tissue, similar to the location at which the disordered phase was observed by Raman spectroscopy. The variable peak positions and profiles support the notion that this is an unstable disordered precursor phase, which conceivably crystallized during the X-ray diffraction measurement. Interestingly, this precursor phase is co-aligned with the c-axes of the mature bone crystals and thus is in intimate relation with the surrounding collagen matrix. We conclude that a major disordered precursor mineral phase containing HPO₄^2- is part of the deposition pathway of the rapidly forming tail fin bones of the zebrafish.
Addadi L., Kerschnitzki M., Akiva A., Ben Shoham S. A., Asscher Y., Wagermaier W., Fratzl P. & Weiner S. 2016. Bone mineralization pathways during the rapid growth of embryonic chicken long bones Journal of Structural Biology. 2016 Jul , 195 (1):82-92.

[Abstract]

The uptake and transport of ions from the environment to the site of bone formation is only partially understood and, for the most part, based on disparate observations in different animals. Here we study different aspects of the biomineralization pathways in one system, the rapidly forming long bones of the chicken embryo. We mainly used cryo-fixation and cryo-electron imaging to preserve the often unstable mineral phases in the tissues. We show the presence of surprisingly large amounts of mineral particles located inside membrane-delineated vesicles in the bone forming tissue between the blood vessels and the forming bone surface. Some of these particles are also located inside mitochondrial networks. The surfaces of the forming bones in the extracellular space contain abundant aggregates of amorphous calcium phosphate particles, but these are not enveloped by vesicle membranes. In the bone resorbing region, osteoclasts also contain many particles in both mitochondrial networks and within vesicles. Some of these particles are present also between cells. These observations, together with the previously reported observation that CaP mineral particles inside membranes are present in blood vessels, leads us to the conclusion that important components of the bone mineralization pathways in rapidly forming chicken bone are dense phase mineral particles bound within membranes. It remains to be determined whether these mineral particles are transported to the site of bone formation in the solid state, fluid state or dissolve and re-precipitate.
Kerschnitzki M., Akiva A., Ben Shoham S. A., Koifman N., Shimoni E., Rechav K., Arraf A. A., Schultheiss T. M., Talmon Y., Zelzer E., Weiner S. & Addadi L. 2016. Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development Bone. 2016 , 83 :65-72.

[Abstract]

During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself.
Khalifa G. M., Kirchenbuechler D., Koifman N., Kleinerman O., Talmon Y., Elbaum M., Addadi L., Weiner S. & Erez J. 2016. Biomineralization pathways in a foraminifer revealed using a novel correlative cryo-fluorescence–SEM–EDS technique Journal of Structural Biology. 2016 Nov , 196 (2):155-163.

[Abstract]

Foraminifera are marine protozoans that are widespread in oceans throughout the world. Understanding biomineralization pathways in foraminifera is particularly important because their calcitic shells are major components of global calcium carbonate production. We introduce here a novel correlative approach combining cryo-SEM, cryo-fluorescence imaging and cryo-EDS. This approach is applied to the study of ion transport processes in the benthic foraminifer genus Amphistegina. We confirm the presence of large sea water vacuoles previously identified in intact and partially decalcified Amphistegina lobifera specimens. We observed relatively small vesicles that were labelled strongly with calcein, and also identified magnesium (Mg)-rich mineral particles in the cytoplasm, as well as in the large sea water vacuoles. The combination of cryo-microscopy with elemental microanalysis and fluorescence imaging reveals new aspects of the biomineralization pathway in foraminifera which are, to date, unique in the world of biomineralization. This approach is equally applicable to the study of biomineralization pathways in other organisms.
Gur D., Pierantoni M., Dov N. E., Hirsh A., Feldman Y. (., Weiner S. & Addadi L. 2016. Guanine Crystallization in Aqueous Solutions Enables Control over Crystal Size and Polymorphism Crystal Growth & Design. 2016 Sept , 16 (9):4975-4980.

[Abstract]

Anhydrous guanine crystals are among the most widespread organic crystals used by organisms to produce structural colors. The main advantage of guanine is its exceptionally high refractive index in the reflecting direction (∼1.8). For the same reason, guanine is a promising candidate material for a variety of different optical applications. Crystallization of guanine is challenging and usually involves using polar aprotic organic solvents such as dimethyl sulfoxide (DMSO). Here, we show that the crystallization of guanine from aqueous solutions is possible under conditions that provide control over crystal polymorphism and size. Using this approach we were able produce large crystals of the elusive guanine monohydrate phase. We were also able to rationalize the formation of the different phases obtained as a function of which tautomer of guanine is stable in solutions of varying pH.
Addadi L., Gal A., Faivre D., Scheffel A. & Weiner S. 2016. Control of Biogenic Nanocrystal Formation in Biomineralization Israel Journal of Chemistry. 2016 Apr , 56 (4):227-241.

[Abstract]

Many organisms build crystals with almost complete control over all aspects of crystal formation, from nucleation to growth, from composition to polymorphic structure, and from morphology to size. In biomineralization, the control is fundamentally always exerted at the level of nanometers, because the building blocks themselves are at the nanoscale. We have chosen to describe in some detail four biological systems that produce nanoscale crystals using different mineralization pathways, different levels of control, and have different functions. These four cases are: bone crystal composites; guanine nanocrystal reflectors; magnetotactic bacteria with single domain magnets; and coccoliths, whose functions have yet to be identified. This is followed by a discussion aimed at identifying possible specific and/or common underlying principles involved in nanocrystal formation in biology.
Gal A., Weiner S. & Addadi L. 2015. A perspective on underlying crystal growth mechanisms in biomineralization: Solution mediated growth versus nanosphere particle accretion CrystEngComm. 2015 Apr , 17 (13):2606-2615.

[Abstract]

Many organisms form crystals from transient amorphous precursor phases. In cases where the precursor phases were imaged, they were seen to consist of nanosphere particles. Interestingly, some mature biogenic crystals also have a nanosphere particle morphology, but some are characterized by crystallographic faces that are smooth at the nanometer level. There are also biogenic crystals that have both crystallographic faces and nanosphere particle morphology. This highlight presents a working hypothesis, stating that some biomineralization processes involve growth by nanosphere particle accretion, where amorphous nanoparticles are incorporated as such into growing crystals and their morphology is preserved upon crystallization. This process produces biogenic crystals with a nanosphere particle morphology. Other biomineralization processes proceed by ion-by-ion growth, and some cases of biological crystal growth involve both processes. We also identify several biomineralization processes which do not seem to fit this working hypothesis. It is our hope that this highlight will inspire studies that will shed more light on the underlying crystallization mechanisms in biology.
Gur D., Palmer B. A., Leshem B., Oron D., Fratzl P., Weiner S. & Addadi L. 2015. The Mechanism of Color Change in the Neon Tetra Fish: A Light-Induced Tunable Photonic Crystal Array Angewandte Chemie (International ed. in English). 2015 Apr , 54 (42):12426-12430.

[Abstract]

The fresh water fish neon tetra has the ability to change the structural color of its lateral stripe in response to a change in the light conditions, from blue-green in the light-adapted state to indigo in the dark-adapted state. The colors are produced by constructive interference of light reflected from stacks of intracellular guanine crystals, forming tunable photonic crystal arrays. We have used micro X-ray diffraction to track in time distinct diffraction spots corresponding to individual crystal arrays within a single cell during the color change. We demonstrate that reversible variations in crystal tilt within individual arrays are responsible for the light-induced color variations. These results settle a long-standing debate between the two proposed models, the "Venetian blinds" model and the "accordion" model. The insight gained from this biogenic light-induced photonic tunable system may provide inspiration for the design of artificial optical tunable systems.
Vidavsky N., Masic A., Schertel A., Weiner S. & Addadi L. 2015. Mineral-bearing vesicle transport in sea urchin embryos Journal of Structural Biology. 2015 Dec , 192 (3):358-365.

[Abstract]

Sea urchin embryos sequester calcium from the sea water. This calcium is deposited in a concentrated form in granule bearing vesicles both in the epithelium and in mesenchymal cells. Here we use in vivo calcein labeling and confocal Raman spectroscopy, as well as cryo-FIB-SEM 3D structural reconstructions, to investigate the processes occurring in the internal cavity of the embryo, the blastocoel. We demonstrate that calcein stained granules are also present in the filopodial network within the blastocoel. Simultaneous fluorescence imaging and Raman spectroscopy show that these granules do contain a calcium mineral. By tracking the movements of these granules, we show that the granules in the epithelium and primary mesenchymal cells barely move, but those in the filopodial network move long distances. We could however not detect any unidirectional movement of the filopodial granules. We also show the presence of mineral containing multivesicular vesicles that also move in the filopodial network. We conclude that the filopodial network is an integral part of the mineral transport process, and possibly also for sequestering calcium and other ions. Although much of the sequestered calcium is deposited in the mineralized skeleton, a significant amount is used for other purposes, and this may be temporarily stored in these membrane-delineated intracellular deposits.
Weiner S., Brumfeld V., Marder O. & Barzilai O. 2015. Heating of flint debitage from Upper Palaeolithic contexts at Manot Cave, Israel: Changes in atomic organization due to heating using infrared spectroscopy Journal of Archaeological Science. 2015 Feb , 54 :45-53.

[Abstract]

The heat treatment of flint is known to change its mechanical properties and improve its fracture behaviour during knapping. Here we examine 20 flint artifacts from Upper Paleolithic contexts from Manot Cave, Israel, using Fourier transform infrared spectroscopy and compare them to geogenic flint beds from the walls inside the cave and from outcrops just above the cave. We show that the 512 and 467cm^-1 peaks are broader in most of the flint debitage pieces as compared to the geogenic flint, and that broadening of these peaks occurs when geogenic flint from the cave wall is heated. We also present an empirical simple method to monitor these changes.
Akiva A., Malkinson G., Masic A., Kerschnitzki M., Bennet M., Fratzl P., Addadi L., Weiner S. & Yaniv K. 2015. On the pathway of mineral deposition in larval zebrafish caudal fin bone Bone. 2015 Jun , 75 :192-200.

[Abstract]

A poorly understood aspect of bone biomineralization concerns the mechanisms whereby ions are sequestered from the environment, concentrated, and deposited in the extracellular matrix. In this study, we follow mineral deposition in the caudal fin of the zebrafish larva in vivo. Using fluorescence and cryo-SEM-microscopy, in combination with Raman and XRF spectroscopy, we detect the presence of intracellular mineral particles located between bones, and in close association with blood vessels. Calcium-rich particles are also located away from the mineralized bone, and these are also in close association with blood vessels. These observations challenge the view that mineral formation is restricted to osteoblast cells juxtaposed to bone, or to the extracellular matrix. Our results, derived from observations performed in living animals, contribute a new perspective to the comprehensive mechanism of bone formation in vertebrates, from the blood to the bone. More broadly, these findings may shed light on bone mineralization processes in other vertebrates, including humans.
Asscher Y., Lehmann G., Rosen S. A., Weiner S. & Boaretto E. 2015. Absolute dating of the late bronze to iron age transition and the appearance of philistine culture in Qubur el-Walaydah, southern Levant Radiocarbon. 2015 , 57 (1):77-97.

[Abstract]

The Late Bronze Age to the Iron Age transition involves profound cultural and political changes in the southern Levant. The transition is dated to the 12th century BC, based on archaeological artifacts and historical documents. A more precise absolute date for this transition for the southern Levant based on radiocarbon is diffcult since the¹⁴C calibration curve reduces precision signifcantly due to wiggles that form an approximately 200-yr-long plateau. This article analyzes¹⁴C samples from the Late Bronze Age to the Iron Age transition at Qubur el-Walaydah. To increase the resolution of¹⁴C dates within the plateau,¹⁴C samples were collected only from well-defned multilayered contexts.¹⁴C dates from 11 contexts were obtained and these were analyzed using a Bayesian model that incorporated the stratigraphic information. Using this integra-tive approach we date the Late Bronze Age III levels at Qubur el-Walydah, containing the initial phase of locally produced Philistine pottery between 1185–1140 BC, and the Late Bronze to Iron Age transition between 1140–1095 BC.
Reznikov N., Chase H., Brumfeld V., Shahar R. & Weiner S. 2015. The 3D structure of the collagen fibril network in human trabecular bone: Relation to trabecular organization Bone. 2015 Feb , 71 :189-195.

[Abstract]

Trabecular bone is morphologically and functionally different from compact bone at the tissue level, but both are composed of lamellae at the micrometer-scale level. We present a three-dimensional study of the collagenous network of human trabecular lamellar bone from the proximal femur using the FIB-SEM serial surface view method. The results are compared to human compact lamellar bone of the femoral shaft, studied by the same method. Both demineralized trabecular and compact lamellar bone display the same overall structural organization, namely the presence of ordered and disordered materials and the confinement of the canalicular network to the disordered material. However, in trabecular bone lamellae a significant proportion of the ordered collagen fibril arrays is aligned with the long axis of the trabecula and, unlike in compact bone, is not related to the anatomical axis of the whole femur. The remaining ordered collagen fibrils are offset from the axis of a trabecula either by about 30° or 70°. Interestingly, at the tissue scale of millimeters, the most abundant angles between any two connected trabeculae - the inter-trabecular angles - center around 30° and 70°. This implies that within a framework of interconnected trabeculae the same lamellar structure will always have a significant component of the fibrils aligned with the long axes of connected trabeculae. This structural complementarity at different hierarchical levels presumably reflects an adaptation of trabecular bone to function.
Naveh G. & Weiner S. 2015. Initial orthodontic tooth movement of a multirooted tooth: A 3D study of a rat molar ORTHODONTICS & CRANIOFACIAL RESEARCH. 2015 Aug , 18 (3):134-142.

[Abstract]

Objective: To elucidate the 3D interactions in the tooth-PDL-bone complex immediately after application of orthodontic forces and their implications on tooth movement and function. Methods: A special visualization method using microCT allows us to directly image in 3D the movements of a multirooted molar tooth inside the alveolar bone as well as the collagenous network of the PDL. Using fresh, unstained rat mandibular 1st molar under mesial loads of 0.5-1 N, we address basic concepts in orthodontics during the initial stages of orthodontic movement. Results: We show that immediately after the application of orthodontic load, direct distinct contacts between the tooth and the bone form in the furcation area. These contacts limit tooth movement and interfere with whole body translation. Only localized sites of highly compressed PDL between the root surfaces and the bone were observed. In general, the collagenous network of the PDL appeared loose and not densely packed in the compressed side. On the tension side, the fibers maintained their overall orientation without any significant extension of the fibers. Conclusions: Localized direct contact areas between the tooth roots and the bone at the furcation already form within a few minutes of orthodontic tooth movement. This direct and localized bone involvement guides the movement trajectory and provides a mechanism for the miscorrelation found between force levels and tooth movement during the initial stages of an orthodontic tooth movement.
Zou Z., Bertinetti L., Politi Y., Jensen A. C. S., Weiner S., Addadi L., Fratzl P. & Habraken W. J. E. M. 2015. Opposite Particle Size Effect on Amorphous Calcium Carbonate Crystallization in Water and during Heating in Air Chemistry of Materials. 2015 Jun , 27 (12):4237-4246.

[Abstract]

Calcium carbonate is a common constituent of many natural materials, such as shells and skeletons of marine animals. While it is well-documented that additives (organic and inorganic) modulate the crystallization of amorphous calcium carbonate (ACC), the effects of the intrinsic physicochemical characteristics of ACC, such as particle size, shape, and water content on the transformation to crystalline polymorphs, are still poorly understood. Here, we investigate the effect of particle size by preparing ACC nanoparticles with an average size ranging from ∼66 to ∼196 nm using a high-resolution titration setup. Our results show that the particle size determined the polymorph selection in solution; an increasing proportion of vaterite to calcite was observed with decreasing particle size. The polymorph selection was ascribed to a higher apparent solubility of ACC with decreasing particle size, a parameter from which we could determine the surface energy of ACC to be ∼0.33 J/m². Upon heating, particle size showed the opposite effect, as smaller particles favored a higher crystallization temperature from ACC into (only) calcite. When the particle size was large enough, crystallization occurred concomitantly with the removal of bulk water at lower temperatures, where the smallest particles transformed at ∼310°C, only after losing the final (surface) water. Our results highlight the importance of particle size as well as the crystallization conditions on the stability and transformation mechanisms of ACC.
Atkins A., Reznikov N., Ofer L., Masic A., Weiner S. & Shahar R. 2015. The three-dimensional structure of anosteocytic lamellated bone of fish Acta Biomaterialia. 2015 Feb , 13 :311-323.

[Abstract]

Fish represent the most diverse and numerous of the vertebrate clades. In contrast to the bones of all tetrapods and evolutionarily primitive fish, many of the evolutionarily more advanced fish have bones that do not contain osteocytes. Here we use a variety of imaging techniques to show that anosteocytic fish bone is composed of a sequence of planar layers containing mainly aligned collagen fibrils, in which the prevailing principal orientation progressively spirals. When the sequence of fibril orientations completes a rotation of around 180°, a thin layer of poorly oriented fibrils is present between it and the next layer. The thick layer of aligned fibrils and the thin layer of non-aligned fibrils constitute a lamella. Although both basic components of mammalian lamellar bone are found here as well, the arrangement is unique, and we therefore call this structure lamellated bone. We further show that the lamellae of anosteocytic fish bone contain an array of dense, small-diameter (1-4 μm) bundles of hypomineralized collagen fibrils that are oriented mostly orthogonal to the lamellar plane. Results of mechanical tests conducted on beams from anosteocytic fish bone and human cortical bone show that the fish bones are less stiff but much tougher than the human bones. We propose that the unique lamellar structure and the orthogonal hypomineralized collagen bundles are responsible for the unusual mechanical properties and mineral distribution in anosteocytic fish bone.
Kababya S., Gal A., Kahil K., Weiner S., Addadi L. & Schmidt A. 2015. Phosphate-water interplay tunes amorphous calcium carbonate metastability: Spontaneous phase separation and crystallization Vs stabilization viewed by solid state NMR Journal of the American Chemical Society. 2015 Jan , 137 (2):990-998.

[Abstract]

Organisms tune the metastability of amorphous calcium carbonates (ACC), often by incorporation of additives such as phosphate ions and water molecules, to serve diverse functions, such as modulating the availability of calcium reserves or constructing complex skeletal scaffolds. Although the effect of additive distribution on ACC was noted for several biogenic and synthetic systems, the molecular mechanisms by which additives govern ACC stability are not well understood. By precipitating ACC in the presence of different PO₄^3- concentrations and regulating the initial water content, we identify conditions yielding either kinetically locked or spontaneously transforming coprecipitates. Solid state NMR, supported by FTIR, XRD, and electron microscopy, define the interactions of phosphate and water within the initial amorphous matrix, showing that initially the coprecipitates are homogeneous molecular dispersions of structural water and phosphate in ACC, and a small fraction of P-rich phases. Monitoring the transformations of the homogeneous phase shows that PO₄^3- and waters are extracted first, and they phase separate, leading to solid-solid transformation of ACC to calcite; small part of ACC forms vaterite that subsequently converts to calcite. The simultaneous water-PO₄^3- extraction is the key for the subsequent water-mediated accumulation and crystallization of hydroxyapatite (HAp) and carbonated hydroxyapatite. The thermodynamic driving force for the transformations is calcite crystallization, yet it is gated by specific combinations of water-phosphate levels in the initial amorphous coprecipitates. The molecular details of the spontaneously transforming ACC and of the stabilized ACC modulated by phosphate and water at ambient conditions, provide insight into biogenic and biomimetic pathways.
Gur D., Leshem B., Pierantoni M., Farstey V., Oron D., Weiner S. & Addadi L. 2015. Structural Basis for the Brilliant Colors of the Sapphirinid Copepods Journal of the American Chemical Society. 2015 Jul , 137 (26):8408-8411.

[Abstract]

Males of sapphirinid copepods use regularly alternating layers of hexagonal-shaped guanine crystals and cytoplasm to produce spectacular structural colors. In order to understand the mechanism by which the different colors are produced, we measured the reflectance of live individuals and then characterized the organization of the crystals and the cytoplasm layers in the same individuals using cryo-SEM. On the basis of these measurements, we calculated the expected reflectance spectra and found that they are strikingly similar to the measured ones. We show that variations in the cytoplasm layer thickness are mainly responsible for the different reflected colors and also that the copepod color strongly depends on the angular orientation relative to the incident light, which can account for its appearance and disappearance during spiral swimming in the natural habitat.
Shepelenko M., Brumfeld V., Cohen S., Klein E., Lubinevsky H., Addadi L. & Weiner S. 2015. The gizzard plates in the Cephalaspidean gastropod Philine quadripartita: Analysis of structure and function Quaternary International. 2015 Dec , 390 :4-14.

[Abstract]

Cephalaspidean gastropods are common marine mollusks with a unique digestive apparatus containing 3 hardened plates of millimeter size inside the muscular esophageal crop (gizzard). The gizzard plates are reported to either grind or crush shelled prey. The current study aims at better understanding the manner in which the gizzard plates of the cephalaspid Philine quadripartita function in the overall digestion process by relating their structural and mechanical properties. Philine quadripartita possesses 3 gizzard plates which have one of the common configurations of cephalaspidean gizzard plates: two paired plates that are mirror images of each other and one smaller unpaired plate. We used micro-CT to characterize the gizzard musculature, the food which is present at different stages of the digestion process and the working surface of the gizzard plates. We show that the gizzard plates are used to crush the shelled prey, and that the functional mode of the small unpaired plate is different from the larger plates. All 3 plates are composed of a mixture of amorphous calcium carbonate and amorphous calcium phosphate embedded in a chitinous matrix. The proportions of these two mineral phases vary systematically within the plate. The plates have a complex layered structure, whose elastic moduli and hardness also vary in a continuous systematic manner. We observed that the stiffest layer is below the working surface, unlike most teeth where the stiffest layer is at the surface. Rigorous analysis of the elasticity indices of the gizzard plates as compared with sea urchin teeth and synthetic calcite provided insights into the connection between the biological function and the mechanical properties of biological composites. Specifically, we show that materials used for grinding require harder surfaces to avoid excessive wear compared to materials for crushing, whereas both of these functions require high toughness.
Finkelstein I., Weiner S. & Boaretto E. 2015. Preface—The iron age in Israel: The exact and life sciences perspectives Radiocarbon. 2015 , 57 (2):197-206.

[Abstract]

In the original proposal entitled Reconstructing Ancient Israel – The Exact and Life Sciences Perspective, two of us (Israel Finkelstein and Steve Weiner) wrote, “If the microscopic data are well integrated into the macroscopic (archaeological) record, they will undoubtedly provide new insights into the study of Ancient Israel.” And this was what this 5-year (2009–2014) European Research Council (ERC) sponsored program (details below) was all about. New ground was broken on three fronts: conceptual, methodological, and in the generation of new data that indeed provide novel insights into the history and material culture of Ancient Israel in particular and the Iron Age Levant in general. The reviews presented in this special volume synthesize some of these new insights. The findings have been published in about 70 papers (see Appendix).
Atkins A., Milgram J., Weiner S. & Shahar R. 2015. The response of anosteocytic bone to controlled loading Journal of Experimental Biology. 2015 Nov , 218 (22):3559-3569.

[Abstract]

The bones of the skeleton of most advanced teleost fish do not contain osteocytes. Considering the pivotal role assigned to osteocytes in the process of modeling and remodeling (the adaptation of external and internal bone structure and morphology to external loads and the repair of areas with micro-damage accumulation, respectively) it is unclear how, and even whether, their skeleton can undergo modeling and remodeling. Here, we report on the results of a study of controlled loading of the anosteocytic opercula of tilapia (Oreochromis aureus). Using a variety of microscopy techniques we show that the bone of the anosteocytic tilapia actively adapts to applied loads, despite the complete absence of osteocytes. We show that in the directly loaded area, the response involves a combination of bone resorption and bone deposition; we interpret these results and the structure of the resultant bone tissue to mean that both modeling and remodeling are taking place in response to load. We further show that adjacent to the loaded area, new bone is deposited in an organized, layered manner, typical of a modeling process. The material stiffness of the newly deposited bone is higher than that of the bone which was present prior to loading. The absence of osteocytes requires another candidate cell for mechanosensing and coordinating the modeling process, with osteoblasts seeming the most likely candidates.
Asscher Y., Cabanes D., Hitchcock L., Maeir A., Weiner S. & Boaretto E. 2015. Radiocarbon dating shows an early appearance of Philistine material culture in Tell es-Safi/Gath, Philistia Radiocarbon. 2015 , 57 (5):825-850.

[Abstract]

The Late Bronze Age to Iron Age transition in the coastal southern Levant involves a major cultural change, which is characterized, among other things, by the appearance of Philistine pottery locally produced in styles derived from outside the Levant. This transition in the coastal southern Levant is conventionally dated to the 12th century BC, based on historical and archaeological artifacts associated with the Philistine pottery. Radiocarbon dating can provide a more precise independent absolute chronology for this transition, but dating for the period under discussion is complicated by the wiggles and relatively flat slope in the calibration curve, which significantly reduce precision. An additional complication is that the stratigraphic record below and above the transition at this site, as well as at most other sites in the region, is far from complete. We thus used a variety of microarchaeological techniques to improve our understanding of the stratigraphy, and to ensure that the locations with datable short-lived materials were only derived from primary contexts, which could be related directly to the associated material culture. The ¹⁴C dates were modeled using Bayesian statistics that incorporate the stratigraphic information. Using this integrative approach, we date the appearance of the Philistine pottery in Tell es-Safi/Gath in the 13th century BC.
Reznikov N., Shahar R. & Weiner S. 2014. Three-dimensional structure of human lamellar bone: The presence of two different materials and new insights into the hierarchical organization Bone. 2014 Feb , 59 :93-104.

[Abstract]

Lamellar bone is the most common bone type in humans. The predominant components of individual lamellae are plywood-like arrays of mineralized collagen fibrils aligned in different directions. Using a dual-beam electron microscope and the Serial Surface View (SSV) method we previously identified a small, but significantly different layer in rat lamellar bone, namely a disordered layer with collagen fibrils showing little or no preferred orientation. Here we present a 3D structural analysis of 12 SSV volumes (25 complete lamellae) from femora of 3 differently aged human individuals. We identify the ordered and disordered motifs in human bone as in the rat, with several significant differences. The ordered motif shows two major preferred orientations, perpendicular to the long axis of the bone, and aligned within 10-20° of the long axis, as well as fanning arrays. At a higher organizational level, arrays of ordered collagen fibrils are organized into 'rods' around 2 to 3. μm in diameter, and the long axes of these 'rods' are parallel to the lamellar boundaries. Human bone also contains a disordered component that envelopes the rods and fills in the spaces between them. The disordered motif is especially well-defined between adjacent layers of rods. The disordered motif and its interfibrillar substance stain heavily with osmium tetroxide and Alcian blue indicating the presence of another organic component in addition to collagen. The canalicular network is confined to the disordered material, along with voids and individual collagen fibrils, some of which are also aligned more or less perpendicular to the lamellar boundaries. The organization of the ordered fibril arrays into rods enveloped in the continuous disordered structure was not observed in rat lamellar bone. We thus conclude that human lamellar bone is comprised of two distinct materials, an ordered material and a disordered material, and contains an additional hierarchical level of organization composed of arrays of ordered collagen fibrils, referred to as rods. This new structural information on human lamellar bone will improve our understanding of structure-mechanical function relations, mechanisms of mechano-sensing and the characterizations of bone pathologies.
Reznikov N., Shahar R. & Weiner S. 2014. Bone hierarchical structure in three dimensions Acta Biomaterialia. 2014 Sept , 10 (9):3815-3826.

[Abstract]

Bone is a complex hierarchically structured family of materials that includes a network of cells and their interconnected cell processes. New insights into the 3-D structure of various bone materials (mainly rat and human lamellar bone and minipig fibrolamellar bone) were obtained using a focused ion beam electron microscope and the serial surface view method. These studies revealed the presence of two different materials, the major material being the well-known ordered arrays of mineralized collagen fibrils and associated macromolecules, and the minor component being a relatively disordered material composed of individual collagen fibrils with no preferred orientation, with crystals inside and possibly between fibrils, and extensive ground mass. Significantly, the canaliculi and their cell processes are confined within the disordered material. Here we present a new hierarchical scheme for several bone tissue types that incorporates these two materials. The new scheme updates the hierarchical scheme presented by Weiner and Wagner (1998). We discuss the structures at different hierarchical levels with the aim of obtaining further insights into structure-function-related questions, as well as defining some remaining unanswered questions.
Vidavsky N., Addadi S., Mahamid J., Shimoni E., Ben-Ezra D., Shpigel M., Weiner S. & Addadi L. 2014. Initial stages of calcium uptake and mineral deposition in sea urchin embryos Proceedings of the National Academy of Sciences of the United States of America. 2014 , 111 (1):39-44.

[Abstract]

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo- SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5-1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20-30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule.
Goldenberg L., Neumann R. & Weiner S. 2014. Microscale distribution and concentration of preserved organic molecules with carbon-carbon double bonds in archaeological ceramics: Relevance to the field of residue analysis Journal of Archaeological Science. 2014 Feb , 42 (1):509-518.

[Abstract]

Biologically produced compounds preserved in ancient ceramics can provide invaluable information on the vessel contents. Analysis and interpretation of these so-called archaeological "residues" is therefore important for understanding and reconstructing aspects of social and cultural behaviors of ancient societies. Based on the reaction of unsaturated compounds with iodine, we developed and apply two new methods. The first is a simple and relatively rapid method for assessing the amounts of unsaturated compounds in archaeological ceramics using X-ray fluorescence (XRF). We show that this method is a reliable indicator for assessing the general preservation state of the organic material and therefore a potential pre-screening method for identifying ceramic samples suitable for organic residue analysis. The second complementary approach, based on the same iodine reaction, makes it possible to map the unsaturated molecules on ceramic surfaces at a scale that enables to correlate organic matter distribution and the underlying mineral grains using a scanning electron microscope (SEM) equipped with an energy dispersive spectrometer (EDS) detector. With this method we show that the extent of adsorption of lipids onto ceramic surfaces varies with the surface properties of the different minerals in the ancient ceramic, with calcium containing minerals showing the highest affinity for compounds with unsaturated bonds. The ceramic substrate therefore influences the types of organic compounds bound and hence preserved in the ceramic. Fundamental information obtained using this method is essential for better interpreting molecular assemblages extracted from archaeological ceramics.
Hirsch A., Azuri I., Addadi L., Weiner S., Yang K., Curtarolo S. & Kronik L. 2014. Infrared absorption spectrum of brushite from first principles Chemistry of Materials. 2014 May , 26 (9):2934-2942.

[Abstract]

Brushite, CaHPO₄·2H₂O, is a crystalline hydrated acidic form of calcium phosphate that occurs in both physiological and pathological biomineralization processes. Additionally, it is biocompatible in humans. Several groups have investigated the experimental Fourier transform infrared vibrational spectrum of brushite. These investigations have led to a long-standing debate concerning the correct assignment for a few of the observed frequencies, particularly, the water stretching and libration modes. Here, we perform a comprehensive first principles theoretical investigation of the vibrational spectrum of brushite with calculations based on dispersion-corrected density functional theory. We obtain both the vibrational frequencies, using the frozen phonon approach, and their corresponding peak intensities, based on the Born effective charge tensor. This allows for an unambiguous assignment of all vibrations, including water vibration and libration modes.
Addadi L. & Weiner S. 2014. Biomineralization: Mineral formation by organisms PHYSICA SCRIPTA. 2014 , 89 (9).

[Abstract]

Organisms form many different types of minerals, with diverse shapes and sizes. These minerals fulfill a variety of functions. Inspired by the late H A Lowenstam, Steve Weiner and Lia Addadi have addressed many questions that relate to the mechanisms by which biological organisms produce these mineral phases and how their structures relate to their functions. Addadi and Weiner have explored the manner in which macromolecules extracted from mineralized tissues can interact with some crystal planes and not others, how these macromolecules can be occluded inside the forming crystals residing preferentially on specific crystal planes, and how they can induce one polymorph of calcium carbonate and not another to nucleate. Addadi and Weiner have also identified a novel strategy used by the sea urchin to form its smooth and convoluted mineralized skeletal elements. The strategy involves the initial production by cells of a highly disordered mineral precursor phase in vesicles, and then the export of this so-called amorphous phase to the site of skeletal formation, where it crystallizes. This strategy is now known to be used by many different invertebrate phyla, as well as by vertebrates to build bones and teeth. One of the major current research aims of the Weiner - Addadi group is to understand the biomineralization pathways whereby ions are extracted from the environment, are transported and deposited inside cells within vesicles, how these disordered phases are then transferred to the site of skeletal formation, and finally how the so-called amorphous phase crystallizes. Biology has clearly evolved unique strategies for forming crystalline minerals. Despite more than 300 years of research in this field, many challenging questions still remain unanswered.
Gal A., Kahil K., Vidavsky N., DeVol R. T., Gilbert P. U. P. A., Fratzl P., Weiner S. & Addadi L. 2014. Particle accretion mechanism underlies biological crystal growth from an amorphous precursor phase Advanced Functional Materials. 2014 Sept , 24 (34):5420-5426.

[Abstract]

Many biogenic minerals are composed of aggregated particles at the nanoscale. These minerals usually form through the transformation of amorphous precursors into single crystals inside a privileged space controlled by the organism. Here, in vitro experiments aimed at understanding the factors responsible for producing such single crystals with aggregated particle texture are presented. Crystallization is achieved by a two-step reaction in which amorphous calcium carbonate (ACC) is first precipitated and then transformed into calcite in small volumes of water and in the presence of additives. The additives used are gel-forming molecules, phosphate ions, and the organic extract from sea urchin embryonic spicules - all are present in various biogenic crystals that grow via the transformation of ACC. Remarkably, this procedure yields faceted single-crystals of calcite that maintain the nanoparticle texture. The crystals grow predominantly by the accretion of ACC nanoparticles, which subsequently crystallize. Gels and phosphate ions stabilize ACC via a different mechanism than sea urchin spicule macromolecules. It is concluded that the unique nanoparticle texture of biogenic minerals results from formation pathways that may differ from one another, but given the appropriate precursor and micro-environment, share a common particle accretion mechanism.
Atkins A., Dean M. N., Habegger M. L., Motta P. J., Ofer L., Repp F., Shipov A., Weiner S., Currey J. D. & Shahar R. 2014. Remodeling in bone without osteocytes: Billfish challenge bone structure-function paradigms Proceedings of the National Academy of Sciences of the United States of America. 2014 Nov , 111 (45):16047-16052.

[Abstract]

A remarkable property of tetrapod bone is its ability to detect and remodel areas where damage has accumulated through prolonged use. This process, believed vital to the long-term health of bone, is considered to be initiated and orchestrated by osteocytes, cells within the bone matrix. It is therefore surprising that most extant fishes (neoteleosts) lack osteocytes, suggesting their bones are not constantly repaired, although many species exhibit long lives and high activity levels, factors that should induce considerable fatigue damage with time. Here, we show evidence for active and intense remodeling occurring in the anosteocytic, elongated rostral bones of billfishes (e.g., swordfish, marlins). Despite lacking osteocytes, this tissue exhibits a striking resemblance to the mature bone of large mammals, bearing structural features (overlapping secondary osteons) indicating intensive tissue repair, particularly in areas where high loads are expected. Billfish osteons are an order of magnitude smaller in diameter than mammalian osteons, however, implying that the nature of damage in this bone may be different. Whereas billfish bone material is as stiff as mammalian bone (unlike the bone of other fishes), it is able to withstand much greater strains (relative deformations) before failing. Our data show that fish bone can exhibit far more complex structure and physiology than previously known, and is apparently capable of localized repair even without the osteocytes believed essential for this process. These findings challenge the unique and primary role of osteocytes in bone remodeling, a basic tenet of bone biology, raising the possibility of an alternative mechanism driving this process.
Magal R. A., Reznikov N., Shahar R. & Weiner S. 2014. Three-dimensional structure of minipig fibrolamellar bone: Adaptation to axial loading Journal of Structural Biology. 2014 May , 186 (2):253-264.

[Abstract]

Fibrolamellar bone is transiently produced by large, fast growing mammals. The fibrolamellar bone unit is initially formed by elaboration of a network of blood vessels. This is followed by the deposition of a thin, porous and hypercalcified layer, then by the infilling of the vascular cavities by the sequential deposition of a relatively thick rapidly forming bone on both sides of the hypercalcified layer, and finally by lamellar bone. We investigated the 3D structure of the collagenous network of fibrolamellar bone from the femora of a young minipig using mainly the FIB-SEM dual beam microscope and the Serial Surface View method. This enabled us to identify the fibril orientation, the canalicular network organization and other structural motifs within each element of the fibrolamellar unit. The first formed primary hypercalcified layer (PHL) is composed of fibril arrays and multiple small pores, and appears to have an isotropic structure. The major bone component is deposited on both sides of the PHL, and is composed of collagen fibrils with a preferred orientation, mainly aligned parallel to the bone long axis. This bone component is therefore parallel-fibered bone and not woven bone. We also observed that the collagen fibers are organized into bundles. The lamellar bone has most of its collagen fibrils aligned with the bone long axis. This study therefore shows that the large majority of collagen fibrils in fibrolamellar bone are aligned with the bone long axis. This anisotropic structure therefore appears to be adapted to loading along the bone long axis.
DeVol R. T., Metzler R. A., Kabalah-Amitai L., Pokroy B., Politi Y., Gal A., Addadi L., Weiner S., Fernandez-Martinez A., Demichelis R., Gale J. D., Ihli J., Meldrum F. C., Blonsky A. Z., Killian C. E., Salling C. B., Young A. T., Marcus M. A., Scholl A., Doran A., Jenkins C., Bechtel H. A. & Gilbert P. U. P. A. 2014. Oxygen spectroscopy and polarization-dependent imaging contrast (PIC)-mapping of calcium carbonate minerals and biominerals Journal Of Physical Chemistry B. 2014 Jul , 118 (28):8449-8457.

[Abstract]

X-ray absorption near-edge structure (XANES) spectroscopy and spectromicroscopy have been extensively used to characterize biominerals. Using either Ca or C spectra, unique information has been obtained regarding amorphous biominerals and nanocrystal orientations. Building on these results, we demonstrate that recording XANES spectra of calcium carbonate at the oxygen K-edge enables polarization-dependent imaging contrast (PIC) mapping with unprecedented contrast, signal-to-noise ratio, and magnification. O and Ca spectra are presented for six calcium carbonate minerals: aragonite, calcite, vaterite, monohydrocalcite, and both hydrated and anhydrous amorphous calcium carbonate. The crystalline minerals reveal excellent agreement of the extent and direction of polarization dependences in simulated and experimental XANES spectra due to X-ray linear dichroism. This effect is particularly strong for aragonite, calcite, and vaterite. In natural biominerals, oxygen PIC-mapping generated high-magnification maps of unprecedented clarity from nacre and prismatic structures and their interface in Mytilus californianus shells. These maps revealed blocky aragonite crystals at the nacre-prismatic boundary and the narrowest calcite needle-prisms. In the tunic spicules of Herdmania momus, O PIC-mapping revealed the size and arrangement of some of the largest vaterite single crystals known. O spectroscopy therefore enables the simultaneous measurement of chemical and orientational information in CaCO₃ biominerals and is thus a powerful means for analyzing these and other complex materials. As described here, PIC-mapping and spectroscopy at the O K-edge are methods for gathering valuable data that can be carried out using spectromicroscopy beamlines at most synchrotrons without the expense of additional equipment.
Gur D., Leshem B., Oron D., Weiner S. & Addadi L. 2014. The Structural Basis for Enhanced Silver Reflectance in Koi Fish Scale and Skin Journal of the American Chemical Society. 2014 Dec , 136 (49):17236-17242.

[Abstract]

Fish have evolved biogenic multilayer reflectors composed of stacks of intracellular anhydrous guanine crystals separated by cytoplasm, to produce the silvery luster of their skin and scales. Here we compare two different variants of the Japanese Koi fish; one of them with enhanced reflectivity. Our aim is to determine how biology modulates reflectivity, and from this to obtain a mechanistic understanding of the structure and properties governing the intensity of silver reflectance. We measured the reflectance of individual scales with a custom-made microscope, and then for each individual scale we characterized the structure of the guanine crystal/cytoplasm layers using high-resolution cryo-SEM. The measured reflectance and the structural-geometrical parameters were used to calculate the reflectance of each scale, and the results were compared to the experimental measurements. We show that enhanced reflectivity is obtained with the same basic guanine crystal/cytoplasm stacks, but the structural arrangement between the stack, inside the stacks, and relative to the scale surface is varied when reflectivity is enhanced. Finally, we propose a model that incorporates the basic building block parameters, the crystal orientation inside the tissue, and the resulting reflectance and explains the mechanistic basis for reflectance enhancement.
Naveh G. R. S., Brumfeld V., Dean M., Shahar R. & Weiner S. 2014. Direct MicroCT imaging of non-mineralized connective tissues at high resolution Connective Tissue Research. 2014 Jan , 55 (1):52-60.

[Abstract]

The 3D imaging of soft tissues in their native state is challenging, especially when high resolution is required. An X-ray-based microCT is, to date, the best choice for high resolution 3D imaging of soft tissues. However, since X-ray attenuation of soft tissues is very low, contrasting enhancement using different staining materials is needed. The staining procedure, which also usually involves tissue fixation, causes unwanted and to some extent unknown tissue alterations. Here, we demonstrate that a method that enables 3D imaging of soft tissues without fixing and staining using an X-ray-based bench-top microCT can be applied to a variety of different tissues. With the sample mounted in a custom-made loading device inside a humidity chamber, we obtained soft tissue contrast and generated 3D images of fresh, soft tissues with a resolution of 1 micron voxel size. We identified three critical conditions which make it possible to image soft tissues: humidified environment, mechanical stabilization of the sample and phase enhancement. We demonstrate the capability of the technique using different specimens: an intervertebral disc, the non-mineralized growth plate, stingray tessellated radials (calcified cartilage) and the collagenous network of the periodontal ligament. Since the scanned specimen is fresh an interesting advantage of this technique is the ability to scan a specimen under load and track the changes of the different structures. This method offers a unique opportunity for obtaining valuable insights into 3D structure-function relationships of soft tissues.
Faingold A., Cohen S. R., Shahar R., Weiner S., Rapoport L. & Wagner D. H. 2014. The effect of hydration on mechanical anisotropy, topography and fibril organization of the osteonal lamellae Journal of Biomechanics. 2014 Jan , 47 (2):367-372.

[Abstract]

The effect of hydration on the mechanical properties of osteonal bone, in directions parallel and perpendicular to the bone axis, was studied on three length scales: (i) the mineralized fibril level (~100. nm), (ii) the lamellar level (~6. μm); and (iii) the osteon level (up to ~30. μm).We used a number of techniques, namely atomic force microscopy (AFM), nanoindentation and microindentation. The mechanical properties (stiffness, modulus and/or hardness) have been studied under dry and wet conditions. On all three length scales the mechanical properties under dry conditions were found to be higher by 30-50% compared to wet conditions. Also the mechanical anisotropy, represented by the ratio between the properties in directions parallel and perpendicular to the osteon axis (anisotropy ratio, designated here by AnR), surprisingly decreased somewhat upon hydration. AFM imaging of osteonal lamellae revealed a disappearance of the distinctive lamellar structure under wet conditions. Altogether, these results suggest that a change in mineralized fibril orientation takes place upon hydration.
Reznikov N., Almany-Magal R., Shahar R. & Weiner S. 2013. Three-dimensional imaging of collagen fibril organization in rat circumferential lamellar bone using a dual beam electron microscope reveals ordered and disordered sub-lamellar structures Bone. 2013 Feb , 52 (2):676-683.

[Abstract]

Lamellar bone is a major component of most mammalian skeletons. A prominent component of individual lamellae are parallel arrays of mineralized type I collagen fibrils, organized in a plywood like motif. Here we use a dual beam microscope and the serial surface view (SSV) method to investigate the three dimensional collagen organization of circumferential lamellar bone from rat tibiae after demineralization and osmium staining. Fast Fourier transform analysis is used to quantitatively identify the mean collagen array orientations and local collagen fibril dispersion. Based on collagen fibril array orientations and variations in fibril dispersion, we identify 3 distinct sub-lamellar structural motifs: a plywood-like fanning sub-lamella, a unidirectional sub-lamella and a disordered sub-lamella. We also show that the disordered sub-lamella is less mineralized than the other sub-lamellae. The hubs and junctions of the canalicular network, which connect radially oriented canaliculi, are intimately associated with the disordered sub-lamella. We also note considerable variations in the proportions of these 3 sub-lamellar structural elements among different lamellae. This new application of Serial Surface View opens the way to quantitatively compare lamellar bone from different sources, and to clarify the 3-dimensional structures of other bone types, as well as other biological structural materials.
Naveh G. R. S., Brumfeld V., Shahar R. & Weiner S. 2013. Tooth periodontal ligament: Direct 3D microCT visualization of the collagen network and how the network changes when the tooth is loaded Journal of Structural Biology. 2013 Feb , 181 (2):108-115.

[Abstract]

The periodontal ligament (PDL), a soft tissue connecting the tooth and the bone, is essential for tooth movement, bone remodeling and force dissipation. A collagenous network that connects the tooth root surface to the alveolar jaw bone is one of the major components of the PDL. The organization of the collagenous component and how it changes under load is still poorly understood. Here using a state-of-the-art custom-made loading apparatus and a humidified environment inside a microCT, we visualize the PDL collagenous network of a fresh rat molar in 3D at 1μm voxel size without any fixation or contrasting agents. We demonstrate that the PDL collagen network is organized in sheets. The spaces between sheets vary thus creating dense and sparse networks. Upon vertical loading, the sheets in both networks are stretched into well aligned arrays. The sparse network is located mainly in areas which undergo compressive loading as the tooth moves towards the bone, whereas the dense network functions mostly in tension as the tooth moves further from the bone. This new visualization method can be used to study other non-mineralized or partially mineralized tissues, and in particular those that are subjected to mechanical loads. The method will also be valuable for characterizing diseased tissues, as well as better understanding the phenotypic expressions of genetic mutants.
Gur D., Politi Y., Sivan B., Fratzl P., Weiner S. & Addadi L. 2013. Guanine-based photonic crystals in fish scales form from an amorphous precursor ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 2013 Jan , 52 (1):388-391.

[Abstract]

Starting from disorder: Anhydrous guanine crystals compose the photonic arrays responsible for the skin and scale iridescence found in Japanese Koi fish. These guanine crystals were found to form in intracellular vesicles (see picture) through an amorphous precursor phase. A combined cryo-SEM and synchrotron radiation X-ray diffraction study showed the evolution of the crystals in great detail.
Gal A., Habraken W., Gur D., Fratzl P., Weiner S. & Addadi L. 2013. Calcite crystal growth by a solid-state transformation of stabilized amorphous calcium carbonate nanospheres in a hydrogel Angewandte Chemie (International ed.). 2013 Apr , 52 (18):4867-4870.

[Abstract]

An ugly duckling grows into a swan: Many organisms grow their crystalline mineral phases through the secondary nucleation of nanospheres made of an amorphous precursor phase. Stable amorphous calcium carbonate biominerals were used to induce a similar transformation in vitro. The amorphous nanospheres underwent a solid‐phase transformation that resulted in highly ordered calcite crystals composed of aggregated particles.
Namdar D., Gilboa A., Neumann R., Finkelstein I. & Weiner S. 2013. Cinnamaldehyde in early iron age phoenician flasks raises the possibility of levantine trade with South East Asia Mediterranean Archaeology & Archaeometry. 2013 , 13 (2):1-19.

[Abstract]

Small ceramic flasks with thick walls and narrow openings were produced in Phoenicia. These flasks were common in Phoenicia, the southern Levant and Cyprus in the early Iron Age, namely in the 11th-mid-9th centuries BCE. Their shape, size, decoration and find-contexts suggest that they contained some precious materials and were part of a commercial network operating in these regions. We analyzed the lipid contents of 27 such containers from 5 archaeological sites in Israel using gas chromatography coupled with mass spectrometry (GC-MS). The organic extractions of 10 of these flasks contained cinnamaldehyde (C9H8O), a major component of cinnamon. In antiquity the cinnamon tree grew only in South and South East Asia. As cinnamaldehyde is found in small quantities in some modern potential contaminants, possible contamination of the small flasks with this compound was carefully assessed. Significantly, two recently excavated small flasks that were not handled directly contained relatively high concentrations of cinnamaldehyde. Other vessel types from the same archaeological sites and in some cases the same contexts did not contain cinnamaldehyde. Thus it is unlikely that the presence of cinnamaldehyde in the flasks results from contamination. This finding raises the intriguing possibility of long distance trade in the early Iron Age, assuming that the extracted cinnamaldehyde is indeed derived from the bark of the cinnamon tree. This is consistent with other suggestions that trade from South/South East Asia to the West took place at such an early date.
Meiri M., Huchon D., Bar-Oz G., Boaretto E., Horwitz L. K., Maeir A. M., Sapir-Hen L., Larson G., Weiner S. & Finkelstein I. 2013. Ancient DNA and population turnover in southern levantine pigs- Signature of the sea peoples migration? Scientific Reports. 2013 Nov , 3 .

[Abstract]

Near Eastern wild boars possess a characteristic DNA signature. Unexpectedly, wild boars from Israel have the DNA sequences of European wild boars and domestic pigs. To understand how this anomaly evolved, we sequenced DNA from ancient and modern pigs from Israel. Pigs from Late Bronze Age (until ca. 1150 BCE) in Israel shared haplotypes of modern and ancient Near Eastern pigs. European haplotypes became dominant only during the Iron Age (ca. 900 BCE). This raises the possibility that European pigs were brought to the region by the Sea Peoples who migrated to the Levant at that time. Then, a complete genetic turnover took place, most likely because of repeated admixture between local and introduced European domestic pigs that went feral. Severe population bottlenecks likely accelerated this process. Introductions by humans have strongly affected the phylogeography of wild animals, and interpretations of phylogeography based on modern DNA alone should be taken with caution.
Foran E., Weiner S. & Fine M. 2013. Biogenic fish-gut calcium carbonate is a stable amorphous phase in the gilt-head seabream, sparus aurata Scientific Reports. 2013 , 3 .

[Abstract]

The main source of calcium carbonate (CaCO₃) in the ocean comes from the shells of calcifying planktonic organisms, but substantial amounts of CaCO₃ are also produced in fish intestines. The precipitation of CaCO₃ assists fish in intestinal water absorption and aids in whole body Ca²⁺ homeostasis. Here we report that the product formed in the intestinal lumen of the gilt-head seabream, Sparus aurata, is an amorphous calcium carbonate (ACC) phase. With FTIR spectroscopy and SEM imaging, our study shows that the fish-derived carbonates from S. aurata are maintained as a stable amorphous phase throughout the intestinal tract. Moreover, intestinal deposits contained up to 54 mol% Mg²⁺, the highest concentration yet reported in biogenic ACC. Mg is most likely responsible for stabilizing this inherently unstable mineral. The fish carbonates also displayed initial rapid dissolution when exposed to seawater, exhibiting a significant increase in carbonate concentration.
Asscher Y., Regev L., Weiner S. & Boaretto E. 2012. Atomic disorder in Fossil TooThand bone mineral: An FTIR study using the grinding curve method ArcheoSciences. 2012 Sept , 35 (1):135-141.

[Abstract]

Bone and tooThmineral generally undergo diagenetic changes. These changes in the carbonate hydroxyapatite structure and composition can affect the signals embedded in the mineral phase, such as migration behavior, age of the specimen and the reconstruction of past environments. Mineral preservation state can be assessed using infrared spectroscopy which provides information on crystal disorder at the atomic level and mineral composition. Here we present a new approach to evaluate carbonate hydroxyapatite atomic disorder using infrared spectroscopy and the standard KBr sample mounting method. We show that by repeated grinding of the sample and then plotting the infrared splitting factor against the widThof the major phosphate absorption peak after each grinding, grinding curves with well defined trend lines can be obtained. The offsets between curves refiect differences in atomic disorder. We show that grinding curve offsets can be used to evaluate the state of preservation of bone, dentine and enamel mineral.
Gal A., Hirsch A., Siegel S., Li C., Aichmayer B., Politi Y., Fratzl P., Weiner S. & Addadi L. 2012. Plant cystoliths: A complex functional biocomposite of four distinct silica and amorphous calcium carbonate phases Chemistry-A European Journal. 2012 Aug , 18 (33):10262-10270.

[Abstract]

Plant cystoliths are mineralized objects that are formed by specialized cells in the leaves of certain plants. The main mineral component of cystoliths by volume is amorphous calcium carbonate (ACC) and the minor component is silica. We show that the silica stalk is formed first and is essential for ACC formation. Furthermore, the cystolith is shown to be composed of four distinct mineral phases with different chemical properties: an almost pure silica phase grades into a Mg-rich silica phase. This Mg-rich silica is overlaid by a relatively stable ACC phase. A bulky and less stable ACC phase encapsulates the first ACC phase. This architecture poses interesting questions about the role of Mg in the silica phase and suggests a strategy for ACC stabilization that takes advantage of a precise regulation of the mineral-growth microenvironment. The fantastic four: Cystoliths are mineralized objects that are mainly composed of amorphous calcium carbonate (ACC), which is found in the leaves of several plants. They have a unique composition and architecture of four distinct amorphous phases. A Mg-rich silica phase is essential for the formation of two distinct ACC phases. The inner ACC phase has inherently higher stability, presumably required by the sequential formation mechanism.
Addadi L., Vidavsky N. & Weiner S. 2012. Transient precursor amorphous phases in biomineralization. In the footsteps of Heinz A. Lowenstam Zeitschrift Fur Kristallographie. 2012 Nov , 227 (11):711-717.

[Abstract]

Heinz A. Lowenstam's discovery in 1967, together with Ken Towe that the magnetite mineral in mature chiton teeth forms from a disordered transient precursor phase, ferrihydrite, remained an isolated curiosity for 30 years. During the last 15 years, many more examples were found in both invertebrates and vertebrates, where the mature crystalline mineral phase is formed through a transient amorphous precursor phase. Here we review this widespread phenomenon, and also describe the details of the transformation process in the formation of the calcitic spicules of the sea urchin larva.We identify many open questions.
Poduska K. M., Regev L., Berna F., Mintz E., Milevski I., Khalaily H., Weiner S. & Boaretto E. 2012. Plaster characterization at the PPNB site of yiftahel (Israel) including the use of ¹⁴C: Implications for plaster production, preservation, and dating Radiocarbon. 2012 , 54 (3-4):887-896.

[Abstract]

The Pre-Pottery Neolithic B (PPNB) site of Yiftahel, Israel, contains abundant plaster floors. We surveyed the states of preservation of the plasters using an infrared spectroscopic assay that characterizes the extent of disorder of the atoms in the calcite crystal lattice. We identified the 3 best-preserved plaster samples that had disorder signatures most similar to modern plaster. We then studied the surface layers, fine-grained matrices, and large aggregates of these samples using micromorphology, Fourier transform infrared (FTIR) microscopy, stable carbon and radiocarbon concentrations. Even though some of the plaster components have a geogenic appearance in micromorphology slides and in FTIR spectra, the ¹⁴C analyses show that all components were exposed to high temperatures and as a result were equilibrated with the ¹⁴C content of the atmosphere ~10,000 yr ago. This implies that the plasters at Yiftahel were produced entirely from heat-altered calcite. We also show that these plasters have undergone significant diagenesis. The plaster component with the most disordered atomic signature, and hence the most similar in this respect to modern plaster, did indeed produce a ¹⁴C date close to the expected age.
Naveh G. R. S., Chattah N. L., Zaslansky P., Shahar R. & Weiner S. 2012. Tooth-PDL-bone complex: Response to compressive loads encountered during mastication - A review Archives of Oral Biology. 2012 Dec , 57 (12):1575-1584.

[Abstract]

The components of the tooth-periodontal ligament (PDL)-alveolar bone complex act in a synergistic manner to dissipate the loads incurred during mastication. The complex incorporates a diverse array of structural features for this purpose. These include the non-mineralized and hence soft PDL that absorbs much of the initial loads. The internal structure of the tooth also includes soft interphases that essentially surround the dentine core. These interphases, although stiffer than the PDL, still are more compliant than the dentine core, and are thus key components that allow the tooth itself to deform and hence help dissipate the compressive loads. There is also direct evidence that even under moderate compressive loads, when the tooth moves in the alveolar bone socket, this movement is guided by specific locations where the tooth comes into contact with the bone surface. The combination of all these responses to load is that each tooth type appears to move and deform in a specific manner when loaded. Much, however, still remains to be learned about these three-dimensional responses to load and the factors that control them. Such an understanding will have major implications for dentistry, that include a better understanding of phenomena such as abfraction, the manner in which tooth implants function even in the absence of a PDL-like tissue and the implications to bone remodelling of the movements imposed during orthodontic interventions.
Gal A., Brumfeld V., Weiner S., Addadi L. & Oron D. 2012. Certain biominerals in leaves function as light scatterers Advanced Materials. 2012 Mar , 24 (10):OP77-OP83.

[Abstract]

Cystoliths are amorphous calcium carbonate bodies that form in the leaves of some plant families. Cystoliths are regularly distributed in the epidermis and protrude into the photosynthetic tissue, the mesophyll. The photosynthetic pigments generate a steep light gradient in the leaf. Under most illumination regimes the outer mesophyll is light saturated, thus the photosynthetic apparatus is kinetically unable to use the excess light for photochemistry. Here we use micro-scale modulated fluorometry to demonstrate that light scattered by the cystoliths is distributed from the photosynthetically inefficient upper tissue to the efficient, but light deprived, lower tissue. The results prove that the presence of light scatterers reduces the steep light gradient, thus enabling the leaf to use the incoming light flux more efficiently. MicroCT and electron microscopy confirm that the spatial distribution of the minerals is compatible with their optical function. During the study we encountered large calcium oxalate druses in the same anatomical location as the cystoliths. These druses proved to have similar light scattering functions as the cystoliths. This study shows that certain minerals in the leaves of different plants distribute the light flux more evenly inside the leaf. Leaf minerals function as internal light scatterers inside leaves. They transfer light from the saturated upper tissue into the light deprived lower tissue. This eases the steep light gradient inside the leaf and improves photosynthetic efficiency on the tissue scale.
Naveh G. R. S., Shahar R., Brumfeld V. & Weiner S. 2012. Tooth movements are guided by specific contact areas between the tooth root and the jaw bone: A dynamic 3D microCT study of the rat molar Journal of Structural Biology. 2012 Feb , 177 (2):477-483.

[Abstract]

Teeth sustain high loads over a lifetime and yet intact tooth failure is rare. The different structures of the tooth, jaw bone and the intervening soft periodontal ligament enable the tooth to endure repeated loading during mastication. Although mechanical and functional properties of the different components are thoroughly investigated, the manner in which the whole tooth functions under load is still enigmatic. A custom-made loading system inside a microCT scanner was used to directly visualize the root movements in relation to the jaw bone as the rat molar tooth was loaded. At low loads no contact was observed between the root surface and the bone, whereas at higher loads three specific contact areas between the root surface and the jaw bone were observed. These contact areas restrict tooth movement in the buccal-lingual direction, but enable the tooth to rock in a "seesaw" like manner in the distal-mesial direction. The contact areas appear to play a role in determining tooth motion and in turn define the manner in which the whole tooth moves when loaded. These observations are important for understanding basic structure-function relations of the tooth-PDL-bone system, and have direct implications for better understanding pathological and therapeutic processes in orthodontics, periodontics and jaw bone regeneration.
Eliyahu-Behar A., Yahalom-Mack N., Shilstein S., Zukerman A., Shafer-Elliott C., Maeir A. M., Boaretto E., Finkelstein I. & Weiner S. 2012. Iron and bronze production in Iron Age IIA Philistia: new evidence from Tell es-Safi/Gath, Israel Journal of Archaeological Science. 2012 Feb , 39 (2):255-267.

[Abstract]

A metallurgically-oriented excavation in Area A at Tell es-Safi/Gath yielded evidence for iron and bronze production dating to the early Iron Age IIA. Two pit-like features, which differed considerably from one another in colour, texture and content, were excavated. Evidence shows that each feature represents a different in situ activity related to iron production, inferred by the presence of hammerscales, slag prills and slag. An upturned crucible was found on top of one of the features. Analysis of the crucible slag showed that it was used for bronze metallurgy. Tuyeres, both round and square in cross-section, were found in and around the two features. The presence of the two industries together presents a unique opportunity to explore the relationship between copper and iron working. This is especially important against the background of the scarcity of evidence for iron production in the Levant during the early phases of the Iron Age. (C) 2011 Elsevier Ltd. All rights reserved.
Cabanes D., Gadot Y., Cabanes M., Finkelstein I., Weiner S. & Shahack-Gross R. 2012. Human impact around settlement sites: A phytolith and mineralogical study for assessing site boundaries, phytolith preservation, and implications for spatial reconstructions using plant remains Journal of Archaeological Science. 2012 Aug , 39 (8):2697-2705.

[Abstract]

Defining the extent of human activity around settlement sites is of particular significance in archaeology as it may define peripheral activity areas and thus the site's boundary. In Near Eastern archaeology, site boundaries are usually defined by the presence of architectural and other macroscopic archaeological remains. Here we use the phytolith concentrations and morphotype assemblages, as well as changes in the mineralogical composition of the sediments in and around the small Iron Age site of Izbet Sartah in central Israel to determine the site boundaries. The site has a shallow stratigraphy and highly bioturbated sediments. Coincidental changes in the clay/quartz ratio and phytolith concentrations define the boundary between high and low impact anthropogenic activities. This boundary is generally some 20 m away from the architectural remains. In addition, we note that the phytoliths in the site's core show clear evidence of having been affected by chemical dissolution (i.e., diagenesis), while those in the vicinity of the site's boundary have undergone severe diagenesis. These observations indicate that phytolith diagenesis will affect site boundaries determination, as well as phytolith-based reconstructions of activity areas. We propose that phytolith preservation depends on the initial amount of available silica, the depth of burial with respect to the active root area of modern vegetation, and the presence of fresh phytoliths in the soil.
Asscher Y., Weiner S. & Boaretto E. 2011. Variations in atomic disorder in biogenic carbonate hydroxyapatite using the infrared spectrum grinding curve method Advanced Functional Materials. 2011 Sept , 21 (17):3308-3313.

[Abstract]

Biogenic carbonate hydroxyapatite crystals are inherently disordered at the atomic level due mainly to the substitutions of various ions in the crystal structure, and, in the case of the bone family of materials, to the fact that these very small crystals have a very large surface-to-bulk ratio. Characterization of the extent of disorder is of much interest, as this relates to the stability and hence solubility of the crystals. Here the infrared spectrometry grinding curve approach developed for calcite, is adapted to carbonate hydroxyapatites. The infrared splitting factor is plotted against the full width at half height of the strong phosphate absorption peak as a function of increased grinding of the sample. By doing so, the contribution of particle size to the shape of the peaks is better separated from the contribution of atomic disorder to peak shape. It is shown that differences in disorder exist between dentine, cementum, and bone crystals which could reflect crystal size and/or atomic defects within the crystal. It is als shown that systematic differences exist between enamel samples from different taxa, which we assume only reflects atomic disorder differences within these large crystals. The method can be used to characterize atomic disorder in natural hydroxyapatites, as well as in the many different types of synthetic hydroxyapatites used for biomedical implants. Carbonated hydroxyapatite is widely used in biomedical applications. Biogenic carbonate hydroxyapatite crystals are inherently disordered at the atomic level. Using the grinding curve approach based on peak broadening in infrared spectrometry, systematic disorder differences between enamel samples from different taxa, and differences in disorder between dentine, cementum and bone crystals are shown.
Schmidt M. W., Torn M. S., Abiven S., Dittmar T., Guggenberger G., Janssens I. A., Kleber M., Kögel-Knabner I., Lehmann J., Manning D. A., Nannipieri P., Rasse D. P., Weiner S. & Trumbore S. E. 2011. Persistence of soil organic matter as an ecosystem property Nature. 2011 Oct , 478 (7367):49-56.

[Abstract]

Globally, soil organic matter (SOM) contains more than three times as much carbon as either the atmosphere or terrestrial vegetation. Yet it remains largely unknown why some SOM persists for millennia whereas other SOM decomposes readily and this limits our ability to predict how soils will respond to climate change. Recent analytical and experimental advances have demonstrated that molecular structure alone does not control SOM stability: in fact, environmental and biological controls predominate. Here we propose ways to include this understanding in a new generation of experiments and soil carbon models, thereby improving predictions of the SOM response to global warming.
Khalifa G. M., Weiner S. & Addadi L. 2011. Mineral and matrix components of the operculum and shell of the barnacle balanus amphitrite: Calcite crystal growth in a hydrogel Crystal Growth & Design. 2011 Nov , 11 (11):5122-5130.

[Abstract]

Sessile barnacles produce two types of mineralized exoskeletons: a cone-shaped shell and an operculum that is used to seal the shell opening. The mineral of both types is calcite. We show that the calcite crystals of the shell and the operculum (specifically the scutum) of the sessile barnacle Balanus amphitrite both fracture with conchoidal cleavage, have surfaces decorated with small rhombohedral shaped calcite crystals, and are poorly oriented. The scutum calcite is significantly more disordered at the atomic level than the shell calcite. We also show that a major component of the inter-crystalline organic matrix of the shell and scutum is a non-proteinaceous sulfate-rich polymer that behaves as a hydrogel, and that the intracrystalline matrix contains highly acidic proteins. The crystal properties and microstructure are consistent with the calcite crystals forming in a hydrogel-like environment. The barnacle shell and operculum have many unique properties indicating that the crystal growth conditions are well controlled and possibly adapted to fulfill mechanical functions, which enable the barnacle to survive in the high energy environment of the intertidal zone.
Weiner S. & Addadi L. 2011. Crystallization Pathways in Biomineralization Annual Review of Materials Research. 2011 , 41 :21-40.

[Abstract]

A crystallization pathway describes the movement of ions from their source to the final product. Cells are intimately involved in biological crystallization pathways. In many pathways the cells utilize a unique strategy: They temporarily concentrate ions in intracellular membrane-bound vesicles in the form of a highly disordered solid phase. This phase is then transported to the final mineralization site, where it is destabilized and crystallizes. We present four case studies, each of which demonstrates specific aspects of biological crystallization pathways: seawater uptake by foraminifera, calcite spicule formation by sea urchin larvae, goethite formation in the teeth of limpets, and guanine crystal formation in fish skin and spider cuticles. Three representative crystallization pathways are described, and aspects of the different stages of crystallization are discussed. An in-depth understanding of these complex processes can lead to new ideas for synthetic crystallization processes of interest to materials science.
Suzuki M., Kogure T., Weiner S. & Addadi L. 2011. Formation of aragonite crystals in the crossed lamellar microstructure of limpet shells Crystal Growth & Design. 2011 Nov , 11 (11):4850-4859.

[Abstract]

The crossed lamellar microstructure is the most commonly formed shell structure in mollusks. Although the mechanical properties and the architecture of the crossed lamellar microstructure have been extensively studied, its formation mechanisms are largely unknown. In this study, we obtained information on the formation processes of the aragonite crystals in the crossed lamellar microstructure using two converging approaches: cryo-scanning electron microscopy performed on the forming shell layer and in vitro crystallization experiments with extracted matrix macromolecules. We show that deposition of the first crystalline particles occurs in a thin granular layer on the growing surface of the mature crossed lamellar microstructure. The granules subsequently grow to form the lamellar prisms. In vitro, the ensemble of organic macromolecules extracted from the crossed lamellar layers induced the formation of only aragonite crystals from solutions containing Ca/Mg ion ratio of 1/2.5 within 6 h, and that the morphology and the crystallographic orientation of the synthetic crystals are the same as those of the biogenic crystals. In the absence of matrix macromolecules, both calcite and aragonite crystals formed. These observations provide insights into the early crystal nucleation and growth events of aragonite crystals in the formation of the crossed lamellar microstructure.
Rebollo N. R., Weiner S., Brock F., Meignen L., Goldberg P., Belfer-Cohen A., Bar-Yosef O. & Boaretto E. 2011. New radiocarbon dating of the transition from the Middle to the Upper Paleolithic in Kebara Cave, Israel Journal of Archaeological Science. 2011 Sept , 38 (9):2424-2433.

[Abstract]

The Middle to Upper Paleolithic transition (MP-UP transition) is considered a major technological and cultural threshold, at the time when modern humans spread "out of Africa", expanded from the Levant into Europe and possibly into central and northern Asia. The dating of this techno-cultural transition has proved to be extremely difficult because it occurred sometime before 40,000 radiocarbon years before present ((14)C years BP), which is close to the end of the effective dating range of radiocarbon. Other dating methods such as Thermoluminescence cm or Electron Spin Resonance (ESR) are not sufficiently precise to date the recorded archaeological MP-UP transition in the Levant. Here we report a consistent set of stratified radiocarbon ages on freshly excavated charcoal from Kebara Cave, Mt. Carmel (Israel), that span the late Middle Paleolithic (MP) and Early Upper Paleolithic (EUP) This study applied novel strategies to improve sample preparation techniques and data analysis to obtain high-resolution radiocarbon models. From this study it is proposed that the MP-UP transition for this site can be placed immediately after 45,200 +/- 700 (14)C years BP and before 43,600 +/- 600 (14)C years BP or from 49/48 to 47/46 radiocarbon calibrated years before present (years Cal BP). (C) 2011 Elsevier Ltd. All rights reserved.
Mahamid J., Addadi L. & Weiner S. 2011. Crystallization pathways in bone Cells Tissues Organs. 2011 Aug , 194 (2-4):92-97.

[Abstract]

Many biomineralization processes involve the sequestering of ions by cells and their translocation through the cells to the final deposition site. In many invertebrate crystallization pathways the cells deposit an initial highly disordered mineral phase with intracellular vesicles, and this mineral is subsequently transported into the final deposition site outside the cell. As this initial mineral phase is metastable, it can easily dissolve or crystallize during sample preparation and examination. A cryogenic electron microscopy study of the forming fin bone of a zebra fish strain with continuously growing fins shows that the cells responsible for bone tissue formation do have mineral-bearing intracellular vesicles and that the mineral phase is a highly disordered calcium phosphate. We also show that globules of disordered calcium phosphate are present in the extracellular collageneous matrix and that they are not membrane bound. Close to the mineralization front these globules appear to penetrate into the collagen fibrils where they crystallize to form mature bone. This crystallization pathway is similar to pathways observed in invertebrates, and it differs from the matrix vesicle pathway documented for a variety of vertebrate mineralizing tissues as the extracellular mineral globules are not membrane bound.
Cabanes D., Weiner S. & Shahack-Gross R. 2011. Stability of phytoliths in the archaeological record: a dissolution study of modern and fossil phytoliths Journal of Archaeological Science. 2011 Sept , 38 (9):2480-2490.

[Abstract]

Opaline phytoliths are important microfossils used in archaeological and ecological research. Relatively little is known about the stability of phytoliths after burial. Under alkaline pH conditions they can dissolve, and mechanical disturbances can cause a loss of their more delicate appendages. Here we present an experimental study of phytolith stability (combination of solubility and abrasion). Modern and fossil phytoliths were extracted from wheat using new methods to minimize dissolution, and by burning in an oven. These assemblages were placed in a solution buffered to pH 10 and maintained under constant temperature and shaking conditions. The silicon concentrations in the solution were monitored once a week for 5 weeks. The phytolith morphologies in each assemblage were determined at the outset of the experiment and after 5 weeks. The results show that there are differences in stability between various assemblages. Modern inflorescence wheat phytolith assemblages are more unstable than those from leaves/stems. Burnt assemblages are less stable than unburnt assemblages, and a fossil phytolith assemblage about 3000 years old is more stable than the modern wheat assemblages. The results also show that individual phytolith morphotypes have different stabilities, and as a result of dissolution and abrasion, some morphotypes may resemble others. This study further shows that archaeological and/or paleo-environmental interpretation of phytolith assemblages may change with the assemblage's state of preservation. (C) 2011 Elsevier Ltd. All rights reserved.
Mahamid J., Sharir A., Gur D., Zelzer E., Addadi L. & Weiner S. 2011. Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: A cryo-electron microscopy study Journal of Structural Biology. 2011 Jun , 174 (3):527-535.

[Abstract]

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates. (C) 2011 Elsevier Inc. All rights reserved.
Regev L., Eckmeier E., Mintz E., Weiner S. & Boaretto E. 2011. Radiocarbon concentrations of wood ash calcite: Potential for dating Radiocarbon. 2011 , 53 (1):117-127.

[Abstract]

Ash is formed when plant calcium oxalate crystals (CaC₂O₄) decompose to form calcite (CaCO₃). We found that ash does retain the original calcium oxalate radiocarbon concentration, but in addition, there is another minor ¹⁴C source. This is shown by the presence of a consistent small shift in the pMC and δ¹³C levels when comparing cellulose and ash from modern and archaeological woods. Possible mechanisms for ¹⁴C exchange during combustion or due to diagenesis are considered in order to define parameters for identifying better-preserved wood ash samples.
Suzuki M., Dauphin Y., Addadi L. & Weiner S. 2011. Atomic order of aragonite crystals formed by mollusks CrystEngComm. 2011 Nov , 13 (22):6780-6786.

[Abstract]

Aragonite is the most common polymorph of calcium carbonate produced by mollusks. The crystals have different shapes and sizes, and are intimately associated with an organic matrix. Here we adapt an infrared spectroscopic method that was developed to characterize atomic disorder in calcite crystals, in order to characterize aragonite crystals produced by mollusks, as well as geogenic and synthetic aragonite crystals. The method involves systematically changing the particle size distribution by repeated grinding of the sample. We show that large single geogenic aragonite crystals are well ordered compared to aragonite formed from boiling water, which in turn is better ordered than synthetic aragonite crystals produced in vitro in the presence of additives. The most ordered biogenic aragonite is from the crossed lamellar structure. Nacre and myostracum are less ordered and the large ligament crystals are even less ordered than nacre. These trends do not correspond to those observed from X-ray diffraction peak broadening, indicating that the two techniques reflect different types of ordering. The differences in atomic order observed by infrared spectroscopy between biogenic aragonite crystals and also between geogenic aragonite crystals presumably reflect different modes of crystal formation.
Poduska K. M., Regev L., Boaretto E., Addadi L., Weiner S., Kronik L. & Curtarolo S. 2011. Decoupling local disorder and optical effects in infrared spectra: Differentiating between calcites with different origins Advanced Materials. 2011 Jan , 23 (4):550-554.

[Abstract]

Infrared spectral peak broadening due to atomic disorder and narrowing due to particle-size-related optical absorption effects can be decoupled experimentally and theoretically. Applied to different sources of polycrystalline calcite, the method provides a powerful diagnostic tool for archaeology, geology, and materials/biomaterials science.
Namdar D., Zukerman A., Maeir A. M., Katz J. C., Cabanes D., Trueman C., Shahack-Gross R. & Weiner S. 2011. The 9th century BCE destruction layer at Tell es-Safi/Gath, Israel: Integrating macro- and microarchaeology Journal of Archaeological Science. 2011 Dec , 38 (12):3471-3482.

[Abstract]

Destruction events in multi-period sites are valuable marker horizons that represent time-synchronous events across the site and sometimes between sites. Destruction layers often preserve rich finds that provide insights into site use. Here we use both macro- and microarchaeological methods to study a destruction event from the late 9th century at Tell es-Safi/Gath in Israel. A major conflagration at this specific location resulted in the consolidation of parts of the roof construction materials, thus enabling us to differentiate between roof, walls and floor materials. We could reconstruct the events which lead to the formation of an approximately 80 cm thick layer. The base of this layer that overlies the floor surface is a thin charred organic material-rich ash layer. As the clays in this layer were not altered by heat and the ceramics still have preserved residues, we conclude that the ash was produced elsewhere and was redistributed to this location. Ceramics that are associated with burnt roof sediments do not have preserved residues. We also estimate the time that each of the accumulation events might have taken, and conclude that this accumulation occurred over decades. The architecture and artifacts found within and beneath the destruction do not allow us to unequivocally identify the function of this area prior to destruction. We do however identify an unusual bin and associated stone pavement, and a corner rich in artifacts, phytoliths and charred organic material. We also show that a wall was built of fired mud bricks; a most unusual occurrence for this time period in the Levant. This study demonstrates well the usefulness of an integrated macro- and microarchaeological approach to understanding the archaeological record, as well as the benefits of using an on-site laboratory.
Chattah N. L., Kupczik K., Shahar R., Hublin J. & Weiner S. 2011. Structure-function relations of primate lower incisors: a study of the deformation of Macaca mulatta dentition using electronic speckle pattern interferometry (ESPI) Journal of Anatomy. 2011 Jan , 218 (1):87-95.

[Abstract]

Teeth adopt a variety of different morphologies, each of which is presumably optimized for performing specific functions during feeding. It is generally agreed that the enamel cap is a crucial element in controlling the mechanical behavior of mammalian teeth under load. Incisors are particularly interesting in terms of structure-function relations, as their role in feeding is that of the 'first bite'. However, little is known how incisor cap morphology is related to tooth deformation. In the present paper we examine the mechanical behavior of mandibular central incisors in the cercopithecine primate Macaca mulatta under loads similar to those encountered during ingestion. We map three-dimensional displacements on the labial surface of the crown as it is compressed, using electronic speckle pattern interferometry (ESPI), an optical metrology method. In addition, micro-computed tomography is used to obtain data regarding the morphology of the enamel cap, which in the M. mulatta lower incisors exhibits missing or very little enamel on the lingual face. The results showed that although compressed along a longitudinal axis, deformation in the incisors mostly occurred in the lingual direction and orthogonal to the direction of the applied load. Both isolated, embedded teeth and teeth in the mandible showed considerable lingual deformation. Incisor deformation in the mandible was generally greater, reflecting the additional freedom of movement enabled by the supporting structures. We show that the association with adjacent teeth in the arch is significant for the behavior of the tooth under load. Finally, loading two teeth simultaneously in the mandible showed that they work as one functional unit. We suggest that these results demonstrate the importance of enamel cap morphology in directing deformation behavior; an ability stemming from the stiffness of the enamel cap overlying the more pliable dentin.
Clode P. L., Lema K., Saunders M. & Weiner S. 2011. Skeletal mineralogy of newly settling Acropora millepora (Scleractinia) coral recruits Coral Reefs. 2011 Mar , 30 (1):1-8.

[Abstract]

Knowledge of skeletogenesis in scleractinian corals is central to reconstructing past ocean and climate histories, assessing and counteracting future climate and ocean acidification impacts upon coral reefs, and determining the taxonomy and evolutionary path of the Scleractinia. To better understand skeletogenesis and mineralogy in extant scleractinian corals, we have investigated the nature of the initial calcium carbonate skeleton deposited by newly settling coral recruits. Settling Acropora millepora larvae were sampled daily for 10 days from initial attachment, and the carbonate mineralogy of their newly deposited skeletons was investigated. Bulk analyses using Raman and infrared spectroscopic methods revealed that the skeletons were predominantly comprised of aragonite, with no evidence of calcite or an amorphous precursor phase, although presence of the latter cannot be discounted. Sensitive selected area electron diffraction analyses of sub-micron areas of skeletal regions further consolidated these data. These findings help to address the uncertainty surrounding reported differences in carbonate mineralogy between larval and adult extant coral skeletons by indicating that skeletons of new coral recruits share the same aragonitic mineralogy as those of their mature counterparts. In this respect, we can expect that skeletogenesis in both larval and mature growth stages of scleractinian corals will be similarly affected by ocean acidification and predicted environmental changes.
Salamon M., Tzur S., Arensburg B., Zias J., Nagar Y., Weiner S. & Boaretto E. 2010. Ancient Mtdna Sequences And Radiocarbon Dating Of Human Bones From The Chalcolithic Caves Of Wadi El-Makkukh Mediterranean Archaeology & Archaeometry. 2010 , 10 (2):1-14.

[Abstract]

DNA from fossil human bones can provide valuable information for understanding intra- and inter-population relationships. Using the DNA preserved inside crystal aggregates from human fossil bones containing relatively large amounts of collagen, we demonstrate the presence of reproducible mtDNA control region sequences. Radiocarbon dates from each bone show that the burial caves were used for up to 600 years during the Chalcolithic period (5^th-4^th millennium BP). A comparison of the ancient DNA sequences with modern mtDNA databases indicates that all samples can most likely be assigned to the R haplogroup sub-clades, which are common in West-Eurasia. In four cases more precise and confident haplogroup identifications could be achieved (H, U3a and H6). The H haplogroup is present in three out of the four assigned ancient samples. This haplogroup is prevalent today in West - Eurasia. The results reported here tend to genetically link this Chalcolithic group of individuals to the current West Eurasian populations.
Gal A., Weiner S. & Addadi L. 2010. The stabilizing effect of silicate on biogenic and synthetic amorphous calcium carbonate Journal of the American Chemical Society. 2010 Sept , 132 (38):13208-13211.

[Abstract]

Silicate ions increase the thermal stability of the unstable amorphous calcium carbonate (ACC). This effect was observed first by comparing ACC from two different species of cystoliths, small calcified bodies formed in the leaves of some plants. The temperature of crystallization to calcite in the silicate-rich cystoliths from M. alba is 100 °C higher than that of the silicate-poor cystoliths from F. microcarpa. The stabilizing effect is confirmed in vitro with synthetic samples differing in their silicate content. With increasing silicate concentration in ACC, the crystallization temperature to calcite also increases. A mechanism of geometric frustration is suggested, whereby the presence of the tetrahedral silicate ion in the flat carbonate lattice prevents organization into crystalline polymorphs.
Dror A. A., Politi Y., Shahin H., Lenz D. R., Dossena S., Nofziger C., Fuchs H., De Angelis M. H., Paulmichl M., Weiner S. & Avraham K. B. 2010. Calcium oxalate stone formation in the inner ear as a result of an Slc26a4 mutation Journal of Biological Chemistry. 2010 Jul , 285 (28):21724-21735.

[Abstract]

Calcium oxalate stone formation occurs under pathological conditions and accounts for more than 80% of all types of kidney stones. In the current study, we show for the first time that calcium oxalate stones are formed in the mouse inner ear of a genetic model for hearing loss and vestibular dysfunction in humans. The vestibular system within the inner ear is dependent on extracellular tiny calcium carbonate minerals for proper function. Thousands of these biominerals, known as otoconia, are associated with the utricle and saccule sensory maculae and are vital for mechanical stimulation of the sensory hair cells.We show that a missense mutation within the Slc26a4 gene abolishes the transport activity of its encoded protein, pendrin. As a consequence, dramatic changes in mineral composition, size, and shape occur within the utricle and saccule in a differential manner. Although abnormal giant carbonate minerals reside in the utricle at all ages, in the saccule, a gradual change in mineral composition leads to a formation of calcium oxalate in adult mice. By combining imaging and spectroscopy tools, we determined the profile of mineral composition and morphology at different time points. We propose a novel mechanism for the accumulation and aggregation of oxalate crystals in the inner ear.
Politi Y., Batchelor D. R., Zaslansky P., Chmelka B. F., Weaver J. C., Sagi I., Weiner S. & Addadi L. 2010. Role of magnesium ion in the stabilization of biogenic amorphous calcium carbonate: A structure-function investigation Chemistry of Materials. 2010 Jan , 22 (1):161-166.

[Abstract]

Magnesium is a key component used by many organisms in biomineralization. One role for magnesium is in stabilizing an otherwise unstable amorphous calcium carbonate (ACC) phase. The way in which this stabilization is achieved is unknown. Here, we address this question by studying the chemical environment around magnesium in biogenic and synthetic ACCs using Mg K-edge X-ray absorption spectroscopy (XAS). We show that although the short-range structure around the Mg ion is different in the various minerals studied, they all involve a shortening of the Mg-O bond length compared to crystalline anhydrous MgCO ₃ minerals. We propose that the compact structure around magnesium introduces distortion in the CaCO₃ host mineral, thus inhibiting its crystallization. This study also shows that despite technical challenges in the soft X-ray energy regime, Mg K-edge XAS is a valuable tool for structural analysis of Mg containing amorphous materials, in biology and materials science.
Katz O., Cabanes D., Weiner S., Maeir A. M., Boaretto E. & Shahack-Gross R. 2010. Rapid phytolith extraction for analysis of phytolith concentrations and assemblages during an excavation: An application at Tell es-Safi/Gath, Israel Journal of Archaeological Science. 2010 Jul , 37 (7):1557-1563.

[Abstract]

A rapid phytolith extraction procedure is described, that allows phytolith concentrations and morphotype assemblages to be analyzed within hours. This procedure enables the results of these analyses to be used during an archaeological excavation, in order to better understand how plants were used. The new procedure was tested using a standard phytolith extract and two experimental phytolith-sediment blends and found to be both accurate and precise. The reliability of partial slide counting was evaluated and found to be as accurate and precise as existing phytolith quantification methods. The new extraction and counting procedures were applied to an archaeological site, Tell es-Safi/Gath, Israel. The results demonstrate how information on phytolith concentrations in sediments that are available from one day to the next, can be used during an excavation to more effectively document the local features of interest and obtain better information.
Mahamid J., Aichmayer B., Shimoni E., Ziblat R., Li C., Siegel S., Paris O., Fratzl P., Weiner S. & Addadi L. 2010. Mapping amorphous calcium phosphate transformation into crystalline mineral from the cell to the bone in zebrafish fin rays Proceedings of the National Academy of Sciences of the United States of America. 2010 Apr , 107 (14):6316-6321.

[Abstract]

The continuously forming fin bony rays of zebrafish represent a simple bone model system in which mineralization is temporally and spatially resolved. The mineralized collagen fibrils of the fin bones are identical in structure to those found in all known bone materials. We study the continuous mineralization process within the tissue by using synchrotron microbeam x-ray diffraction and small-angle scattering, combined with cryo-scanning electron microscopy. The former provides information on the mineral phase and the mineral particles size and shape, whereas the latter allows high-resolution imaging of native hydrated tissues. The integration of the two techniques demonstrates that new mineral is delivered and deposited as packages of amorphous calcium phosphate nanospheres, which transform into platelets of crystalline apatite within the collagen matrix.
Zolotoyabko E., Caspi E. N., Fieramosca J. S., Von Dreele D. R. B., Marin F., Mor G., Addadi L., Weiner S. & Politi Y. 2010. Differences between bond lengths in biogenic and geological calcite Crystal Growth & Design. 2010 Mar , 10 (3):1207-1214.

[Abstract]

We used high-resolution neutron powder diffraction to accurately measure the atomic positions and bond lengths in biogenic and geological calcite. A special procedure for data analysis was developed in order to take into account the considerable amounts of magnesium present in all the investigated samples. As a result, in biogenic calcite we found some atomic bonds to have significantly different lengths as compared to those in geological calcite, after the contribution of magnesium is accounted for. The maximum effect (elongation up to 0.7%) was found for the C-O bonds. We also analyzed changes in frequencies and spectral widths of normal vibrations of carbonate groups in biogenic calcite (as compared to geological calcite) measured by Raman and Fourier transform IR techniques. Surprisingly, the frequency shifts after subtracting the magnesium contribution are close to zero. At the same time, substantial spectral broadening (up to 1.2%) in biogenic calcite as compared to geological samples was detected. Possible explanations for the experimental findings are discussed.
Levy-Lior A., Shimoni E., Schwartz O., Gavish-Regev E., Oron D., Oxford G., Weiner S. & Addadi L. 2010. Guanine-Based biogenic photonic-crystal arrays in fish and spiders Advanced Functional Materials. 2010 Jan , 20 (2):320-329.

[Abstract]

Biological photonic systems composed of anhydrous guanine crystals evolved separately in several taxonomie groups. Here, two such systems found in fish and spiders, both of which make use of anhydrous guanine crystal plates to produce structural colors, are examined. Measurements of the photoniccrystal structures using cryo-SEM show that the crystal plates in both fish skin and spider integument are ∼20-nm thick. The reflective unit in the fish comprises stacks of single plates alternating with ∼ 230-nm-thick cytoplasm layers. In the spiders the plates are formed as doublet crystals, cemented by 30-nm layers of amorphous guanine, and are stacked with ∼200nm of cytoplasm between crystal doublets. They achieve light reflective properties through the control of crystal morphology and stack dimensions, reaching similar efficiencies of light reflectivity in both fish skin and spider integument. The structure of guanine plates in spiders are compared with the more common situation in which guanine occurs in the form of relatively unorganized prismatic crystals, yielding a matt white coloration.
Barak M. M., Weiner S. & Shahar R. 2010. The contribution of trabecular bone to the stiffness and strength of rat lumbar vertebrae Spine. 2010 Oct , 35 (22):E1153-E1159.

[Abstract]

Study Design: In vitro compressive load-displacement experiments on intact rat lumbar vertebrae and on the same vertebrae after part of their trabecular bone was removed. Objective: To determine the contribution of the trabecular bone component to the stiffness and strength of rat lumbar vertebrae. Summary Of Background Data: Vertebral fractures are common in the aging population, possibly resulting from the deterioration of the mechanical properties of vertebral bone. Studies of the contribution of trabecular bone to the mechanical behavior of whole vertebra were published, but yielded mixed results. Here, we propose a novel optical metrology approach to address this important question. Methods: The bodies of intact rat lumbar vertebrae and the bodies of the same vertebrae after part of their trabecular bone was removed were loaded within their elastic region in a wet environment. The amount of trabecular bone removed was determined by micro-computer tomography scanning. Deformation maps of the dorsal vertebral surface of the intact and manipulated vertebrae were obtained using an optical metrology method, and compared. Intact and manipulated vertebrae were also loaded to failure in compression and their strengths and stiffness were compared. Results: The preferred trabecular orientation was found to be along the anterior-posterior axis, which is similar to humans. Removal of up to 42% of the trabecular tissue in the intact vertebrae did not significantly affect lumbar vertebral stiffness. However, removal of even smaller amounts of the intact trabecular tissue significantly reduced vertebral strength. Conclusion: Trabeculae in rat lumbar vertebrae fulfill an important role in failure resistance (strength), but have little or no effect on the deformational behavior (stiffness) of the bone. These results differ from previous results we reported for rat femora, where removal of trabecular bone surprisingly increased the stiffness of the whole bone, and suggest that trabecular tissue may have different functions depending on anatomic location, bone function and morphology, and mode of loading.
Regev L., Zukerman A., Hitchcock L., Maeir A. M., Weiner S. & Boaretto E. 2010. Iron Age hydraulic plaster from Tell es-Safi/Gath, Israel Journal of Archaeological Science. 2010 Dec , 37 (12):3000-3009.

[Abstract]

Hydraulic plasters or mortars prior to the Roman period are rare. Here, we report the identification and characterization of 3000 year old (Iron Age) hydraulic plaster surfaces from the site of Tell es-Safi/Gath. This site, located in central Israel, was occupied almost continuously from prehistoric through modern times, and is identified as the Canaanite and Philistine city of Gath. A survey using an on-site Fourier transform infrared spectrometer (FTIR) identified the presence of amorphous silicates, in addition to calcite, in each of two superimposed plaster layers. This suite of minerals is characteristic of hydraulic plaster. An in-depth characterization of the plasters using FTIR, acid dissolution, X-ray fluorescence (XRF), X-ray powder diffractometry (pXRD), heating experiments and scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (SEM-EDS), shows that special silicate-containing minerals were brought from some distance to the site in order to produce these plaster surfaces. We therefore conclude that the plasters were deliberately produced, and were not the result of a fortuitous addition of local silicate minerals. A layer of around 150μm thick enriched in carbonate hydroxylapatite was found in both plaster surfaces, and the same mineral was identified by infrared spectrometry in the surrounding sediments. This suggests that organic materials were used on these surfaces. These surfaces are among the oldest hydraulic plasters known.
Regev L., Poduska K. M., Addadi L., Weiner S. & Boaretto E. 2010. Distinguishing between calcites formed by different mechanisms using infrared spectrometry: Archaeological applications Journal of Archaeological Science. 2010 Dec , 37 (12):3022-3029.

[Abstract]

Infrared spectrometry is a well-established method for the identification of minerals. Due to its simplicity and the short time required to obtain a result, it can be practiced on-site during excavation using portable infrared spectrometers. However, the identification of a mineral may not be sufficient. For example, a lime plaster floor and a crushed chalk surface have a similar appearance and are composed of the same mineral - calcite. Here we exploit differences in the infrared spectra of geogenic, biogenic and pyrogenic calcites for the identification of each calcite type. The infrared calcite spectrum has three characteristic peaks in the region of 400-4000 cm^-1, designated ν₂, ν₃, and ν₄. When a calcite sample is ground, as part of the measurement preparation procedure, some grinding dependent changes will be revealed in the infrared spectrum. With additional grinding, the ν₃ peak narrows and the heights of the ν₂ and ν₄ peaks decrease, when both are normalized to the ν₃ height. By plotting the normalized heights of the ν₂ versus the ν₄ of several grindings of the same sample, a characteristic trend line is formed for each calcite type. The trend lines of geogenic calcites have the shallowest slopes and highest ν₄ values when compared to pyrogenic calcites, which can be further divided to ash and plaster/mortar samples. This method can assist in distinguishing between the various calcites, and provide insights into homogeneity and preservation state of the calcitic materials in question.
Fratzl P. & Weiner S. 2010. Bio-Inspired Materials - Mining the Old Literature for New Ideas Advanced Materials. 2010 Nov , 22 (41):4547-4550.

[Abstract]

Historical scientific books describe a wealth of organisms that could be useful to materials scientists in search of "bio-inspiration". Very often these books are not written in English, and are not easy for the materials science community to access. We propose that translating and re-editing such books or making them available electronically could be of extreme value to present-day researchers. (Side view of a diatom with a siliceous skeleton drawn by Hackel in 1904, reproduced with permission from Marixverlag GmbH, Wiesbaden.)
Boaretto E., Wu X., Yuan J., Bar-Yosef O., Chu V., Pan Y., Liu K., Cohen D., Jiao T., Li S., Gu H., Goldberg P. & Weiner S. 2009. Radiocarbon dating of charcoal and bone collagen associated with early pottery at Yuchanyan Cave, Hunan Province, China Proceedings of the National Academy of Sciences of the United States of America. 2009 Jun , 106 (24):9595-9600.

[Abstract]

Yuchanyan Cave in Daoxian County, Hunan Province (People's Republic of China), yielded fragmentary remains of 2 or more ceramic vessels, in addition to large amounts of ash, a rich animal bone assemblage, cobble and flake artifacts, bone tools, and shell tools. The artifacts indicate that the cave was a Late Paleolithic foragers' camp. Here we report on the radiocarbon ages of the sediments based on analyses of charcoal and bone collagen. The best-preserved charcoal and bone samples were identified by prescreening in the field and laboratory. The dates range from around 21,000 to 13,800 cal BP. We show that the age of the ancient pottery ranges between 18,300 and 15,430 cal BP. Charcoal and bone collagen samples located above and below one of the fragments produced dates of around 18,000. These ceramic potsherds therefore provide some of the earliest evidence for pottery making in China.
Ma Y., Aichmayer B., Paris O., Fratzl P., Meibom A., Metzler R. A., Politi Y., Addadi L., Gilbert P. U. P. A. & Weiner S. 2009. The grinding tip of the sea urchin tooth exhibits exquisite control over calcite crystal orientation and Mg distribution Proceedings of the National Academy of Sciences of the United States of America. 2009 Apr , 106 (15):6048-6053.

[Abstract]

The sea urchin tooth is a remarkable grinding tool. Even though the tooth is composed almost entirely of calcite, it is used to grind holes into a rocky substrate itself often composed of calcite. Here, we use 3 complementary high-resolution tools to probe aspects of the structure of the grinding tip: X-ray photoelectron emission spec-tromicroscopy (X-PEEM), X-ray microdiffraction, and NanoSIMS. We confirm that the needles and plates are aligned and show here that even the high Mg polycrystalline matrix constituents are aligned with the other 2 structural elements when imaged at 20-nm resolution. Furthermore, we show that the entire tooth is composed of 2 cooriented polycrystalline blocks that differ in their orientations by only a few degrees. A unique feature of the grinding tip is that the structural elements from each coaligned block interdigitate. This interdigitation may influence the fracture process by creating a corrugated grinding surface. We also show that the overall Mg content of the tooth structural elements increases toward the grinding tip. This probably contributes to the increasing hardness of the tooth from the periphery to the tip. Clearly the formation of the tooth, and the tooth tip in particular, is amazingly well controlled. The improved understanding of these structural features could lead to the design of better mechanical grinding and cutting tools.
Barak M. M., Currey J. D., Weiner S. & Shahar R. 2009. Are tensile and compressive Young's moduli of compact bone different? Journal of the Mechanical Behavior of Biomedical Materials. 2009 Jan , 2 (1):51-60.

[Abstract]

This study examines the question of whether the stiffness (Young's modulus) of secondary osteonal cortical bone is different in compression and tension. Electronic speckle pattern interferometry (ESPI) is used to measure concurrently the compressive and tensile strains in cortical bone beams tested in bending. ESPI is a non-contact method of measuring surface deformations over the entire region of interest of a specimen, tested wet. The measured strain distributions across the beam, and the determination of the location of the neutral axis, demonstrate in a statistically-robust way that the tensile Young's modulus is slightly (6%), but significantly greater than that of the compressive Young's modulus. It is also shown that within a relatively small bone specimen there are considerable variations in the modulus, presumably caused by structural inhomogeneities.
Boaretto E., Barkai R., Gopher A., Berna F., Kubik P. W. & Weiner S. 2009. Specialized flint procurement strategies for hand axes, scrapers and blades in the late lower paleolithic: A ¹⁰Be study at Qesem Cave, Israel Human Evolution. 2009 Jan , 24 (1):1-12.

[Abstract]

The procurement and selection of raw materials for producing different stone tools in the past provide invaluable insights into hominid technological capabilities and behavior. Flint has been extensively studied to document its sources, tool production, use, and recycling. Less is known about the procurement strategies used for obtaining the raw materials. Our approach is based on the concentration of cosmogenic in situ produced ¹⁰Be within the flint. As this is depth dependent, flint material collected from the surface can be differentiated from fint collected at depths or from special environments which protected the flint from cosmic radiation. ¹⁰Be concentrations in different tool types from the Lower Paleolithic strata of Qesem cave showed that the raw materials for large scrapers and hand-axes were obtained from deep buried material or recently exposed material. The smaller blades showed a larger distribution of ¹⁰Be that resembles the concentrations of ¹⁰Be in flint nodules collected from the soil surface around the cave. This is consistent with the observation that the large scrapers and handaxes were re-sharpened. Therefore some 400,000 years ago the Qesem cave inhabitants possessed a detailed knowledge of the resources, and the capability to procure appropriate raw materials for specific tool types.
Chattah N. L., Sharir A., Weiner S. & Shahar R. 2009. Determining the elastic modulus of mouse cortical bone using electronic speckle pattern interferometry (ESPI) and micro computed tomography: A new approach for characterizing small-bone material properties Bone. 2009 Jul , 45 (1):84-90.

[Abstract]

Mice phenotypes are invaluable for understanding bone formation and function, as well as bone disease. The elastic modulus is an important property of bones that can provide insights into bone quality. The determination of the elastic modulus of mouse cortical bone is complicated by the small dimensions of the bones. Whole bone bending tests are known to under estimate the elastic modulus compared to nanoindentation tests. The latter however provides information on extremely localized areas that do not necessarily correspond to the bulk elastic modulus under compression. This study presents a novel method for determining the bulk or effective elastic modulus of mouse cortical bone using the femur. We use Electronic Speckle Pattern Interferometry (ESPI), an optical method that enables the measurement of displacements on the bone surface, as it is compressed under water. This data is combined with geometric information obtained from micro-CT to calculate the elastic modulus. Roughly tubular cortical bone segments (2 mm) were cut from the diaphyses of femora of four week old C57BL/6 (B6) female mice and compressed axially using a mechanical tension-compression device. Displacements in the loading direction were mapped on the bone surface after loading the specimen. A linear regression of the displacement vs. axial-position enabled the calculation of the effective strain. Effective stress was calculated using force (N) data from the system's load cell and the mean cross-sectional area of the sample as determined by micro-CT. The effective elastic modulus (E) was calculated from the stress to strain ratio. The method was shown to be accurate and precise using a standard material machined to similar dimensions as those of the mouse femoral segments. Diaphyses of mouse femora were shown to have mean elastic moduli of 10.4 ± 0.9 GPa for femora frozen for eight months, 8.6 ± 1.4 GPa for femora frozen for two weeks and 8.9 ± 1.1 GPa for the fresh femora. These values are much higher than those measured using three-point bending, and lower than values reported in the literature based on nanoindentation tests from mice bones of the same age. We show that this method can be used to accurately and precisely measure the effective elastic modulus of mouse cortical bone.
Namdar D., Neumann R., Goren Y. & Weiner S. 2009. The contents of unusual cone-shaped vessels (cornets) from the Chalcolithic of the southern Levant Journal of Archaeological Science. 2009 Mar , 36 (3):629-636.

[Abstract]

Cornets are cone-shaped ceramic vessels, characteristic of the Chalcolithic period (ca. 4700-3700 BC) in Israel and Jordan. Their contents and use are unknown. Gas chromatography with flame ionization and mass-selective detection, showed that extracts of cornets from five different sites with different related activities (domestic, habitation cave and a cultic complex) all contain the same assemblage of mainly n-alkanes adsorbed within their walls. This assemblage differs from those found in other types of ceramic vessels from the same sites, as well as from the residues found within the associated sediments. The assemblage of odd and even-numbered n-alkanes found in the cornets is almost identical to that found in the residues of beeswax heated on modern ceramic fragments, as well as in a beehive from the Iron Age IIA strata at Tel Rehov, Israel. Thus the cornets are most likely to have contained beeswax. The presence of beeswax in the cornets contributes to our understanding of the Chalcolithic period; a time when secondary products such as milk, olive oil and wine are thought to have come into use.
Elbaum R., Melamed-Bessudo C., Tuross N., Levy A. & Weiner S. 2009. New methods to isolate organic materials from silicified phytoliths reveal fragmented glycoproteins but no DNA Quaternary International. 2009 Jan , 193 (1-2):11-19.

[Abstract]

Phytoliths are abundant in many archaeological sites, and can provide information on past vegetation. Very few analyses of their chemical composition have been made. Our measurements of the δ¹³C composition of modern wheat phytoliths suggest the presence of relatively large amounts of sugars and/or proteins in the water-soluble fraction, and lipids in the insoluble fraction. Other experimental approaches demonstrate that modern wheat phytoliths contain large quantities of glyco-conjugated proteins in a degraded state. One open question is whether or not phytoliths contain original DNA of the mother plant. Extracting protected ancient DNA from phytoliths would open many opportunities for progress in archaeobotanical studies. In order to address this question, we developed a method to dissolve phytoliths under conditions that do not degrade naked DNA, and showed that only a minimal amount of DNA was lost during the procedure. A hypersensitive assay did not, however, detect any DNA in extracts of phytoliths from an unburned phytolith-rich layer in Iron-Age sediments from Tel Dor, Israel. Extractions from modern phytolith samples of wheat also failed to provide any indication of DNA. We conclude that DNA is absent or not routinely recoverable in a random assembly of siliceous phytoliths.
Eliyahu-Behar A., Regev L., Shilstein S., Weiner S., Shalev Y., Sharon I. & Berg J. 2009. Identifying a roman casting pit at Tel Dor, Israel: Integrating field and laboratory research Journal of Field Archaeology. 2009 , 34 (2):135-151.

[Abstract]

The functional identification of industrial features is difficult when few characteristic artifacts are preserved. We studied a Roman-period pyrotechnological feature at Tel Dor, Israel, where the only possibly diagnostic ceramic artifacts were found in fill between the walk and above the floor, rendering their association with the feature itself uncertain. The ceramic artifacts included coarse slabs and fragments of utilitarian vessels, some vitrified with adhering bronze droplets or slag-like residues. Analysis of the sediments within and around the industrial feature, using an X-ray fluorescence spectrometer on-site, revealed high concentrations of copper and lead, indicating metallurgical activity. Lead isotope analyses showed that the slag-like residue adhering to a ceramic fragment had the same isotopic values as the sediments, implying that the fragment was associated with the activities carried out in the feature. Microscopic and chemical analyses of the slag-like residue demonstrated that it was produced from melting leaded bronze. Some of the ceramic fragments contained elongated impressions on their inner surfaces, similar to those of casting molds found at other sites. We propose that the feature was used as a casting pit for bronze objects.
Tsartsidou G., Lev-Yadun S., Efstratiou N. & Weiner S. 2009. Use of space in a Neolithic village in Greece (Makri): phytolith analysis and comparison of phytolith assemblages from an ethnographic setting in the same area Journal of Archaeological Science. 2009 Oct , 36 (10):2342-2352.

[Abstract]

Phytolith analyses were conducted in a Pottery Neolithic village (Makri) of Northern Greece in order to reconstruct aspects of past human activities as a function of both space and time. The analyses of phytolith assemblages were based on a reference collection of modern plant phytoliths (Tsartsidou et al., 2007), as well as an ethnographic study in an agropastoral community (Sarakini) in the same area that showed that many phytolith assemblages are characteristic of the activities carried out in different locations within and around the village (Tsartsidou et al., 2008). The same approach was used for studying the phytolith assemblages in the Neolithic village of Makri, namely measuring phytolith concentrations, diversities of phytolith assemblages relative to control samples collected from samples outside the village and detailed analysis of various phytolith morphotypes. At Makri samples from floors and various constructions (i.e. pit, platforms) were analysed, as well as sediments from an open area inside the village. The results show that Neolithic Makri was a society with a mixed agricultural and pastoral economy. Wheat and barley were cultivated for food and fodder and free-range animals were raised in a village inhabited year round. Indoor areas were not clearly differentiated from outdoor areas inside the village. The phytolith assemblages in only one series of floors produced at a specific location over an extended period of time reflected the use of that space for cereal storage or food processing. The phytolith assemblages from all the other floors examined did not reflect the local activities, but rather the constructional materials used for producing the floors.
Barak M. M., Geiger S., Chattah N. L. T., Shahar R. & Weiner S. 2009. Enamel dictates whole tooth deformation: A finite element model study validated by a metrology method Journal of Structural Biology. 2009 Dec , 168 (3):511-520.

[Abstract]

In order to understand whole tooth behavior under load the biomechanical role of enamel and dentin has to be determined. We approach this question by comparing the deformation pattern and stiffness of intact teeth under load with the deformation pattern and stiffness of the same teeth after the enamel has been mechanically compromised by introducing a defect. FE models of intact human premolars, based on high resolution micro-CT scans, were generated and validated by in vitro electronic speckle pattern interferometry (ESPI) experiments. Once a valid FE model was established, we exploit the flexibility of the FE model to gain more insight into whole tooth function. Results show that the enamel cap is an intrinsically stiff biological structure and its morphology dictates the way a whole tooth will mechanically behave under load. The mechanical properties of the enamel cap were sufficient to mechanically maintain almost its entire stiffness function under load even when a small defect (cavity simulating caries) was introduced into its structure and breached the crown integrity. We conclude that for the most part, that enamel and not dentin dictates the mechanical behavior of the whole tooth.
Chattah N. L., Shohar R. & Weiner S. 2009. Design strategy of minipig molars using electronic speckle pattern interferometry: Comparison of deformation under load between the tooth-mandible complex and the isolated tooth Advanced Materials. 2009 Jan , 21 (4):413-418.

[Abstract]

The design strategy of minipig molars using electronic speckle pattern interferometry through comparison of deformation under load between the tooth-mandible complex and the isolated tooth was studied. The experiment measured the mechanical behavior of whole teeth using strain gauges/extensometers to measure local displacements and strains. The study used the ESPI to map the displacement of whole molars loaded in two distinct regimes. Healthy minipigs' tooth were coated with a thin coat of white spray, a common procedure in optical testing. After testing, the section was extracted from its holder and the bone was carefully removed from the root using a water-cooled dental drill. The study showed that the minipig molar is capable of deforming and rotating at low loads, and that the intrinsic reaction of the crown to eccentric loading is complemented and even enhanced by the structures supporting the teeth.
Albert R. M., Shahack-Gross R., Cabanes D., Gilboa A., Lev-Yadun S., Portillo M., Sharon I., Boaretto E. & Weiner S. 2008. Phytolith-rich layers from the Late Bronze and Iron Ages at Tel Dor (Israel): mode of formation and archaeological significance Journal of Archaeological Science. 2008 Jan , 35 (1):57-75.

[Abstract]

The presence of many phytolith-rich layers in late Bronze and Iron Age deposits at Tel Dor, Israel, are indicative of specific locations where plants were concentrated. Detailed studies of six of these phytolith-rich layers and associated sediments from Tel Dor show that the phytoliths were derived mainly from wild and domestic grasses. The most common domestic grass was the cereal Triticum aestivum (bread wheat). Three of these layers have a microlaminated microstructure, associated dung spherulites and phosphate nodules; characteristics that all point to the phytolith-rich layers having formed from dung in animal enclosures. In two of the layers, the microlaminated structure is absent while dung spherulites and phosphate nodules are present, suggesting that these too originate from dung that was not deposited in an enclosure. The sixth layer is microlaminated but does not contain spherulites. We thus cannot suggest a parsimonious explanation of its observed properties. Concentrations of burnt phytoliths are present in three locations, implying that dung was either burnt in situ or the ashes from burnt dung were redeposited. The transformation of dung accumulations into phytolith-rich layers involves a loss of organic material and hence a significant reduction in sediment volume, which is clearly apparent in the stratigraphy of some of the locations examined. The volume reduction can be observed in the macrostratigraphy and has important implications with regard to macrostratigraphic interpretation. The presence of abundant phytolith-rich layers on the tell has significant implications for the concept of 'urbanism' during these periods.
Chu V., Regev L., Weiner S. & Boaretto E. 2008. Differentiating between anthropogenic calcite in plaster, ash and natural calcite using infrared spectroscopy: implications in archaeology Journal of Archaeological Science. 2008 Apr , 35 (4):905-911.

[Abstract]

Infrared spectroscopy provides information not only on the type of calcium carbonate polymorph, but also on the extent of atomic order. In calcite, three major infrared absorption peaks are identified: ν₃, ν₂, and ν₄. It was shown that the ratio between ν₂ and ν₄ bands reflects the order of the calcite crystal structure. In this paper we analyse this ratio in geologically formed calcites, archaeological plasters, modern plasters and experimentally prepared plasters. For the geological calcite, the values of the ν₂/ν₄ ratio are around 3, whereas for the experimentally prepared plasters, the values are around 6.5. The ν₂/ν₄ ratio for archaeological plasters varies from 3 to 6. This shows that a high ratio is indicative of disorder in the crystal, and implies that the calcite was formed from calcium oxide at high temperatures. It also implies that this disorder can be preserved for at least 14,000 years. The ν₂/ν₄ ratio of calcite from archaeological sites can thus be used to differentiate between anthropogenic calcite, such as in plaster, mortar and wood ash, from geogenic calcite, such as in limestone. The ratio may also be used to identify plaster or ash that still retains its original crystals and therefore carbon-14 content.
Politi Y., Metzler R. A., Abrecht M., Gilbert B., Wilt F. H., Sagi I., Addadi L., Weiner S. & Gilbert P. U. P. A. 2008. Transformation mechanism of amorphous calcium carbonate into calcite in the sea urchin larval spicule Proceedings of the National Academy of Sciences of the United States of America. 2008 Nov , 105 (45):17362-17366.

[Abstract]

Sea urchin larval spicules transform amorphous calcium carbonate (ACC) into calcite single crystals. The mechanism of transformation is enigmatic: the transforming spicule displays both amorphous and crystalline properties, with no defined crystallization front. Here, we use X-ray photoelectron emission spectromicroscopy with probing size of 40-200 nm. We resolve 3 distinct mineral phases: An initial short-lived, presumably hydrated ACC phase, followed by an intermediate transient form of ACC, and finally the biogenic crystalline calcite phase. The amorphous and crystalline phases are juxtaposed, often appearing in adjacent sites at a scale of tens of nanometers. We propose that the amorphous-crystal transformation propagates in a tortuous path through preexisting 40- to 100-nm amorphous units, via a secondary nucleation mechanism.
Mahamid J., Sharir A., Addadi L. & Weiner S. 2008. Amorphous calcium phosphate is a major component of the forming fin bones of zebrafish: Indications for an amorphous precursor phase Proceedings of the National Academy of Sciences of the United States of America. 2008 Sept , 105 (35):12748-12753.

[Abstract]

A fundamental question in biomineralization is the nature of the first-formed mineral phase. In vertebrate bone formation, this issue has been the subject of a long-standing controversy. We address this key issue using the continuously growing fin bony rays of the Tuebingen long-fin zebrafish as a model for bone mineralization. Employing high-resolution scanning and transmission electron microscopy imaging, electron diffraction, and elemental analysis, we demonstrate the presence of an abundant amorphous calcium phosphate phase in the newly formed fin bones. The extracted amorphous mineral particles crystallize with time, and mineral crystallinity increases during bone maturation. Based on these findings, we propose that this amorphous calcium phosphate phase may be a precursor phase that later transforms into the mature crystalline mineral.
Ma Y., Cohen S., Addadi L. & Weiner S. 2008. Sea urchin tooth design: An "all-calcite" polycrystalline reinforced fiber composite for grinding rocks Advanced Materials. 2008 Apr , 20 (8):1555-1559.

[Abstract]

A study was conducted to investigate how different kinds of calcite crystals in the sea urchin tooth work together as an effective grinding tool. The study showed that the polycrystalline matrix has a higher elastic modulus and hardness than the single crystalline needles and plates, and that the working surface is smooth. It ascribed the unique properties of the matrix to a combination of a very high Mg content, the lack of orientation of the nanocrystals, and their very small size. The single crystals contain relatively high concentrations of Mg, and presumably like other echinoderm crystals, occluded proteins that reduce the brittleness of the calcite and allow it to deform in a plastic manner and fracture with conchoidal cleavage. The study proposed that it is the unusually high hardness and modulus of the Mg-enriched matrix that is mainly responsible for the ability to grind the rocky substrate, whereas the arrays of calcite needles and plates act as a support framework for the polycrystalline matrix.
Eliyahu-Behar A., Shilstein S., Raban-Gerstel N., Goren Y., Gilboa A., Sharon I. & Weiner S. 2008. An integrated approach to reconstructing primary activities from pit deposits: iron smithing and other activities at Tel Dor under Neo-Assyrian domination Journal of Archaeological Science. 2008 Nov , 35 (11):2895-2908.

[Abstract]

Secondary pit deposits in historical occupations of Near Eastern mounds are usually regarded as uninteresting and are seldom analyzed. We used an integrated approach to study all the artifacts as well as the sediments in a pit at Tel Dor, on Israel's Carmel coast, dating to the 7th c. BCE - a period when the site served as an Assyrian administrative center. This pit was unusually large, had a peculiar ceramic assemblage, and many macroscopic metallurgical wastes. A detailed excavation and analysis revealed that the pit served intermittently as a waste disposal site for an iron smithy and for pottery that was presumably involved in maritime trading. On two occasions the area was also used for animal penning. Despite the obvious importance of the iron industry to the Neo-Assyrian Empire, actual workshops are rare in its archaeological record. Hence the new information regarding an Iron Age iron smithy in the southern Levant contributes to the study of this industry, and also to the history of Dor in this period.
Levy-Lior A., Pokroy B., Levavi-Sivan B., Leiserowitz L., Weiner S. & Addadi L. 2008. Biogenic guanine crystals from the skin of fish may be designed to enhance light reflectance Crystal Growth & Design. 2008 Feb , 8 (2):507-511.

[Abstract]

The metallic luster from the skin of fish is due to a photonic crystal system composed of multilayer stacks of cytoplasm and crystals. The crystals are described as thin (50-100 nm) plates of guanine, with no reference to their hydration state. We established through X-ray diffraction that their crystal structure is that of anhydrous guanine. We noted that their crystal structurefunction relationship is exceptional compared to other purines with similar molecular stacking of the crystal structure. These elongate in the direction of molecular stacking, in contrast to the biogenic anhydrous guanine crystals whose smallest dimension is in the stacking direction. On the basis of the known crystal structure of anhydrous guanine, theoretical growth morphology was calculated. These calculations predict crystals elongated in the direction of the molecular stacking. The exposed molecular plane of the biogenic crystals is the (102) plane, which is composed of densely packed H-bonded guanine molecules. It is known that the in-plane polarizability of guanine molecules is significantly higher than the direction perpendicular to the molecular plane, most likely causing anisotropy of the crystals refractive index. It is therefore conceivable that the unique morphology observed in crystals from the skin of fish is designed to enhance their light reflective properties.
Rebollo N. R., Cohen Ofri O. I., Popovitz-Biro R., Bar-Yosef O., Meignen L., Goldberg P., Weiner S. & Boaretto E. 2008. Structural characterization of charcoal exposed to high and low pH: Implications for ¹⁴C sample preparation and charcoal preservation Radiocarbon. 2008 , 50 (2):289-307.

[Abstract]

Chemical and structural similarities between poorly preserved charcoal and its contaminants, as well as low radiocarbon concentrations in old samples, complicate ¹⁴C age determinations. Here, we characterize 4 fossil charcoal samples from the late Middle Paleolithic and early Upper Paleolithic strata of Kebara Cave, Israel, with respect to the structural and chemical changes that occur when they are subjected to the acid-base-acid (ABA) treatment. Differential thermal analysis and TEM show that acid treatment disrupts the structure, whereas alkali treatment results in the reformation of molecular aggregates. The major changes are ascribed to the formation of salt bridges at high pH and the disruption of the graphite-like crystallites at low pH. Weight losses during the treatments are consistently greater for older samples, implying that they are less well preserved. Based on the changes observed in vitro due to pH fluctuations, various methods for removing contamination, as well as a mechanism for preferential preservation of charcoal in nature, are proposed.
Weiner S. 2008. Biomineralization: A structural perspective Journal of Structural Biology. 2008 Sept , 163 (3):229-234.

[Abstract]

Biomineralization is an inherently structural subject; the structure of the mineral phase, the structure of the matrix composed of macromolecules and especially the structure of the interphase zone between them. Studies of the dynamics of mineral formation have revealed that a widespread strategy used by many organisms is to first form a disordered mineral phase. Only when it is in place and has adopted its appropriate shape, is it induced to crystallize. Matrix studies have highlighted the importance of a unique group of proteins that are rich in aspartic acid. These are involved in controlling mineral formation. Relating structure to function in mineralized tissues, often involves an understanding of mechanical properties in terms of not only the hierarchical structure of the tissue, but also the graded structure that varies from one location to another. Structure is thus in many respects the foundation upon which the field of biomineralization rests.
Nudelman F., Shimoni E., Klein E., Rousseau M., Bourrat X., Lopez E., Addadi L. & Weiner S. 2008. Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: An environmental- and cryo-scanning electron microscopy study Journal of Structural Biology. 2008 May , 162 (2):290-300.

[Abstract]

A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer.
Mazar A., Namdar D., Panitz-Cohen N., Neumann R. & Weiner S. 2008. Iron Age beehives at Tel Rehov in the Jordon valley Antiquity. 2008 Sept , 82 (317):629-639.

[Abstract]

Beehives were discovered in a densley built area in the Iron Age city of Rehov (tenth-ninth century BC). They consisted of hollow clay cylinders, each with a little hole at one end (for the bee) and a removable lid at the other (for the bee keeper). These beehives, the earliestfound in the Near East, were identified by analogy with examples pictured on Egyptian tombs and in use by traditional peoples. The suggested identification was confirmed by chemical analysis.
Addadi L., Politi Y., Nudelman F. & Weiner S. 2008. Biomineralization design strategies and mechanisms of mineral formation: Operating at the edge of instability Engineering Of Crystalline Materials Properties: State Of The Art In Modeling, Design And Applications. 2008 , :1-15.

[Abstract]

The biological approach to forming crystals is proving to be most surprising. Three strategies evolved by organisms to build their mineralized materials will be discussed. These are: 1) Building mineralized structures with stable amorphous phases; 2) building mineralized structures with single crystals; 3) building mineralized structures with polycrystalline organized arrays. Interestingly, all appear to involve at some stage the use of an amorphous mineral phase.
Utku F. S., Klein E., Saybasili H., Yucesoy C. A. & Weiner S. 2008. Probing the role of water in lamellar bone by dehydration in the environmental scanning electron microscope Journal of Structural Biology. 2008 Jun , 162 (3):361-367.

[Abstract]

Water, collagen and mineral are the three major components of bone. The structural organization of water and its functions within the bone were investigated using the environmental scanning electron microscope and by analyzing dimensional changes that occur when fresh equine osteonal bone is dehydrated and then rehydrated. These changes are attributed mainly to loss of bulk and weakly bound water. In longitudinal sections a contraction of 1.2% was observed perpendicular to the lamellae, whereas no contraction occurred parallel to the lamellae. In transverse sections a contraction of 1.4% was observed both parallel and perpendicular to the lamellae. SEM back scattered electron images showed that about half of an individual lamella is less mineralized, and thus has more water than the other half. We therefore propose that contractions perpendicular to lamellae are due to the presence of more water-filled rather than mineral-filled channels within the mineralized collagen fibril arrays. As these channels are also aligned with the crystal planes, the crystal arrays, oriented as depicted in the rotated plywood model for lamellar bone, facilitate or hinder contraction in different directions.
Barak M. M., Weiner S. & Shahar R. 2008. Importance of the integrity of trabecular bone to the relationship between load and deformation of rat femora: An optical metrology study Journal of Materials Chemistry. 2008 , 18 (32):3855-3864.

[Abstract]

Bone is a composite hierarchical structure composed of a cortical shell and inner trabecular tissue. One of the most basic questions in whole-bone function is the relative contributions of cortical and trabecular bone tissues to the loaded whole bone. In this study, the manner in which the cortical surfaces of an intact proximal rat femur deform under load is compared to the same femur after some of the trabecular bone in the distal femoral neck was removed. The surface displacements were measured by electronic speckle pattern interferometry (ESPI) and the extent of trabecular bone removed was determined by high resolution micro-CT scanning. The results show that after damaging the trabecular bone tissue in the distal femoral neck, the manner in which compressive loads are transformed to other regions of the femoral neck changed. The whole bone behaved in a 'stiffer' manner. This demonstrates the importance of connectivity of the trabeculae and that beyond a certain threshold of damage the normal load-transferring mechanism is impaired. Since these experiments were carried out in a non-contact non-destructive manner in a wet environment and the rat femur was loaded in a close-to physiological manner, we postulate that our results have a direct relevance to the in vivo biomechanical behavior of the femoral neck.
Tsartsidou G., Lev-Yadun S., Efstratiou N. & Weiner S. 2008. Ethnoarchaeological study of phytolith assemblages from an agro-pastoral village in Northern Greece (Sarakini): development and application of a Phytolith Difference Index Journal of Archaeological Science. 2008 Mar , 35 (3):600-613.

[Abstract]

Phytolith assemblages are analysed in an ethnographic agro-pastoral community in Northern Greece. A new method for analyzing the data, combined with the concentrations of phytoliths per gram sediment, helps to differentiate diverse uses of space in the village. The Phytolith Difference Index (PDI) contrasts the phytolith assemblages in sediment samples from the region around the village least affected by human activities with those in the village and its immediate surroundings. The PDI reveals that many of the samples are dominated by the input of the stalks of the domestic cereal, rye, which is used for food, animal fodder and roof thatching. The PDI also differentiates between dung from mules or donkeys with dung from free ranging cows and goats. Activity areas analysed include storage areas, stabling areas, animal enclosures, floors from living areas that were repeatedly swept, hearths and open areas between structures. The combined use of the PDI, together with phytolith concentrations and phytolith morphotype analyses, may prove to be useful for deciphering activity areas in archaeological sites of not only agro-pastoralists, but also pastoralists and hunter-gatherers.
Mercier N., Valladas H., Froget L., Joron J., Reyss J., Weiner S., Goldberg P., Meignen L., Bar-Yosef O., Belfer-Cohen A., Chech A., Kuhn S. L., Stiner M. C., Tillier A., Arensburg B. & Vandermeersch B. 2007. Hayonim Cave: a TL-based chronology for this Levantine Mousterian sequence Journal of Archaeological Science. 2007 Jul , 34 (7):1064-1077.

[Abstract]

The thermoluminescence dating method was applied to 77 heated flints from the Mousterian layers of Hayonim Cave in order to provide a precise TL-based chronology for this important Levantine sequence. A detailed dosimetric study was performed by using 76 dosimeter capsules and revealed strong spatial dose-rate variations. In parallel, Fourier transform infrared spectrometry enabled the identification of various mineral assemblages in the sediments of the cave and to localize the boundaries of these assemblages. By comparing these two data sets, it is shown that low dose-rate values (∼500 μGy/a) are systematically recorded in areas where the calcite-dahllite (CD) assemblage is preserved, whereas higher values (up to 1300 μGy/a) are associated with the leucophosphite, montgomeryite, variscite and siliceous aggregates (LMVS) assemblage. The dosimetric and mineralogical information was combined in order to assess, where possible, the dose-rate experienced by each flint during its burial. Some of the flint samples analyzed were too close to mineral assemblage boundaries and were therefore discarded. This rigorous selection led to TL ages ranging from 230 to 140 ka for the lower part of the Mousterian sequence (layers F and Lower E), which contains lithic industries characterized by blade production using the laminar method.
Politi Y., Mahamid J., Goldberg H., Weiner S. & Addadi L. 2007. Asprich mollusk shell protein: In vitro experiments aimed at elucidating function in CaCO₃ crystallization CrystEngComm. 2007 , 9 (12):1171-1177.

[Abstract]

Acidic proteins are key components of the organic matrix of many biologically formed minerals and are therefore thought to play an important role in their formation. Here we study the effect of one unusually acidic protein of the Asprich family, associated with mollusk shell prismatic layer, on the precipitation of CaCO₃ in vitro. We show that Asprich induces and transiently stabilizes the deposition of amorphous calcium carbonate (ACC). Asprich also induces the formation of ACC when adsorbed onto chitin, a major component of the intracrystalline organic matrix of the prismatic layer. Based on this evidence, combined with previous studies on the forming prisms in the shell layer, we suggest that the in vivo function of Asprich is inducing and stabilizing ACC particles and inhibiting their uncontrolled crystallization until they undergo secondary nucleation on the growing prisms.
Tsartsidou G., Lev-Yadun S., Albert R., Miller-Rosen A., Efstratiou N. & Weiner S. 2007. The phytolith archaeological record: strengths and weaknesses evaluated based on a quantitative modern reference collection from Greece Journal of Archaeological Science. 2007 Aug , 34 (8):1262-1275.

[Abstract]

The phytolith record from archaeological strata is a powerful tool for reconstructing aspects of past human behaviour and ecology. Considerable insights into the problems and potentials of this record can be obtained by studying phytoliths in modern plants. We prepared a phytolith reference collection of modern plants mostly from northern Greece that includes quantitative information on phytolith concentrations (number of phytoliths per gram dry organic material) and morphological assemblages. Here we analyse this reference collection with the aim of evaluating the strengths and weaknesses of the archaeological phytolith record. The reference collection comprises 62 different plant taxa. Of them, 28 are trees, 1 conifer, 10 shrubs and 1 herb, 3 domesticated annual legumes and 19 monocotyledons. We demonstrate quantitatively the extent to which woody species, legumes and fruits of dicots contribute only small amounts of phytoliths to the sediments per unit tissue dry weight, while leaves of trees and shrubs produce significant amounts of phytoliths and grasses are prolific phytolith producers. We compare the data of this reference collection with a similar reference collection from another Mediterranean ecosystem (Israel). The comparison indicates that some aspects of phytolith production are probably genetically controlled, whereas others are environmentally controlled. We note that despite the fact that woody species produce few phytoliths per gram of tissue, their record can be most informative when taking into account other properties of the sediments. We also note that jigsaw puzzle-shaped phytoliths normally form in plants that grow in humid conditions, but may form in arid environments when the plants are irrigated. This study clearly shows the extent to which some plants can be under-represented and others over-represented in the phytolith record. Knowing the extent of this bias can greatly improve our interpretation of the phytolith record.
Berna F., Behar A., Shahack-Gross R., Berg J., Boaretto E., Gilboa A., Sharon I., Shalev S., Shilstein S., Yahalom-Mack N., Zorn J. R. & Weiner S. 2007. Sediments exposed to high temperatures: reconstructing pyrotechnological processes in Late Bronze and Iron Age Strata at Tel Dor (Israel) Journal of Archaeological Science. 2007 Mar , 34 (3):358-373.

[Abstract]

Many of the sediments analysed from Tel Dor (Israel) show structural alterations indicating that they were exposed to high temperatures. This observation is consistent with the abundant evidence for use of pyrotechnology from the earliest exposed Middle Bronze Age strata through the Roman period. Such structurally altered sediments may well represent one of the more widespread and durable records of pyrotechnology, and as such could be invaluable for reconstructing past human activities. The specific aims of this research are therefore to develop the means for identifying local sediments that were altered by different pyrotechnological activities and to elucidate the varying circumstances whereby sediments were exposed to high temperatures in a Late Bronze and Iron Age 1 section. We first characterize natural sediments sampled on and in the proximity of the tell and monitor their transformations due to exposure to high temperatures in an oven and in open fires, focusing in particular on the transformations of the clay mineral components of mud-brick materials. The analytical techniques used include micromorphology, Fourier transform infrared spectrometry (FTIR), X-ray powder diffractometry (XRD) and X-ray fluorescence (XRF) spectrometry. Using the temperature "calibrated" data, we confirm that large volumes of sediments at Tel Dor were exposed to high temperatures. In Area G, we identify three fundamentally different ways that heat-affected sediments were produced and accumulated: (1) In the Late Bronze Age (Phases 11-12) the sediments were heated to temperatures between 800 and 900 degrees C and were then deposited in the area under investigation. A plausible scenario is that these sediments were exposed to heat from ovens or kilns; (2) During the early Iron Age (Phase 10) the heat-affected sediments (heated above 1000 degrees C) formed in close association with casting pits for the working of copper-containing (bronze) objects. (3) During Phase 9 of the Iron Ag
Gueta R., Natan A., Addadi L., Weiner S., Refson K. & Kronik L. 2007. Local atomic order and infrared spectra of biogenic calcite ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 2007 , 46 (1-2):291-294.

[Abstract]

Shining light on local order: The relations between local crystalline order and peak intensities in the infrared spectra of calcite are explained in terms of different sensitivities to Ca-O and O⋯O distances (see picture) by comparing ab initio phonon spectra for ideal and distorted calcite unit cells with experimental spectra of various biogenic and geological calcites. (Figure Presented).
Sone E. D., Weiner S. & Addadi L. 2007. Biomineralization of limpet teeth: A cryo-TEM study of the organic matrix and the onset of mineral deposition Journal of Structural Biology. 2007 Jun , 158 (3):428-444.

[Abstract]

The continuously growing limpet radula contains teeth at various stages of maturity and thus provides an excellent opportunity for studying the processes and mechanisms of their mineralization. We report here on our structural investigations of the pre-formed chitin matrix and the initial deposition and growth of goethite (α-FeOOH) crystals within the matrix. By using cryo-techniques, in which unstained sections of the teeth are examined in a frozen-hydrated state in a transmission electron microscope (TEM), we were able to characterize the process without introducing artifacts normally associated with the staining, dehydration, and embedding required for conventional TEM. The unmineralized matrix consists of relatively well ordered, densely packed arrays of chitin fibers, with only a few nanometers between adjacent fibers. There are clearly no pre-formed compartments that control goethite crystal size and shape; rather, crystals must push aside or engulf the fibers as they grow. By examining teeth nearly row-by-row around the onset of mineralization, we were able to image the first-formed mineral within the chitin matrix. These linear deposits of goethite appear to nucleate on the chitin fibers, which thus control the orientation of the crystals. Crystal growth, on the other hand, is apparently not influenced by the matrix, in contrast to many other biomineralization systems.
Ma Y., Weiner S. & Addadi L. 2007. Mineral deposition and crystal growth in the continuously forming teeth of sea urchins Advanced Functional Materials. 2007 Oct , 17 (15):2693-2700.

[Abstract]

The early stages of formation of the crystalline elements in the continuously forming sea urchin teeth were studied using polarized light microscopy, SEM, TEM and calcite overgrowth. Transient amorphous calcium carbonate (ACC) is initially deposited in the needles and subsequently transforms into a single crystal of calcite. ACC exists in the center of both the "primary plates and the needles, even though the surfaces are already well crystallized. TEM images and electron diffraction patterns, as well as calcite crystal overgrowth shows that the highly convoluted primary plate-lamellar needle complex grows into a single crystal of calcite. The calcite crystal c axis is approximately parallel to the slightly curved surface of the primary plate. The needles grow along the unusual [102] direction of calcite. This study demonstrates that highly complex shaped single crystals are produced using a transient amorphous precursor phase. The beneficial properties of the single crystals are then combined with those of a polycrystalline matrix to yield a sophisticated composite functional material. This strategy could conceivably be applied to the production of synthetic materials.
Nudelman F., Chen H. H., Goldberg H. A., Weiner S. & Addadi L. 2007. Spiers Memorial Lecture: Lessons from biomineralization: Comparing the growth strategies of mollusc shell prismatic and nacreous layers in Atrina rigida Faraday Discussions. 2007 , 136 :9-25.

[Abstract]

The mollusc shell prismatic layer of Atrina rigida is composed of an assemblage of large and relatively perfect single calcite crystals, embedded in an organic matrix. A key to elucidating basic mechanisms of mineralization is understanding the structures of the matrix, the mineral and the relations between them. The matrix that envelopes each prism (the inter-prismatic matrix) is composed mainly of glycine-rich proteins, while the matrix inside each prism (intra-crystalline matrix) is composed of a network of chitin fibers. Prisms grow by deposition of mineral particles on the chitin fibers. The mineral particles are associated with highly acidic proteins from the Asprich family, which presumably stabilize an amorphous mineral precursor. We infer that once in contact with the already formed crystalline prism, the particles crystallize by epitaxial nucleation. In nacre, sheets of β-chitin are interspaced by silk-like proteins in a hydrated gel-like state. β-Chitin forms a scaffold onto which the acidic proteins are adsorbed. Some of these are organized into a crystal nucleation site, where nucleation of aragonite, supposedly from colloidal amorphous calcium carbonate particles, is induced. Comparing the mechanisms of growth of the nacreous and prismatic layers can help to understand the underlying strategies of formation of mineralized structures.
Namdar D., Neumann R., Sladezki Y., Haddad N. & Weiner S. 2007. Alkane composition variations between darker and lighter colored comb beeswax Apidologie. 2007 Sept , 38 (5):453-461.

[Abstract]

Beeswax is composed of fatty acids, odd numbered n-alkanes and wax esters. Focusing on the most stable components of beeswax, namely the n-alkanes, we have found by gas chromatography and gas chromatography-mass spectrometry analyses of combs from twelve colonies from Israel and Jordan that as beeswax ages and darkens its n-alkane composition changes. The amount of even numbered n-alkanes ( C-22-C-32) is significantly higher in darker colored beeswax as compared to light colored beeswax. We attribute this in part to the accumulation of cuticular residues found in the darker colored comb cells. Cuticular residues are known to contain C-23-C-32 odd and even numbered n-alkanes.
Cohen Ofri O. I., Popovitz-Biro R. & Weiner S. 2007. Structural characterization of modern and fossilized charcoal produced in natural fires as determined by using electron energy loss spectroscopy Chemistry-A European Journal. 2007 , 13 (8):2306-2310.

[Abstract]

Charcoal produced in natural fires is widespread, but surprisingly little is known about its structure and stability. TEM and electron energy loss spectroscopy (EELS) were used to characterize the organized graphitelike microcrystallites and amorphous nonorganized phases of modern charcoal that had been produced in natural fires. In addition, a semiordered structure was identified in two modern charcoal samples. Fossilized charcoal contains fewer graphite-like microcrystallites than modern samples. EELS spectra confirmed that the dominant structure in fossilized charcoal is amorphous carbon. EELS measurements also revealed that only the nonorganized phase contains oxygen, which indicates that the degradation of the fossilized charcoal structure occurs mainly through oxidation processes. The few graphite-like microcrystallites found in fossilized charcoal were composed of onion-like structures that are probably less prone to oxidation owing to their rounded structures.
Shahar R. & Weiner S. 2007. Insights into whole bone and tooth function using optical metrology Journal of Materials Science. 2007 Nov , 42 (21):8919-8933.

[Abstract]

Understanding the relations between the mechanical responses of whole entities, their materials properties and their structures, is a challenge. This challenge is greatly enhanced when the material itself is complex, and when the entity it forms has a convoluted shape. It is for these reasons that it is still beyond the state-of-the-art to predict and fully understand the mechanical functions of whole biological entities such as bones and teeth. Recent advances in optical metrology open up new opportunities as they enable the precise and accurate mapping of the manner in which the entire surface of a whole stiff mineralized tissue deforms. Furthermore these data can be obtained non-destructively and without contact with the sample. Data of this kind create the exciting possibility of relating the complex distribution of mechanical properties of loaded biological materials such as bone and teeth and their microstructures to deformations and strains. Such studies could improve our understanding of normal physiological processes such as skeletal aging, as well as disease processes such as osteoporosis. They also provide opportunities for engineers designing bio-inspired materials to study the principles, advantages, and characteristics of the behavior of hierarchical and multifunctional materials. In this manuscript we review optical metrology methods, highlight studies of whole body function for bones and teeth, and in particular those studies that provide insights into structure-function relations. We also outline the potential for future studies.
Shahar R., Zaslansky P., Barak M. M., Friesem A., Currey J. D. & Weiner S. 2007. Anisotropic Poisson's ratio and compression modulus of cortical bone determined by speckle interferometry Journal of Biomechanics. 2007 , 40 (2):252-264.

[Abstract]

Young's modulus and Poisson's ratios of 6 mm-sized cubes of equine cortical bone were measured in compression using a micro-mechanical loading device. Surface displacements were determined by electronic speckle pattern-correlation interferometry. This method allows for non-destructive testing of very small samples in water. Analyses of standard materials showed that the method is accurate and precise for determining both Young's modulus and Poisson's ratio. Material properties were determined concurrently in three orthogonal anatomic directions (axial, radial and transverse). Young's modulus values were found to be anisotropic and consistent with values of equine cortical bone reported in the literature. Poisson's ratios were also found to be anisotropic, but lower than those previously reported. Poisson's ratios for the radial-transverse and transverse-radial directions were 0.15 ± 0.02, for the axial-transverse and axial-radial directions 0.19 ± 0.04, and for the transverse-axial and radial-axial direction 0.09 ± 0.02 (mean±SD). Cubes located only millimetres apart had significantly different elastic properties, showing that significant spatial variation occurs in equine cortical bone.
Cohen-Ofri I., Weiner L., Boaretto E., Mintz G. & Weiner S. 2006. Modern and fossil charcoal: Aspects of structure and diagenesis Journal of Archaeological Science. 2006 Mar , 33 (3):428-439.

[Abstract]

The structures and compositions of modern and fossil charcoal samples were compared in order to evaluate charcoal degradation processes in archaeological sites. Modern charcoal samples produced in campfires contain two major phases: graphite-like microcrystallites and a non-organized phase. These phases create a mosaic-like structure with differing relative proportions depending on the taxonomic source of the wood used. Fossil charcoal samples (Tel Dor, Israel: 3000 years BP and Kebara Cave, Israel: 40,000 years BP) also contained the graphite-like microcrystallites and the non-organized phases, but were clearly altered compared to modern charcoal. The graphite-like phase of the fossil charcoal has much higher electrical resistivity, and its ESR properties show that it has markedly altered surface electronic states. Infrared spectra show the presence of additional carboxylate groups. Oxidation has therefore altered the structure. This appears to be a "self-humification" process that affects the graphitic component, and probably the non-organized phase as well.
Zaslansky P., Shahar R., Barak M. M., Friesem A. & Weiner S. 2006. Tooth and bone deformation: Structure and material-properties by ESPI Speckle06: Speckles, From Grains To Flowers. 2006 , 6341 .

[Abstract]

In order to understand complex-hierarchical biomaterials such as bones and teeth, it is necessary to relate their structure and mechanical-properties. We have adapted electronic speckle pattem-correlation interferometry (ESPI) to make measurements of deformation of small water-immersed specimens of teeth and bones. By combining full-field ESPI with precision mechanical loading we mapped sub-micron displacements and determined material-properties of the samples. By gradually and elastically compressing the samples, we compensate for poor S/N-ratios and displacement differences of about 100nm were reliably determined along samples just 2 similar to 3mm long. We produced stress-strain curves well within the elastic performance range of these materials under biologically relevant conditions. For human tooth-dentin, Young's modulus in inter-dental areas of the root is 40% higher than on the outer sides. For cubic equine bone samples the compression modulus of axial orientations is about double the modulus of radial and tangential orientations (20 GPa versus 10 GPa respectively). Furthermore, we measured and reproduced a surprisingly low Poisson's ratio, which averaged about 0.1. Thus the non-contact and non-destructive measurements by ESPI produce high sensitivity analyses of mechanical properties of mineralized tissues. This paves the way for mapping deformation-differences of various regions of bones, teeth and other biomaterials.
Politi Y., Levi-Kalisman Y., Raz S., Wilt F., Addadi L., Weiner S. & Sagi I. 2006. Structural characterization of the transient amorphous calcium carbonate precursor phase in sea urchin embryos Advanced Functional Materials. 2006 Jul , 16 (10):1289-1298.

[Abstract]

Sea urchin embryos form their calcitic spicular skeletons via a transient precursor phase composed of amorphous calcium carbonate (ACC). Transition of ACC to calcite in whole larvae and isolated spicules during development has been monitored using X-ray absorption spectroscopy (XAS). Remarkably, the changing nature of the mineral phase can clearly be monitored in the whole embryo samples. More detailed analyses of isolated spicules at different stages of development using both XAS and infrared spectroscopy demonstrate that the short-range order of the transient ACC phase resembles calcite, even though infrared spectra show that the spicules are mostly composed of an amorphous mineral phase. The coordination sphere is at first distorted but soon adopts the octahedral symmetry typical of calcite. Long-range lattice rearrangement follows to form the calcite single crystal of the mature spicule. These studies demonstrate the feasibility of real-time monitoring of mineralized-tissue development using XAS, including the structural characterization of transient amorphous phases at the atomic level.
Addadi L., Joester D., Nudelman F. & Weiner S. 2006. Mollusk shell formation: A source of new concepts for understanding biomineralization processes Chemistry-A European Journal. 2006 Jan , 12 (4):980-987.

[Abstract]

The biological approach to forming crystals is proving to be most surprising. Mollusks build their shells by using a hydrophobic silk gel, very acidic aspartic acid rich proteins, and apparently also an amorphous precursor phase from which the crystals form. All this takes place in a highly structured chitinous framework. Here we present ideas on how these disparate components work together to produce the highly structured pearly nacreous layer of the mollusk shell.
Nudelman F., Gotliv B., Addadi L. & Weiner S. 2006. Mollusk shell formation: Mapping the distribution of organic matrix components underlying a single aragonitic tablet in nacre Journal of Structural Biology. 2006 Feb , 153 (2):176-187.

[Abstract]

Control over mineral formation in mollusk shells is exerted by the macromolecules of the organic matrix. Using histochemical methods, we mapped the carboxylates and sulfates of proteins and polysaccharides on the surfaces of decalcified interlamellar matrices from the nacreous shell layer of the cephalopod Nautilus pompilius, expanding upon an earlier study by Crenshaw and Ristedt [Crenshaw, M.A., Ristedt, H., 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Watabe, N., Wilbur, K.M. (Ed.), The Mechanisms of Mineralization in the Invertebrates and Plants. University of South Carolina Press, Colombia, pp. 355-367]. We observed four different zones underlying a single crystal: (1) a central spot rich in carboxylates; (2) a central ring-shaped area rich in sulfates; (3) an area between the central nucleation region and the imprint periphery containing carboxylates, and (4) the intertabular matrix, rich in carboxylates and sulfates. We also mapped matrix functional groups on the nacreous matrix surfaces of the bivalve Atrina rigida, but did not identify well-defined zones. Immuno-mapping of the constituents of the aragonite-nucleating protein fraction from Atrina nacre showed that these macromolecules are located both in the intertabular matrix and in the center of the crystal imprints for both Atrina and Nautilus matrix surfaces. Their presence at the latter location is consistent with their purported role in aragonite nucleation. The observed differentiation in the distribution of matrix components and their functional groups shows that the different stages of single crystal growth are highly controlled by the matrix.
Zaslansky P., Friesem A. & Weiner S. 2006. Structure and mechanical properties of the soft zone separating bulk dentin and enamel in crowns of human teeth: Insight into tooth function Journal of Structural Biology. 2006 Feb , 153 (2):188-199.

[Abstract]

The 200-300 mu m soft zone of dentin, found beneath enamel in crowns of human teeth, is thought to fulfill important roles in tooth function. but little is known about its structure-mechanical relations. Scanning electron microscopy images of fracture Surfaces showed that near the dentino-enamel junction (DEJ), a porous reticulate matrix of intertubular-dentin contains tubules with no peritubular lining. Peritubular-dentin however is found at some distance from the DEJ, and it gradually thickens kith increasing depth into the bulk dentin. Concurrently, tighter packing of the collagen fibers is observed with a gradual increase in mineral deposits on and between the fibers. This structurally graded zone is known to be softer when tested for rnicro-hardness. It undergoes greater strain compared to bulk dentin, when measured using Moire interferometry. We investigated the deformation and stiffness of this zone by means of non-contact laser-speckle interferometry (ESPI), and nanometer-scale deformations were tracked during compression-testing performed in water. We report a significantly reduced stiffness of this zone compared to bulk dentin. with mid-buccal regions or teeth averaging 3.5 GPa compared with 9.7 GPa in mid-lingual regions. Our results support and expand upon the hypothesis that the durability of the whole tooth relies upon a bucco-lingual asymmetric matching of stiffness by means of all interphase: a cushioning soft layer between enamel and bulk dentin. (C) 2005 Elsevier Inc. All rights reserved.
Zaslansky P., Shahar R., Friesem A. & Weiner S. 2006. Relations between shape, materials properties, and function in biological materials using laser speckle interferometry: In situ tooth deformation Advanced Functional Materials. 2006 Oct , 16 (15):1925-1936.

[Abstract]

The manner in which stiff biological objects, such as whole bones and teeth, deform under load can provide direct insight into their in vivo functions, while highlighting the relations between their structure and materials properties. A new approach for studying the mechanical functions of such objects, using as an example the crowns of human teeth, is developed. Tooth-crown deformation under a compressive load is determined in water using laser speckle interferometry. The deformation patterns are analyzed using a novel procedure that reveals the relative magnitudes of 3D displacements of the outer surface. Nanometer-scale deformations of natural teeth were compared to deformations of identical acrylic replicas, in order to differentiate between contributions of the structure-material properties from contributions of morphology. It is shown that human premolars deform in a manner that is largely controlled by shape; in natural teeth, the enamel cap appears to displace mainly as a rigid body, undergoing moderate deformation. These observations contribute to the understanding of whole-tooth performance under load. The approach for analyzing the deformation of loaded whole objects is directly applicable to the study of many stiff biological specimens, including comparisons between normal and altered (repaired or genetically modified) bones.
Wang L., Nancollas G. H., Henneman Z. J., Klein E. & Weiner S. 2006. Nanosized particles in bone and dissolution insensitivity of bone mineral Biointerphases. 2006 Sept , 1 (3):106-111.

[Abstract]

Most of the mineral crystals in bone are platelets of carbonated apatite with thicknesses of a few nanometers embedded in a collagen matrix. We report that spherical to cylindrical shaped nanosized particles are also an integral part of bone structure observed by high resolution scanning electron microscopy. High resolution back scattered electron imaging reveals that the spherical particles have a contrast similar to the crystal platelets, suggesting that they are thus likely to have similar mineral properties. By means of constant composition (CC) dissolution of bone, similar sized nanoparticles are shown to be insensitive to demineralization and are thought to be dynamically stabilized due to the absence of active pits/defects on the crystallite surfaces. Similar reproducible self-inhibited dissolution was observed with these nanoparticles during CC dissolution of synthetic carbonated apatite. This result rules out the possible influence of complicating biological factors such as the possible presence of organic matrix components and other impurities. This phenomenon can be explained by a unique dissolution model involving size considerations at the nanoscale. The unexpected presence of nanoparticles in mature bone may also be due to the stabilization of some nanosized particles during the formation process in a fluctuating biological milieux. (c) 2006 American Vacuum Society.
Weiner S., Nudelman F., Sone E., Zaslansky P. & Addadi L. 2006. Mineralized biological materials: A perspective on interfaces and interphases designed over millions of years Biointerphases. 2006 Jun , 1 (2):P12-P14.
Weiner S. & Addadi L. 2006. Response [7] Science. 2006 Mar , 311 (5767):1555.
Elbaum R., Melamed-Bessudo C., Boaretto E., Galili E., Lev-Yadun S., Levy A. & Weiner S. 2006. Ancient olive DNA in pits: Preservation, amplification and sequence analysis Journal of Archaeological Science. 2006 Jan , 33 (1):77-88.

[Abstract]

The olive tree (Olea europaea) was domesticated by vegetative propagation of selected wild individuals with superior fruit. Later, new cultivars were established repeatedly from feral trees or from crosses between wild, feral, and domesticated trees. Thus the genetic background of many contemporary domesticated lines is a mixture of ancient cultivars and local wild trees. Ancient DNA may illuminate the complicated process of olive domestication because such DNA sequences provide data about ancient genomes that existed closer to the domestication events. Well preserved DNA must be available for such studies, even though in the Mediterranean region, where olive cultivation took place, the climatic conditions are not favorable for DNA preservation. To select for well preserved pits we measured their proportions of lignin by IR spectroscopy, and correlated this with parameters of DNA quality such as template length in an olive-specific repeat array, and template quantity as determined by real-time PCR amplification. Archaeological pits that passed these tests did contain high quality ancient DNA. We present the first ancient olive DNA sequences and compare them to modern wild, feral and domesticated lines.
Weiner S. 2006. Transient precursor strategy in mineral formation of bone Bone. 2006 Sept , 39 (3):431-433.

[Abstract]

The strategy in biomineralization of initially depositing a less ordered mineral and then transforming it into a more crystalline mature phase is probably widespread among invertebrates. The report in this issue by N.J. Crane, V. Popescu, M.D. Morris, P. Steenhuis, M.A. Ignelzi, Raman spectroscopic evidence for octacalcium phosphate and other mineral species deposited during intramembraneous mineralization. Bone (In press), using micro-Raman spectroscopy to study early mineral deposits in mice calvaria, provides strong evidence that the transient precursor strategy also occurs in vertebrates.
Verri G., Barkai R., Gopher A., Hass M., Kubik P., Paul M., Ronen A., Weiner S. & Boaretto E. 2005. Flint procurement strategies in the Late Lower Palaeolithic recorded by in situ produced cosmogenic ¹⁰Be in Tabun and Qesem Caves (Israel) Journal of Archaeological Science. 2005 Feb , 32 (2):207-213.

[Abstract]

The in situ produced cosmogenic beryllium isotope, ¹⁰Be, in flint artifacts from different layers in prehistoric caves can provide information on flint procurement. The buildup of ¹⁰Be in a flint matrix is related to the exposure time of the flint to cosmic rays. Although this exposure history can be complex, the ¹⁰Be content of flint assemblages can show whether the raw material was obtained from shallow mining and/or surface collection as opposed to sediments two or more meters below the surface. Flint artifact assemblages from two Palaeolithic caves in Israel, Tabun and Qesem, were analyzed. In Tabun cave the flint artifacts from Lower Layer E (Acheulo-Yabrudian, around 400 000-200 000 yr) contain very small amounts of ¹⁰Be, which is consistent with flint procured from sediments two or more meters deep. Artifacts from above and below Tabun Lower Layer E show a more complex distribution, as do artifacts from all layers of Qesem cave (Acheulo-Yabrudian). This is probably due to the fact that they were surface collected and/or mined from shallow (less than 2 m) depths. We show here that artifact assemblages have different concentrations of ¹⁰Be, indicating different raw material procurement strategies.
Salamon M., Tuross N., Arensburg B. & Weiner S. 2005. Relatively well preserved DNA is present in the crystal aggregates of fossil bones Proceedings of the National Academy of Sciences of the United States of America. 2005 Sept , 102 (39):13783-13788.

[Abstract]

DNA from fossil human bones could provide invaluable information about population migrations, genetic relations between different groups and the spread of diseases. The use of ancient DNA from bones to study the genetics of past populations is, however, very often compromised by the altered and degraded state of preservation of the extracted material. The universally observed postmortem degradation, together with the real possibility of contamination with modern human DNA, makes the acquisition of reliable data, from humans in particular, very difficult. We demonstrate that relatively well preserved DNA is occluded within clusters of intergrown bone crystals that are resistant to disaggregation by the strong oxidant NaOCl. We obtained reproducible authentic sequences from both modern and ancient animal bones, including humans, from DNA extracts of crystal aggregates. The treatment with NaOCl also minimizes the possibility of modern DNA contamination. We thus demonstrate the presence of a privileged niche within fossil bone, which contains DNA in a better state of preservation than the DNA present in the total bone. This counterintuitive approach to extracting relatively well preserved DNA from bones significantly improves the chances of obtaining authentic ancient DNA sequences, especially from human bones.
Weiner S., Sagi I. & Addadi L. 2005. Choosing the crystallization path less traveled Science. 2005 Aug , 309 (5737):1027-1028.
Gotliv B., Kessler N., Sumerel J., Morse D., Tuross N., Addadi L. & Weiner S. 2005. Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida ChemBioChem. 2005 Feb , 6 (2):304-314.

[Abstract]

Almost all mineralized tissues contain proteins that are unusually acidic. As they are also often intimately associated with the mineral phase, they are thought to fulfill important functions in controlling mineral formation. Relatively little is known about these important proteins, because their acidic nature causes technical difficulties during purification and characterization procedures. Much effort has been made to overcome these problems, particularly in the study of mollusk-shell formation. To date about 16 proteins from mollusk-shell organic matrices have been sequenced, but only two are unusually rich in aspartic and glutamic acids. Here we screened a cDNA library made from the mRNA of the shelf-forming cells of a bivalve, Atrina rigida, using probes for short Asp-containing repeat sequences, and identified ten different proteins. Using more specific probes designed from one subgroup of conserved sequences, we obtained the full sequences of a family of seven aspartic acid-rich proteins, which we named "Asprich"; a subfamily of the unusually acidic shell-matrix proteins. Polyclonol antibodies raised against a synthetic peptide of the conserved acidic 1 domain of these proteins reacted specifically with the matrix components of the calcitic prismatic layer, but not with those of the aragonitic nacreous foyer, Thus the Asprich proteins are constituents of the prismatic layer shell matrix. We con identify different domains within these sequences, including a signal peptide characteristic of proteins destined for extracellular secretion, a conserved domain rich in aspartic acid that contains a sequence very similar to the calcium-binding domain of Calsequestrin, and another domain rich in aspartic acid, that varies between the seven sequences. We also identified a domain with DEAD repeats that may have Mg-binding capabilities. Although we do not know, as yet, the function of these proteins, their generally conserved sequences do indicate that they might well
Sone E., Weiner S. & Addadi L. 2005. Morphology of goethite crystals in developing limpet teeth: Assessing biological control over mineral formation Crystal Growth & Design. 2005 Nov , 5 (6):2131-2138.

[Abstract]

The continuously forming mineralized teeth of some mollusks can be conveniently studied during their formation. The mature teeth of limpets (Gastropoda) are composed of goethite (α-FeOOH) crystals embedded in silica. A chitinous organic matrix defines the microenvironment of mineral deposition and presumably controls crystal growth. We studied the newly formed goethite crystals in the teeth. They are diverse both in size and in morphology, traits not often found in mineralized biological materials. Many of the goethite crystals express relatively stable faces commonly found in synthetic goethite, implying that their growth is not under strict biological control. Some control is certainly exercised, however, in the biogenic crystals that are hollow with very thin walls (down to 2 nm) and those that are triangular in cross-section, which is not anticipated from the crystal symmetry. Furthermore, goethite crystals with well-defined habits are not generally formed under ambient conditions. These observations reflect the fascinating and complex conditions of growth for biogenic goethite crystals, which we are only beginning to understand.
Yizhaq M., Mintz G., Cohen I., Khalaily H., Weiner S. & Boaretto E. 2005. Quality controlled radiocarbon dating of bones and charcoal from the early Pre-Pottery Neolithic B (PPNB) of Motza (Israel) Radiocarbon. 2005 , 47 (2):193-206.

[Abstract]

Radiocarbon dating of early Pre-Pottery Neolithic B (PPNB) deposits at the site of Motza, Israel, was achieved by first prescreening many charcoal and bone samples in order to identify those that are in the most suitable state of preservation for dating. For assessing bone preservation, we determined the collagen contents, and by infrared spectroscopy the collagen purity. The collagen samples of the best preserved bones were then further characterized by their C/N ratios and amino acid compositions. Prescreening of the charcoal samples involved monitoring the changes in infrared and Raman spectra during the acid-alkali-acid treatments. In some samples, we noted that the clay content increased with additional alkali treatments. These samples were rejected, as this could result in erroneous dates. No differences were observed in the ¹⁴C dates between charcoal and bone collagen samples. The dates range from 10,600-10,100 cal BP, which is consistent with dates for the early PPNB from other sites. This is of much interest in terms of better understanding where and when domestication of animals began in this period, and how agriculture spread throughout the Levant.
Zaslansky P., Currey J., Friesem A. & Weiner S. 2005. Phase shifting speckle interferometry for determination of strain and Young's modulus of mineralized biological materials: A study of tooth dentin compression in water Journal of Biomedical Optics. 2005 Mar , 10 (2).

[Abstract]

Mineralized biological materials have complex hierarchical graded structures. It is therefore difficult to understand the relations between their structure and mechanical properties. We report the use of electronic speckle pattern-correlation interferometry (ESPI) combined with a mechanical compression apparatus to measure the strain and Young's modulus of root dentin compressed under water. We describe the optomechanical instrumentation, experimental techniques and procedures needed to measure cubes as small as 1×1×2 mm. Calibration of the method is performed using aluminum, which shows that the measurements are accurate within 3% of the compression modulus reported for standard aluminum 6061. Our results reveal that the compression moduli of root dentin from the buccal and lingual sides of the root are quite different from the moduli of the interproximal sides. Root dentin from interproximal locations is found to have an average modulus of 21.3 GPa, which is about 40% stiffer than root dentin from the buccal and lingual locations, found to have a modulus of 15.0 GPa. Our approach can be used to map deformations on irregular surfaces, and measure strain on wet samples of varying sizes. This can be extended to the study of other biological materials including bone and synthetic biomaterials.
Shahack-Gross R., Albert R., Gilboa A., Nagar-Hilman O., Sharon I. & Weiner S. 2005. Geoarchaeology in an urban context: The uses of space in a Phoenician monumental building at Tel Dor (Israel) Journal of Archaeological Science. 2005 Sept , 32 (9):1417-1431.

[Abstract]

Interpretation of past urban societies in the Near East, whose settlements are known mostly as tell sites, is largely based on macro-stratigraphy and on the association of architecture with macroscopic artifacts. Analyses of sediments, common in prehistoric sites, are rare in tell sites. Here we show the results of a detailed geoarchaeological study of the micro-stratigraphy of a sedimentary sequence associated with early Iron Age Phoenician monumental architecture. The study involves mineralogical, micromorphological and phytolith analyses and provides new insights into the stratigraphic sequence and the use of architectural spaces. The sedimentary sequence examined comprises alternating layers of gray 'fill' deposits and white 'floors'. We show that 'floors' made from local calcareous sandstone in the lower part of the sedimentary sequence were heated and are thus in effect 'plaster floors'. A concentration of micro-laminated, trampled fish remains above the most elaborate of these plaster 'floors' indicates activities related to fish processing. Fine white layers in the upper part of the sedimentary sequence that were considered as plaster based on macroscopic examination are in fact composed almost entirely of opaline grass phytoliths. The phytoliths appear in an undulating micro-laminated structure and are associated with dung spherulites and phosphate nodules, thus probably reflecting livestock penning. The formation of 'phytolith floors' involves extensive volume reduction due to the degradation of the organic material and this may result in 'floor' subsidence, a phenomenon that is often observed in archaeological sites. Most 'fill' deposits include macroscopic and microscopic remains of wood ash, bones, phytoliths, charcoal, ceramics, plaster and mollusk shells, reflecting the debris produced from household activities. This study shows how a combination of macro-stratigraphy with microscopic and mineralogical analyses of the sediments within architectural spaces can provide information on the varying ways in which the space was used through time, and also contributes to solving macro-stratigraphic problems.
Berna F., Matthews A. & Weiner S. 2004. Solubilities of bone mineral from archaeological sites: The recrystallization window Journal of Archaeological Science. 2004 Jul , 31 (7):867-882.

[Abstract]

Bone mineral solubility is an important parameter for understanding the preservation of bones in the archaeological and palaeontological records. In this study we have measured the solubility of the carbonated hydroxyl apatite of sub-recent and fossil bones, as well as synthetic hydroxyl apatite in deionized water and in pH-buffered solutions. The solutions were open to the atmosphere and the pH values were around neutral; measurement conditions that are relevant to bone mineral preservation in nature, but that were absent from most previous studies. We obtained internally consistent results from both the water and the buffered experiments supporting the notion that we are measuring an inherent property of the mineral phase. We found that bone mineral is much more soluble than synthetic hydroxyl apatite. We measured the ionic activity products at "steady state" conditions and we identify a recrystallization window between pH 7.6 and 8.1, which defines the conditions under which bone crystals dissolve and reprecipitate as a more insoluble form of carbonated hydroxyl apatite. As these conditions are common in nature, most fossil bones will not maintain their original crystals with time. We also found that bones that contained small amounts of calcite did not dissolve at all during our experiments. These results provide a basis for better understanding the conditions in sediments under which bones are preserved and the relative states of preservation of bone. They also have important implications for the selection of the most appropriate bone samples for paleoenvironmental and paleodiet analyses and dating.
Estroff L., Addadi L., Weiner S. & Hamilton A. 2004. An organic hydrogel as a matrix for the growth of calcite crystals Organic & Biomolecular Chemistry. 2004 Jan , 2 (1):137-141.

[Abstract]

The growth of calcite in an aqueous gel of 1 was studied and the appearance of the crystals was found to change over time. Crystals removed from the gel at progressively longer times showed severely affected surfaces resulting from dissolution. If crystals were removed from the gel after 3.5 hours, at which point there were no etch pits, and then placed in either buffer or pure water, etch pits, similar to those observed on crystals that are left in the gel, were observed. Control calcite crystals exposed to similar conditions (water or buffer) show no significant dissolution after equivalent times. A probable cause of the altered dissolution is the non-specific occlusion of gelator aggregates at sites of imperfection. The gel appears to provide a microenvironment in which the molecules that form the matrix also participate in the crystallization. This system allows the study of the unique properties of a gel for influencing the nucleation and growth of inorganic crystals, some of which may be important for better understanding biomineralization.
Politi Y., Arad T., Klein E., Weiner S. & Addadi L. 2004. Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase Science. 2004 Nov , 306 (5699):1161-1164.

[Abstract]

The skeletons of adult echinoderms comprise large single crystals of calcite with smooth convoluted fenestrated morphologies, raising many questions about how they form. By using water etching, infrared spectroscopy, electron diffraction, and environmental scanning electron microscopy, we show that sea urchin spine regeneration proceeds via the initial deposition of amorphous calcium carbonate. Because most echinoderms produce the same type of skeletal material, they probably all use this same mechanism. Deposition of transient amorphous phases as a strategy for producing single crystals with complex morphology may have interesting implications for the development of sophisticated materials.
Trueman C., Behrensmeyer A., Tuross N. & Weiner S. 2004. Mineralogical and compositional changes in bones exposed on soil surfaces in Amboseli National Park, Kenya: Diagenetic mechanisms and the role of sediment pore fluids Journal of Archaeological Science. 2004 Jun , 31 (6):721-739.

[Abstract]

Bones exposed on tropical savannah grasslands of Amboseli National Park, Kenya undergo extensive post-mortem, alteration within 40 years. A combined analytical approach involving TEM microscopy, trace metal analysis, FTIR spectroscopy, and petrographic analysis has revealed a complex, dynamic diagenetic environment operating within exposed bones, driven by evaporative transport of soil water from the bone/soil interface to the upper exposed surface of the bone. This process results in extensive bone/soil-water interaction, and is responsible for increases in the concentrations of trace elements such as Ba and La of 100 - >1000% within 15 years. The maximum and mean size of bone crystallites increases with continued exposure. This change in mean crystallite length is correlated positively with increases in bone crystallinity, which in turn is associated with degradation of the bone protein. Microbial decomposition is rarely observed in the Amboseli bones, but where present resulted in severe dissolution-reprecipitation of bone mineral. Many bones showed extensive permineralization of the larger vascular spaces with calcite and barite and, to a lesser extent, crandallite. Permineralization of unburied bones may account for 95% reduction in macro (micron-millimeter scale) porosity in the bone within 2 years of death. We produce a model for pre-burial diagenesis of bone in and tropical environments that highlights extensive alteration of bone chemistry within 1-40 years post-mortem.
Verri G., Barkai R., Bordeanu C., Gopher A., Hass M., Kaufman A., Kubik P., Montanari E., Paul M., Ronen A., Weiner S. & Boaretto E. 2004. Flint mining in prehistory recorded by in situ-produced cosmogenic Be-10 Proceedings of the National Academy of Sciences of the United States of America. 2004 May , 101 (21):7880-7884.

[Abstract]

The development of mining to acquire the best raw materials for producing stone tools represents a breakthrough in human technological and intellectual development. We present a new approach to studying the history of flint mining, using in situ-produced cosmogenic Be-10 concentrations. We show that the raw material used to manufacture flint artifacts approximate to300,000 years old from Qesem Cave (Israel) was most likely surf ace-collected or obtained from shallow quarries, whereas artifacts of the same period from Tabun Cave (Israel) were made of flint originating from layers 2 or more meters deep, possibly mined or quarried by humans.
Karkanas P., Koumouzelis M., Kozlowski J., Sitlivy V., Sobczyk K., Berna F. & Weiner S. 2004. The earliest evidence for clay hearths: Aurignacian features in Klisoura Cave 1, southern Greece Antiquity. 2004 Sept , 78 (301):513-525.

[Abstract]

The authors describe clay features dating from c. 34-23 000 years ago discovered in a stratified occupation sequence in a Greek cave. The clay was brought from outside the cave, puddled with water and shaped into shallow basins. Laboratory analyses have shown that these clay features were burnt. This together with the occurrence of fragments of wood ash and phytoliths lying on their surfaces suggest that these features were hearths used for cooking, including the roasting of wild grasses.
Shahack-Gross R., Berna F., Karkanas P. & Weiner S. 2004. Bat guano and preservation of archaeological remains in cave sites Journal of Archaeological Science. 2004 Sept , 31 (9):1259-1272.

[Abstract]

The formation of authigenic minerals in cave sediments can be used to reconstruct the paleochemical conditions that prevailed in the past, and in this way to assess the completeness of the archaeological record. Previous studies indicated that the major driving mechanisms for chemical diagenesis in prehistoric caves that result in the formation of authigenic minerals, are the degradation of bat guano and the local hydrology. We therefore investigated contemporary bat guano deposits in caves and the sediments directly below such deposits. The emerging patterns show that the formation of authigenic minerals occurs under acidic conditions within tens of years. The availability of phosphate, Al, K, and Fe increases with increasing organic matter degradation, while the availability of nitrogen and sulfur decreases. Insectivorous bat guano contains larger amounts of phosphate and is more acidic than fruit bat guano. In the first stages of diagenesis micro-chemical environments form within the degrading bat guano that result in the formation of a wide variety of authigenic minerals. During advanced stages of diagenesis steady state conditions are achieved resulting in authigenic mineral assemblages being dominated by one mineral type at the same locale. This is reminiscent of known distributions of authigenic minerals in prehistoric cave sediments. Under the acidic conditions produced by degrading bat guano deposits, calcareous artifacts and bones are not expected to persist for long periods of time, unless there is a lot of calcite in the sediments that could buffer the sediment water pH. Guano itself is not likely to be preserved. Periods of cave abandonment can be inferred from the presence of authigenic minerals in the sediments as it is during these times that bats mainly occupy the cave. The occurrence of certain authigenic minerals in open-air sites may serve as evidence for areas rich in organic matter containing phosphate (i.e., animal enclosures, latrines, sewers, trash pits) and/or bones. As the degradation process is rapid, the use of authigenic minerals to identify these loci is relevant to almost all archaeological, as well as historical sites.
Shahack-Gross R., Marshall F., Ryan K. & Weiner S. 2004. Reconstruction of spatial organization in abandoned Maasai settlements: Implications for site structure in the Pastoral Neolithic of East Africa Journal of Archaeological Science. 2004 Oct , 31 (10):1395-1411.

[Abstract]

The analysis of spatial organization in archaeological sites is important for the interpretation of economic and social issues. In East Africa, the appearance of mobile herders, adoption of pastoralism by some hunter-gatherers, and spread of competing pastoral groups, create a complex archaeological record and interpretive problems associated with the beginnings of food production. Spatial analyses could contribute to their resolution, but are difficult because most sites lack macroscopic features. We present a geo-ethnoarchaeological study of abandoned pastoral Maasai settlements that allows us to evaluate the "archaeological visibility" of ephemeral features such as hearths, trash pits, gates, houses and fences. Micromorphology, mineralogy and phytolith analyses show that features containing ash have the highest visibility. Livestock enclosures, a feature studied by us previously, can also be identified based on this suite of techniques. Large livestock gates have poor visibility but may be recognized. Small gates, fences and house floors could not be detected using the methods applied here. Identifying livestock enclosures, trash pits and cooking hearths based on this approach, and houses based on post-hole positions, will contribute to a better understanding of the spread of food production in Africa. Our findings will also contribute to studies of pastoralists in other regions of the world.
Rink W., Schwarcz H., Weiner S., Goldberg P., Meignen L. & Bar-Yosef O. 2004. Age of the Mousterian industry at Hayonim Cave, Northern Israel, using electron spin resonance and ²³⁰Th/²³⁴U methods Journal of Archaeological Science. 2004 Jul , 31 (7):953-964.

[Abstract]

Electron spin resonance (ESR) analysis was done on teeth excavated at Hayonim between 1993 and 1997. ²³⁰Th/²³⁴U analysis was done on two of these teeth and one calcite speleothem excavated in 1992. Small amounts of uranium uptake into teeth provided nearly ideal conditions for ESR age determinations. The combined ESR/²³⁰Th/²³⁴U analyses on teeth confirmed that uranium uptake occurred according to values constrained by early (EU) and linear (LU) uptake ESR model age estimates, suggesting that EU and LU ESR model ages on other teeth were reliable burial ages. A speleothem stratigraphically above all the teeth yielded a ²³⁰Th/²³⁴U age of 155.3 ± ^2.9 _1.4 ka. The EU and LU ESR mean model ages for teeth in layers just below the speleothem were 177 ± 12 and 182 ± 15 ka respectively. Teeth in the lowest part of the section in the Central Area yielded younger ages, apparently due to the increase in radioactive potassium content associated with the formation of siliceous aggregate mineral assemblages nearby. These results were rejected. The ages of the reliably dated portion of the section were contemporaneous with levels at Tabun Cave containing both Tabun C-type and D-type lithics, but they were significantly younger than the levels at Tabun which contain only the D-type lithics. The use of infrared spectroscopy to identify mineral assemblages in close proximity to teeth were important to be able to select only those teeth not affected by changes in radioactive dose rate through time.
Weiner S. & Zaslansky P. 2004. Structure-mechanical function relations in bones and teeth Learning From Nature How To Design New Implantable Biomaterials: From Biomineralization Fundamentals To Biomimetic Materials And Processing Routes. 2004 , 171 :3-13.

[Abstract]

Keywords: LAMELLAR BONE; HUMAN DENTIN; CRYSTAL ORGANIZATION; PROPERTY GRADIENTS; COLLAGEN FIBRILS; MICROHARDNESS; MICROSTRUCTURE; STRENGTH; FRACTURE
Raz S., Hamilton P., Wilt F., Weiner S. & Addadi L. 2003. The transient phase of amorphous calcium carbonate in sea urchin larval spicules: The involvement of proteins and magnesium ions in its formation and stabilization Advanced Functional Materials. 2003 Jun , 13 (6):480-486.

[Abstract]

Amorphous calcium carbonate (ACC) is a precursor phase of calcite in the formation of the sea urchin larval spicule. The goal of this research is to study the formation and stabilization mode of this transient phase. We first characterized the mineralogy of the spicules from the sea urchin Strongylocentrotus purpuratus. We then examined the role of the macromolecules extracted from the spicules at different growth stages in the formation of transient ACC in vitro. The biogenic amorphous transient phase is shown to be both structurally and compositionally different from the known stable ACC phases. It does not contain bound water, and is thus the first dehydrated ACC phase to be detected. The macromolecules that were extracted at early stages of spicule growth, when the amorphous content of the biogenic mineral is high, induced the formation of transient ACC in vitro in the presence of magnesium ions. In contrast, the macromolecules extracted at a later stage, when the spicules are completely crystalline, induced the formation of single crystals of low magnesian calcite. We therefore deduce that the macromolecules from the sea urchin larval spicules together with magnesium ions, mediate the transient formation of ACC as a precursor to calcite. These observations may well provide novel ideas for improved materials synthesis.
Addadi L., Raz S. & Weiner S. 2003. Taking advantage of disorder: Amorphous calcium carbonate and its roles in biomineralization Advanced Materials. 2003 Jun , 15 (12):959-970.

[Abstract]

Amorphous calcium carbonate (ACC) in its pure form is highly unstable, yet some organisms produce stable ACC, and cases are known in which ACC functions as a transient precursor of more stable crystalline aragonite or calcite. Studies of biogenic ACC show that there are significant structural differences, including the observation that the stable forms are hydrated whereas the transient forms are not. The many different ways in which ACC can be formed in vitro shed light on the possible mechanisms involved in stabilization, destabilisation, and transformation of ACC into crystalline forms of calcium carbonate- We show here that ACC is a fascinating form of calcium carbonate that may well be of much interest to materials science and biomineralization.
Gotliv B., Addadi L. & Weiner S. 2003. Mollusk shell acidic proteins: In search of individual functions ChemBioChem. 2003 Jun , 4 (6):522-529.

[Abstract]

Acidic proteins play a major role in the biomineralization process. These proteins are generally thought to control mineral formation and growth. Thus, characterization of individual acidic protein is important as a first step toward linking function to individual proteins, which is our ultimate goal. In order to characterize the protein(s) responsible for the assemblage of biominerals, a new gel many, if not all of the acidic protein were visualized on the gel for the first time. In an in vitro assay we show that proteins extracted from an aragonitic shell layer induce the formation of amorphous calcium carbonate prior to its transformation into the aragonitic crystalline form. This study removes some major obstacles in the characterization of acidic proteins and sheds more light on the functions of these proteins in the biomineralization process.
Weiner S. & Dove P. 2003. An overview of biomineralization processes and the problem of the vital effect Biomineralization. 2003 , 54 :1-29.

[Abstract]

Keywords: AMORPHOUS CALCIUM-CARBONATE; SEA-URCHIN EMBRYOS; ORGANIC MATRIX; STABLE-ISOTOPES; PLANKTONIC-FORAMINIFERA; DIATOM BIOSILICA; CORAL SKELETONS; CRYSTAL TEXTURE; PRECURSOR PHASE; NACREOUS LAYER
Weiner S., Levi-Kalisman Y., Raz S. & Addadi L. 2003. Biologically formed amorphous calcium carbonate Connective Tissue Research. 2003 , 44 (SUPPL. 1):214-218.

[Abstract]

Many organisms from a wide variety of taxa produce amorphous calcium carbonate (ACC), despite the fact that it is inherently unstable and relatively soluble in its pure state. These properties also make it difficult to detect and characterize ACC. Raman spectroscopy is a particularly useful method for investigating ACC because the sample can be examined wet, and extended X-ray absorption fine structure (EXAFS) analysis can provide detailed information on the short-range order. Other methods for characterizing ACC include infrared spectroscopy, thermogravimetric analysis and differential thermal analysis (TGA and DTA), transmission electron microscopy (TEM), and electron and X-ray diffraction. Because of the difficulties involved, we suspect that ACC is far more widely distributed than is presently known, and a comparison of EXAFS spectra shows that different biogenic ACC phases have different short-range order structures. We also suspect that ACC fulfils many different functions, including as a transient precursor phase during the formation of crystalline calcium carbonate.
Elbaum R., Weiner S., Albert R. & Elbaum M. 2003. Detection of burning of plant materials in the archaeological record by changes in the refractive indices of siliceous phytoliths Journal of Archaeological Science. 2003 Feb , 30 (2):217-226.

[Abstract]

The most resilient remains of plants in most archaeological sites are the siliceous phytoliths-special cells that are partially or completely silicified during the plant's life. These cells have characteristic morphologies, and thus the phytoliths can often be used to identify the taxonomic affinities of plants brought to an archaeological site. In order to determine what they were used for, other means of analysis are needed. We present here a method to distinguish burnt from unburnt phytolith assemblages. The method is based on measuring the refractive index (RI) of individual phytoliths. The phytoliths even from a single plant have a range of RI values. Burning a phytolith sample causes a shift to higher RI. Comparing burnt and unburnt samples we demonstrate that it is possible to differentiate between them based on the fraction of phytoliths with RI higher than 1·440. This serves as a basis for a simplified mode of measurement that requires only the use of a petrographic light microscope and a mineral oil of R1 1·440. We apply the simplified method to two Natufian samples from Hayonim cave (Western Galilee, Israel).
Aizenberg J., Weiner S. & Addadi L. 2003. Coexistence of amorphous and crystalline calcium carbonate in skeletal tissues Connective Tissue Research. 2003 , 44 (SUPPL. 1):20-25.

[Abstract]

We describe a new type of composite skeletal tissues in which calcite and stabilized amorphous calcium carbonate (ACC) coexist in well-defined domains. The organisms that form such structures are widely separated in the animal kingdom phylogenetic tree: calcareous sponges and ascidians. This paper compares the microstructures of their composite skeletal elements: The triradiate spicules from the sponge Clathrina are composed of a core of calcite embedded in a thick layer of ACC and covered by a thin calcitic envelope; the tunic spicules from the ascidian Pyura pachydermatina are composed of a core of ACC enveloped by an insoluble organic sheath and covered by a thick calcitic layer. We compare and contrast the macromolecules associated with different amorphous and crystalline phases and their ability to induce the formation of stabilized ACC in vitro.
Wood J. D., Wang R., Weiner S. & Pashley D. H. 2003. Mapping of tooth deformation caused by moisture change using moiré interferometry Dental Materials. 2003 May , 19 (3):159-166.

[Abstract]

Bonding of composite restorative materials to teeth usually involves surface etching and drying of both dentin and enamel. These two hard tissues have very different water contents. Objectives: The purpose of this study was to evaluate the effects of changes in humidity on the dimensional changes in dentin disks constrained by enamel and in unconstrained dentin. Methods: Changes in humidity were used to induce changes in the moisture level of the hard tissues, which created a mechanical load across the dentin-enamel junction (DEJ). High-sensitivity moiré interferometry was used to measure the magnitudes of the strains. Diffraction gratings (with a sensitivity of 2400lines/mm) were transferred to moist polished sections of human teeth, which were subsequently dehydrated and then hydrated again. The fringe patterns from moiré interferometry recorded the change in deformation between the moisture states. Results: The results indicated that there were wide variations in strain between the two specimen geometries. The strains were fairly constant through the bulk of the dentin in both specimen types. However, the strain gradients were very high across the dentin-enamel interface into the dentin in the unconstrained geometry, and nearly zero in the constrained geometry. Significance: Moiré interferometry is a powerful tool to study the deformation of materials that are not isotropic and are not linearly elastic. It provides full-field, high-resolution deformation fields in tooth specimens. Deformations observed in this study indicate that the DEJ zone is a unique material interface that needs to be better understood in terms of normal tooth function, and that its material properties be taken into account when natural hard tissues are restored.
Falini G., Weiner S. & Addadi L. 2003. Chitin-silk fibroin interactions: Relevance to calcium carbonate formation in invertebrates Calcified Tissue International. 2003 May , 72 (5):548-554.

[Abstract]

In mineralized tissues chitin is almost always associated with proteins, many of which are known to have chitin recognition consensus sequences. It has been observed in some mollusk shells that there is a well-defined spatial relation between the crystallographic axes of the crystals and the chitin fibrils. This implies that the chitin functions directly or indirectly as a template for nucleation of the mineral phase. It is thus of much interest to understand the exact nature of the interface between the chitin and the proteins at the molecular level in mineralized tissues. Chitin/silk fibroin interactions were studied in vitro at the molecular level using homogenous films composed of the two macromolecules. The results show that the silk fibroin intercalates between the molecular planes of the chitin, and that the interactions are mainly through the chitin acetyl groups. Published X-ray diffraction patterns and infrared spectra of mineralized tissue organic matrices, as well as infrared spectra reported here of the squid pen and lobster cuticle, all show that in vivo the chitin and protein are not intimately mixed, but exist as two phases. We deduce that there is an interfacial plane between them in which the interactions are through the amide groups.
Shahack-Gross R., Marshall F. & Weiner S. 2003. Geo-ethnoarchaeology of pastoral sites: The identification of livestock enclosures in abandoned Maasai settlements Journal of Archaeological Science. 2003 Apr , 30 (4):439-459.

[Abstract]

The earliest food producers in Africa were mobile pastoralists who left limited archaeological traces. As a result archaeologists studying the spread of food production in the region have difficulty distinguishing early pastoralists from hunter-gatherers with whom they interacted. This geo-ethnoarchaeological study contributes to the resolution of the problem through identification of sediments distinctive of livestock enclosures, and thus of pastoral settlements. Sediments were sampled in and around currently occupied and recently abandoned Maasai livestock enclosures ranging in age between one and 40 years. Twenty to thirty years after site abandonment, there is no visible difference between enclosure and regional sediments. Micromorphological, mineralogical, and phytolith analyses, of enclosure sediments, however, allow differentiation of enclosure from regional sediments. Our results show that a unique undulating microlaminated structure is distinctive of enclosure sediments. Enclosure sediments, especially small stock, also contain a rare mineral, monohydrocalcite (CaCO₃ . H₂O). In addition, large amounts of opal (SiO₂ . nH₂O), in the form of phytoliths, are found in enclosure relative to regional sediments. These differences are likely to be preserved in the archaeological record, and this approach will allow better understanding of the spread of pastoralism in Africa and elsewhere.
Levy-Lior A., Weiner S. & Addadi L. 2003. Achiral Calcium-Oxalate Crystals with Chiral Morphology from the Leaves of Some Solanacea Plants Helvetica Chimica Acta. 2003 , 86 (12):4007-4017.

[Abstract]

The leaves of some plants, particularly among the Solanacea, contain crystals of calcium oxalate with a peculiar chiral pseudo-tetrahedral morphology, even though the calcium oxalate crystal structure is centrosymmetric, hence achiral. We studied the morphology of these crystals extracted from the leaves of three Solanacea plants: the potato, the hot pepper, and a species of wild Solanum. The crystal morphology was the same in all three species. Based on the examination of more than 100 crystals from each plant, we showed that the crystal morphology is chiral with invariant chirality. We suggest that morphological chirality is induced by macromolecules during nucleation from a specific, genetically encoded crystal plane, and is further established during subsequent controlled crystal growth. This is one of few examples where it is possible to deduce a molecular mechanism for biologically induced breaking of morphological symmetry in organisms. A very high level of recognition is required by the macromolecules to allow them to distinguish between symmetry-related crystal planes. It is also surprising that this finely controlled mechanism of crystal formation, including the chiral morphology, has been conserved during evolution.
Treves K., Traub W., Weiner S. & Addadi L. 2003. Aragonite formation in the chiton (Mollusca) girdle Helvetica Chimica Acta. 2003 , 86 (4):1101-1112.

[Abstract]

In the chitons (Polyplacophora, Mollusca), the body is not entirely protected by the shell. Mineralized spicules or scales often, but not always, decorate the exposed part of the girdle. Here, we report a study on the composition and ultrastructural organization of these mineralized skeletal parts in four different chiton species. In all specimens, the mineral component (97 - 98 wt-%) is aragonite, and the organic matrix (2 - 3 wt-%) consists of highly glycosylated proteins. X-Ray diffraction and scanning electron microscopy show that the organic matrix fibers are aligned, morphologically and crystallographically, with the prismatic aragonite crystals. Matrix and mineral are thus clearly related. The matrix-mineral composite bundles are, however, assembled in the various skeletal parts examined with widely different degrees of alignment and order. In the same organism, the crystals are aligned within a range of +/- 15degrees in one type of spicule, while they are randomly oriented in another type. The wide heterogeneity in shape, density, and ultrastructure suggests that the girdle mineralized tissues do not fulfill a fundamental role necessary for the survival of the organism. This, together with the lack of chitin in the organic matrix, supports the hypothesis that they evolved separately from the other chiton mineralized tissues, namely the shell plates and teeth.
Estroff L., Leiserowitz L., Addadi L., Weiner S. & Hamilton A. 2003. Characterization of an organic hydrogel: A cryo-transmission electron microscopy and X-ray diffraction study Advanced Materials. 2003 Jan , 15 (1):38-42.

[Abstract]

The nanoscale structure and molecular organization of an organic hydrogel in its native state are evaluated. The data collected allows to understand the molecular assembly processes that lead to the gelation of water by organic molecules. Various spectroscopic, microscopic and X ray techniques are used to study the molecular organization and structure of gels.
Albert R., Bar-Yosef O., Meignen L. & Weiner S. 2003. Quantitative phytolith study of hearths from the Natufian and Middle Palaeolithic levels of Hayonim Cave (Galilee, Israel) Journal of Archaeological Science. 2003 Apr , 30 (4):461-480.

[Abstract]

A study of the mineralogy and phytolith assemblages of hearts and their associated sediments in Hayonim Cave, Israel, shows that wood ash is a major component of the sediments of both the Natufian (approximately 12,000 BP) and the Mousterian deposits (approximately 100 to 250,000 BP). Interestingly, phytolith analyses show that in the Natufian deposits grasses are the second most abundant form of vegetation preserved, whereas in the Mousterian deposits leaves of dicotyledonous plants are the second most abundant form of vegetation preserved. Significant differences were repeatedly observed in phytolith assemblages from closely associated but different stratigraphic layers, showing that vertical mixing of intact sediments is not intensive, and that there was a diverse input of vegetation over relatively short time periods. No consistent differences were observed in the mineralogy and the phytolith assemblages between hearths and their associated sediments. Both contain abundant ash components. This study demonstrates that quantitative analyses of phytoliths together with mineralogical analyses, can provide much information on the use of plants for fuel and other purposes by the cave occupants.
Weiner S., Goldberg P. & Bar-Yosef O. 2002. Three-dimensional distribution of minerals in the sediments of Hayonim Cave, Israel: Diagenetic processes and archaeological implications Journal of Archaeological Science. 2002 Nov , 29 (11):1289-1308.

[Abstract]

The mineral components of the sediments that accumulate in an archaeological site constitute a potentially rich source of information on the diagenesis, and in turn the archaeology of the site. This detailed three-dimensional study of the mineral assemblages in mainly the Mousterian sediments of Hayonim Cave incorporates more than 2100 infrared analyses performed on-site during the excavation, as well as diverse analyses in the laboratory. Three major mineral assemblages are identified: the calcite-dahllite (CD) assemblage, the assemblage comprising mainly montgomeryite, leucophosphite and siliceous aggregates (LMVS), and a highly altered sediment in which the clays have broken down and silica was released. The boundaries between these assemblages were mapped in detail. The overall picture is one of extreme heterogeneity with sharp variations occuring over distances of a few centimetres. The relation between the CD and LMVS assemblages shows that it is a product of post-depositional diagenesis, whereas the altered clay assemblage formed beneath an erosional unconformity. The CD and LMVS assemblages were derived primarily from an accumulation several metres thick of ash deposits produced by humans. Ash is thus shown to be a major component of the sediments of this cave. The distribution of the CD assemblage reflects to a large extent the locations of two active springs/seepages in the cave. The distribution of the CD assemblage also faithfully maps the distribution of bones in the cave, showing that their distribution is a function of preservational conditions and not human activities (Stiner et al., 2001). The conditions that produced the erosional unconformity, also resulted in severe alteration of the clays and other mineral components of these sediments. The erosion process and the fact that the thickness of this altered zone decreases towards the centre of the cave, indicates that the diagenetic driving force was probably climatic. The three-dimensional distributions of the mineral assemblages have a direct bearing on the dating of the cave by thermoluminescence and electron spin resonance.
Aizenberg J., Lambert G., Weiner S. & Addadi L. 2002. Factors involved in the formation of amorphous and crystalline calcium carbonate: A study of an ascidian skeleton Journal of the American Chemical Society. 2002 Jan , 124 (1):32-39.

[Abstract]

The majority of invertebrate skeletal tissues are composed of the most stable crystalline polymorphs of CaCO₃, calcite, and/or aragonite. Here we describe a composite skeletal tissue from an ascidian in which amorphous and crystalline calcium carbonate coexist in well-defined domains separated by an organic sheath. Each biogenic mineral phase has a characteristic Mg content (5.9 and 1.7 mol %, respectively) and concentration of intramineral proteins (0.05 and 0.01 wt %, respectively). Macromolecular extracts from various biogenic amorphous calcium carbonate (ACC) skeletons are typically glycoproteins, rich in glutamic acid and hydroxyamino acids. The proteins from the crystalline calcitic phases are aspartaterich. Macromolecules extracted from biogenic ACC induced the formation of stabilized ACC and/or inhibited crystallization of calcite in vitro. The yield of the synthetic ACC was 15-20%. The presence of Mg facilitated the stabilization of ACC: the protein content in synthetic ACC was 0.12 wt % in the absence of Mg and 0.07 wt % in the presence of Mg (the Mg content in the precipitate was 8.5 mol %). In contrast, the macromolecules extracted from the calcitic layer induced the formation of calcite crystals with modified morphology similar to that expressed by the original biogenic calcite. We suggest that specialized macromolecules and magnesium ions may cooperate in the stabilization of intrinsically unstable amorphous calcium carbonate and in the formation of complex ACC/calcite tissues in vivo.
Weiss I., Tuross N., Addadi L. & Weiner S. 2002. Mollusc larval shell formation: Amorphous calcium carbonate is a precursor phase for aragonite Journal of Experimental Zoology. 2002 Oct , 293 (5):478-491.

[Abstract]

The larval shells of the marine bivalves Mercenaria mercenaria and Crassostrea gigas are investigated by polarized light microscopy, infrared spectroscopy, Raman imaging spectroscopy, and scanning electron microscopy. Both species contain similar shell ultrastructures. We show that larval shells contain amorphous calcium carbonate (ACC), in addition to aragonite. The aragonite is much less crystalline than nonbiogenic aragonite. We further show that the initially deposited mineral phase is predominantly ACC that subsequently partially transforms into aragonite. The postset juvenile shell, as well as the adult shell of Mercenaria also contains aragonite that is less crystalline than nonbiogenic aragonite. We conclude that ACC fulfills an important function in mollusc larval shell formation. It is conceivable that ACC may also be involved in adult shell formation.
Karkanas P., Rigaud J., Simek J., Albert R. & Weiner S. 2002. Ash bones and guano: A study of the minerals and phytoliths in the sediments of Grotte XVI, Dordogne, France Journal of Archaeological Science. 2002 , 29 (7):721-732.

[Abstract]

Very few prehistoric cave deposits in Western Europe contain visible hearth remains, even though there is abundant evidence of fire use by cave inhabitants. Grotte XVI (Dordogne, France) is exceptional in this respect, in that it contains a most conspicuous layer (Couche C) characterized by a series of brightly coloured bedded sediments. The formation of Couche C has previously been ascribed to the ash and charcoal produced by the cave's Mousterian occupants burning mainly lichens. The Mousterian sedimentary layers below Couche C, and the Upper Palaeolithic sediments unconformably overlying Couche C, are quite different in texture and colour, and show no visible evidence of ash deposits. Analyses of the minerals, microstructures and phytoliths in Couche C are consistent with the notion that these sediments were derived mainly from ash produced by burning wood and to a lesser extent grass. The original calcite component of the ash has been diagenetically altered to carbonated apatite around the cave periphery, and to even more insoluble phosphate minerals in the centre. One such mineral, newberyite, most likely formed by the reaction between guano degradation products and the ash. Under these circumstances bone preservation is also affected. The overlying Upper Palaeolithic sediments contain calcite. One of these layers (Abb) contains much more calcite than all the other layers, as well as abundant phytoliths derived from wood and grass. This layer also contained many artifacts and bones. It is thus concluded that ash is a major component of Layer Abb, even though it is not visible to the maked eye in the form of hearths.
Garty J., Kunin P., Delarea J. & Weiner S. 2002. Calcium oxalate and sulphate-containing structures on the thallial surface of the lichen Ramalina lacera: Response to polluted air and simulated acid rain Plant Cell And Environment. 2002 Dec , 25 (12):1591-1604.

[Abstract]

The formation of calcium-containing structures on the thallial surface of the lichen Ramalina lacera (With.) J.R. Laund. in response to air pollution and to simulated acid rain, was studied in in situ and transplanted thalli. In situ thalli were collected from an unpolluted site and transplanted to heavily polluted and less polluted sites for a 10 month period. Additional thalli were treated either with double distilled water or with simulated acid rain. Scanning electron microscopy and infrared spectrometry revealed that thallial surfaces of in situ R. lacera samples collected in unpolluted sites were covered with two kinds of calcium oxalate crystals: whewellite and weddellite. These aggregates of calcium oxalate crystals appear to disintegrate and provide a crystal layer on the thallial surface. Infrared spectroscopy of powder scraped from thallial surfaces of transplants, retrieved from non-polluted sites, showed the presence of whewellite and weddellite, whereas powders obtained from thalli retrieved from polluted sites contained whewellite, weddellite and gypsum. It is suggested that a certain fraction of the gypsum detected in crater-like structures in transplants from polluted sites and in thalli treated with simulated acid rain is endogenous and should be considered a biomineral.
Alon D., Mintz G., Cohen I., Weiner S. & Boaretto E. 2002. The use of raman spectroscopy to monitor the removal of humic substances from charcoal: Quality control for ¹⁴C dating of charcoal Radiocarbon. 2002 , 44 (1):1-11.

[Abstract]

One of the largest sources of uncertainty in radiocarbon dating stems from the sample pretreatment procedures used to minimize contamination. A major source of carbon contamination in charcoal from archaeological sites is humic substances carried by groundwater. Here we present a method, independent of ¹⁴C dating itself, to evaluate the effectiveness of the cleaning procedure of charcoal. Raman spectra of mixtures of humic substances (HS) and laboratory prepared charcoal indicate that Raman spectroscopy can be used as a semi-quantitative measure of the amount of humic substances associated with archaeological charcoal. Raman spectral analysis of archaeological charcoal samples subjected to different cleaning regimes supports this contention. Such measurements can provide quality control for charcoal preparation procedures and may assist in the interpretation of carbon-dating results.
Levi-Kalisman Y., Raz S., Weiner S., Addadi L. & Sagi I. 2002. Structural differences between biogenic amorphous calcium carbonate phases using X-ray absorption spectroscopy Advanced Functional Materials. 2002 Jan , 12 (1):43-48.

[Abstract]

We compare the organization of the first coordination shells around the calcium ion in biogenic ACC phases from three different sources. The results show that although the three biogenic samples have the same chemical composition, which is referred to collectively under the name "amorphous calcium carbonate", they are structurally different from one another. These differences may be attributed to the diverse modes of formation of such biogenic materials and may account for their known variations in stability.
Raz S., Testeniere O., Hecker A., Weiner S. & Luquet G. 2002. Stable amorphous calcium carbonate is the main component of the calcium storage structures of the crustacean Orchestia cavimana Biological Bulletin. 2002 Dec , 203 (3):269-274.

[Abstract]

Amorphous calcium carbonate (ACC) is the least stable form of the six known phases of calcium carbonate. It is, however, produced and stabilized by a variety of organisms. In this study we examined calcium storage structures from the terrestrial crustacean Orchestia cavimana, in order to better understand their formation mode and function. By using X-ray diffraction, infrared and Raman spectroscopy, thermal analysis and elemental analysis, we determined that the mineral comprising these storage structures is amorphous calcium carbonate with small amounts of amorphous calcium phosphate (5%). We suggest that the use of amorphous calcium carbonate might be advantageous for these storage structures, which function as reservoirs of ions during the animal molting period. Its high solubility is beneficial for temporary storage of calcium carbonate ions that are subsequently dissolved and used elsewhere. Stabilization of these amorphous minerals is probably due to macromolecular constituents of the organic matrix, and to the magnesium and phosphate present in the mineral phase.
Weiner S. & Addadi L. 2002. Biomineralization: At the cutting edge Science. 2002 Oct , 298 (5592):375-376.
Albert R. & Weiner S. 2001. Study of phytoliths in prehistoric ash layers from Kebara and Tabun Caves using a quantitative approach Phytoliths: Applications In Earth Sciences And Human History. 2001 , :251-266.

[Abstract]

Phytoliths are common in hearths and ash layers from archaeological sites. They can potentially provide valuable information about the type of wood used to make the fire and possibly, which trees and which parts of the trees were used. These results can also differentiate between ash layers of anthropological origin and those due to spontaneous combustion based on the presence of wood phytoliths found in caves. To carry out this study, two archaeological sites were studied, Tabun and Kebara Caves, (Mt. Carmel, Israel). Tabun Cave belongs to the Lower and Middle Paleolithic periods and Kebara Cave to the Middle and Upper Paleolithic periods. Thirty different plant taxa common in the area were analyzed, among them woody dicots, herbaceous dicots and grasses. These analyses were based on the phytolith concentrations in the different species and on the morphological characteristics of these phytoliths. The amounts of phytoliths present in the different species were obtained by counting the phytoliths from the Acid Insoluble Fraction. In this way all the more soluble minerals were removed with strong acid, to minimize the effect of weight changes of the samples due to diagenesis. The results showed that phytoliths in grasses are about 20 times more abundant than in wood and bark of woody dicots. The morphology of the phytoliths showed that in wood and bark, phytoliths with variable morphologies are the most common, while in the leaves of the same species and in grasses, phytoliths with consistent morphologies are the most abundant. The variations in the ratio of phytolith types of these two groups, in both the reference collection and the archaeological samples, together with the different proportions of acid insoluble fraction and the different extent of production of phytoliths, can indicate whether the phytoliths present in the archaeological samples are derived from the wood and bark of trees. The morphology of the phytoliths with consistent morphology were st
Levi-Kalisman Y., Falini G., Addadi L. & Weiner S. 2001. Structure of the nacreous organic matrix of a bivalve mollusk shell examined in the hydrated state using Cryo-TEM Journal of Structural Biology. 2001 , 135 (1):8-17.

[Abstract]

During mollusk shell formation, the mineral phase forms within an organic matrix composed of β-chitin, silk-like proteins, and acidic glycoproteins rich in aspartic acid. The matrix is widely assumed to play an important role in controlling mineralization. Thus, understanding its structure is of prime importance. Cryo-transmission electron microscopy (Cryo-TEM) studies of the matrix of the bivalve Atrina embedded in vitrified ice show that the interlamellar sheets are composed mainly of highly ordered and aligned β-chitin fibrils. The silk, which is quantitatively an important component of the matrix, could not be imaged within the sheets. Organic material was, however, observed between sheets. We infer that this is the location of the silk. As this material reveals no regular structure, we suggest that at least prior to mineralization the silk is in the form of a hydrated gel. This is supported by cryo-TEM structural observations of an artificial assembly of β-chitin with and without silk. This view of the nacreous organic matrix significantly changes previous models of the matrix structure and hence hypotheses pertaining to the mechanisms by which mineral formation occurs.
Aizenberg J., Tkachenko A., Weiner S., Addadi L. & Hendler G. 2001. Calcitic microlenses as part of the photoreceptor system in brittlestars Nature. 2001 Aug , 412 (6849):819-822.

[Abstract]

Photosensitivity in most echinoderms has been attributed to 'diffuse' dermal receptors. Here we report that certain single calcite crystals used by brittlestars for skeletal construction are also a component of specialized photosensory organs, conceivably with the function of a compound eye. The analysis of arm ossicles in Ophiocoma showed that in light-sensitive species, the periphery of the labyrinthic calcitic skeleton extends into a regular array of spherical microstructures that have a characteristic double-lens design. These structures are absent in light-indifferent species. Photolithographic experiments in which a photoresist film was illuminated through the lens array showed selective exposure of the photoresist under the lens centres. These results provide experimental evidence that the microlenses are optical elements that guide and focus the light inside the tissue. The estimated focal distance (4-7 μm below the lenses) coincides with the location of nerve bundles - The presumed primary photoreceptors. The Yens array is designed to minimize spherical aberration and birefringence and to detect light from a particular direction. The optical performance is further optimized by phototropic chromatophores that regulate the dose of illumination reaching the receptors. These structures represent an example of a multifunctional biomaterial that fulfills both mechanical and optical functions.
Gorring-Morris A. N., Weiner S. & Boaretto E. 2001. Radiometric Dating of the PPNB Mortuary Site of Kfar HaHoresh, Lower Galilee, Israel: Problems and Preliminary Results Mitekufat Haeven: Journal of the Israel Prehistoric Society. 2001 , 31 :213-217.
Stiner M., Kuhn S., Surovell T., Goldberg P., Meignen L., Weiner S. & Bar-Yosef O. 2001. Bone preservation in Hayonim Cave (Israel): A macroscopic and mineralogical study Journal of Archaeological Science. 2001 , 28 (6):643-659.

[Abstract]

Understanding the cause of patchy bone distributions in archaeological sites requires that one distinguish bone decomposition in place from "empty" areas where bones were never present. Marked horizontal variations in bone abundance are found in the thick Mousterian layer (E) of Hayonim Cave, a large Paleolithic site in northern Israel. Infra-red analyses of minerals in the sediments identify zones of advanced diagenesis and decomposition alongside zones whose chemistry clearly favoured the preservation of bones and wood ash. These differences adhere closely to the distribution of recognizable bones in the deposits, indicating that spatial variation in bone abundance is essentially a product of differential preservation conditions. However, the few bones present in the bone-poor units are in surprisingly good condition. The higher degree of abrasion damage and more random orientations of these bones indicate that small amounts of recent material were introduced into older layers by small burrowing animals and perhaps localized trampling. Tha ratio of post-Mousterian to Mousterian artifacts in layer E, and the numeric contrasts in bone abundance among stratigraphic units, indicate that time-averaging from mechanical intrusion was quantitatively unimportant (2-5%) throughout this>2·4 m thick layer. Our findings support Karkanas et al. (2000) suggestion that bone and ash mineral diagenesis in caves follow step-wise rather than gradual transformations in geological time. Good preservation environments can be distinguished from poorer ones on the basis of mineral assemblages in sediments, and deposits that once contained bone and wood ash can be identified long after the visible traces of these materials have disappeared.
Goldberg P., Weiner S., Bar-Yosef O., Xu Q. & Liu J. 2001. Site formation processes at Zhoukoudian, China Journal of Human Evolution. 2001 , 41 (5):483-530.

[Abstract]

Zhoukoudian is often cited for its human remains and the early evidence of fire. Yet, since its first excavations over 70 years ago, detailed studies of processes responsible for the accumulation of anthropogenic and geogenic sediments in the site have been sparse. This paper provides some details of site formation processes mainly through field observations of the extant section at Locality 1, and the use of soil micromorphology and Fourier Transform Infrared Spectrometry (FTIR) analyses of the sediments. Samples from Layers 10 through 3 show extensive water deposition of fine silt-sized material (reworked loess), including fine-grained organic matter. The dark organic-rich unit in Layer 10-often cited as one of the earliest evidence of fire-is a water-laid accumulation. Much of the fine-grained sediment was derived from outside Locality 1, implying that the site was open to varying extents throughout most of its depositional history. The 4-6 m accumulation of "ashes" in Layer 4 represents subaerial water-laid silt deposits derived from the loess-covered hillslopes surrounding the site. They presumably accumulated in an open depression that formed after the collapse of the brecciated roof deposits represented by Layer 6. Diagenesis is present in many of the Layers, and is exemplified by calcite precipitation and dissoulution, and localized apatite (dahllite) replacement of calcite. In Layer 4 diagenesis is more advanced, including calcite/dahllite precipitation, subaerial weathering of the loess and associated precipitation of hematite, alteration of clay and the neoformation of quartz. Many of our conclusions concur with those of Teilhard de Chardin and Young published over 70 years ago.
Addadi L. & Weiner S. 2001. Crystals, asymmetry and life Nature. 2001 Jun , 411 (6839):753-755.

[Abstract]

Understanding the formation of asymmetrical shapes during the growth of symmetrical crystalline structures is a first step towards understanding asymmetry in biology.
Bouropoulos N., Weiner S. & Addadi L. 2001. Calcium oxalate crystals in tomato and tobacco plants: Morphology and in vitro interactions of crystal-associated macromolecules Chemistry-A European Journal. 2001 May , 7 (9):1881-1888.

[Abstract]

Plants form calcium oxalate crystals with unique morphologies under well-controlled conditions. We studied the morphology of single calcium oxalate monohydrate (whewellite) crystals extracted from tomato and tobacco leaves. These crystals have a pseudotetrahedral shape. We identified the ((1) over bar 01), (101) or (102), (1(0) over bar 2), and ((h) over bar(k) over bar0) faces as stable faces. The morphology is chiral with unique handedness. We also show that calcium oxalate monohydrate crystals isolated from tomato, tobacco, and bougainvillea leaves contain macromolecules rich in Gly, Glx, and Ser. Crystal-associated macromolecules extracted from tomato and tobacco influence the morphology of calcium oxalate monohydrate crystals grown in vitro, promoting preferential development of the {120} faces. Furthermore, crystal-associated macromolecules from tobacco promote nucleation of calcium oxalate monohydrate crystals, whereas model polypeptides do not have any significant effect on nucleation. These results imply an active role of the crystal-associated macromolecules in the formation of pseudotetrahedral shapes in vitro, and these properties may in part be responsible for the unique chiral morphology of the natural pyramidal-shaped crystals.
Raz S., Weiner S. & Addadi L. 2000. Formation of high-magnesian calcites via an amorphous precursor phase: Possible biological implications Advanced Materials. 2000 , 12 (1):38-42.

[Abstract]

High-magnesian calcite can be formed through an amorphous precursor phase under ambient conditions and in the absence of additives. The presence of magnesium ions in the crystallization solution causes an increase in the degree of supersaturation. This results in the precipitation of metastable particles, and in particular amorphous particles. These particles are usually spherical, as expected from a liquid phase that minimizes its surface contact with the surroundings. This amorphous phase subsequently transforms into calcite, aragonite, and high-magnesian calcite, depending upon the conditions of precipitation.
Karkanas P., Bar-Yosef O., Goldberg P. & Weiner S. 2000. Diagenesis in prehistoric caves: The use of minerals that form in situ to assess the completeness of the archaeological record Journal of Archaeological Science. 2000 , 27 (10):915-929.

[Abstract]

An interpretation of the archaeological record, in particular that of a prehistoric cave site, is complicated by the diversity of depositional and post-depositional processes that affect the material deposited. Here we propose to use the authigenic minerals that form in situ within the cave sediments to reconstruct the ancient chemical environments in the sediments. This can be done by experimentally determining the conditions under which each of the authigenic minerals are stable. Although this information is not available to date for minerals formed in a prehistoric cave, we present calculated stability field data for the relevant minerals. The results clearly demonstrate the feasibility of this approach. This information, particularly if based on measurements of real authigenic cave minerals, will facilitate an assessment of the completeness of the cave archaeological record. This is particularly important for determining whether or not the distributions of archaeologically important materials, such as bones, teeth, plant phytoliths, charcoal and ash, reflect their original burial distributions or were altered as a result of secondary diagenetic processes. (C) 2000 Academic Press.
Levi-Kalisman Y., Raz S., Weiner S., Addadi L. & Sagi I. 2000. X-Ray absorption spectroscopy studies on the structure of a biogenic "amorphous" calcium carbonate phase Journal Of The Chemical Society-Dalton Transactions. 2000 , 2000 (21):3977-3982.

[Abstract]

Amorphous calcium carbonate is formed by a surprisingly large number of organisms, bearing in mind how unstable it is. Organisms use amorphous calcium carbonate as temporary storage sites for ions, as precursor phases that transform into more stable crystalline calcium carbonate polymorphs, or in a stabilized form for mechanical purposes. Here one example is examined that fulfils the latter function; the so-called antler shaped spicules formed by the ascidian Pyura pachydermatina. These spicules are composed of amorphous calcium carbonate containing 16% (w/w) water and 14 mole% phosphate. A detailed X-ray absorption spectroscopy (XAS) study of the calcium K-edge in the spicules shows a first co-ordination shell with seven/eight oxygen atoms, and a second co-ordination shell with four/five carbon atoms. The best fit was obtained using monohydrocalcite as model, and is consistent with a slightly expanded hydrated structure. It is noteworthy that the XAS spectrum of these spicules is quite different from that reported previously for the amorphous calcium carbonate of plant cystoliths. This raises the intriguing possibility that the biogenic amorphous phases differ structurally from each other, and that the differences can account for their diverse modes of formation and function.
Beniash E., Traub W., Veis A. & Weiner S. 2000. A transmission electron microscope study using vitrified ice sections of predentin: Structural changes in the dentin collagenous matrix prior to mineralization Journal of Structural Biology. 2000 , 132 (3):212-225.

[Abstract]

The assembly of the collagenous organic matrix prior to mineralization is a key step in the formation of bones and teeth. This process was studied in the predentin of continuously forming rat incisors, using unstained vitrified ice sections examined in the transmission electron microscope. Progressing from the odontoblast surface to the mineralization front, the collagen fibrils thicken to ultimately form a dense network, and their repeat D-spacings and banding patterns vary. Using immunolocalization, the most abundant noncollagenous protein in dentin, phosphophoryn, was mapped to the boundaries between the gap and overlap zones along the fibrils nearest the mineralization front. It thus appears that the premineralized collagen matrix undergoes dynamic changes in its structure. These may be mediated by the addition and interaction with the highly anionic noncollagenous proteins associated with collagen. These changes presumably create a collagenous framework that is able to mineralize.
Boaretto E., Berkovits D., Hass M., Hui S., Kaufman A., Paul M. & Weiner S. 2000. Dating of prehistoric caves sediments and flints using ¹⁰Be and ²⁶Al in quartz from Tabun Cave (Israel): Progress report Nuclear Instruments & Methods In Physics Research Section B-Beam Interactions With Materials And Atoms. 2000 , 172 (1-4):767-771.

[Abstract]

There is an important need to develop additional dating methods beyond the ¹⁴C limit and independent of thermoluminescence (TL) and electron spin resonance (ESR). We propose to apply the method of burial dating to prehistoric sites using the decay of in situ produced radioisotopes ¹⁰Be and ²⁶Al. The Tabun Cave, Mt. Carmel (Israel) has a sedimentary sequence which represents the type section for about the last 800,000 years in the Levant. The sediments in the cave are mainly of aeolian origin and are rich in quartz. Flint tools are also found in the sediments. Sediment samples and flint tools were selected from the same layer. Physical and chemical procedures to extract ¹⁰Be and ²⁶Al atoms from the quartz fraction of the sediments and from the flint samples were developed, while measuring the natural Al levels as a monitor of the atmospheric component of the cosmogenic nuclides. AMS measurements were performed at the 14UD Pelletron Koffler Accelerator Laboratory, Weizmann Institute, and sensitivities of the order of 1 × 10^-14, in isotopic abundances for both ¹⁰Be and ²⁶Al respectively (corresponding to ∼5×10⁵ atoms) were obtained. First, measurements of a number of Tabun Cave sediment samples and flints show that ¹⁰Be and ²⁶Al analyses have the potential for dating prehistoric cave sediments, provided problems relating to the presence of relatively large amounts of stable Al can be solved, as well as obtaining a better understanding of the burial history of the flints prior to being brought into the cave.
Weiner S. & Wagner H. D. 2000. Letter to the Editor [Bone vol. 27/6 Dec 2000:875–876] Bone. 2000 , 27 (6):875-876.

[Abstract]

Why does a bone fracture? We should be able to answer that in a nontrivial manner, but we cannot. This is an embarrassing admission in an age when treatments are available that can reverse the tendency toward bone loss and thus lessen the danger of fracture. Furthermore, if we knew much more about the relation between the material bone and its fracture properties, we might be able to optimize treatments to improve the quality of the material, or at least predict when the chance of a fracture increases, taking into account also the state of the material. We have been working on aspects of this problem for 15 years. We were recently much humbled by some observations we made,2 which illustrate just how little is still known about the relations between bone microstructure and its mechanical properties. As these results are probably of interest to many in the bone community, and especially researchers focussing on osteoporosis, we thought it would be appropriate to briefly summarize the results and highlight major open questions. [First paragraph]
Liu D., Wagner D. H. & Weiner S. 2000. Bending and fracture of compact circumferential and osteonal lamellar bone of the baboon tibia Journal Of Materials Science-Materials In Medicine. 2000 , 11 (1):49-60.

[Abstract]

Lamellar bone is common among primates, either in the form of extended planar circumferential arrays, or as cylindrically shaped osteons. Osteonal bone generally replaces circumferential lamellar bone with time, and it is therefore of much interest to compare the mechanical properties and fracture behavior of these two forms of lamellar bone. This is, however, difficult as natural specimens of circumferential lamellar bone large enough for standard mechanical tests are not available. We found that as a result of treatment with large doses of alendronate, the lateral sides of the diaphyses of baboon tibia contained fairly extensive regions of circumferential lamellar bone, the structure of which appears to be indistinguishable from untreated lamellar bone. Three-point bending tests were used to determine the elastic and ultimate properties of almost pure circumferential lamellar bone and osteonal bone in four different orientations relative to the tibia long axis. After taking into account the differences in porosity and extent of mineralization of the two bone types, the flexural modulus, bending strength, fracture strain and nominal work-to-fracture properties were similar for the same orientations, with some exceptions. This implies that it is the lamellar structure itself that is mainly responsible for these mechanical properties. The fracture behavior and morphologies of the fracture surfaces varied significantly with orientation in both types of bone. This is related to the microstructure of lamellar bone. Osteonal bone exhibited quite different damage-related behavior during fracture as compared to circumferential lamellar bone. Following fracture the two halves of osteonal bone remained attached whereas in circumferential lamellar bone they separated. These differences could well provide significant adaptive advantages to osteonal bone function. (C) 2000 Kluwer Academic Publishers.
Weiner S., Addadi L. & Wagner H. D. 2000. Materials design in biology Materials Science & Engineering C-Biomimetic And Supramolecular Systems. 2000 , 11 (1):1-8.

[Abstract]

The materials formed by organisms are often the products of hundreds of millions of years of fine-tuning by evolution. They thus incorporate some neat solutions to complex structural problems. Here, we examine four different mineralized biological materials that all appear to fulfill a multipurpose function. They are the crossed lamellar structure of mollusk shells, the skeleton of sea urchins, the lamellar bone type of vertebrates, and the biogenic silica deposited by a wide variety of organisms. They all have very different structures and compositions, but are all structurally designed to reduce the extent of mechanical anisotropy. The strategies for reducing anisotropy are manifold, including features that to date have not been incorporated into synthetic materials.
Mann S. & Weiner S. 1999. Biomineralization: Structural questions at all length scales: Editorial Journal of Structural Biology. 1999 Jun , 126 (3):179-181.
Weiner S., Traub W. & Wagner H. D. 1999. Lamellar bone: Structure-function relations Journal of Structural Biology. 1999 Jun , 126 (3):241-255.

[Abstract]

The term 'bone' refers to a family of materials that have complex hierarchically organized structures. These structures are primarily adapted to the variety of mechanical functions that bone fulfills. Here we review the structure-mechanical relations of one bone structural type, lamellar bone. This is the most abundant type in many mammals, including humans. A lamellar unit is composed of five sublayers. Each sublayer is an array of aligned mineralized collagen fibrils. The orientations of these arrays differ in each sublayer with respect to both collagen fibril axes and crystal layers, such that a complex rotated plywood-like structure is formed. Specific functions for lamellar bone, as opposed to the other bone types, could not be identified. It is therefore proposed that the lamellar structure is multifunctional-the 'concrete' of the bone family of materials. Experimentally measured mechanical properties of lamellar bone demonstrate a clear-cut anisotropy with respect to the axis direction of long bones. A comparison of the elastic and ultimate properties of parallel arrays of lamellar units formed in primary bone with cylindrically shaped osteonal structures in secondary formed bone shows that most of the intrinsic mechanical properties are built into the lamellar structure. The major advantages of osteonal bone are its fracture properties. Mathematical modeling of the elastic properties based on the lamellar structure and using a rule-of-mixtures approach can closely simulate the measured mechanical properties, providing greater insight into the structure-mechanical relations of lamellar bone.
Albert R., Lavi O., Estroff L., Weiner S., Tsatskin A., Ronen A. & Lev-Yadun S. 1999. Mode of occupation of Tabun Cave, Mt Carmel, Israel during the Mousterian Period: A study of the sediments and phytoliths Journal of Archaeological Science. 1999 Oct , 26 (10):1249-1260.

[Abstract]

Alternative modes of occupation of Tabun Cave during the deposition of the Mousterian Levels B and C have been proposed. Garrod and Bate (1937, Excavations at the Wady El-Mughara, Volume 1. Oxford: Clarendon Press) interpreted the archaeological record of both levels as being indicative of domestic occupational activities. Jelinek et al. (1973, Palaeorient 1, 151-183) proposed that the presence of articulated limb bones of Dama mesopotamica in the Level B sediments below the cave chimney indicated that the cave was used as a natural game trap. They also noted that the white ash layers in Level C extended across the whole cave, and proposed that this was due to the burning of natural vegetation in the cave. This study aims at clarifying the modes of occupation during these periods. Level B sediments closely resemble the terra rossa soil that is common in this region. Burning activity is inferred from charcoal fragments seen in thin sections. A minor wood ash component is present based on the preponderance of phytoliths with a variable morphology as compared to those with a consistent morphology, as well as phytoliths with shapes characteristics of those formed in wood and bark of local trees. Thus fires were produced in the cave during this period. The cave may also have been used as a game trap. Level C is composed of multiple layers of brown, black and white sediments. Micromorphology, mineralogy and phytolith analyses all show that these layers are mixtures of terra rossa soil and ash, with the latter being abundant in the white layers. The phytoliths in these layers are derived almost entirely from wood and bark, and not from grasses. These observations are consistent with a domestic occupational mode.
Karkanas P., Kyparissi-Apostolika N., Bar-Yosef O. & Weiner S. 1999. Mineral assemblages in Theopetra, Greece: A framework for understanding diagenesis in a prehistoric cave Journal of Archaeological Science. 1999 Sept , 26 (9):1171-1180.

[Abstract]

Prehistoric cave sediments are often subjected to severe mechanical and chemical alteration, making it difficult to interpret aspects of their archaeology. Theopetra cave offers unique opportunities to resolve aspects of this problem, particularly in relation to chemical changes, because it has a relatively well defined stratigraphy and its older deposits have been subjected to unusually severe diagenesis. A study of the mineralogy and micromorphology of the sediments, and in particular the phosphatic minerals that formed in the sediments after deposition, shows that each stratigraphic unit has its own pattern of authigenic mineral distribution. In some units these patterns vary sequentially in both lateral and vertical directions. These variations reflect changing degrees of diagenesis and the observed patterns imply that every unit obtained its diagenetic fingerprint fairly soon after burial. The prominent ash layers in the older sediments were subjected to unusually severe diagenetic alteration, such that most of the relatively stable siliceous components of ash decomposed into amorphous silica. An intimate association between ash minerals and the K, Fe-phosphate mineral leucophosphite was also observed. The sequential changes in authigenic mineral assemblages in Theopetra are basically similar to those observed in several caves in Israel, raising the possibility that common processes are involved and that information obtained from these detailed studies can be applied to other caves in diverse geographic regions.
Beniash E., Addadi L. & Weiner S. 1999. Cellular control over spicule formation in sea urchin embryos: A structural approach Journal of Structural Biology. 1999 Mar , 125 (1):50-62.

[Abstract]

The spicules of the sea urchin embryo form in intracellular membrane- delineated compartments. Each spicule is composed of a single crystal of calcite and amorphous calcium carbonate. The latter transforms with time into calcite by overgrowth of the preexisting crystal. Relationships between the membrane surrounding the spiculogenic compartment and the spicule mineral phase were studied in the transmission electron microscope (TEM) using freeze-fracture. In all the replicas observed the spicules were tightly surrounded by the membrane. Furthermore, a variety of structures that are related to the material exchange process across the membrane were observed. The spiculogenic cells were separated from other cell types of the embryo, frozen, and freeze-dried on the TEM grids. The contents of electron-dense granules in the spiculogenic cells were shown by electron diffraction to be composed of amorphous calcium carbonate. These observations are consistent with the notion that the amorphous calcium carbonate-containing granules contain the precursor mineral phase for spicule formation and that the membrane surrounding the forming spicule is involved both in transport of material and in controlling spicule mineralization.
Addadi L., Aizenberg J., Beniash E. & Weiner S. 1999. On the concept of a single crystal in biomineralization Crystal Engineering: From Molecules And Crystals To Materials. 1999 , 538 :1-22.

[Abstract]

Keywords: CALCITIC SPONGE SPICULES; PROTEIN INTERACTIONS; DESIGN STRATEGIES; BRAGG REFLECTION; MACROMOLECULES; MORPHOGENESIS; CARBONATE; MAGNETITE; ELEMENTS; TEXTURE
Liu D. M., Weiner S. & Wagner D. H. 1999. Anisotropic mechanical properties of lamellar bone using miniature cantilever bending specimens Journal of Biomechanics. 1999 Jul , 32 (7):647-654.

[Abstract]

The flexural modulus and work-to-fracture properties of circumferential lamellar bone from baboon tibia are presented, based on experiments with miniature cantilever bending specimens. Data is provided for specimens in three orthogonal directions that show marked anisotropy. The advantages of such miniature specimens (about 150 μm in diameter and 2 mm long) include the possibility of sampling very small volumes within a heterogeneous structure such as osteonal bone, or studying biological materials that are not available in large enough volumes for conventional mechanical analysis.
Addadi L. & Welner S. 1999. Micro-printing with crystal inks Nature. 1999 Apr , 398 (6727):461-462.

[Abstract]

Patterning a surface is the key to growing crystals on some areas of a substrate and not others. This can now be done, bringing the prospect of crystalline materials, patterned on nanometre scales, a step closer to reality.
Weiner S., Veis A., Beniash E., Arad T., Dillon J., Sabsay B. & Siddiqui F. 1999. Peritubular dentin formation: Crystal organization and the macromolecular constituents in human teeth Journal of Structural Biology. 1999 Jun , 126 (1):27-41.

[Abstract]

Peritubular dentin (PTD) is a relatively dense mineralized tissue that surrounds the tubules of coronal tooth dentin. It is composed mainly of crystals of carbonated apatite together with a small amount of collagen. Its mode of formation has been investigated by studying the relatively dense particles isolated from a powdered preparation. Electron microscopic examination of the PTD particles, including 3-dimensional image reconstruction and electron diffraction, shows that the organization of the crystals of PTD is very similar to that of the adjacent intertubular dentin (ITD). The latter contains relatively large amounts of collagen and the carbonated apatite crystals are closely associated with the collagen matrix. The proteins present in the PTD particles are soluble after decalcification and stain with Stains All. The principal protein has higher molecular weight and a quite different amino acid composition than the phosphophoryns of the intertubular dentin. The interface between the PTD and the ITD shows structural continuity. These data show how two distinct carbonated apatite- based mineralized tissues can be organized and formed contiguously within the same organ by utilizing different sets of matrix proteins.
Wang R. & Weiner S. 1998. Human root dentin: Structural anisotropy and Vickers microhardness isotropy Connective Tissue Research. 1998 , 39 (4):269-279.

[Abstract]

The demanding mechanical functions and the variable structure of dentin make it an invaluable material for studying the structure-mechanical function relations of a mineralized collagen-containing tissue. The mineralized collagen fibril axes in human root dentin are mainly located on the incremental plane. Within this plane there is a preferred orientation in the general root-crown direction. The apatite crystals are aligned in three dimensions within an individual collagen fibril, but this orientation does not necessarily extend to the neighboring fibrils. Crystals are also present as aggregates without any preferred orientation. The structure is therefore clearly anisotropic with respect to the collagen fibril orientation, but less so with respect to overall crystal orientation. Vickers microhardness measurements of the root dentin are essentially the same on the three orthogonal planes with respect to the incremental plane. Knoop microhardness measurements are also the same on all three orthogonal planes when the major diagonal is aligned perpendicular to the collagen fibril axis preferred orientation direction. In-plane variations of up to 20% are observed in the orthogonal direction. The material is thus isotropic in the three main directions with respect to Vickers microhardness, but anisotropic in structure. This paradoxical situation is attributed mainly to the variable modes of crystal organization.
Weiner S., Xu Q., Goldberg P., Liu J. & Bar-Yosef O. 1998. Evidence for the use of fire at Zhoukoudian, China Science. 1998 Jul , 281 (5374):251-253.

[Abstract]

Zhoukoudian is widely regarded as having the oldest reliable evidence for the controlled use of fire by humans. A reexamination of the evidence in Layer 10, the earliest archaeological horizon in the site, shows that burned and unburned bones are present in the same layer with stone tools. However, no ash or charcoal remnants could be detected. Hence, although indirect evidence for burning is present, there is no direct evidence for in situ burning.
Weiner S. & Wagner H. D. 1998. The material bone: Structure-mechanical function relations Annual Review of Materials Science. 1998 , 28 (1):271-298.

[Abstract]

The term bone refers to a family of materials, all of which are built up of mineralized collagen fibrils. They have highly complex structures, described in terms of up to 7 hierarchical levels of organization. These materials have evolved to fulfill a variety of mechanical functions, for which the structures are presumably fine-tuned. Matching structure to function is a challenge. Here we review the structure-mechanical relations at each of the hierarchical levels of organization, highlighting wherever possible both underlying strategies and gaps in our knowledge. The insights gained from the study of these fascinating materials are not only important biologically, but may well provide novel ideas that can be applied to the design of synthetic materials.
Levi Y., Albeck S., Brack A., Weiner S. & Addadi L. 1998. Control over aragonite crystal nucleation and growth: An in vitro study of biomineralization Chemistry-A European Journal. 1998 Mar , 4 (3):389-396.

[Abstract]

Calcite and aragonite are the two most stable polymorphs of calcium carbonate. Even though thy have very similar structures, many organisms are able to selectively deposit one polymorph and not the other. Recent in vitro studies have shown that one or more mollusk shell matrix macromolecules are capable of specific polymorph nucleation, provided they are in an appropriate microenvironment.([1]) In this study we examine aspects of the structure and function of some of the components of this β-chitin-silk fibroin in vitro system. We also show, by scanning electron microscopy, that the chitin framework is very porous, thus facilitating the diffusion of ions and macromolecules into the structure. Fluorescent light microscopy and scanning electron microscopy (SEM) demonstrate that the silk fibroin is intimately associated with the chitin framework. One particular fraction purified from the assemblage of mollusk-shell macromolecules extracted from an aragonitic shell layer is able to specifically induce aragonite crystal formation in vitro. These crystals are needle-shaped, whereas the aragonite crystals induced by the total assemblage of macromolecules are shortened into ellipsoids. This implies that other components in the assemblage modulate crystal growth. Finally, testing of a series of Asp and Leu or Glu and Leu containing synthetic peptides in the in vitro assay system shows that only (Asp-Leu)(n) is capable of specifically inducing aragonite formation. All these observations demonstrate that polymorph specificity is dependent upon the amino acid sequence, the conformation of specific protein(s) in the mollusk shell and the microenvironment in which crystal nucleation and growth takes place.
Zylberberg L., Traub W., De Buffrenil V., Allizard F., Arad T. & Weiner S. 1998. Rostrum of a toothed whale: Ultrastructural study of a very dense bone Bone. 1998 Sept , 23 (3):241-247.

[Abstract]

The rostral bones of the toothed whale, Mesoplodon densirostris, consist mainly of hypermineralized secondary osteons and have yielded among the highest values for density (2.6 g/cm³) and mineral content (86.7%) yet reported for any bone. Scanning and transmission electron microscopy show parallel rods of mineral oriented along the length of the rostrum. These consist of platey crystals of carbonated hydroxyapatite, which, judging from electron diffraction, are extremely well and coherently aligned. The collagen component of the rostral bone consists largely of very thin fibrils aligned in longitudinal register to form tubular networks. The collagen fibrils are also aligned with the lengths of the mineral rods, which are apparently accomodated in the tubular spaces of the collagenous network. This peculiar ultrastructure clearly differs from the densely packed mineralized fibrils commonly observed in vertebrate lamellar osseous tissues, although histological examination has indicated some vestiges of 'normal' primary bone surrounding the secondary osteons. Thus, the bone tissue in the rostrum is characterized by a remarkably sparse collagenous component. This ultrastructure can explain the high density, stiffness, and brittleness of the rostrum that have been observed. It also raises interesting questions about possible modes of crystal growth during ongoing mineralization in normal bone, and may have some relevance in the mechanical behavior of dense bones in pathological conditions.
Aksay I. & Weiner S. 1998. Biomaterials - Is this really a field of research? Current Opinion In Solid State & Materials Science. 1998 Jun , 3 (3):219-220.
Akiva U., Wagner H. D. & Weiner S. 1998. Modelling the three-dimensional elastic constants of parallel-fibred and lamellar bone Journal of Materials Science. 1998 , 33 (6):1497-1509.

[Abstract]

The complex hierarchical structure of lamellar bone makes understanding structure mechanical function relations, very difficult. We approach the problem by first using the relatively simple structure of parallel-fibred bone to construct a mathematical model for calculating Young's moduli in three-dimensions. Parallel-fibred bone is composed essentially of arrays of mineralized collagen fibrils, which are also the basic structural motif of the individual lamellae of lamellar bone. Parallel-fibred bone structure has orthotropic symmetry. As the sizes and shapes of crystals in bone are not well known, the model is also used to compare the cases of platelet-, ribbon- and sheet-reinforced composites. The far more complicated rotated plywood structure of lamellar bone results in the loss of the orthotropic symmetry of individual lamellae. The mathematical model used circumvents this problem by sub-dividing the lamellar unit into a thin lamella, thick lamella, transition zone between them, and the recently observed "back-flip" lamella. Each of these is regarded as having orthotropic symmetry. After the calculation of their Young's moduli they are rotated in space in accordance with the rotated plywood model, and then the segments are combined to present the overall modulus values in three-dimensions. The calculated trends compare well with the trends in microhardness values measured for circumferential lamellar bone. Microhardness values are, as yet, the only measurements available for direct comparison. Although the model is not directly applicable to osteonal bone, which is composed of many hollow cylinders of lamellar bone, the range of calculated modulus values and the trends observed for off-axis calculations, compare well with measured values.
Addadi L. & Weiner S. 1997. Biomineralization - A pavement of pearl Nature. 1997 Oct , 389 (6654):912-&.
Wang R. Z. & Weiner S. 1997. Strain-structure relations in human teeth using Moire fringes Journal of Biomechanics. 1997 May , 31 (2):135-141.

[Abstract]

Teeth are subjected to stress during normal function. The manner in which the resulting strain is distributed within the tooth is related to its structure. The Moire fringe technique was used to map the in-plane strain distribution in slices from human tooth crowns under compression. The strain inside enamel is much less than in dentin, and there is a roughly 200 μm thick zone in dentin beneath the dentin-enamel junction which undergoes larger strain than the central coronal dentin. This zone is softer and less mineralized than the bulk of the dentin. The strain distribution in this zone along the dentin-enamel junction shows localized maxima on both the lingual and the labial sides. This study is consistent with the hypothesis that within the dentin there are structural adaptations for transferring and minimizing stress.
Wang R. Z., Addadi L. & Weiner S. 1997. Design strategies of sea urchin teeth: Structure, composition and micromechanical relations to function PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES. 1997 , 352 (1352):469-480.

[Abstract]

The teeth of sea urchins comprise a variety of different structural entities, all of which are composed of magnesium-bearing calcite together with a small amount of organic material. The teeth are worn down continuously, but in such a way that they remain sharp and functional. Here we describe aspects of the structural, compositional and micromechanical properties of the teeth of Paracentrotus lividus using scanning electron microscopy, infrared spectrometry, atomic absorption, X-ray diffraction and microindentation. The S-shaped single crystalline calcitic fibres are one of the main structural elements of the tooth. They extend from the stone part to the keel. The diameter of the fibres increases gradually from less than 1 mμ at the stone tip to about 20 μm at the keel end, while their MgCO₃ contents decrease from about 13 mol% to about 4.5 mol%. Each fibre is coated by a thin organic sheath and surrounded by polycrystalline calcitic discs containing as much as 35 mol% MgCO₃. This structure constitutes a unique kind of gradient fibre-reinforced ceramic matrix composite, whose microhardness and toughness decrease gradually from the stone part to the keel. Primary plates are also important structural elements of the tooth. Each primary plate has a very unusual sandwich-like structure with a calcitic envelope surrounding a thin apparently amorphous CaCO₃ layer. This central layer, together with the primary plate/disc interface, improves the toughness of this zone by stopping and blunting cracks. The self-sharpening function of the teeth is believed to result from the combination of the geometrical shape of the main structural elements and their spatial arrangement, the interfacial strength between structural elements, and the hardness gradient extending from the working stone part to the surrounding zones. The sea urchin tooth structure possesses an array of interesting functional design features, some of which may possibly be applicable to materials science.
Beniash E., Aizenberg J., Addadi L. & Weiner S. 1997. Amorphous calcium carbonate transforms into calcite during sea urchin larval spicule growth Proceedings Of The Royal Society B-Biological Sciences. 1997 , 264 (1380):461-465.

[Abstract]

Sea urchin larvae form an endoskeleton composed of a pair of spicules. For more than a century it has been stated that each spicule comprises a single crystal of the CaCO₃ mineral, calcite. We show that an additional mineral phase, amorphous calcium carbonate, is present in the sea urchin larval spicule, and that this inherently unstable mineral transforms into calcite with time. This observation significantly changes our concepts of mineral formation in this well-studied organism.
Weiner S. & Addadi L. 1997. Design strategies in mineralized biological materials Journal of Materials Chemistry. 1997 May , 7 (5):689-702.

[Abstract]

Organisms have been producing mineralized skeletons for the past 550 million years. They have evolved many different strategies for improving these materials at almost all hierarchical levels from Angstroms to millimetres. Key components of biological materials are the macromolecules, which are intimately involved in controlling nucleation, growth, shaping and adapting mechanical properties of the mineral phase to function. One interesting tendency that we have noted is that organisms have developed several strategies to produce materials that have more isotropic properties. Much can still be learned from studying the principles of structure-function relations of biological materials. Some of this information may also provide new ideas for improved design of synthetic materials.
Weiner S., Arad T., Sabanay I. & Traub W. 1997. Rotated plywood structure of primary lamellar bone in the rat: Orientations of the collagen fibril arrays Bone. 1997 Jun , 20 (6):509-514.

[Abstract]

A basic structural motif of lamellar bone is the arrays of parallel collagen fibrils, with successive arrays having different orientations to form a plywood-like structure. Measurements of the angles between adjacent arrays from cryomicrotomed and vitrified thin sections of demineralized rat bone, cut approximately parallel to the lamellar boundary plane, show that most angles are around 30°, although a subset are around 70°. A structural model for collagen organization based on these measurements is proposed in which an individual lamellar unit (thick and thin lamellae together with transition zones) is composed of five arrays of parallel collagen fibrils, each offset by 30°.
Aizenberg J., Hanson J., Koetzle T., Weiner S. & Addadi L. 1997. Control of macromolecule distribution within synthetic and biogenic single calcite crystals Journal of the American Chemical Society. 1997 Feb , 119 (5):881-886.

[Abstract]

The ability of organisms to exercise control over crystal growth is wonderfully exemplified by skeleton formation in echinoderms. A sea urchin spine is a unique composite of a single crystal of calcite and glycoproteins intercalated inside the crystal during its growth. Here we performed a detailed morphological and high-resolution synchrotron X-ray diffraction study of the textures of synthetic and biogenic calcite crystals. We show that the intracrystalline macromolecules from sea urchin spines, when allowed to interact with growing calcite crystals in vitro, selectively reduce the coherence lengths and degrees of alignment of the perfect domains in specific crystallographic directions. These directions also correspond to the newly-developed stable faces. In contrast, the defect distribution of young sea urchin spines composed entirely of spongy stereomic structure is much more isotropic. In mature spines containing secondarily filled-in wedges of calcite, the degree of anisotropy is intermediate between that of the synthetic crystals and the young spines. The macromolecules extracted from young and mature spines are, however, very similar. These observations demonstrate the inherent capability of occluded matrix macromolecules to finely differentiate between crystal planes by stereochemical recognition processes. They also show that in biologically-produced calcite crystals this process can be overridden to produce a more isotropic material.
Shahack-Gross R., BarYosef O. & Weiner S. 1997. Black-coloured bones in Hayonim Cave, Israel: Differentiating between burning and oxide staining Journal of Archaeological Science. 1997 May , 24 (5):439-446.

[Abstract]

Identification of fossil burned bones is commonly based on their black colour. While colour-based identification is applicable to recent burned bones, fossil bones may be black due to mineral staining, such as by black manganese oxides. We therefore developed an analytical method that purifies oxides from bones. The end product of the method was analysed by means of Fourier transform infra-red (FTIR) spectroscopy. The method can be used for differentiating between black fossil bones that are burned and unstained, burned and stained, and stained but not burned. A survey of 30 black bones from the Mousterian and Aurignacian deposits of Hayonim Cave (Israel) showed that the majority of the bones were indeed burned, of which a few were burned and stained. Several bones were stained and unburned. This method can be readily applied to other archaeological sites where the proportions of burned and stained bones may be quite different.
Ziv V., Wagner H. D. & Weiner S. 1996. Microstructure-microhardness relations in parallel-fibered and lamellar bone Bone. 1996 May , 18 (5):417-428.

[Abstract]

Understanding the mechanical function of bone material in relation to its structure is a fascinating but very complicated problem to resolve. Part of the complexity arises from the hierarchical structural organization of bone. Microhardness measurements, initially on relatively simply structured parallel-fibered bone, show a marked anisotropy in three orthogonal directions. This may, in part, be due to the highly anisotropic structure of the basic building block of bone, the mineralized collagen fibril. Microhardness measurements made face-on to the layers of crystals and collagen triple helical molecules, show much lower values than those made edge-on to these layers. Microhardness measurements of the much more complex 'rotated-plywood' structure of lamellar bone, reveal the well-known general tendency toward anisotropy in relation to the long axis of the bone. A detailed examination of microhardness-microstructure relations of lamellar bone, however, shows that only in certain orientations can microhardness values be related directly to a specific attribute of the lamellar structure. Clearly, the gradual tilting and rotating of the mineralized collagen fibrils that form this structure produce a material that tends toward having isotropic microhardness properties, even though its basic building block is highly anisotropic. This may be an important structural attribute that allows lamellar bone to withstand a variety of mechanical challenges.
Aizenberg J., Lambert G., Addadi L. & Weiner S. 1996. Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates Advanced Materials. 1996 Mar , 8 (3):222-&.

[Abstract]

Communication: Composite structures in which amorphous and crystalline phases coexist are formed by some organisms. The spicules of the sponge Clathrina are shown to be composed of a crystalline calcitic core and an outer layer of amorphous CaCO3 (see figure). The latter material, normally a highly unstable mineral, is stabilized in vitro by macromolecules extracted from the outer sponge spicule layer. The combination of crystalline and stabilized amorphous phases may have important implications for materials applications.
Albeck S., Weiner S. & Addadi L. 1996. Polysaccharides of intracrystalline glycoproteins modulate calcite crystal growth in vitro Chemistry-A European Journal. 1996 , 2 (3):278-284.

[Abstract]

Assemblies of glycoproteins from within the mineralized tissues of sea urchins and mollusks both interact in vitro in a similar manner with growing calcite crystals. A protein-rich fraction, a polysaccharide-rich fraction, and a fraction composed of densely glycosylated peptide cores were obtained by chemical and enzymatic treatment of the glycoproteins from sea-urchin spines. Each fraction was partially purified and characterized (amino acid composition, FTIR and NMR spectroscopy). A comparison of the interactions of these fractions with growing calcite crystals in vitro shows that the polysaccharide moieties of these glycoproteins are intimately involved in the interaction with growing calcite crystals on planes approximately parallel to the c crystallographic axis. Presumably the polysaccharides in the mollusk-shell glycoproteins are likewise responsible for the similar interactions of these macromolecules with calcite. We suggest that structured polysaccharide moieties of glycoproteins are important in controlling aspects of crystal growth in vivo as well.
Falini G., Albeck S., Weiner S. & Addadi L. 1996. Control of aragonite or calcite polymorphism by mollusk shell macromolecules Science. 1996 Jan , 271 (5245):67-69.

[Abstract]

Many mineralizing organisms selectively form either calcite or aragonite, two polymorphs of calcium carbonate with very similar crystalline structures. Understanding how these organisms achieve this control has represented a major challenge in the field of biomineralization. Macromolecules extracted from the aragonitic shell layers of some mollusks induced aragonite formation in vitro when preadsorbed on a substrate of β-chitin and silk fibroin. Macromolecules from calcitic shell layers induced mainly calcite formation under the same conditions. The results suggest that these macromolecules are responsible for the precipitation of either aragonite or calcite in vivo.
Aizenberg J., Ilan N., Weiner S. & Addadi L. 1996. Intracrystalline macromolecules are involved in the morphogenesis of calcitic sponge spicules Connective Tissue Research. 1996 , 35 (1-4):17-23.

[Abstract]

Control over the shapes of biologically formed crystals is generally not well understood. We have studied the morphogenesis of the different-shaped calcareous sponge spicules using high-resolution synchrotron X-ray diffraction. We show that a remarkable correlation exists between the distribution of defects within spicule crystals at the nanometer level and their macroscopic morphologies at the millimeter level. These defects are produced by controlled intercalation of specialized macromolecules into the crystals. We also show that such intracrystalline macromolecules are involved in the regulation of the shapes of synthetic crystals grown de novo from solution, and epitaxially overgrown on the spicule surfaces. We conclude that intracrystalline macromolecules play an important role in modulation of the morphologies of the forming biogenic crystals. Possible mechanisms that may account for the observed growth patterns are supported by fluorescence labeling experiments in vivo.
Albeck S., Addadi L. & Weiner S. 1996. Regulation of calcite crystal morphology by intracrystalline acidic proteins and glycoproteins Connective Tissue Research. 1996 , 35 (1-4):365-370.

[Abstract]

Many biologically formed calcite crystals contain intracrystalline macromolecules. The ways in which they interact with growing calcite crystals were evaluated by monitoring changes in the morphology of calcite crystals grown in vitro in their presence. Macromolecules were extracted from within isolated prisms from the prismatic layer of the shell of the mollusk Atrina rigida and from spines of the sea urchin Paracentrotus lividus. Two modes of interaction were identified; the interaction of highly acidic proteins with calcite planes perpendicular to the c crystallographic axis and the interaction of glycoproteins with planes roughly parallel to the c axis. By different preparative procedures we demonstrated that the polysaccharide moieties of the sea urchin spine glycoproteins are directly involved in the latter mode of interactions. We suggest that organisms utilize the abilities of these macromolecules to interact in different ways with calcite crystals, and in so doing fine-tune aspects of the control of crystal growth in vivo.
Shahack-Gross R., Shemesh A., Yakir D. & Weiner S. 1996. Oxygen isotopic composition of opaline phytoliths: Potential for terrestrial climatic reconstruction Geochimica et Cosmochimica Acta. 1996 Oct , 60 (20):3949-3953.

[Abstract]

Opaline mineralized bodies are produced by many terrestrial plants and accumulate in certain soils and archaeological sites. Analyses of the oxygen isotopic compositions of these so-called phytoliths from stems and leaves of wheat plants grown in a greenhouse showed a linear relationship with stem and leaf water isotopic compositions and hence, indirectly, rain water isotopic composition. Analyses of wheat plants grown in fields showed that stem phytoliths isotopic composition directly reflects the seasonal air temperature change, whereas leaf phytoliths isotopic composition reflects both temperature and relative humidity. Temperature and the oxygen isotopic composition of stem phytoliths were related by an equation similar to that proposed for marine opal. Oxygen isotopic compositions of fossil phytoliths, and in particular those from stems, could be valuable for reconstructing past terrestrial climate change.
Schiegl S., Goldberg P., BarYosef O. & Weiner S. 1996. Ash deposits in Hayonim and Kebara caves, Israel: Macroscopic, microscopic and mineralogical observations, and their archaeological implications Journal of Archaeological Science. 1996 Sept , 23 (5):763-781.

[Abstract]

A study of the hearths and ash layers preserved in Hayonim and Kebara caves, Israel, showed that they are composed of one or several different minerals. These include the polymorph of CaCO₃, calcite, a variety of phosphate minerals and a suite of siliceous minerals. The major component of the latter are siliceous aggregates that are present in wood. Detailed studies of the mineral associations in these fossil ash layers, as well as in fresh wood ash, using optical and scanning electron microscopy in the back scattered electron mode together with elemental analyses, showed that ash in these caves undergoes a series of diagenetic changes. The best preserved ash layers resemble fresh ash and are composed mainly of calcite with minor amounts of siliceous minerals. The calcite reacts with phosphate-rich solutions to form carbonated apatite and with time the latter dissolves and a variety of other phosphate-containing minerals form. With each diagenetic change, some of the more soluble minerals are lost and ultimately only the ash-derived siliceous minerals remain. In both caves there are local accumulations of such siliceous minerals that are metres thick. In fact ash-derived minerals in general are major components of the sediments in both Kebara and Hayonim caves. Their diagenesis involves a hugh reduction in volume, which in turn may have important effects on the geological structures and stratigraphy of the sediments. The changing mineralogy of the sediments due to diagenesis complicates the use of present-day estimates of radiation content of the sediments for correcting age estimates by thermoluminescence and electron spin resonance. Finally, the presence of the relatively stable ash-derived siliceous minerals in a sedimentary layer in other caves, could be a useful means of identifying the presence of ash in other archaeological sites where macroscopic hearth features are absent.
Ziv V., Sabanay I., Arad T., Traub W. & Weiner S. 1996. Transitional structures in lamellar bone Microscopy Research and Technique. 1996 Feb , 33 (2):203-213.

[Abstract]

Scanning electron micrographs of fractured surfaces of mineralized bone show a lamellar structure with alternating smooth and rough regions. These have been interpreted as corresponding to two distinct collagen fibril and mineral crystal orientations in a rotated plywood structure. However, in various bones, there are clear indications of transition zones between lamellae in which the fibrils, as well as the plate-like crystals, have intermediate orientations. Strong evidence for intermediate collagen fibril orientations comes from vitrified cryo-sections of demineralized bone. These show zones of fibril segments graded in length between more homogenous regions of fibrils roughly parallel to the specimen section. Evidence for intermediate crystal orientations comes from transmission electron micrographs and electron diffraction patterns of crushed bone fragments. A tentative scheme is presented for an interlamellar transition zone, involving rotation about the collagen fibril axis as well as tilting of this axis parallel to the plane of the interlamellar boundary. Although it may be convenient to think of the structure of lamellar bone as being composed of alternating thick and thin lamellae, it is probably more correct and biologically more relevant to consider one pair of lamellae as the product of a single depositional cycle of varyingly oriented collagen fibrils that subsequently mineralize.
STINER M., KUHN S., Weiner S. & BARYOSEF O. 1995. Differential Burning, Recrystallization, and Fragmentation of Archaeological Bone Journal of Archaeological Science. 1995 Mar , 22 (2):223-237.

[Abstract]

This paper presents research on the conditions under which progressive levels of burning may occur to archaeological bone, and how burning damage changes bones’ crystal structure and susceptibility to fragmentation (a.k.a. friability). Experiments were conducted to simulate common patterns of high-temperature bone diagenesis and fragmentation previously documented in Paleolithic shelter sites. Bones buried up to 6 cm below the coal beds of the experimental fires were carbonized, but calcination occurred only with direct exposure to live coals. Analysis by infra-red spectroscopy reveals that marked changes in crystallinity accompany the macroscopic transformations in colour and friability of modern, fire-altered bone; specifically, a monotonic, non-linear decrease in mean fragment length across six colour categories was observed when samples were agitated or trampled, and a concordant decline in bone identifiability, first with respect to skeletal element and ultimately the recognizability of bone tissue itself. These findings help qualify the behavioural and taphonomic implications of fragmented, burned bones in archaeological sites, especially with regard to potential stratigraphic associations between artefacts and hearth features in sites and the intensity of space use by human occupants. The identification of burning damage on archaeological bone is a separate issue, however. It was found that the molecular signatures of recrystallization in modern burned bones partly overlap with recrystallization caused by weathering after only 1 to 2 years of exposure in an arid setting and by partial fossilization of archaeological bones over the long term. While infra-red and X-ray diffraction techniques effectively describe heat-induced changes in modern bone mineral and are an important aid for modelling diagenetic processes, these techniques did not reliably identify burning damage to archaeological bones. Cross-referencing readily visible colour phases with HCl-insoluble fraction data proves much more effective and economically feasible for the latter purpose.
AIZENBERG J., HANSON J., KOETZLE T., Leiserowitz L., Weiner S. & Addadi L. 1995. Biologically Induced Reduction in Symmetry: A Study of Crystal Texture of Calcitic Sponge Spicules Chemistry-A European Journal. 1995 Oct , 1 (7):414-422.

[Abstract]

Organisms can exert a remarkable degree of control over crystal growth. One way of achieving this is by the adsorption of specialized macromolecules on specific planes of the growing crystals. With continued growth of the crystal, the macromolecules are incorporated inside the crystal bulk. Their presence does not change the crystal structure, but creates discontinuities in the perfect lattice. Here we study in detail three unusual cases of reduction in symmetry at the level of crystal domain shapes, induced by this controlled intercalation. We examined sponge spicules, which are single crystals of Mg‐bearing calcite. They were specifically chosen for this study, because their morphologies do not reflect the hexagonal symmetry of calcite. Their crystal textures (coherence lengths and angular spreads) were characterized by high‐resolution X‐ray diffraction with well‐collimated synchrotron radiation. The results are compared to analogous studies of synthetic calcite and Mg‐bearing calcite. In all the selected spicules reduction in symmetry is observed in the coherence lengths among symmetry‐related crystallographic directions. The reconstructed shapes of the domains of perfect structure closely match the specific spicule morphologies. The synthetic crystals show no such reduction in symmetry. Although the manner by which such exquisite control is achieved is not known, we envisage it involving a combination of oriented nucleation with either physical or stereochemically driven adsorption.
AIZENBERG J., HANSON J., ILAN M., Leiserowitz L., KOETZLE T., Addadi L. & Weiner S. 1995. Morphogenesis of calcitic sponge spicules: A role for specialized proteins interacting with growing crystals FASEB Journal. 1995 , 9 (2):262-268.

[Abstract]

Crystals formed in biological tissues often adopt remarkable morphologies that are thought to be determined mainly by the shapes of the confined spaces in which they grow. Another possible way of controlling crystal shape, demonstrated only in vitro, is by means of specialized proteins preferentially interacting with certain crystal faces. In so doing, they reduce the rate of growth in these directions and consequently change the overall crystal shape. In an X-ray diffraction study of the distribution of defects within the lattice of calcite crystals produced by certain sponges, we show that a remarkable correlation exists between the defect patterns or crystal texture and the macroscopic morphology of the spicules. This was observed in two cases in which proteins are present within the spicule crystal, but not in a third case where such intracrystalline proteins are absent. Furthermore, one of the spicules exhibited marked differences in texture even within families of structurally identical crystal planes, demonstrating that the organisms exert exquisite control over the microenvironment in which crystals grow. We conclude that highly controlled intercalation of specialized proteins inside the crystals is an additional means by which organisms control spicule growth.
Addadi L., Aizenberg J., Albeck S., Falini G. & Weiner S. 1995. Structural control over the formation of calcium carbonate mineral phases in biomineralization Supramolecular Stereochemistry. 1995 , 473 :127-139.

[Abstract]

Keywords: Chemistry, Organic; Chemistry, Physical
Weiner S., SCHIEGL S., GOLDBERG P. & BARYOSEF O. 1995. Mineral Assemblages in Kebara and Hayonim Caves, Israel: Excavation Strategies, Bone Preservation, and Wood Ash Remnants Israel Journal of Chemistry. 1995 , 35 (2):143-154.

[Abstract]

The mineral assemblages in prehistoric sites can provide essential information on several important topics in archaeology. One of the key analytical tools used is Fourier‐transform infrared spectrometry, which is operated both on‐site during the excavation and in the laboratory. Three topics are reviewed here based on studies of Hayonim and Kebara caves in Israel. (1) Reaction rims form on calcitic and dolomitic rocks buried in the sediments. They are normally the result of interaction of the rock minerals with phosphate‐rich groundwater. The mineralogical nature of these reaction rims can be indicative of prevailing chemical conditions in the sediments, which in turn provide information on whether or not bones are preserved at the site, or are preserved at specific locales within the site. Thus, a survey of reaction rims can be helpful in determining the potential of a future excavation site, or areas within a site, and for optimizing excavation strategies during an ongoing project. (2) The preservational states of the bones themselves, together with information on the mineralogical nature of some of the more labile sediment components, can be used for ascertaining whether or not the bone distribution patterns as excavated represent the original burial locations, or have been affected by secondary dissolution of bones in some areas and not others. (3) The ashes from wood fires are a major component of the sediments in both caves studied. Understanding and monitoring the complex series of diagenetic changes that ashes undergo in these environments can provide invaluable information, not only on the manner in which fire was used by the cave occupants, but also on a series of important processes that affect the depositional history of the site itself. The study of mineral assemblages in prehistoric cave sites, as illustrated here with 3 examples, and possibly in open‐air sites as well, has the potential for contributing significantly to a better understanding of many archaeological problems.
MERCIER N., VALLADAS H., JORON J., SCHIEGL S., BARYOSEF O. & Weiner S. 1995. Thermoluminescence Dating and the Problem of Geochemical Evolution of Sediments — A Case Study: The Mousterian Levels at Hayonim Israel Journal of Chemistry. 1995 , 35 (2):137-141.

[Abstract]

The thermoluminescence dating of burnt flints has become a major tool in the elaboration of the chronology of human settlements of the last half a million years. The reliability of an age estimate depends to a great extent on the accuracy with which the internal and external radiation dose rates prevailing during the duration of burial can be determined. While determining the internal dose rate is relatively straightforward, the same cannot be said of the external, particularly if the sediment surrounding the flints is mineralogically heterogeneous or has undergone diagenetic changes during prolonged burial. In this article we show some examples of the problems confronted when one attempts to determine the external dose rates experienced by flints buried in a relatively heterogeneous environment of sediments, such as those sampled at the Middle Paleolithic occupation levels at Hayonim (Israel) which show evidence of past mineralogical evolution. The impact of changes produced by leaching and chemical reactions between the components of hearth ashes on the radioisotopic composition of the sediment and consequently the environmental dose rates were examined by subjecting several distinct sediment layers to thorough mineralogical, radiochemical, and dosimetric analyses. The problems of external dose‐rate determinations in mineralogically heterogeneous sediments are examined, particularly in those consisting of variable amounts of siliceous aggregates, apatite, and other phosphorus‐rich minerals produced by diagenetic reactions with bone residues.
Rosen A. M. & Weiner S. 1994. Identifying ancient irrigation: A new method using opaline phytoliths from emmer wheat Journal of Archaeological Science. 1994 Jan , 21 (1):125-132.

[Abstract]

A vital factor in the rise of the first state societies is the development of complex farming systems employing labour-intensive irrigation. Ancient irrigation is, however, difficult to recognize archaeologically. Here we report a new method for identifying ancient irrigation based on recognizing the increased deposition of silica in irrigated cereals. Our experiments showed that emmer wheat grown with irrigation in semi-arid plots produced phytoliths with greater numbers of silicified cells per phytolith than wheat that was dry-farmed. The presence of such large multi-celled phytoliths in semi-arid archaeological sites can be used as evidence for prehistoric irrigation.
Ziv V. & Weiner S. 1994. Bone crystal sizes: A comparison of transmission electron microscopic and x-ray diffraction line width broadening techniques Connective Tissue Research. 1994 , 30 (3):165-175.

[Abstract]

Bone crystals are particularly small and hence estimating their sizes have proved to be difficult and values obtained inconsistent. Here we use rat bone crystals of different ages, as well as different synthetic carbonate apatite crystals, to compare two methods commonly used for determining bone crystal sizes. One method involves direct measurement of crystal lengths and widths, but not thicknesses, from transmission electron microscope (TEM) photographs of dispersed crystals, The second method utilizes X-ray diffraction line width broadening to estimate the average length of crystals. We conclude, that line width broadening values tend to reflect crystal coherence lengths rather than the physical dimensions of the whole crystal. TEM measurements provide reliable estimates of average crystal lengths and widths and their ranges. Sample preparation procedures, however, cause breakage of the fragile crystals, which probably results in underestimates of in vivo crystal sizes.
Wachtel E. & Weiner S. 1994. Small‐angle x‐ray scattering study of dispersed crystals from bone and tendon Journal of Bone and Mineral Research. 1994 Oct , 9 (10):1651-1655.

[Abstract]

A small‐angle x‐ray scattering study of dispersed crystals from rat bone and mineralized turkey tendon shows that the particles in both preparations have the same scattering behavior. The data are very similar to those reported by Fratzl et al.¹³ for intact turkey tendon and are consistent with the crystals being plate shaped. These observations have important implications for understanding both the structure and mineralization processes of these tissues.
VANDERMEERSCH B., ARENSBURG B., TILLIER A., RAK Y., Weiner S., SPIERS M. & ASPILLAGA E. 1994. MIDDLE PALEOLITHIC DENTAL BACTERIA FROM KEBARA, ISRAEL Comptes Rendus De L Academie Des Sciences Serie Ii. 1994 Sept , 319 (6):727-731.

[Abstract]

This study reports one aspect of the oral health displayed by a Middle Palaeolithic skeleton dated to ca. 60,000 years BP in Kebara cave (Israel). The excellent state of preservation of the Kebara 2 skeleton, due to the body's decomposition in a filled grave permits an original palaeobiological approach considering the time period. The preliminary results of the first scanning electron microscope study of dental calculus deposit are presented. Evidence of fossil bacteria from the Kebara 2 individual leads us to suggest that the role of diet might be secondary in importance to the individual immunological response to the same stimuli.
AIZENBERG J., Albeck S., Weiner S. & Addadi L. 1994. Crystal-protein interactions studied by overgrowth of calcite on biogenic skeletal elements Journal of Crystal Growth. 1994 Sept , 142 (1-2):156-164.

[Abstract]

A key parameter in the biological control of crystal formation is the interaction of a group of acidic macromolecules with the mineral phase. Here we study protein-calcite interactions using epitaxial overgrowth of synthetic calcite crystals under conditions in which local release of occluded macromolecules from the biogenic substrate occurs. The macromolecules subsequently interact with the newly formed overgrown crystals, resulting in modified calcite morphology. This novel method provides a means of mapping crystal-protein interactions under conditions that minimally affect the conformational states of the acidic macromolecules. We show that proteins released from calcitic sponge spicules and mollusc prisms specifically interact with {001} and {01l} faces of calcite, whereas proteins released from echinoderm skeletal elements only interact with {01l} faces. The extent to which the overgrown crystals are affected by the proteins varies even in the same organism and within the same element, depending on the site and crystallographic orientation of the skeletal elements.
Traub W., Arad T., Vetter U. & Weiner S. 1994. Ultrastructural studies of bones from patients with osteogenesis imperfecta Matrix Biology. 1994 Aug , 14 (4):337-345.

[Abstract]

Bone samples from patients suffering from osteogenesis imperfecta (OI) types I, II, III and IV, as well as normal controls, were studied by scanning (SEM) and transmission electron microscopy (TEM). SEM views of normal bone at low magnification show coherent structure, with regular striations due to a lamellar plywood like arrangement of the mineralized collagen fibrils. Compact lamellar bone was also found in various OI specimens, but in limited disconnected regions separated by open spaces. Furthermore, some OI, but not normal, bones have regions of loose unconnected fibers and others of apparently abnormally dense mineral deposition. High resolution TEM studies of OI bone fragments have served to elucidate the structures of these different textures. There appears to be a substantial, though reduced, proportion of normal lamellar bone even in quite severe OI. However, the regions of loose fibers are largely unmineralized and probably contain abnormal collagen. Other regions are overmineralized, with generally small unorganized apatite crystals deposited onto fibril surfaces or in separate clusters. These structural abnormalities, together with the paucity of normal bone, may explain the fragility of OI bones.
Wagner H. D. & Weiner S. 1994. Authors' response [On the relationship between the microstructure of bone and its mechanical stiffness] Journal of Biomechanics. 1994 Jul , 27 (7):995.

[Abstract]

Zioupos and Currey criticize our study in three different respects. The first point of contention arises from a misunderstanding of the text, which could probably have been more clearly written. We modelled lamellar bone that is folded into cylinders. This could be the midshaft region of the long bone of a small animal, mature Haversian bone, or that part of fibrolamellar (plexiform) bone that is cylindrical (the primary osteons). We did indeed compare our theoretical data to the experimental data of Bonfield and Grynpas (1977), who used bovine fibrolamellar bone. Zioupos and Currey are correct in pointing out that this is by no means all composed of ‘cylindrical’ bone. We compromised in this way in order to compare our data to that of others, who also attempted to model the same set of experimental data. We clearly stated, however, that the true test must ‘await more experimental measurements of the type performed by Bonfield and Grynpas (1977), but under conditions where the bone is carefully oriented in all three dimensions with respect to its structure’ (our study, p. 1318). [First paragraph]
Addadi L., AIZENBERG J., Albeck S., BERMAN A., Leiserowitz L. & Weiner S. 1994. Controlled occlusion of proteins: A tool for modulating the properties of skeletal elements Molecular Crystals and Liquid Crystals. 1994 Jun , 248 (1):185-198.

[Abstract]

Composite materials in which the organic host is stiffened by guest particles, are widely used in nature and are produced synthetically by man. Organisms also produce a different type of composite in which the host is a crystal and the guests are macromolecules occluded in an orderly fashion within the crystal. The best studied examples, to date, are biogenic calcite crystals, and in particular those formed by the echinoderms. In vitro experiments with calcite crystals grown in the presence of echinoderm intracrystalline proteins, show that these macromolecules are occluded inside the crystal on specific planes, and their presence alters the mechanical properties of the crystal host. Furthermore, the proteins also influence the crystal textural properties. Model studies using crystals of dicarboxylic acid salts grown in the presence of intracrystalline proteins show that the coherence length is reduced in directions perpendicular to the planes on which the proteins adsorb. We found anisotropic effects in almost all the biogenic calcite crystals we examined. Furthermore, we noted an interesting relationship between the variations in coherence length in the different crystallographic directions and the gross morphology of the single crystal elements, suggesting that these proteins may also function in determining the morphology of the crystal during growth. These novel single crystal-protein composites may be just one example of strategies used in nature for producing materials with special properties.
FUREDIMILHOFER H., MORADIANOLDAK J., Weiner S., VEIS A., MINTZ K. & Addadi L. 1994. Interactions of matrix proteins from mineralized tissues with octacalcium phosphate Connective Tissue Research. 1994 , 30 (4):251-264.

[Abstract]

Acidic matrix macromolecules, present in many mineralized tissues, including those of vertebrates, are thought to be involved in controlling crystal formation. Little, however, is known about their in vivo functions, particularly in relation to calcium-phosphate-containing crystals. The manner in which a variety of synthetic and natural acidic macromolecules interact in vitro with crystals of octacalcium phosphate (OCP) has been studied. Interactions were assessed by examining changes in morphology of the crystals resulting from preferential interaction of the additive with some crystal faces and not others. Macromolecules rich in acidic amino acids, with or without polysaccharides, such as polyaspartate and mollusk shell proteins respectively, were shown to interact preferentially with rows of Ca ions exposed on the hydrated plate surface of OCP crystals. In contrast, the phosphorylated proteins, phosphophoryn and phosvitin, interacted specifically with the apatite-like motifs on the OCP side faces. BSP did not interact specifically with OCP, under the experimental conditions used. The observation that these classes of acidic macromolecules recognize different crystal faces should be taken into account when evaluating functions of acidic matrix macromolecules in mineralized tissues.
BERMAN A., HANSON J., Leiserowitz L., KOETZLE T., Weiner S. & Addadi L. 1993. Biological control of crystal texture: A widespread strategy for adapting crystal properties to function Science. 1993 , 259 (5096):776-779.

[Abstract]

Textures of calcite crystals from a variety of mineralized tissues belonging to organisms from four phyla were examined with high-resolution synchrotron x-ray radiation. Significant differences in coherence length and angular spread were observed between taxonomic groups. Crystals from polycrystalline skeletal ensembles were more perfect than those that function as single-crystal elements. Different anisotropic effects on crystal texture were observed for sea urchin and mollusk calcite crystals, whereas none was found for the foraminifer, Patellina, and the control calcite crystals. These results show that the manipulation of crystal texture in different organisms is under biological control and that crystal textures in some tissues are adapted to function. A better understanding of this apparently widespread biological phenomenon may provide new insights for improving synthetic crystal-containing materials.
Weiner S., GOLDBERG P. & BARYOSEF O. 1993. BONE PRESERVATION IN KEBARA CAVE, ISRAEL USING ON-SITE FOURIER-TRANSFORM INFRARED SPECTROMETRY Journal of Archaeological Science. 1993 Nov , 20 (6):613-627.
Albeck S., AIZENBERG J., Addadi L. & Weiner S. 1993. Interactions of Various Skeletal Intracrystalline Components with Calcite Crystals Journal of the American Chemical Society. 1993 Dec , 115 (25):11691-11697.

[Abstract]

The calcitic skeletal elements of many organisms contain small amounts of proteins and glycoproteins which are incorporated within single crystals of calcite. Extraction and partial purification of the intracrystalline macromolecules allowed the identification of their interactions with synthetic calcite crystals. Specific interactions were inferred from induced morphological modifications associated with the development of new faces on calcite crystals grown in the presence of these macromolecules in solution. Partially purified macromolecules extracted from within sea urchin spines interacted only with faces roughly parallel to the c crystallographic axis of calcite, producing well-developed {01l} faces. Macromolecules extracted from within mollusk shell prisms separated into two fractions each having a characteristic amino acid composition and displayed distinct specific interactions with growing calcite crystals: A highly acidic fraction interacts with the {001} set of faces, while a different fraction, which is only moderately acidic and has an amino acid composition similar to that of the sea urchin spines, interacts specifically with the {01l} set of faces. Magnesium, a major ionic component of the sea urchin spines, causes the development of the {011} set of crystal faces. The existing knowledge on the direction of intercalation of the macromolecules in the biogenic crystals agrees with the interacting planes in the synthetic crystals, suggesting the relevance of our in vitro assay to the system in vivo. We suggest that each separate mode of interaction may infer a specific role in the regulation of biological crystal growth.
Geiger S. B. & Weiner S. 1993. Fluoridated carbonatoapatite in the intermediate layer between glass ionomer and dentin Dental Materials. 1993 Jan , 9 (1):33-36.

[Abstract]

The intermediate layer between a glass ionomer restoration and dentin was examined morphologically by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). SEM showed strong bonding between the dentin and glass ionomer and formation of an intermediate layer between them. FTIR spectra of successive scrapings through the ionomer/dentin interface zone showed that the intermediate layer is composed primarily of mineral fluoridated carbonatoapatite. The presence of this sparingly soluble mineral at the interface between the tooth and the restoration may provide high resistance to secondary caries and may, thus, be of great clinical importance.
BERMAN A., HANSON J., Leiserowitz L., KOETZLE T., Weiner S. & Addadi L. 1993. Crystal-protein interactions: Controlled anisotropic changes in crystal microtexture Journal of Physical Chemistry. 1993 , 97 (19):5162-5170.

[Abstract]

A unique set of proteins extracted from a variety of invertebrate calcitic mineralized tissues is able to selectively interact in vitro with certain crystal faces and not others. This was previously demonstrated by observing changes in morphology of crystals grown in the presence of proteins as compared to those grown in the absence of proteins. Following interaction, the proteins are overgrown by the crystal and are subsequently occluded within the crystal itself. Here we address the fundamental question of whether or not the proteins also alter the crystal texture in an anisotropic manner. For this purpose we used high-resolution synchrotron X-ray diffraction to monitor changes in coherence length and angular spread. We studied the interactions of proteins extracted from the mineralized skeletal hard parts of sea urchins and mollusks, with crystals of two calcium dicarboxylic acids, calcium fumarate and calcium malonate, as well as the polymorph of CaCO₃ calcite. For the calcium dicarboxylate crystals, we did demonstrate that the coherence lengths are reduced in the directions perpendicular to the planes onto which the proteins preferentially adsorb. In contrast the calcite crystals grown in the presence of the proteins exhibited an increase in angular spread compared to the controls, but no anisotropic effect in coherence length was detected. A biologically produced calcite crystal, on the other hand, showed a preferential reduction in coherence length in the direction of the c axis. Clearly in the case of calcite, the processes controlling crystal texture in the biological environment are more sophisticated than those in vitro. The detection of a reduction in coherence length in the directions perpendicular to the planes onto which the proteins preferentially adsorbed represents one of very few direct demonstrations that an additive that is able to selectively alter crystal morphology also affects crystal texture in an anisotropically specific manner. An understanding of this phenomenon may, in the future, improve our ability to control crystal texture in synthetic materials.
Weiner S. & Traub W. 1992. Bone structure: From ångstroms to microns FASEB Journal. 1992 Feb , 6 (3):879-885.

[Abstract]

Bone has a complex hierarchical structure, which despite much investigation, is still not well understood. Here we bring together pieces of this complicated puzzle, albeit from different sources, to present a tentative overview of bone structure. The basic building blocks are the extremely small plate-shaped crystals of carbonate apatite, just hundreds of ångstroms long and wide and some 20-30 Å thick. They are arranged in parallel layers within the collagenous framework. At the next hierarchical level these mineral-filled collagen fibrils are ordered into arrays in which the fibril axes and the crystal layers are all organized into a 3-dimensional structure that makes up a single layer or lamella of bone a few microns thick. The orientations of the collagen fibrils and the crystal layers in alternating lamellae of rat bone differ such that in the thinner lamellae, the fibrils and the crystal layers are parallel to the lamellar boundaries. In the thicker lamellae the fibrils are parallel to the boundary, but the crystal layers are rotated out of the plane of the boundary. In many bones these alternating lamellae are organized into even larger ordered structures to produce what is truly a remarkably ordered material, all the way from the molecular scale to the macroscopic product.
Addadi L. & Weiner S. 1992. Control and Design Principles in Biological Mineralization Angewandte Chemie-International Edition In English. 1992 Feb , 31 (2):153-169.

[Abstract]

The control of crystal formation has been developed to a remarkable degree by many organisms. Oriented nucleation, control over crystal morphology, formation of unique composites of proteins and single crystals, and the production of ordered multicrystal arrays, are all well within the realm of biological capability. Understanding the control and design principles in biomineralization is a fascinating subject that may well contribute to the improved fabrication of synthetic materials on the one hand, and to the solution of many serious pathological problems involving mineralization, on the other.
Wagner H. D. & Weiner S. 1992. On the relationship between the microstructure of bone and its mechanical stiffness Journal of Biomechanics. 1992 Nov , 25 (11):1311-1320.

[Abstract]

A recent study of bone structure shows that the plate-shaped carbonate apatite crystals in individual lamellae are arranged in layers across the lamellae, and that the orientation of these layers are different in alternate lamellae. Based on these findings, a new micromechanical model for the Young's modulus of bone is proposed, which accounts for the anisotropy and geometrical characteristics of the material. The model incorporates the platelet-like geometry of the basic reinforcing unit, the presence of alternating thin and thick lamellae, and the orientations of the crystal platelets in the lamellae. The thin and thick lamellae are modeled as orthotropic composite layers made up of thin rectangular apatite platelets within a collagen matrix, and classical orthotropic elasticity theory is used to calculate the Young's modulus of the lamellae. Bone is viewed as an assembly of such orthotropic lamellae bent into cylindrical structures, and having a constant, alternating angle between successive lamellae. The micromechanical model employs a modified rule-of-mixtures to account for the two types of lamellae. The model provides a curve similar to the published experimental data on the angular dependence of Young's modulus, including a local maximum at an angle between 0 and 90°. A rigorous testing of the model awaits additional experimental data.
MORADIANOLDAK J., FROLOW F., Addadi L. & Weiner S. 1992. Interactions between acidic matrix macromolecules and calcium phosphate ester crystals: Relevance to carbonate apatite formation in biomineralization Proceedings Of The Royal Society B-Biological Sciences. 1992 , 247 (1318):47-55.

[Abstract]

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Earlier studies on the interactions between acidic macromolecules and carboxylate- and carbonate-containing crystals showed that the proteins recognize a specific stereochemical motif on the interacting plane. Here we show that a similar stereochemical motif is recognized by acidic mollusc shell macromolecules interacting with four different organic calcium phosphate-containing crystals. In addition, an acidic protein from vertebrate tooth dentin was also observed to recognize a similar structural motif in one of the crystals. The characteristic motif recognized is composed of rows of calcium ions and phosphates arranged in a plane defined by two free oxygens and a phosphorous atom emerging perpendicular to the affected face. These observations may have a direct bearing on the manner in which control over crystal growth is exerted on carbonate apatite crystals commonly found in vertebrate tissues.
BARYOSEF O., VANDERMEERSCH B., ARENSBURG B., BELFERCOHEN A., GOLDBERG P., LAVILLE H., MEIGNEN L., RAK Y., SPETH J., TCHERNOV E., TILLIER A. & Weiner S. 1992. THE EXCAVATIONS IN KEBARA CAVE, MT CARMEL Current Anthropology. 1992 Dec , 33 (5):497-550.

[Abstract]

This paper summarizes the results of recent excavations (1982-90) at the Middle Paleolithic site of Kebara Cave. Work at the cave by earlier excavators is also discussed. Although analysis of the Kebara materials is still far from completed, an overview of the current stage of the investigations includes (1) a synthesis of the site's complex stratigraphy and dating; (2) a description of the spatial patterning of hearths, ash lenses, and bone and artifact concentrations; (3) results of in situ mineralogical studies of cave sediments to determine whether the observed spatial distribution of fossil animal bones is an accurate reflection of past human and/or scavenger activities in the cave or an artifact of differential postdepositional bone loss through groundwater dissolution; (4) a synthesis of the Mousterian stone tool assemblages focusing on.the technology of tool production as reflected in chaines operatoires (a brief summary of the site's Upper Paleolithic assemblages is also provided); (5) an in-depth taphonomic
Traub W., Arad T. & Weiner S. 1992. GROWTH OF MINERAL CRYSTALS IN TURKEY TENDON COLLAGEN-FIBERS Connective Tissue Research. 1992 , 28 (2-Jan):99-111.
Traub W., Arad T. & Weiner S. 1992. ORIGIN OF MINERAL CRYSTAL-GROWTH IN COLLAGEN FIBRILS Matrix. 1992 Aug , 12 (4):251-255.

[Abstract]

Collagen fibrils from young turkey-leg tendons, just beginning to mineralize, were stained with uranyl acetate and examined by electron microscopy. Small needle-like mineral crystals were observed and located, in relation to the collagen banding pattern, as originating at the e band in the gap region and near the surface of the fibrils. These are evidently the sites of crystal nucleation. They lie near binding locations on collagen fibrils of two glycosylated proteins believed to be implicated in the mineralization process, as well as the sites of early crystals in embryonic fowl bones.
Lowenstam H. A. & Weiner S. 1992. Phosphatic shell plate of the barnacle Ibla (Cirripedia): A bone-like structure Proceedings of the National Academy of Sciences of the United States of America. 1992 , 89 (22):10573-10577.

[Abstract]

The carbonate apatite crystals and the segmented structure of the shell plates of the barnacles in the genus Ibla distinguish them from the shell plates of all other barnacles, which are coherent calcitic structures. A detailed study of the hierarchical organization of one of the two shell plate types, the tergum, reveals a remarkably complex structure. Cylinders composed of a chitin-protein complex and nodes of plate-shaped crystals constitute the basic building blocks. The crystals are organized into layered stacks in which the c crystallographic axes are all aligned perpendicular to a 25-nm banded structure. The cylinders are, in turn, ordered in arrays such that parts of each cylinder are aligned in a plane, and parts form arcuate out-of-plane structures. The overall result is a lamellar structure, with a plywood-like motif, that is present throughout an individual segment. A series of segments forms an interlocking mineralized core, which is enclosed within a thick organic envelope. The flexible and complex skeletal structure of the tergum shows some marked similarities to the structure of lamellar bone. Although this is undoubtably a product of convergent evolution, the iblid tergum provides a unique perspective on bone structure, which was heretofore unavailable.
Weiner S. & Addadi L. 1991. Acidic macromolecules of mineralized tissues: The controllers of crystal formation Trends in Biochemical Sciences. 1991 , 16 (C):252-256.

[Abstract]

The deposition of minerals within many biological tissues is a controlled process. Among the most active agents that control biological mineralization are a group of unusually acidic proteins and glycoproteins. These can interact specifically with some crystal faces but not others, induce oriented nucleation, or intercalate in a regular manner into the crystal lattice itself.
Landis W. J., Moradian-Oldak J. & Weiner S. 1991. Topographic imaging of mineral and collagen in the calcifying Turkey tendon Connective Tissue Research. 1991 , 25 (3-4):181-196.

[Abstract]

Topographic imaging, a method of providing a direct view of ultrastructure in three dimensions, has been newly applied to a study of mineral crystals and collagen from calcifying turkey leg tendon. Individual crystals obtained from intact tendon were observed as thin platelets of irregular shape having a relatively smooth surface. Mineralized collagen fibrils isolated singly or examined in thin tissue sections were found to exhibit the characteristic 64-70 nm period and were associated with platelets and needle-like mineral. The crystals were disposed in numerous fashions, notably as small groups of platelets within individual collagen hole zones, as a number of needle-like densities arranged parallel to one another, or as a combination of platelets and needles over entire stretches of single collagen fibrils. The topographic observations of crystals and crystal-collagen interaction clearly demonstrate the plate-like habit of the mineral in calcifying turkey tendon and suggest that these crystals are located both within and on the surface of collagen fibrils. In certain sites such as the collagen hole zones, the crystals appear organized in a specific manner, possibly with a preferred c-axial orientation. Crystals of hydroxyapatite prepared in vitro and examined topographically are similar in habit and texture to the crystals from tendon. When interpretation of this method is corroborated by other independent microscopic techniques, topographic imaging has widespread potential application in many fields of study in which structural surface features of biological tissues or non-biological materials are of interest at the electron microscope level.
Addadi L., Moradian-Oldak J. & Weiner S. 1991. Macromolecule-Crystal Recognition in Biomineralization: Studies Using Synthetic Polycarboxylate Analogs . 1991 , 444 :13-27.

[Abstract]

Control over crystal formation in biology is determined, to a great extent, by the specific recognition of a variety of acidic glycoproteins for crystal surfaces. Understanding the manner in which this occurs can be facilitated by growing crystals in the presence of simple synthetic analogs to these proteins, such as polyaspartic and polyglutamic acids, which can be either in solution or adsorbed on a solid substrate. These experiments demonstrate that the polymers are much more effective at influencing crystal nucleation and growth than the monomers, that the beta-sheet conformation is essential for obtaining specific interactions, that rigid substrates are much better nucleators than fluid-like substrates, and that the different ligands associated with the macromolecules can cooperate to induce crystal nucleation or modulation.
Moradian-Oldak J., Weiner S., Addadi L., Landis W. J. & Traub W. 1991. Electron imaging and diffraction study of individual crystals of bone, mineralized tendon and synthetic carbonate apatite Connective Tissue Research. 1991 , 25 (3-4):219-228.

[Abstract]

A transmission electron microscope study of carbonate apatite crystals isolated from bone and mineralizing tendon, as well as those produced synthetically under approximated-physiological conditions, shows that they are thin irregular shaped plates. Electron diffraction patterns of individual crystals confirm that the large developed crystal face is (100), and that the longest dimensions of the biogenic crystals are aligned with the crystallographic c axes. As the latter are also aligned with collagen fibril axes, the observations provide additional information on the tissue organization itself. The marked similarity between the biologic and synthetic crystals suggests that the biological environment in which the crystals form may not be primarily responsible for controlling their shape.
Weiner S., Arad T. & Traub W. 1991. Crystal organization in rat bone lamellae FEBS Letters. 1991 Jul , 285 (1):49-54.

[Abstract]

The plate-shaped crystals of rat bone are arranged in parallel layers that form coherent structures up to the level of individual lamellae. The crystal layers of the thin lamellae are parallel to the lamellar boundary, whereas those of the thicker lamellae are oblique to the boundary. The basic structure of rat bone can be described as 'rotated plywood'; a structure hitherto unrecognized in either biologic or synthetic materials.
SABANAY I., Arad T., Weiner S. & Geiger B. 1991. Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron microscopy Journal of Cell Science. 1991 Sept , 100 (1):227-236.

[Abstract]

We describe the development and application of a novel approach to high-resolution ultrastructural analysis of cells and tissues. It is based on the preparation of ultrathin frozen sections of fixed tissues, rinsing of the sections, followed by their embedding on the grid in a layer of vitrified ice, and direct observation with a cryoelectron microscope. Examination of smooth muscle, kidney and heart tissues showed that although no heavy metal staining was used, high-contrast images are obtained. Fine details of cytoplasmic filaments and organelles, membranes and membrane-associated structures, as well as connective-tissue elements are all visible. The new method is suitable for immunolabeling, including high resolution localization of specific molecules within the cytoplasm.
Elster H., Gil-Av E. & Weiner S. 1991. Amino acid racemization of fossil bone Journal of Archaeological Science. 1991 Sept , 18 (5):605-617.

[Abstract]

A study of aspartic acid racemization in different fractions of 51 fossil bones of various ages, shows that the collagen-rich fractions do manifest increasing D-aspartic acid contents with increasing age. No such correlation exists for the fractions rich in non-collagenous proteins. More collagen-rich samples, particularly from bones in older sites, are needed to verify the generality of this trend.
BERMAN A., Addadi L., KVICK A., Leiserowitz L., NELSON M. & Weiner S. 1990. Intercalation of sea urchin proteins in calcite: Study of a crystalline composite material Science. 1990 Nov , 250 (4981):664-667.

[Abstract]

Sea urchin skeletal elements are composed of single crystals of calcite. Unlike their synthetic counterparts, these crystals do not have well-developed cleavage and are consequently much more resistant to fracture. This phenomenon is due in part to the presence of acidic glycoproteins occluded within the crystals. By means of x-ray diffraction with synchrotron radiation, it is shown that the presence of the protein in synthetic calcite only slightly decreases the coherence length but significantly increases the angular spread of perfect domains of the crystals. In biogenic calcite, the coherence length is 1/3 to 1/4 as much as that in synthetic calcite and the angular spread is 20 to 50 times as wide. It is proposed that the presence of macromolecules concentrated at mosaic boundaries that are oblique to cleavage planes is responsible for the change in fracture properties. These results may be important in the material sciences, because of the unusual nature of this material, namely, a composite based on the controlled intercalation of macromolecules inside single-crystal lattices.
Heywood B. R., Sparks N. H., Shellis R. P., Weiner S. & Mann S. 1990. Ultrastructure, morphology and crystal growth of biogenic and synthetic apatites Connective Tissue Research. 1990 , 25 (2):103-119.

[Abstract]

The morphology, structure and crystal growth of apatite crystals isolated from calcified turkey tendon and synthetic carbonated apatites have been examined using high resolution transmission electron microscopy. The biogenic apatite consisted of small (35 × 20 × 5 nm) platelike crystals. Despite their irregular shape and ill-defined edges, individual particles were single domain crystals. Lattice images recorded from isolated turkey tendon crystals indicated that the crystallographic oaxis (0001) of apatite lies in the plane of the pjate and parallel to the length of the crystallites. Lattice images suggested that the top face corresponds to the (1100) face of carbonated apatite. Lattice fringes observed in platelike crystallites viewed from the side corresponded to the projection of the apatite structure viewed along the [1120] direction. Thus, it can be argued that crystal growth is constrained along the [1100] direction, extends laterally along the [1120] direction, and is maximal along the [0001] direction. This latter direction is aligned with the collagen fiber axis. A mean length to width ratio (1.7) was determined by systematically measuring the maximum distances parallel and perpendicular to the c-axis identified from lattice images of the crystals. Similar information was obtained from lattice images of crystals located in collagen fibres. This confirmed that the morphological and structural features of isolated turkey tendon apatite crystals correlate directly with the in vivo crystallochemical characteristics of apatite. Crystals of synthetic carbonated apatite prepared at 37°C were also platelike and, although generally much larger, had length to width ratios comparable with the turkey tendon apatite. The synthetic carbonated apatites were noticeably more sensitive to radiolytic damage than the turkey tendon crystals. The crystallographic c-axis of the inorganic particles was aligned parallel with the long, physical axis of the plate and the top face was identified as (1100). Similar data were also obtained from non-carbonated synthetic apatite samples. The results of the present study offer critical information about the crystal growth of individual carbonated apatite crystals in calified turkey tendon and its relationship to the morphology of the crystallites. As similar growth characteristics are expressed in synthetic analogues, the data bring into question the putative regulatory role of the collagen-based matrix upon the nucleation and growth of biogenic apatite. It is suggested that the crystallographic co-alignment of the crystals and collagen fibers may be indicative of interactions between the organic matrix and the nascent crystal lattice during nucleation while the later growth of the crystals is regulated only by the imposition of spatial constraints rather than through specific matrix-crystal interactions.
Weiner S. & BARYOSEF O. 1990. States of preservation of bones from prehistoric sites in the Near East: A survey Journal of Archaeological Science. 1990 Mar , 17 (2):187-196.

[Abstract]

A survey of the states of preservation of organic material in 30 fossil bones from 16 different prehistoric sites in the Near East shows that whereas almost all the bones have little or no collagen preserved, they do, with few exceptions, contain non-collagenous proteins. These macromolecules, therefore, represent an important reservoir of indigenous fossil bone constituents.
Addadi L., BERMAN A., MORADIANOLDAK J. & Weiner S. 1990. TUNING OF CRYSTAL NUCLEATION AND GROWTH BY PROTEINS - MOLECULAR-INTERACTIONS AT SOLID-LIQUID INTERFACES IN BIOMINERALIZATION Croatica Chemica Acta. 1990 Dec , 63 (3):539-544.

[Abstract]

The mineralized tissues of a bivalve mollusk and a sea urchin are both composed of calcium carbonate crystals that are intimately associated with acidic glycoproteins. In vitro studies in which carboxylate-, carbonate- and phosphate-containing crystals are grown in the presence of partially purified acidic glycoproteins from these two tissues show that some of these macro-molecules are able to interact specifically with certain crystal faces. Significantly all the affected crystal faces contain a common stereochemical motif. Interesting differences, however, were observed in the modes of interaction between the mollusk and sea urchin derived acidic glycoproteins. Only the former can induce oriented calcite nucleation in vitro and only the latter can interact from solution with specific calcite crystal faces. These differences are ascribed in part to the fact that the mollusk macromolecules are much more acidic than those from the sea urchin. Some of the acidic glycoproteins are also occluded inside the growing crystals. In the case of the sea urchin, and not of the mollusk, the proteins are preferentially located at specific crystal planes and their presence influences the mechanical properties of the crystal. A detailed study of these composite crystals by X-ray synchrotron radiation shows how the presence of the protein influences the crystal mosaicity. The interactions revealed by these studies follow well defined stereochemical rules, tuned by electrostatic forces. They, in turn, provide new insight into some of the basic underlying processes occurring in biomineralization.
Traub W., Arad T. & Weiner S. 1989. Three-dimensional ordered distribution of crystals in turkey tendon collagen fibers Proceedings of the National Academy of Sciences of the United States of America. 1989 , 86 (24):9822-9826.

[Abstract]

The organization of apatite crystals and collagen fibrils in mineralized turkey tendon has been studied by electron microscopy and electron diffraction. To minimize artifactual distortions the tissue was examined, for the first time, as isolated fibrils in an aqueous environment of vitreous ice, as well as in conventionally prepared sections. The electron micrographs show that the plate-shaped apatite crystals are arranged in parallel arrays across the collagen fibrils. This provides direct evidence for highly asymmetric assembly in collagen fibrils, and, indeed, the fibrils were observed to be elongated rather than round in cross-section. There is, furthermore, a pronounced tendency for the layers of crystals to be coherently aligned in adjacent fibrils. These observations may also be important for understanding the mechanical behavior of bone at the molecular level, as such extended, aligned aggregates of flat crystals could develop into natural fracture planes in mature bone.
Weiner S. & Traub W. 1989. Crystal size and organization in bone Connective Tissue Research. 1989 , 21 (1-4):259-265.

[Abstract]

Size measurements of dispersed rat bone crystals show that with age a greater proportion exceeds 400 A in length. The surface fractures of more heavily mineralized bones tend to be smooth and stepped, whereas those of less mineralized bones are fibrous. These observations combined with information reported elsewhere on the crystal-collagen relations in adjacent fibrils in turkey tendon suggest that some crystals grow out of the confines of the collagen gap regions to form extended aggregates of large flat crystals with well developed fracture planes.
Addadi L., BERMAN A., OLDAK J. & Weiner S. 1989. Structural and stereochemical relations between acidic macromolecules of organic matrices and crystals Connective Tissue Research. 1989 , 21 (1-4):127-135.

[Abstract]

Detailed studies of the molecular organization of various mineralized tissues highlight the role played by acidic glycoproteins in controlling crystal formation. In vitro experiments show that these macromolecules are able to interact with specific faces of different crystals, influencing both nucleation and crystal growth. Significantly a comman stereochemical motif can be recognized in all the interacting faces studies to date.
WUTHIER R., BOSKEY A., RODAN G., GLIMCHER M., VEIS A., NANCI A., YESINOWSKI J., EVANS J., DONNELLY R., WARSHAWSKY H., GRYNPAS M., FISHER L. & Weiner S. 1989. THE CHEMISTRY AND BIOLOGY OF MINERALIZED TISSUES - PROCEEDINGS OF THE 3RD INTERNATIONAL-CONFERENCE ON THE CHEMISTRY AND BIOLOGY OF MINERALIZED TISSUES - HELD IN CHATHAM, MASSACHUSETTS ON OCTOBER 16-21, 1988 - GENERAL DISCUSSION Connective Tissue Research. 1989 , 21 (4-Jan):613-626.
DeNiro M. J. & Weiner S. 1988. Chemical, enzymatic and spectroscopic characterization of "collagen" and other organic fractions from prehistoric bones Geochimica et Cosmochimica Acta. 1988 Sept , 52 (9):2197-2206.

[Abstract]

Three organic fractions from 9 modern bones were prepared: collagen, the HC1 insoluble fraction, and the HC1 soluble fraction. The fraction that has the same solubility characteristics as collagen (referred to herein as "collagen") and the HC1 soluble and insoluble fractions from 44 prehistoric bones were also prepared. Of these 44,17 had "collagen" with δ¹³C and δ¹⁵N values and C N ratios within the ranges displayed by collagen from modern animals that ate the same types of food, whereas "collagen" in the remaining samples displayed clear evidence of diagenetic isotopic and/ or elemental alteration based on comparisons with modern collagen. The bones were characterized in terms of the amino acid compositions and infrared spectra of the three organic fractions, and the amino acid compositions of the low molecular weight products of the reaction between collagenase and "collagen." For all three analytical procedures, prehistoric samples whose "collagen" had not suffered diagenetic isotopic and/or elemental alteration produced results similar to those obtained from modern samples, whereas altered prehistoric samples did not. Application of the chemical, enzymatic and spectroscopic methods presented here should permit unequivocable identification of prehistoric samples that have suffered postmortem alteration of their "collagen" isotopic compositions, including those that have undergone more subtle isotopic shifts than can be identified by changes in the "collagen" C N ratio, the only criterion applied to date for identifying altered samples. Some of the techniques we used to identify altered samples can be applied to fractions more readily prepared than "collagen", thereby facilitating screening of large numbers of samples in order to eliminate those unsuitable for isotopic paleodietary analysis. The same criteria we have established to identify "collagen" samples that have not suffered postmortem stable isotopic alteration could be applied to characterize samples prepared for radiocarbon analysis, leading to more accurate dating of bone.
BERMAN A., Addadi L. & Weiner S. 1988. Interactions of sea-urchin skeleton macromolecules with growing calcite crystals- a study of intracrystalline proteins Nature. 1988 , 331 (6156):546-548.

[Abstract]

The exoskeletons of sea urchins are composed of magnesiumbearing calcite. Individual test plates and spines behave as single crystals in polarized light or when examined by X-ray diffraction^1-3. They do not, however, cleave like inorganic calcite crystals along the {104} hexagonal cleavage planes, but have conchoidal fracture surfaces reminiscent of amorphous glass. Discussion of this paradox revolves around whether the phase is monocrystalline^2,4,5, multicrystalline⁶, or some combination thereof⁷, but provides no explanation for the phenomenon. To address this question we grew crystals of calcite in the presence of acidic glycoproteins extracted from within the mineralized hard parts of sea-urchin tests^8-10. As a control we used analogous proteins from the calcitic layer of a mollusc shell which are known to be nucleators of calcite when adsorbed on a rigid substrate, but inhibitors when in solution^11-13. We show that the sea urchin, but not the mollusc macromolecules selectively adsorb onto specific calcite crystal planes and with continued crystal growth are occluded inside the solid phase. These synthetic crystals fracture with a conchoidal cleavage similar to that observed in sea-urchin calcite. Thus intracrystalline proteins may be responsible for this phenomenon in biology and the manner in which they affect the mechanical properties of the crystals may also have interesting implications to the materials sciences.
WILLIAMS R., Baron R., RAISZ L., OSDOBY P., KRANE S., PROCKOP D., TERMINE J., VEIS A., Weiner S., HAUSCHKA P., HANAOKA H., CHAMBERS T., CAPLAN A., TESTA N. & URIST M. 1988. OSTEOCLAST ACTIVITY Ciba Foundation Symposia. 1988 , 136 :125-130.
Deniro M. J. & Weiner S. 1988. Organic matter within crystalline aggregates of hydroxyapatite: A new substrate for stable isotopic and possibly other biogeochemical analyses of bone Geochimica et Cosmochimica Acta. 1988 Oct , 52 (10):2415-2423.

[Abstract]

The portion of organic matrix in bones that is present within fused aggregates of hydroxyapatite crystals was isolated by oxidizing the rest of the bone organic matter with sodium hypochlorite. The aggregate organic matter from a variety of modern and prehistoric bones was subjected to elemental and stable C and N isotopic analysis. For comparison, collagen from modern bones and the fraction from prehistoric bones with the same solubility characteristics as collagen (referred to herein as "collagen") were subjected to the same analytical procedures. Collagen and aggregate organic matter in modern bones have similar δ¹⁵N values but dissimilar δ¹³C values. The difference may be caused by the presence of non-collagenous proteins (NCPs) in the aggregate organic matter, because the NCPs have δ¹³C values different from those of collagen from the same bone. The organic matter in aggregates is not subject to the same diagenetic processes that can alter the isotope ratios of collagen, and appears to retain an in vivo isotope signal even in cases in which that of "collagen" has been altered. These conclusions apply to samples that were burned prehistorically as well as to those that suffered postmortem alteration only in the depositional environment. The organic matter in aggregates represents a new substrate that should prove useful for stable isotopic studies and possibly for radiocarbon and other biogeochemical analyses of bone.
FLEISCH H., NIJWEIDE P., RUCH J., GLOWACKI J., OWEN M., TERMINE J., SLAVKIN H., HAUSCHKA P., OSDOBY P., Baron R., WILLIAMS R., TESTA N., Weiner S., VIES A., GLIMCHER M., GAZI D., CAPLAN A. & PROCKOP D. 1988. FUTURE PERSPECTIVES Ciba Foundation Symposia. 1988 , 136 :288-296.
DENIRO M. & Weiner S. 1988. USE OF COLLAGENASE TO PURIFY COLLAGEN FROM PREHISTORIC BONES FOR STABLE ISOTOPIC ANALYSIS Geochimica et Cosmochimica Acta. 1988 Oct , 52 (10):2425-2431.
Constantz B. & Weiner S. 1988. Acidic macromolecules associated with the mineral phase of scleractinian coral skeletons Journal of Experimental Zoology. 1988 Dec , 248 (3):253-258.

[Abstract]

Macromolecules intimately associated with the mineral phase of two species of scleractinian coral skeletons are extracted in different ways and partially characterized in some detail for the first time. They are shown to contain an assemblage of glycoproteins in which the protein moiety is rich in acidic amino acids and the carbohydrate moiety contains sulfated polysaccharides. In the presence of calcium, the proteins partially adopt the β‐sheet conformation. The properties of these macromolecules are all consistent with their fulfilling some as yet unidentified roles in controlling mineralization. Furthermore, their properties are similar in many respects to macromolecules present in the mineralized tissues of diverse organisms.
JODAIKIN A., Weiner S., TALMON Y., GROSSMAN E. & Traub W. 1988. Mineral-organic-matrix relations in tooth enamel International Journal of Biological Macromolecules. 1988 Dec , 10 (6):349-352.

[Abstract]

X-ray and electron diffraction patterns show that β-pleated-sheet polypeptide chains are predominantly oriented approximately perpendicular to the c-axes of developing enamel apatite crystallites. This spatial relation suggests a specific role for enamelins in controlling crystal growth.
Addadi L., MORADIAN J., Shay E., MAROUDAS N. & Weiner S. 1987. A CHEMICAL-MODEL FOR THE COOPERATION OF SULFATES AND CARBOXYLATES IN CALCITE CRYSTAL NUCLEATION - RELEVANCE TO BIOMINERALIZATION Proceedings of the National Academy of Sciences of the United States of America. 1987 May , 84 (9):2732-2736.
Jodaikin A., Weiner S., Perl-Treves D., Traub W. & Termine J. D. 1987. Developing enamel matrix proteins: a conformation study of enamelins and amelogenins International Journal of Biological Macromolecules. 1987 Jun , 9 (3):166-168.

[Abstract]

Conformations of the organic matrix proteins in rat and bovine enamel were examined using X-ray diffraction and fourier transform infrared spectroscopy. These were compared with the extracted and purified proteins. The acidic enamelins, both in situ and in the purified form, are in the β-sheet conformation. The hydrophobic amelogenins, on the other hand, do not show any identifiable regular conformations in situ or when purified.
Weiner S. & Traub W. 1986. Organization of hydroxyapatite crystals within collagen fibrils FEBS Letters. 1986 Oct , 206 (2):262-266.

[Abstract]

Transmission electron micrographs of individual mineralized collagen fibrils show that hydroxyapatite crystals are located mainly within the fibrils at the level of the gap regions. The plate-shaped crystals are observed to be more or less uniformly stacked across the fibril diameter. We therefore suggest that the crystals are primarily located in 'grooves' created by contiguous adjacent gaps. The proposal is consistent with the observed crystal distribution in the fibril and with their average widths, which are almost 10-times greater than an individual gap diameter.
Weiner S. & Lowenstam H. 1986. Organization of extracellularly mineralized tissues: A comparative study of biological crystal growt Critical Reviews in Biochemistry and Molecular Biology. 1986 , 20 (4):365-408.
Worms D. & Weiner S. 1986. Mollusk shell organic matrix: Fourier transform infrared study of the acidic macromolecules Journal of Experimental Zoology. 1986 Jan , 237 (1):11-20.

[Abstract]

Ion‐binding ligands and the conformations of some of the acidic matrix macromolecules present in the shells of a gastroped, a bivalve, and a cephalopod, are investigated in vitro by Fourier transform infrared spectroscopy. The complex assemblage of matrix constituents present in the EDTA‐soluble fraction is first separated into two quite different classes of macromolecules by a reversed‐phase chromatographic procedure. Infrared spectra indicate that the constituents of one class, which are proteins rich in aspartic acid, adopt the β‐sheet conformation upon binding calcium to the protein carboxylate groups. The second class of matrix constituents contains proteins rich in serine that appear to be associated with relatively large amounts of polysaccharide. They also bind calcium and upon doing so undergo a conformational change.
Jodaikin A., Traub W. & Weiner S. 1986. Protein conformation in rat tooth enamel Archives of Oral Biology. 1986 , 31 (10):685-689.

[Abstract]

Orientated 0.47 nm reflections in X-ray diffraction patterns of developing rat tooth enamel were confirmed using a wide range of specimen preparations and experimental conditions. This indicates that some of the organic matrix proteins adopt a β-sheet conformation.
Weiner S., Kustanovich Z., Gil-Av E. & Traub W. 1986. State of preservation of the Dead Sea Scrolls (reply) Nature. 1986 , 321 (6066):121-122.
Addadi L. & Weiner S. 1986. INTERACTIONS BETWEEN ACIDIC MACROMOLECULES AND STRUCTURED CRYSTAL-SURFACES - STEREOCHEMISTRY AND BIOMINERALIZATION Molecular Crystals And Liquid Crystals. 1986 , 134 (4-Jan):305-322.
Addadi L. & Weiner S. 1985. Interactions between acidic proteins and crystals: Stereochemical requirements in biomineralization Proceedings of the National Academy of Sciences of the United States of America. 1985 , 82 (12):4110-4114.

[Abstract]

Acidic matrix macromolecules are intimately involved in biological crystal growth. In vitro experiments, in which crystals of calcium dicarboxylate salts were grown in the presence of aspartic acid-rich proteins, revealed a stereochemical property common to all the interacting faces. Calcite crystals are nucleated on stereochemically analogous faces when proteins are adsorbed onto a rigid substrate. The importance of this property in biomineralization is discussed.
LOWENSTAM H. & Weiner S. 1985. TRANSFORMATION OF AMORPHOUS CALCIUM-PHOSPHATE TO CRYSTALLINE DAHLLITE IN THE RADULAR TEETH OF CHITONS Science. 1985 , 227 (4682):51-53.
Weiner S. 1985. Organic matrixlike macromolecules associated with the mineral phase of sea urchin skeletal plates and teeth Journal of Experimental Zoology. 1985 Apr , 234 (1):7-15.

[Abstract]

The skeletal plates and teeth of the echinoid Paracentrotus lividus contain a heterogeneous assemblage of macromolecules that are not part of the connective tissue, but are presumably intimately associated with the mineral phase. Upon dissolution of the Mg‐calcite mineral phase, some of these molecules are insoluble. The insoluble fractions of the teeth and skeletal plates are quite different, the former being predominantly protein and the latter, primarily some unknown nonproteinaceous material. The soluble constituents are similar in both tissues. These hydrophilic macromolecules have been partially separated and characterized. In both hard parts, two distinct classes of macromolecules are present, as indicated by the amino acid compositions of their protein constituents. These two classes of macromolecules are also present in the shells of a foraminifer and in various mollusks, both of which are formed by the “organic matrix‐mediated” biomineralization process. The locations of these macromolecules in the teeth and skeletal plates are not known, nor whether they form coherent structures. It is therefore premature to conclude that these macromolecules do function as an organic matrix, although the results presented are in agreement with such an interpretation.
Weiner S. 1984. Organization of organic matrix components in mineralized tissues Integrative and Comparative Biology. 1984 , 24 (4):945-951.

[Abstract]

The organic matrix is thought to play an important role in controlling crystal growth during the formation of skeletal hard parts. The structural organization of the matrix macromolecular constituents can provide a key to understanding the nature of the control processes. Although the data are limited, both vertebrate and invertebrate organic matrices appear to be organized according to the same "basic motif," namely a core of relatively hydrophobic structural macromolecules (usually proteins) and surface layers of acidic proteins and polysaccharides. Analyses of the latter from different invertebrate phyla using reversed phase high performance liquid chromatography, reveal that the same two classes of macromolecules are present in each of the three cases studied, emphasizing the fundamental importance of these components in crystal growth. Substantial information, at the molecular level, on the conformations and orientations of matrix constituents in relation to the mineral crystal lattice, is available only for mollusk shells, and to some extent on vertebrate tooth enamel. In these cases the major matrix constituents are aligned with one or more mineral crystallographic axes. These observations suggest that the matrix performs active, specific roles in crystal growth. Although it is still premature to assess the importance of various basic crystal growth mechanisms, the data available do not preclude the possibility that epitaxial crystal growth is an important factor.
LOWENSTAM H., Traub W. & Weiner S. 1984. NAUTILUS HARD PARTS - A STUDY OF THE MINERAL AND ORGANIC CONSTITUTENTS Paleobiology. 1984 , 10 (2):268-279.
Jodaikin A., Weiner S. & Traub W. 1984. Enamel rod relations in the developing rat incisor Journal of Structural Biology. 1984 Dec , 89 (3):324-332.

[Abstract]

Scanning electron microscopic and X-ray diffraction studies have shown that mandibular rat incisor teeth have two sets of rods which decussate at angles between 60 and 80° in both the most immature zone and the zone just beyond the opaque margin. A less well oriented interrod enamel component was found at right angles to both sets of rods. The information provides additional views of this complex tissue. Furthermore, it has been shown that the Wistar rat incisal enamel ultrastructure facilitates the use of X-ray diffraction to determine molecular relations between the crystals and matrix constituents as the rods are not all at 90° to one another.
Weiner S. & Traub W. 1984. MACROMOLECULES IN MOLLUSK SHELLS AND THEIR FUNCTIONS IN BIOMINERALIZATION PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES. 1984 , 304 (1121):425-&.
Weiner S. & EREZ J. 1984. Organic matrix of the shell of the foraminifer, Heterostegina depressa. Journal of Foraminiferal Research. 1984 , 14 (3):206-212.

[Abstract]

The organic matrix of the benthonic foraminifer, Heterostegina depressa is composed of a heterogeneous assemblage of proteins and polysaccharides. The fraction which is insoluble after decalcification using EDTA is composed almost entirely of over-sulfated glycosaminoglycans. Protein comprises about 0.5% by weight of this fraction. The EDTA-soluble matrix constituents can be separated into different fractions using reversed phase chromatography. Two major classes of matrix macromolecules are present: one containing mainly proteins rich in aspartic acid and the other proteins rich in serine and glycine, possibly linked to polysaccharides. Many of the biochemical properties of the numerous matrix macromolecules present are similar to those of organic matrices from other phyla. -Authors
Weinstein S. & Weiner S. 1984. Enantiomeric analysis of a mixture of the common protein amino acids as their Dns derivatives. Single-analysis reversed-phase high-performance liquid chromatographic procedure using a chiral mobile phase additive Journal of Chromatography A. 1984 , 303 (C):244-250.
Weiner S. 1983. Mollusk Shell Formation: Isolation of Two Organic Matrix Proteins Associated with Calcite Deposition in the Bivalve Mytilus californianus Biochemistry. 1983 , 22 (17):4139-4145.

[Abstract]

The role of the organic matrix during shell formation is investigated by comparing the soluble protein constituents in two shell layers of the bivalve Mytilus californianus. The two shell layers differ primarily with respect to ultrastructure and mineralogy. The proteins are separated by using ion-exchange chromatography followed by high-performance liquid chromatography. They are further characterized by an analysis of their mild acid hydrolysis cleavage products which reveals information on aspartic acid containing amino acid sequences present in the proteins. Of the approximately 40 matrix constituents separated, only two proteins present in the outer prismatic (calcite) layer contain the amino acid sequence -(Asp-Pro-Thr-Asp)-. These proteins, which have been purified to homogeneity, may in part be responsible for determining the particular type of calcium carbonate polymorph deposited in the outer shell layer.
Weiner S., Talmon Y. & Traub W. 1983. Electron diffraction of mollusc shell organic matrices and their relationship to the mineral phase International Journal of Biological Macromolecules. 1983 Dec , 5 (6):325-328.

[Abstract]

Electron diffraction patterns showing orientation of the chitin and protein constituents of the insoluble organic matrix of mollusc shell nacreous layers have been obtained, using low dose conditions and samples cooled to -100°C. Diffraction patterns of the aragonite crystals were also observed. In a gastropod and a bivalve the spatial relationship between the organic matrix constituents and the aragonite crystallographic axes were shown to be the same as was previously observed for a cephalopod using X-ray diffraction, supporting the notion that mineral crystal growth occurs epitaxially upon a matrix template.
Weiner S. 1982. Separation of acidic proteins from mineralized tissues by reversed-phase high-performance liquid chromatography Journal of Chromatography A. 1982 Aug , 245 (1):148-154.
BUCHARDT B. & Weiner S. 1981. DIAGENESIS OF ARAGONITE FROM UPPER CRETACEOUS AMMONITES - A GEOCHEMICAL CASE-STUDY Sedimentology. 1981 , 28 (3):423-438.
Weiner S. & Tishbee A. 1981. Separation of Dns-amino acids using reversed-phase high-performance liquid chromatography: a sensitive method for determining N-termini of peptides and proteins Journal of Chromatography A. 1981 Sept , 213 (3):501-506.
Weiner S. & Traub W. 1980. X-ray diffraction study of the insoluble organic matrix of mollusk shells FEBS Letters. 1980 Mar , 111 (2):311-316.
Weiner S. 1980. MOLECULAR EVOLUTION FROM THE FOSSIL RECORD - A DREAM OR A REALITY Paleobiology. 1980 , 6 (1):4-5.
Weiner S., Kustanovich Z., Gil-Av E. & Traub W. 1980. Dead sea scroll parchments: Unfolding of the collagen molecules and racemization of aspartic acid Nature. 1980 , 287 (5785):820-823.

[Abstract]

The parchments of the Dead Sea Scrolls were prepared from the skins of animals, mainly sheep and goats^1,2. They were copied and hidden between the beginning of the first century BC and AD 70³. Since then and until their discovery in the late 1940s by local Bedouins, they were located in caves close to the northwestern shore of the Dead Sea⁴. Having been handled by various intermediaries for periods of up to 8 yr, most of the intact scrolls are housed in the Israel Museum, Jerusalem. We have examined small pieces (≲25 mm²) from these scrolls, together with random fragments without any writing, to study some of the chemical changes which have occurred in the past 2,000 yr or so. Using X-ray diffraction and racemization analyses, we found that a major degradative change is due to the unfolding of the collagen molecules to form gelatin and that the degradation generally occurred many centuries ago.
Weiner S., LOWENSTAM H., TABOREK B. & HOOD L. 1979. Fossil mollusk shell organic matrix components preserved for 80 million years Paleobiology. 1979 , 2 :144-150.

[Abstract]

The organic matrix components of a fossil ammonoid shell from the Upper Cretaceous, can be separated into sub-fractions which are generally comparable to those found in extant Nautilus, using ion exchange chromatography. This suggests that at least portions of these components are sufficiently well preserved to interact characteristically with the ion exchange resin. Amino acid compositions of these sub-fractions, however, do not resemble Nautilus organic matrix sub-fractions, indicating that considerable diagenetic alteration of this material has taken place.
Weiner S., NOFF D., MEYER M., WEISMAN Y. & EDELSTEIN S. 1979. Metabolism of cholecalciferol in land snails Biochemical Journal. 1979 , 184 (1):157-161.
Weiner S. 1979. Aspartic acid-rich proteins: Major components of the soluble organic matrix of mollusk shells Calcified Tissue International. 1979 Dec , 29 (1):163-167.

[Abstract]

DEAE-cellulose ion-exchange chromatography separates soluble organic matrix components of three mollusk shells, each from a different taxonomic class, into analogous subfractions. The proteins of all subfractions are enriched in acidic and polar amino acids. In each chromatogram, however, the subfraction which contains the major portion of total protein also contains the highest concentration of aspartic acid. Thus the major components of the soluble organic matrix are aspartic acid-rich proteins. The identification of these proteins in mollusks, together with the known occurrence of aspartic acid and phosphoserine-rich proteins in vertebrate tooth dentin, emphasizes the fundamental importance of such acidic proteins in the processes of mineralization.

BOOKS

Lowenstam, H.A. and Weiner, S. 1989. On Biomineralization. Oxford University Press.
Weiner, S. 2010. Microarchaeology. Beyond the Visual Archaeological Record. Cambridge University Press, New York.

EDITED BOOKS

Weiner, S. and Dove, P.M. 2003. Biomineralization. Reviews in Mineralogy & Geochemistry. American Mineralogical Society, Washington, DC.
Reis, R.L. and Weiner, S. 2004. Learning From Nature How to Design New Implantable Biomaterials. 171, NATO Science Series, Kluwer Publishers, Dordrecht.

Publications

BOOKS

EDITED BOOKS

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