Studies on three matrix molecules in bone and dentin

Abstract: The biomineralization of bone and dentin is a complex and dynamic process, involving the formation of an organic framework in which mineral crystals are deposited. The noncollagenous proteins (NCPs) of the organic phase play a central role in the extracellular matrix (ECM) organization and mineralization regulation. To facilitate the understanding of the mechanisms of biomineralization, it is crucial to expand our knowledge regarding the functions of the NCPs in this process. Therefore, this current study aimed to examine the protein composition of bone and dentin and to identify new components and elucidate their potential functions. Three principal matrix components were identified and subsequently investigated within this thesis; osteoadherin (OSAD), nucleobindin (Nuc) and calreticulin (CRT), and all appear to have a significant role in bone and dentin mineralization. A combination of biochemical, morphological, cellular and molecular biological methods was used to characterize OSAD, Nuc and CRT within bone and dental tissues. OSAD was identified in extracts of bovine dentin and localized in odontoblasts, mineralized dentin matrix, cementum and surrounding alveolar bone, and ultrastructurally, it appeared to be associated with collagen fibrils. Nuc was detected within the tooth in odontoblasts, ameloblasts and dentin matrix, and within bone in the osteoid of newly formed bone, in compact bone and in the various bone cells. In addition, the role of Nuc during the mineralization process was investigated in rat calvaria-derived primary osteoblasts, grown under osteogenic conditions. Low levels of Nuc were detected during cell proliferation, which were subsequently upregulated during differentiation, nodule formation and initial mineralization, followed by a decrease in expression as the mineralization process proceeded. CRT was identified in extracts of bovine dentin and localized within odontoblasts, ameloblasts and, for the first time, extracellularly in predentin matrix. Additionally, to investigate potential functions of the proteins, an in vitro long bone organ culture system was established, using antisense oligonucleotides with the aim of knocking down gene expression and study possible effects on bone formation. From the investigations performed herein, the three proteins studied appear to have significant functions during biomineralization. It could be speculated that OSAD might bind to collagen fibrils and possible act as a regulator of nucleation. Nuc might also play a role as a regulator in the biomineralization of bone and dentin. CRT, on the other hand, appears to play an important role in dentinogenesis and together with Nuc may contribute to the accumulation and transport of Ca2+ ions through the odontoblasts to the mineralization front before hydroxyapatite (HAP) deposition. In conclusion, the studies carried out within this thesis have contributed to the ever emerging picture of the role of NCPs in the dynamic formation of mineralized tissues.