Guinea fowl eggshell structural analysis at different scales reveals how organic matrix induces microstructural shifts that enhance its mechanical properties.

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. STATEMENT OF SIGNIFICANCE: 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.

Biological stenciling of mineralization in the skeleton: Local enzymatic removal of inhibitors in the extracellular matrix.

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. Calcium­carbonate 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.

Bone toughness at the molecular scale: A model for fracture toughness using crosslinked osteopontin on synthetic and biogenic mineral substrates.

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.

Defective Mineralization in X-Linked Hypophosphatemia Dental Pulp Cell Cultures.

X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activity. Here, we evaluated the local role of PHEX in a 3-dimensional model of extracellular matrix (ECM) mineralization. Dense collagen hydrogels were seeded either with human dental pulp cells from patients with characterized PHEX mutations or with sex- and age-matched healthy controls and cultured up to 24 d using osteogenic medium with standard phosphate concentration. Calcium quantification, micro-computed tomography, and histology with von Kossa staining for mineral showed significantly lower mineralization in XLH cell-seeded scaffolds, using nonparametric statistical tests. While apatitic mineralization was observed along collagen fibrils by electron microscopy in both groups, Raman microspectrometry indicated that XLH cells harboring the PHEX mutation produced less mineralized scaffolds having impaired mineral quality with less carbonate substitution and lower crystallinity. In the XLH cultures, immunoblotting revealed more abundant osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) than controls, as well as the presence of fragments of these proteins not found in controls, suggesting a role for PHEX in SIBLING protein degradation. Immunohistochemistry revealed altered OPN and DMP1 associated with an increased alkaline phosphatase staining in the XLH cultures. These results are consistent with impaired PHEX activity having local ECM effects in XLH. Future treatments for XLH should target both systemic and local manifestations.

Total hip arthroplasty following failure of core decompression and tantalum rod implantation.

AIMS: One method of femoral head preservation following avascular necrosis (AVN) is core decompression and insertion of a tantalum rod. However, there may be a high failure rate associated with this procedure. The purpose of this study was to document the clinical and radiological outcomes following total hip arthroplasty (THA) subsequent to failed tantalum rod insertion. PATIENTS AND METHODS: A total of 37 failed tantalum rods requiring total hip arthroplasty were identified from a prospective database. There were 21 hips in 21 patients (12 men and nine women, mean age 37 years, 18 to 53) meeting minimum two year clinical and radiographic follow-up whose THAs were carried out between November 2002 and April 2013 (mean time between tantalum rod implantation and conversion to a THA was 26 months, 6 to 72). These were matched by age and gender to individuals (12 men, nine women, mean age 40 years, 18 to 58) receiving THA for AVN without prior tantalum rod insertion. RESULTS: There were no functional outcome differences between the two groups. Tantalum residue was identified on all post-operative radiographs in the tantalum group. Linear wear rates were comparable between groups with no evidence of catastrophic wear in either group. CONCLUSION: In the short term, tantalum rod implantation does not demonstrate an adverse effect on subsequent total joint replacement surgery. There is however, a high rate of retained tantalum debris on post-operative radiographs and thus there is an unknown risk of accelerated articular wear necessitating longer term study. Cite this article: Bone Joint J 2016;98-B:1175-9.

Craniofacial and Dental Defects in the Col1a1Jrt/+ Mouse Model of Osteogenesis Imperfecta.

Certain mutations in the COL1A1 and COL1A2 genes produce clinical symptoms of both osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS) that include abnormal craniofacial growth, dental malocclusion, and dentinogenesis imperfecta. A mouse model (Col1a1(Jrt)/+) was recently developed that had a skeletal phenotype and other features consistent with moderate-to-severe OI and also with EDS. The craniofacial phenotype of 4- and 20-wk-old Col1a1(Jrt)/+ mice and wild-type littermates was assessed by micro-computed tomography (µCT) and morphometry. Teeth and the periodontal ligament compartment were analyzed by µCT, light microscopy/histomorphometry, and electron microscopy. Over time, at 20 wk, Col1a1(Jrt)/+ mice developed smaller heads, a shortened anterior cranial base, class III occlusion, and a mandibular side shift with shorter morphology in the masticatory region (maxilla and mandible). Col1a1(Jrt)/+ mice also had changes in the periodontal compartment and abnormalities in the dentin matrix and mineralization. These findings validate Col1a1(Jrt)/+ mice as a model for OI and EDS in humans.

Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.

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 20nm-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, 100nm-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.

Abnormal osteopontin and matrix extracellular phosphoglycoprotein localization, and odontoblast differentiation, in X-linked hypophosphatemic teeth.

Mutations in phosphate-regulating gene (PHEX) lead to X-linked hypophosphatemic rickets (XLH), a genetic disease characterized by impaired mineralization in bones and teeth. In human XLH tooth dentin, calcospherites that would normally merge as part of the mineralization process are separated by unmineralized interglobular spaces where fragments of matrix proteins accumulate. Here, we immunolocalized osteopontin (OPN) in human XLH teeth, in a three-dimensional XLH human dental pulp stem cell-collagen scaffold culture model and in a rat tooth injury repair model treated with acidic serine- and aspartate-rich motif peptides (ASARM). In parallel, matrix extracellular phosphoglycoprotein (MEPE) immunolocalization and alkaline phosphatase (ALP) activity were assessed in XLH teeth. OPN was expressed by odontoblasts in the XLH models, and localized to the abnormal calcospherites of XLH tooth dentin. In addition, ALP activity and MEPE localization were abnormal in human XLH teeth, with MEPE showing an accumulation in the unmineralized interglobular spaces in dentin. Furthermore, XLH odontoblasts failed to form a well-polarized odontoblast layer. These data suggest that both MEPE and OPN are involved in impaired tooth mineralization associated with XLH, possibly through different effects on the mineralization process.

Mineralization of dense collagen hydrogel scaffolds by human pulp cells.

While advances in biomineralization have been made in recent years, unanswered questions persist on bone- and tooth-cell differentiation, on outside-in signaling from the extracellular matrix, and on the link between protein expression and mineral deposition. In the present study, we validate the use of a bioengineered three-dimensional (3D) dense collagen hydrogel scaffold as a cell-culture model to explore these questions. Dental pulp progenitor/stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into an extracellular matrix-like collagen gel whose fibrillar density was increased through plastic compression. SHED viability, morphology, and metabolic activity, as well as scaffold mineralization, were investigated over 24 days in culture. Additionally, measurements of alkaline phosphatase enzymatic activity, together with immunoblotting for mineralized tissue cell markers ALPL (tissue-non-specific alkaline phosphatase), DMP1 (dentin matrix protein 1), and OPN (osteopontin), demonstrated osteo/odontogenic cell differentiation in the dense collagen scaffolds coincident with mineralization. Analyses of the mineral phase by electron microscopy, including electron diffraction and energy-dispersive x-ray spectroscopy, combined with Fourier-transform infrared spectroscopy and biochemical analyses, were consistent with the formation of apatitic mineral that was frequently aligned along collagen fibrils. In conclusion, use of a 3D dense collagen scaffold promoted SHED osteo/odontogenic cell differentiation and mineralization.

Compounded PHOSPHO1/ALPL deficiencies reduce dentin mineralization.

Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized tissues, and double ablation of Alpl and Phospho1 in mice leads to a complete absence of skeletal mineralization. Here, we describe mineralization abnormalities in the teeth of Phospho1(-/-) mice, and in compound knockout mice lacking Phospho1 and one allele of Alpl (Phospho1(-/-);Alpl(+/-) ). In wild-type mice, PHOSPHO1 and TNAP co-localized to odontoblasts at early stages of dentinogenesis, coincident with the early mineralization of mantle dentin. In Phospho1 knockout mice, radiography, micro-computed tomography, histology, and transmission electron microscopy all demonstrated mineralization abnormalities of incisor dentin, with the most remarkable findings being reduced overall mineralization coincident with decreased matrix vesicle mineralization in the Phospho1(-/-) mice, and the almost complete absence of matrix vesicles in the Phospho1(-/-);Alpl(+/-) mice, whose incisors showed a further reduction in mineralization. Results from this study support prominent non-redundant roles for both PHOSPHO1 and TNAP in dentin mineralization.

Latissimus dorsi tendon transfer for irreparable tears of the rotator cuff: An anatomical study to assess the neurovascular hazards and ways of improving tendon excursion.

Latissimus dorsi tendon transfer (LDTT) is technically challenging. In order to clarify the local structural anatomy, we undertook a morphometric study using six complete cadavers (12 shoulders). Measurements were made from the tendon to the nearby neurovascular structures with the arm in two positions: flexed and internally rotated, and adducted in neutral rotation. The tendon was then transferred and measurements were taken from the edge of the tendon to a reference point on the humeral head in order to assess the effect of a novel two-stage release on the excursion of the tendon. With the shoulder flexed and internally rotated, the mean distances between the superior tendon edge and the radial nerve, brachial artery, axillary nerve and posterior circumflex artery were 30 mm (26 to 34), 28 mm (17 to 39), 21 mm (12 to 28) and 15 mm (10 to 21), respectively. The mean distance between the inferior tendon edge and the radial nerve, brachial artery and profunda brachii artery was 18 mm (8 to 27), 22 mm (15 to 32) and 14 mm (7 to 21), respectively. Moving the arm to a neutral position reduced these distances. A mean of 15 mm (8 to 21) was gained from a standard soft-tissue release, and 32 mm (20 to 45) from an extensile release. These figures help to define further the structural anatomy of this region and the potential for transfer of the latissimus dorsi tendon.

Local regulation of tooth mineralization by sphingomyelin phosphodiesterase 3.

Sphingomyelin phosphodiesterase 3 (Smpd3) encodes a membrane-bound enzyme that cleaves sphingomyelin to generate several bioactive metabolites. A recessive mutation called fragilitas ossium (fro) in the Smpd3 gene leads to impaired mineralization of bone and tooth extracellular matrix (ECM) in fro/fro mice. In teeth from fro/fro mice at various neonatal ages, radiography and light and electron microscopy showed delayed mantle dentin mineralization and a consequent delay in enamel formation as compared with that in control +/fro mice. These tooth abnormalities progressively improved with time. Immunohistochemistry showed expression of SMPD3 by dentin-forming odontoblasts. SMPD3 deficiency, however, did not affect the differentiation of these cells, as shown by osterix and dentin sialophosphoprotein expression. Using a transgenic mouse rescue model (fro/fro; Col1a1-Smpd3) in which Smpd3 expression is driven by a murine Col1a1 promoter fragment active in osteoblasts and odontoblasts, we demonstrate a complete correction of the tooth mineralization delays. In conclusion, analysis of these data demonstrates that Smpd3 expression in odontoblasts is required for tooth mineralization.

Tooth root dentin mineralization defects in a mouse model of hypophosphatasia.

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PP(i) ), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PP(i) , resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl(-/-) mice, a model for infantile HPP. In Alpl(-/-) mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl(-/-) mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl(-/-) molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl(-/-) dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl(-/-) mice, was present in the generally unmineralized Alpl(-/-) mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN-encoding Spp1 gene in Alpl(-/-) mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral-targeting human TNAP (ENB-0040) to Alpl(-/-) mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PP(i) during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients.

Cohesive behavior of soft biological adhesives: experiments and modeling.

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.

Enzyme replacement therapy prevents dental defects in a model of hypophosphatasia.

Hypophosphatasia (HPP) occurs from loss-of-function mutation in the tissue-non-specific alkaline phosphatase (TNALP) gene, resulting in extracellular pyrophosphate accumulation that inhibits skeletal and dental mineralization. TNALP-null mice (Akp2(-/-)) phenocopy human infantile hypophosphatasia; they develop rickets at 1 week of age, and die before being weaned, having severe skeletal and dental hypomineralization and episodes of apnea and vitamin B(6)-responsive seizures. Delay and defects in dentin mineralization, together with a deficiency in acellular cementum, are characteristic. We report the prevention of these dental abnormalities in Akp2(-/-) mice receiving treatment from birth with daily injections of a mineral-targeting, human TNALP (sALP-FcD(10)). sALP-FcD(10) prevented hypomineralization of alveolar bone, dentin, and cementum as assessed by micro-computed tomography and histology. Osteopontin--a marker of acellular cementum--was immuno-localized along root surfaces, confirming that acellular cementum, typically missing or reduced in Akp2(-/-) mice, formed normally. Our findings provide insight concerning how acellular cementum is formed on tooth surfaces to effect periodontal ligament attachment to retain teeth in their osseous alveolar sockets. Furthermore, they provide evidence that this enzyme-replacement therapy, applied early in post-natal life--where the majority of tooth root development occurs, including acellular cementum formation--could prevent the accelerated tooth loss seen in individuals with HPP.

Ultrastructure of avian eggshell during resorption following egg fertilization.

For skeletal mineralization, the avian embryo mobilizes calcium from its calcitic eggshell. This occurs through dissolution of specific interior regions of the shell in a process that also weakens the shell to allow hatching. Here, we have examined eggshell ultrastructure during dissolution occurring between laying of a fertilized egg (with incubation) and hatching of the chick (Gallus gallus). We have focused on changes in shell mammillae where the majority of dissolution takes place. Using scanning electron microscopy, we describe differences in matrix-mineral structure and relationships not observed in unfertilized eggs (unresorbed eggshell). We document changes in the calcium reserve body - an essential sub-compartment of mammillae - consistent with it being an early, primary source of calcium essential for embryonic skeletal growth. Dissolution events occurring in the calcium reserve sac and in the base plate of the calcium reserve body, and similar changes in surrounding bulk mammillae structure, all correlate with advancing skeletal embryonic calcification. The changes in mammillae sub-structures can generally be characterized as mineral dissolutions revealing fine surface topographies on remaining mineral surfaces and the exposure of an extensive, intracrystalline (occluded) organic matrix network. We propose that this mineral-occluded network regulates how shell mineral is dissolved by providing dissolution channels facilitating calcium release for the embryonic skeleton.

In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization.

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.

Avian eggshell structure and osteopontin.

The avian eggshell primarily consists of calcium carbonate mineral (calcite) and matrix proteins. Here we review matrix-mineral relationships in the eggshell at the ultrastructural level using scanning and transmission electron microscopy, and describe the distribution of osteopontin (OPN) as determined by colloidal gold immunolabeling for OPN. A rich protein network integrated within the calcitic structure of the eggshell shows variable, region-specific organization that included layered fibrous planar sheets of matrix, thin filamentous threads, thin film-like surface coatings, vesicular structures and isolated proteins residing on cleaved {104} crystallographic faces of the eggshell calcite. Except for the vesicular structures, these matrix structures all immunolabeled strongly for OPN. Given the potent mineralization- inhibiting function of OPN, we discuss how this protein might regulate eggshell growth rate and inhibit calcification at specific compartmental boundaries to provide eggshell form.

Matrix Gla protein inhibition of tooth mineralization.

Extracellular matrix (ECM) mineralization is regulated by mineral ion availability, proteins, and other molecular determinants. To investigate protein regulation of mineralization in tooth dentin and cementum, and in alveolar bone, we expressed matrix Gla protein (MGP) ectopically in bones and teeth in mice, using an osteoblast/odontoblast-specific 2.3-kb Col1a1 promoter. Mandibles were analyzed by radiography, micro-computed tomography, light microscopy, histomorphometry, and transmission electron microscopy. While bone and tooth ECMs were established in the Col1a1-Mgp mice, extensive hypomineralization was observed, with values of unmineralized ECM from four- to eight-fold higher in dentin and alveolar bone when compared with that in wild-type tissues. Mineralization was virtually absent in tooth root dentin and cellular cementum, while crown dentin showed "breakthrough" areas of mineralization. Acellular cementum was lacking in Col1a1-Mgp teeth, and unmineralized osteodentin formed within the pulp. These results strengthen the view that bone and tooth mineralization is critically regulated by mineralization inhibitors.

Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro.

We investigated matrix-mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix-mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {104} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {104} calcite faces. OPN inhibited calcite growth in vitro at the {104} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell.