Bone sialoprotein (BSP) is a multifunctional extracellular matrix protein found in mineralized tissues, including bone, cartilage, tooth root cementum (both acellular and cellular types), and dentin. In order to define the role BSP plays in the process of biomineralization of these tissues, we analyzed cementogenesis, dentinogenesis, and osteogenesis (intramembranous and endochondral) in craniofacial bone in Bsp null mice and wild-type (WT) controls over a developmental period (1-60 days post natal; dpn) by histology, immunohistochemistry, undecalcified histochemistry, microcomputed tomography (microCT), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and quantitative PCR (qPCR). Regions of intramembranous ossification in the alveolus, mandible, and calvaria presented delayed mineralization and osteoid accumulation, assessed by von Kossa and Goldner's trichrome stains at 1 and 14 dpn. Moreover, Bsp−/− mice featured increased cranial suture size at the early time point, 1 dpn. Immunostaining and PCR demonstrated that osteoblast markers, osterix, alkaline phosphatase, and osteopontin were unchanged in Bsp null mandibles compared to WT. Bsp−/− mouse molars featured a lack of functional acellular cementum formation by histology, SEM, and TEM, and subsequent loss of Sharpey's collagen fiber insertion into the tooth root structure. Bsp−/− mouse alveolar and mandibular bone featured equivalent or fewer osteoclasts at early ages (1 and 14 dpn), however, increased RANKL immunostaining and mRNA, and significantly increased number of osteoclast-like cells (2-5 fold) were found at later ages (26 and 60 dpn), corresponding to periodontal breakdown and severe alveolar bone resorption observed following molar teeth entering occlusion. Dentin formation was unperturbed in Bsp−/− mouse molars, with no delay in mineralization, no alteration in dentin dimensions, and no differences in odontoblast markers analyzed. No defects were identified in endochondral ossification in the cranial base, and craniofacial morphology was unaffected in Bsp−/− mice. These analyses confirm a critical role for BSP in processes of cementogenesis and intramembranous ossification of craniofacial bone, whereas endochondral ossification in the cranial base was minimally affected and dentinogenesis was normal in Bsp−/− molar teeth. Dissimilar effects of loss of BSP on mineralization of dental and craniofacial tissues suggest local differences in the role of BSP and/or yet to be defined interactions with site-specific factors.

Collagen biomineralization is a complex process and the controlling factors at the molecular level are still not well understood. A particularly high level of spatial control over collagen mineralization is evident in the anchorage of teeth to the jawbone by the periodontal ligament. Here, unmineralized ligament collagen fibrils become mineralized at an extremely sharp mineralization front in the root of the tooth. A model of collagen biomineralization based on demineralized cryosections of mouse molars in the bone socket is presented. When exposed to metastable calcium and phosphate‐containing solutions, mineral re‐deposits selectively into the natively mineralized tissues with high fidelity, demonstrating that the extracellular matrix retains sufficient information to control the rate of mineralization at the tissue level. While solutions of simulated bodily fluid produce amorphous calcium phosphate within the tissue section, a more highly supersaturated solution stabilized with polyaspartic acid produces oriented, crystalline calcium phosphate with diffraction patterns consistent with hydroxyapatite. The model thus replicates both spatial control of mineral deposition, as well as the matrix‐mineral relationships of natively mineralized collagen fibrils, and can be used to elucidate roles of specific biomolecules in the highly controlled process of collagen biomineralization. This knowledge will be critical in the design of collagen‐based scaffolds for tissue engineering of hard‐soft tissue interfaces.

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Formation of hydroxyapatite (HAP) within collagen fibrils, as found in bone, dentine and cementum, is thought to be mediated by proteins rich in aspartate (Asp) and glutamate such as osteopontin and bone sialoprotein, respectively. Indeed polyaspartate ( pAsp), a homopolymer analogue of such proteins, has been shown to induce intrafibrillar mineralization of collagen from solutions of calcium and phosphate that are supersaturated with respect to HAP. To elucidate the role of pAsp in mineralization of collagen, we explored the effect of pAsp chain length on in vitro HAP deposition in demineralized mouse periodontal tissue sections. Through characterization of both tissue sections and mineralizing solution, we show that chain length contributes to the effectiveness of pAsp in mediating intrafibrillar mineralization. This function appears to be associated with inhibition of otherwise kinetically favoured crystallization in the bulk solution, which allows for intrafibrillar crystallization, though this does not preclude the possibility of a more active role for pAsp in addition. Inhibition of crystallization in solution by pAsp occurs by slowing the growth of amorphous calcium phosphate and stabilization of this phase, rather than by sequestration of Ca 2þ ions. These results suggest that the length of Asp-rich sequences of mineralizing proteins may be essential to their function, and could also be useful in optimization of mineralized tissue replacement synthesis.

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