Mineral tessellation in bone and the stenciling principle for extracellular matrix mineralization.

We review here the Stenciling Principle for extracellular matrix mineralization that describes a double-negative process (inhibition of inhibitors) that promotes mineralization in bone and other mineralized tissues, whereas the default condition of inhibition alone prevents mineralization elsewhere in soft connective tissues. The stenciling principle acts across multiple levels from the macroscale (skeleton/dentition vs soft connective tissues), to the microscale (for example, entheses, and the tooth attachment complex where the soft periodontal ligament is situated between mineralized tooth cementum and mineralized alveolar bone), and to the mesoscale (mineral tessellation). It relates to both small-molecule (e.g. pyrophosphate) and protein (e.g. osteopontin) inhibitors of mineralization, and promoters (enzymes, e.g. TNAP, PHEX) that degrade the inhibitors to permit and regulate mineralization. In this process, an organizational motif for bone mineral arises that we call crossfibrillar mineral tessellation where mineral formations - called tesselles - geometrically approximate prolate ellipsoids and traverse multiple collagen fibrils (laterally). Tesselle growth is directed by the structural anisotropy of collagen, being spatially restrained in the shorter transverse tesselle dimensions (averaging 1.6 × 0.8 × 0.8 µm, aspect ratio 2, length range 1.5-2.5 µm). Temporo-spatially, the tesselles abut in 3D (close ellipsoid packing) to fill the volume of lamellar bone extracellular matrix. Poorly mineralized interfacial gaps between adjacent tesselles remain discernable even in mature lamellar bone. Tessellation of a same, small basic unit to form larger structural assemblies results in numerous 3D interfaces, allows dissipation of critical stresses, and enables fail-safe cyclic deformations. Incomplete tessellation in osteomalacia/odontomalacia may explain why soft osteomalacic bones buckle and deform under loading.

Crossfibrillar mineral tessellation in normal and Hyp mouse bone as revealed by 3D FIB-SEM microscopy.

In bone, structural components such as mineral extend across length scales to provide essential biomechanical functions. Using X-ray micro-computed tomography (µCT), and focused ion beam scanning electron microscopy (FIB-SEM) in serial-surface-view mode, together with 3D reconstruction, entire mouse skeletons and small bone tissue volumes were examined in normal wildtype (WT) and mutant Hyp mice (an animal model for X-linked hypophosphatemia/XLH, a disease with severe hypomineralization of bone). 3D thickness maps of the skeletons showed pronounced irregular thickening and abnormalities of many skeletal elements in Hyp mice compared to WT mice. At the micro- and nanoscale, near the mineralization front in WT tibial bone volumes, mineralization foci grow as expanding prolate ellipsoids (tesselles) to abut and pack against one another to form a congruent and contiguous mineral tessellation pattern within collagen bundles that contributes to lamellar periodicity. In the osteomalacic Hyp mouse bone, mineralization foci form and begin initial ellipsoid growth within normally organized collagen assembly, but their growth trajectory aborts. Mineralization-inhibiting events in XLH/Hyp (low circulating serum phosphate, and increased matrix osteopontin) combine to result in decreased mineral ellipsoid tessellation - a defective mineral-packing organization that leaves discrete mineral volumes isolated in the extracellular matrix such that ellipsoid packing/tessellation is not achieved. Such a severely altered mineralization pattern invariably leads to abnormal compliance, other aberrant biomechanical properties, and altered remodeling of bone, all of which indubitably lead to macroscopic bone deformities and anomalous mechanical performance in XLH/Hyp. Also, we show the relationship of osteocytes and their cell processes to this mineralization pattern.

Multiscale structural evolution of citrate-triggered intrafibrillar and interfibrillar mineralization in dense collagen gels.

The mineralized extracellular matrix of bone is an organic-inorganic nanocomposite consisting primarily of carbonated hydroxyapatite, fibrous type I collagen, noncollagenous proteins, proteoglycans, and diverse biomolecules such as pyrophosphate and citrate. While much is now known about the mineralization-regulating role of pyrophosphate, less is known about the function of citrate. In order to assess the effect of negatively charged citrate on collagen mineralization, citrate-functionalized, bone osteoid-mimicking dense collagen gels were exposed to simulated body fluid for up to 7 days to examine the multiscale evolution of intra- and interfibrillar collagen mineralization. Here, we show by increases in methylene blue staining that the net negative charge of collagen can be substantially augmented through citrate functionalization. Structural and compositional analyses by transmission and scanning electron microscopy (including X-ray microanalysis and electron diffraction), and atomic force microscopy, all demonstrated that citrate-functionalized collagen fibrils underwent extensive intrafibrillar mineralization within 12 h in simulated body fluid. Time-resolved, high-resolution transmission electron microscopy confirmed the temporal evolution of intrafibrillar mineralization of single collagen fibrils. Longer exposure to simulated body fluid resulted in additional interfibrillar mineralization, all through an amorphous-to-crystalline transformation towards apatite (assessed by X-ray diffraction and attenuated total reflection-Fourier-transform infrared spectroscopy). Calcium deposition assays indicated a citrate concentration-dependent temporal increase in mineralization, and micro-computed tomography confirmed that >80 vol% of the collagen in the gels was mineralized by day 7. In conclusion, citrate effectively induces mesoscale intra- and interfibrillar collagen mineralization, a finding that advances our understanding of the role of citrate in mineralized tissues.

Deep learning for 3D imaging and image analysis in biomineralization research.

Biomineralization research examines structure-function relations in all types of exo- and endo-skeletons and other hard tissues of living organisms, and it relies heavily on 3D imaging. Segmentation of 3D renderings of biomineralized structures has long been a bottleneck because of human limitations such as our available time, attention span, eye-hand coordination, cognitive biases, and attainable precision, amongst other limitations. Since recently, some of these routine limitations appear to be surmountable thanks to the development of deep-learning algorithms for biological imagery in general, and for 3D image segmentation in particular. Many components of deep learning often appear too abstract for a life scientist. Despite this, the basic principles underlying deep learning have many easy-to-grasp commonalities with human learning and universal logic. This primer presents these basic principles in what we feel is an intuitive manner, without relying on prerequisite knowledge of informatics and computer science, and with the aim of improving the reader's general literacy in artificial intelligence and deep learning. Here, biomineralization case studies are presented to illustrate the application of deep learning for solving segmentation and analysis problems of 3D images ridden by various artifacts, and/or which are plainly difficult to interpret. The presented portfolio of case studies includes three examples of imaging using micro-computed tomography (µCT), and three examples using focused-ion beam scanning electron microscopy (FIB-SEM), all on mineralized tissues. We believe this primer will expand the circle of users of deep learning amongst biomineralization researchers and other life scientists involved with 3D imaging, and will encourage incorporation of this powerful tool into their professional skillsets and to explore it further.

Nanostructure of mouse otoconia.

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical for maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (~50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (~10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy - both methods being sensitive to the detection of crystalline and amorphous forms in the sample - showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. These findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.

Modulation of calcium oxalate dihydrate growth by phosphorylated osteopontin peptides.

Osteopontin (OPN) is a significant component of kidney stone matrix and a key modulator of stone formation. Here, we investigated the effects of different phosphorylated states of a synthesized peptide of OPN (the ASARM peptide; acidic, serine- and aspartate-rich motif) on calcium oxalate dihydrate (COD) crystals, a major mineral phase of kidney stones. In vitro, phosphorylated OPN-ASARM peptides strongly inhibited COD crystal growth in solution as compared to the nonphosphorylated state, with increasing inhibitory potency correlating with the degree of peptide phosphorylation. Scanning electron microscopy revealed that the inhibition from the phosphopeptides resulted in distinctive, rosette-like crystal aggregates called spherulites. The OPN-ASARM peptides preferentially bound and specifically inhibited the {1 1 0} crystallographic faces of COD, as identified by combining atomic force microscopy and computational simulation approaches. These {1 1 0} surfaces of COD have high lattice calcium occupancy (exposure), providing preferential binding sites for the highly acidic peptides; binding and inhibition by OPN-ASARM peptides at the {1 1 0} faces led to crystal aggregation and intergrowth. The crystal spherulite formations obtained in vitro when using the most phosphorylated form of OPN-ASARM peptide at a high concentration, resembled crystal morphologies observed in vivo in a rat model of urolithiasis, in which crystal deposits in the kidney contain abundant OPN as revealed by immunogold labeling. A mechanistic model for spherulite formation is proposed based on the symmetry and crystallographic structure of COD, where the phosphate groups of OPN-ASARM bind to calcium atoms at [1 1 1] step risers on the COD {1 1 0} surface, inducing the periodic emergence of new COD crystals to form spherulites.

Matrix Gla protein deficiency impairs nasal septum growth, causing midface hypoplasia.

Genetic and environmental factors may lead to abnormal growth of the orofacial skeleton, affecting the overall structure of the face. In this study, we investigated the craniofacial abnormalities in a mouse model for Keutel syndrome, a rare genetic disease caused by loss-of-function mutations in the matrix Gla protein (MGP) gene. Keutel syndrome patients show diffuse ectopic calcification of cartilaginous tissues and impaired midface development. Our comparative cephalometric analyses of micro-computed tomography images revealed a severe midface hypoplasia in Mgp-/- mice. In vivo reporter studies demonstrated that the Mgp promoter is highly active at the cranial sutures, cranial base synchondroses, and nasal septum. Interestingly, the cranial sutures of the mutant mice showed normal anatomical features. Although we observed a mild increase in mineralization of the spheno-occipital synchondrosis, it did not reduce the relative length of the cranial base in comparison with total skull length. Contrary to this, we found the nasal septum to be abnormally mineralized and shortened in Mgp-/- mice. Transgenic restoration of Mgp expression in chondrocytes fully corrected the craniofacial anomalies caused by MGP deficiency, suggesting a local role for MGP in the developing nasal septum. Although there was no up-regulation of markers for hypertrophic chondrocytes, a TUNEL assay showed a marked increase in apoptotic chondrocytes in the calcified nasal septum. Transmission electron microscopy confirmed unusual mineral deposits in the septal extracellular matrix of the mutant mice. Of note, the systemic reduction of the inorganic phosphate level was sufficient to prevent abnormal mineralization of the nasal septum in Mgp-/-;Hyp compound mutants. Our work provides evidence that modulation of local and systemic factors regulating extracellular matrix mineralization can be possible therapeutic strategies to prevent ectopic cartilage calcification and some forms of congenital craniofacial anomalies in humans.

Persistence of Vascular Calcification after Reversal of Uremia.

The extent to which vascular calcification is reversible and the possible mechanisms are unclear. To address this, calcified aortas from uremic mice were transplanted orthotopically into normal mice, and the calcium content, histology, and minerals of the allografts were compared with the nontransplanted donor aorta. Calcium content decreased immediately after transplantation but remained constant thereafter, with 68% ± 12% remaining after 34 weeks. X-ray diffraction showed the presence of apatite in both donor aortas and allografts. Osteoclasts were absent in the allografts and there was no expression of the macrophage marker CD11b, the osteoclast marker tartrate-resistant acid phosphatase, or carbonic anhydrase II. The initial loss of calcium was less in heavily calcified aortas and was associated with an increase in the Ca/P ratio from 1.49 to 1.63, consistent with a loss of nonapatitic calcium. The results indicate that vascular calcification persists after reversal of uremia, because of a lack of active resorption of apatite. This failure to resorb established calcifications may contribute to the severity of vascular calcification and suggests that therapy should be aimed at prevention.

Impaired mineral quality in dentin in X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is a skeletal disorder arising from mutations in the PHEX gene, transmitted in most cases as an X-linked dominant trait. PHEX deficiency leads to renal phosphate wasting and hypophosphatemia, as well as impaired mineralization of bone and dentin, resulting in severe skeletal and dental complications. Dentin mineralization defects appear as characteristic, large interglobular spaces resulting from the lack of fusion of calculospherites in the circumpulpal region during the mineralization process. Here, we examined changes in the composition and structure of dentin using Raman spectroscopy on XLH human teeth, and using transmission electron microscopy on the dentin of Hyp mice (the murine model of XLH). The dentin of patients with XLH showed changes in the quality of the apatitic mineral, with greater carbonate substitution and lower crystallinity compared to the dentin of age-matched control teeth. In addition, ultrastructural analysis by transmission electron microscopy revealed a major disorganization of the peri- and intertubular structure of the dentin, with odontoblast processes residing within an unmineralized matrix sheath in the Hyp mouse. Taken together, these results indicate that like for bone and tooth cementum, there are impaired mineral quality and matrix changes in XLH dentin reflecting high sensitivity to systemic serum phosphate levels and possibly other local changes in the dentin matrix.

Constitutive nuclear expression of dentin matrix protein 1 fails to rescue the Dmp1-null phenotype.

Dentin matrix protein 1 (DMP1) plays multiple roles in bone, tooth, phosphate homeostasis, kidney, salivary gland, reproductive cycles, and the development of cancer. In vitro studies have indicated two different biological mechanisms: 1) as a matrix protein, DMP1 interacts with αvß3 integrin and activates MAP kinase signaling; and 2) DMP1 serves as a transcription co-factor. In vivo studies have demonstrated its key role in osteocytes. This study attempted to determine whether DMP1 functions as a transcription co-factor and regulates osteoblast functions. For gene expression comparisons using adenovirus constructs, we targeted the expression of DMP1 either to the nucleus only by replacing the endogenous signal peptide with a nuclear localization signal (NLS) sequence (referred to as (NLS)DMP1) or to the extracellular matrix as the WT type (referred to as (SP)DMP1) in MC3T3 osteoblasts. High levels of DMP1 in either form greatly increased osteogenic gene expression in an identical manner. However, the targeted (NLS)DMP1 transgene driven by a 3.6-kb rat Col 1α1 promoter in the nucleus of osteoblasts and osteocytes failed to rescue the phenotyope of Dmp1-null mice, whereas the (SP)DMP1 transgene rescued the rickets defect. These studies support the notion that DMP1 functions as an extracellular matrix protein, rather than as a transcription co-factor in vivo. We also show that DMP1 continues its expression in osteoblasts during postnatal development and that the deletion of Dmp1 leads to an increase in osteoblast proliferation. However, poor mineralization in the metaphysis indicates a critical role for DMP1 in both osteoblasts and osteocytes.

Critical role for αvß6 integrin in enamel biomineralization.

Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with the ameloblast plasma membrane. Although it is known that integrins mediate cell-matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. We hypothesized that αvß6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both ß6 integrin mRNA and protein. The maxillary incisors of Itgb6(-/-) mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6(-/-) mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6(-/-) enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6(-/-) mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, in which αvß6 integrin was not an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvß6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition and/or turnover and subsequent enamel biomineralization.

Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia.

As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth - where calcium phosphate crystals are deposited and grow within an extracellular matrix - is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth-suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue-nonspecific alkaline phosphatase and phosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X-linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X-linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating small integrin-binding ligand N-linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine- and aspartate-rich-motif peptide, may accumulate locally to impair mineralization in this disease.

Attaching organic fibers to mineral: The case of the avian eggshell.

Bird eggs possess a mineralized eggshell with a soft underlying fibrous membrane. These dissimilar material layers successfully evolved a structural attachment to each other as a conserved avian reproduction strategy essential to avian embryonic development, growth, and hatching of the chick. To understand how organic membrane fibers attach to shell mineral (calcite), 3D multiscale imaging including X-ray and electron tomography coupled with deep learning-based feature segmentation was used to show how membrane fibers are organized and anchored into shell mineral. Whole fibers embed into mineral across the microscale, while fine mineral projections (granules/spikes) insert into fiber surfaces at the nanoscale, all of which provides considerable surface area and multiscale anchorage at the organic-inorganic interface between the fibrous membrane and the shell. Such a reciprocal anchorage system occurring at two different length scales between organic fibers and inorganic mineral provides a secure attachment mechanism for avian eggshell integrity across two dissimilar materials.

Mineralization of Bone Extracellular Matrix-like Scaffolds Fabricated as Silk Sericin-Functionalized Dense Collagen-Fibrin Hybrid Hydrogels.

The design of hydrogels that combine both the biochemical cues needed to direct seeded cellular functions and mineralization to provide the structural and mechanical properties approaching those of mineralized native bone extracellular matrix (ECM) represents a significant challenge in bone tissue engineering. While fibrous hydrogels constituting of collagen or fibrin (and their hybrids) can be considered as scaffolds that mimic to some degree native bone ECM, their insufficient mechanical properties limit their application. In the present study, an automated gel aspiration-ejection (automated GAE) method was used to generate collagen-fibrin hybrid gel scaffolds with micro-architectures and mechanical properties approaching those of native bone ECM. Moreover, the functionalization of these hybrid scaffolds with negatively charged silk sericin accelerated their mineralization under acellular conditions in simulated body fluid and modulated the proliferation and osteoblastic differentiation of seeded MC3T3-E1 pre-osteoblastic cells. In the latter case, alkaline phosphatase activity measurements indicated that the hybrid gel scaffolds with seeded cells showed accelerated osteoblastic differentiation, which in turn led to increased matrix mineralization. In summary, the design of dense collagen-fibrin hybrid gels through an automated GAE process can provide a route to tailoring specific biochemical and mechanical properties to different types of bone ECM-like scaffolds, and can provide a model to better understand cell-matrix interactions in vitro for bioengineering purposes.

Mineral tessellation in mouse enthesis fibrocartilage, Achilles tendon, and Hyp calcifying enthesopathy: A shared 3D mineralization pattern.

The hallmark of enthesis architecture is the 3D compositional and structural gradient encompassing four tissue zones - tendon/ligament, uncalcified fibrocartilage, calcified fibrocartilage and bone. This functional gradient accommodates the large stiffness differential between calcified bone and uncalcified tendon/ligament. Here we analyze in 3D the organization of the mouse Achilles enthesis and mineralizing Achilles tendon in comparison to lamellar bone. We use correlative, multiscale high-resolution volume imaging methods including µCT with submicrometer resolution and FIB-SEM tomography (both with deep learning-based image segmentation), and TEM and SEM imaging, to describe ultrastructural features of physiologic, age-related and aberrant mineral patterning. We applied these approaches to murine wildtype (WT) Achilles enthesis tissues to describe in normal calcifying fibrocartilage a crossfibrillar mineral tessellation pattern similar to that observed in lamellar bone, but with greater variance in mineral tesselle morphology and size. We also examined Achilles enthesis structure in Hyp mice, a murine model for the inherited osteomalacic disease X-linked hypophosphatemia (XLH) with calcifying enthesopathy. In Achilles enthesis fibrocartilage of Hyp mice, we show defective crossfibrillar mineral tessellation similar to that which occurs in Hyp lamellar bone. At the cellular level in fibrocartilage, unlike in bone where enlarged osteocyte mineral lacunae are found as peri-osteocytic lesions, mineral lacunar volumes for fibrochondrocytes did not differ between WT and Hyp mice. While both WT and Hyp aged mice demonstrate Achilles tendon midsubstance ectopic mineralization, a consistently defective mineralization pattern was observed in Hyp mice. Strong immunostaining for osteopontin was observed at all mineralization sites examined in both WT and Hyp mice. Taken together, this new 3D ultrastructural information describes details of common mineralization trajectories for enthesis, tendon and bone, which in Hyp/XLH are defective.

Contributions of increased osteopontin and hypophosphatemia to dentoalveolar defects in osteomalacic Hyp mice.

X-linked hypophosphatemia (XLH) is an inherited disorder caused by inactivating mutations in the PHEX gene leading to renal phosphate wasting, rickets and osteomalacia. XLH is also associated with dentoalveolar mineralization defects in tooth enamel, dentin and cementum, and in alveolar bone, which lead to an increased prevalence of dental abscesses, periodontal disease and tooth loss. Genetic mouse experiments, and deficiencies in XLH patient therapies where treatments do not fully ameliorate mineralization defects, suggest that other pathogenic mechanisms may exist in XLH. The mineralization-inhibiting, secreted extracellular matrix phosphoprotein osteopontin (OPN, gene Spp1) is a substrate for the PHEX enzyme whereby extensive and inactivating degradation of inhibitory OPN by PHEX facilitates mineralization. Conversely, excess OPN accumulation in skeletal and dental tissues - for example in XLH where inactivating mutations in the PHEX gene limit degradation of inhibitory OPN, or as occurs in Fgf23-null mice - contributes to mineralization defects. We hypothesized that Spp1/OPN ablation in Hyp mice (a mouse model for XLH) would reduce dentoalveolar mineralization defects. Immunostaining revealed increased OPN in Hyp vs. wild-type (WT) alveolar bone, particularly in osteocyte lacunocanalicular networks where Hyp mice have characteristic hypomineralized peri-osteocytic lesions (POLs). Micro-computed tomography and histology showed that ablation of Spp1 in Hyp mice (Hyp;Spp1-/-) on a normal diet did not ameliorate bulk defects in enamel, dentin, or alveolar bone. On a high-phosphate diet, both Hyp and Hyp;Spp1-/- mice showed improved mineralization of enamel, dentin, and alveolar bone. Silver staining indicated Spp1 ablation did not improve alveolar or mandibular bone osteocyte POLs in Hyp mice; however, they were normalized by a high-phosphate diet in both Hyp and Hyp;Spp1-/- mice, although inducing increased OPN. Collectively, these data indicate that despite changes in OPN content in the dentoalveolar mineralized tissues, there exist other compensatory mineralization mechanisms that arise from knockout of Spp1/OPN in the Hyp background.

ATP acts as a survival signal and prevents the mineralization of aortic valve.

Calcific aortic valve disease (CAVD) is a disorder related to progressive mineralization of valvular tissue that is a leading cause of heart disease. Thus far, there is no medical treatment to prevent the mineralization of aortic valves. It is generally thought that pathologic mineralization is linked to apoptosis of vascular cells. However, the role of apoptosis during mineralization as well as the survival signals for valvular interstitial cells (VICs), the main cellular component of aortic valves, remains to be identified. Here, through several lines of evidence, we show that bioavailability of extracellular ATP is a signal which determines survival or apoptosis of VICs and, in doing so, plays a major role in the development of CAVD. Specifically, in CAVD and in VIC cultures undergoing mineralization, we found a high level of the ectonucleotidase ENPP1. In addition, a genetic polymorphism in the intron 9 of the ENPP1 gene was associated with CAVD in a case-control cohort as well as with mRNA expression levels of ENPP1 in aortic valves. A high level of ENPP1 in CAVD promoted apoptosis-mediated mineralization of VICs by depleting the extracellular pool of ATP. We then documented that release of ATP by VICs promoted cell survival via the P2Y(2) receptor and the PI3K/Akt signaling pathway. Hence, our results show that level of ENPP1 modulates extracellular concentration of ATP, which is an important survival signal for VICs. These findings may help to develop novel pharmacological treatment for CAVD.

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization.