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Guinea fowl eggshells have an unusual structural arrangement that is different from that of most birds, consisting of two distinct layers with different microstructures. This bilayered organization, and distinct microstructural characteristics, provides it with exceptional mechanical properties. The inner layer, constituting about one third of the eggshell thickness, contains columnar calcite crystal units arranged vertically as in most bird shells. However, the thicker outer layer has a more complex microstructural arrangement formed by a switch to smaller calcite domains with diffuse/interlocking boundaries, partly resembling the interfaces seen in mollusk shell nacre. The switching process that leads to this remarkable second-layer microstructure is unknown. Our results indicate that the microstructural switching is triggered by changes in the inter- and intracrystalline organic matrix. During production of the outer microcrystalline layer in the later stages of eggshell formation, the interactions of organic matter with mineral induce an accumulation of defects that increase crystal mosaicity, instill anisotropic lattice distortions in the calcite structure, interrupt epitaxial growth, reduce crystallite size, and induce nucleation events which increase crystal misorientation. These structural changes, together with the transition between the layers and each layer having different microstructures, enhance the overall mechanical strength of the Guinea fowl eggshell. Additionally, our findings provide new insights into how biogenic calcite growth may be regulated to impart unique functional properties. Avian eggshells are mineralized to protect the embryo and to provide calcium for embryonic chick skeletal development. Their thickness, structure and mechanical properties have evolved to resist external forces throughout brooding, yet ultimately allow them to crack open during chick hatching. One particular eggshell, that of the Guinea fowl, has structural features very different from other galliform birds – it is bilayered, with an inner columnar mineral structure (like in most birds), but it also has an outer layer with a complex microstructure which contributes to its superior mechanical properties. This work provides novel and new fundamental information about the processes and mechanisms that control and change crystal growth during the switch to microcrystalline domains when the second outer layer forms. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: Extracellular proteins play a key role in generating and maintaining cohesion and adhesion in biological tissues. These “natural glues” are involved in vital biological processes such as blood clotting, wound healing and maintaining the structural integrity of tissues. Macromolecular assemblies of proteins can be functionally stabilized in a variety of ways in situ that include ionic interactions as well as covalent crosslinking to form protein networks that can extend both within and between tissues. Within tissues, myriad cohesive forces are required to preserve tissue integrity and function, as are additional appropriate adhesive forces at interfaces both within and between tissues of differing composition. While the mechanics of some key structural adhesive proteins have been characterized in tensile experiments at both the macroscopic and single protein levels, the fracture toughness of thin proteinaceous interfaces has never been directly measured. Here, we describe a novel and simple approach to measure the cohesive behavior and toughness of thin layers of proteinaceous adhesives. The test is based on the standard double-cantilever beam test used for engineering adhesives, which was adapted to take into account the high compliance of the interface compared with the beams. This new “rigid double-cantilever beam” method enables stable crack propagation through an interfacial protein layer, and provides a direct way to measure its full traction–separation curve. The method does not require any assumption of the shape of the cohesive law, and the results provide abundant information contributing to understanding the structural, chemical and molecular mechanisms acting in biological adhesion. As an example, results are presented using this method for thin films of fibrin—a protein involved in blood clotting and used clinically as a tissue bio-adhesive after surgery—with the effects of calcium and crosslinking by Factor XIII being examined. Finally, a simple model is proposed, demonstrating how a bell-shaped cohesive law forms during the failure of the fibrin interface based on an eight-chain model whose structure degrades and changes configuration with stress. [Copyright &y& Elsevier]

Summary: Objective: We have previously shown that microfractured ovine defects are repaired with more hyaline cartilage when the defect is treated with in situ-solidified implants of chitosan–glycerol phosphate (chitosan–GP) mixed with autologous whole blood. The objectives of this study were (1) to characterize chitosan–GP/blood clots in vitro, and (2) to develop a rabbit marrow stimulation model in order to determine the effects of the chitosan–GP/blood implant and of debridement on the formation of incipient cartilage repair tissue. Methods: Blood clots were characterized by histology and in vitro clot retraction tests. Bilateral 3.5×4mm trochlear defects debrided into the calcified layer were pierced with four microdrill holes and filled with a chitosan–GP/blood implant or allowed to bleed freely as a control. At 1 day post-surgery, initial defects were characterized by histomorphometry (n =3). After 8 weeks of repair, osteochondral repair tissues between or through the drill holes were evaluated by histology, histomorphometry, collagen type II expression, and stereology (n =16). Results: Chitosan–GP solutions structurally stabilized the blood clots by inhibiting clot retraction. Treatment of drilled defects with chitosan–GP/blood clots led to the formation of a more integrated and hyaline repair tissue above a more porous and vascularized subchondral bone plate compared to drilling alone. Correlation analysis of repair tissue between the drill holes revealed that the absence of calcified cartilage and the presence of a porous subchondral bone plate were predictors of greater repair tissue integration with subchondral bone (P <0.005), and of a higher total O''Driscoll score (P <0.005 and P <0.01, respectively). Conclusions: Chitosan–GP/blood implants applied in conjunction with drilling, compared to drilling alone, elicited a more hyaline and integrated repair tissue associated with a porous subchondral bone replete with blood vessels. Concomitant regeneration of a vascularized bone plate during cartilage repair could provide progenitors, anabolic factors and nutrients that aid in the formation of hyaline cartilage. [Copyright &y& Elsevier]

The most prominent structural components in bone are collagen and mineral. However, bone additionally contains a substantial amount of noncollagenous proteins (most notably of the SIBLING protein family), some of which may act as cohesive/adhesive “binders” for the composite hybrid collagen/mineral scaffolding, whether in the bulk phase of bone, or at its interfaces. One such noncollagenous protein – osteopontin (OPN) – appears to be critical to the deformability and fracture toughness of bone. In the present study, we used a reconstructed synthetic mineral-OPN-mineral interface, and a biogenic (natural tooth dentin) mineral/collagen-OPN-mineral/collagen interface, to measure the fracture toughness of OPN on mineralized substrates. We used this system to test the hypothesis that OPN crosslinking by the enzyme tissue transglutaminase 2 (TG2) that is found in bone enhances interfacial adhesion to increase the fracture toughness of bone. For this, we prepared double-cantilever beam substrates of synthetic pure hydroxyapatite mineral, and of narwhal dentin, and directly apposed them to one another under different intervening OPN/crosslinking conditions, and fracture toughness was tested using a miniaturized loading stage. The work-of-fracture of the OPN interface was measured for different OPN formulations (monomer vs. polymer), crosslinking states, and substrate composition. Noncrosslinked OPN provided negligible adhesion on pure hydroxyapatite, whereas OPN crosslinking (by the chemical crosslinker glutaraldehyde, and TG2 enzyme) provided strong interfacial adhesion for both hydroxyapatite and dentin using monomeric and polymeric OPN. Pre-coating of the substrate beams with monomeric OPN further improved the adhesive performance of the samples, likely by allowing effective binding of this nascent OPN form to mineral/matrix components, with this pre-attachment providing a protein layer for additional crosslinking between the substrates. [ABSTRACT FROM AUTHOR]

Abstract: In trabecular bone fracture repair in vivo, osteogenesis occurs through endochondral ossification under hypoxic conditions, or through woven bone deposition in the vicinity of blood vessels. In vitro osteogenesis assays are routinely used to test osteoblastic responses to drugs, hormones, and biomaterials for bone and cartilage repair applications. These cell culture models recapitulate events that occur in woven bone synthesis, and are carried out using primary osteoblasts, osteoblast precursors such as bone marrow-derived mesenchymal stromal cells (BMSCs), or various osteoblast cell lines. With time in culture, cell differentiation is typically assessed by examining levels of alkaline phosphatase activity (an early osteoblast marker) and by evaluating the assembly of a collagen (type I)-containing fibrillar extracellular matrix that mineralizes. In this review, we have made a comparative analysis of published osteogenic assays using calvarial cells, calvaria-derived cell lines, and bone marrow stromal cells. In all of these cell types, alkaline phosphatase activity shows similar progression over time using a variety of osteogenic and mineralizing media conditions; however, levels of alkaline phosphatase activity are not proportional to observed mineralization levels. [Copyright &y& Elsevier]

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20 nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100 nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone. [ABSTRACT FROM AUTHOR]

Biomineralization is remarkably diverse and provides myriad functions across many organismal systems. Biomineralization processes typically produce hardened, hierarchically organized structures usually having nanostructured mineral assemblies that are formed through inorganic-organic (usually protein) interactions. Calcium‑carbonate biomineral predominates in structures of small invertebrate organisms abundant in marine environments, particularly in shells (remarkably it is also found in the inner ear otoconia of vertebrates), whereas calcium-phosphate biomineral predominates in the skeletons and dentitions of both marine and terrestrial vertebrates, including humans. Reconciliation of the interplay between organic moieties and inorganic crystals in bones and teeth is a cornerstone of biomineralization research. Key molecular determinants of skeletal and dental mineralization have been identified in health and disease, and in pathologic ectopic calcification, ranging from small molecules such as pyrophosphate, to small membrane-bounded matrix vesicles shed from cells, and to noncollagenous extracellular matrix proteins such as osteopontin and their derived bioactive peptides. Beyond partly knowing the regulatory role of the direct actions of inhibitors on vertebrate mineralization, more recently the importance of their enzymatic removal from the extracellular matrix has become increasingly understood. Great progress has been made in deciphering the relationship between mineralization inhibitors and the enzymes that degrade them, and how adverse changes in this physiologic pathway (such as gene mutations causing disease) result in mineralization defects. Two examples of this are rare skeletal diseases having osteomalacia/odontomalacia (soft bones and teeth) – namely hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH) – where inactivating mutations occur in the gene for the enzymes tissue-nonspecific alkaline phosphatase (TNAP, TNSALP, ALPL) and phosphate-regulating endopeptidase homolog X-linked (PHEX), respectively. Here, we review and provide a concept for how existing and new information now comes together to describe the dual nature of regulation of mineralization – through systemic mineral ion homeostasis involving circulating factors, coupled with molecular determinants operating at the local level in the extracellular matrix. For the local mineralization events in the extracellular matrix, we present a focused concept in skeletal mineralization biology called the Stenciling Principle – a principle (building upon seminal work by Neuman and Fleisch) describing how the action of enzymes to remove tissue-resident inhibitors defines with precision the location and progression of mineralization. Unlabelled Image • A review on extracellular matrix mineralization in the skeleton. • Extracellular matrix composition and structure of bone is described. • Mechanisms of mineralization in bone are described. • TNAP enzyme degrades mineralization inhibitor PP i (reference to hypophosphatasia). • PHEX enzyme degrades mineralization inhibitor OPN (reference to X-linked hypophosphatemia). [ABSTRACT FROM AUTHOR]