| Odontoblastic differentiation and dentin mineralization is a complex procedure modified by many factors, but the detailed and intrinsic mechanism was unclear.Serine protease high-temperature requirement protein Al (HtrA1) is implicated in bone mineralization. It contributes to the development of rheumatic disorders and spinal disk degeneration, it also inhibits or promotes physiological bone mineralization. Bone morphogenetic protein-2(BMP-2), one member of TGF-? family, is associated with the promotion of odontoblastic differentiation and dentin mineralization, while matrix Gla protein (MGP) is an inhibitor of dentin mineralization. HtrAl has been shown to inhibit the expression and/or signal transference of TGF-? family and cleave MGP. The role of HtrAl in dentin formation was still unknown. We speculated that HtrA1might regulate dentin formation through modifying the expression of BMP-2and MGP.The present study was divided into two parts for investigating the effects of HtrAl in odontoblastic differentiation and dentin mineralization.Part one:Expression of HtrAl in tooth development, pulp-dentin complex and during reparative dentin formationExperiment one:Immunohistochemical localization of HtrAl during mouse tooth developmentObjective:The aim of present study was to investigate the expression of HtrAl during mouse tooth development.Methods:Samples were collected from Kun-ming mouse at various time points. Immunohistochemistry technique was used to detect the expression of HtrAl during mouse first molar development.Results:From embryonic day13.5(E13.5) to E16.5, HtrA1was expressed in the new born alveolar bone matrix. On E18.5, HtrAl could be detected in the alveolar bone matrix, dentin matrix and dental follicle cells. On the post-natal day0(PO), HtrAl was detected in the alveolar bone matrix, dentin matrix, dental follicle cells and cervical loop epithelial cells. On P5, HtrAl was expressed in the unclacified alveolar bone matrix, dentin matrix, dental follicle cells, Hertwigs root sheath (HERS) and the ectomesenchymal cells adjacent to the HERS. On P14, HtrA1was present in predentin, dental follicle cells, periodontal ligament and the uncalcified alveolar bone, but absent from the calcified alveolar bone and dentin. On P90, HtrA1showed positive staining in predentin, periodontal ligament cells and dental pulp cells.Conclusion:The temporal and spatial expression pattern of HtrA1during mouse tooth development suggested that HtrA1might play a role in mouse periodontal tissue development and dentin formation.Experiment two:Expression of HtrA1, BMP-2and MGP in human pulp-dentin complexObjective:To investigate the presence of HtrA1, BMP-2and MGP in pulp-dentin complex.Methods:Extracted healthy human teeth were collected and prepared for the detection of HtrA1, BMP-2and MGP proteins by immnohistochemistry.Results:HtrA1, BMP-2and MGP showed positive staining in the odontoblasts of human pulp-dentin complex tissue.Conclusion:HtrA1, BMP-2and MGP were co-localized in odontoblasts; they might interact with each other to regulate the odontoblastic differentiation as well as dentin mineralization.Experiment three:The expression of HtrA1and MGP during induced reparative dentin formationObjective:The purpose of the present study was to investigate the expression of HtrA1and MGP during rat induced reparative dentin formation.Methods:Rats were randomly sacrificed after direct pulp-capping on days0,7,14and21. Maxillary segments were obtained and routinely prepared for histological analysis, immunohistochemistry, quantum dots-based double immunofluorescence and CRi's Nuance imaging system-based quantitative determination.Results:The square measure values of reparative dentin significantly increased on day7and continued to increase until day21. HtrA1, MGP, nestin and bone sialoprotein (BSP) were positively stained and co-localized in the odontoblasts and/or odontoblast-like cells zone and the uncalcified reparative dentin during induced reparative dentin formation. The expressions of HtrAl and MGP were significantly enhanced after direct pulp-capping on day7and did not significantly change between days7,14, and day21. Both expressions of HtrAl and MGP were positively correlated with the square measure values of reparative dentin. However, no correlation was found between the expressions of HtrAl and MGP.Conclusion:HtrAl could be observed and might possibly be involved in the process of reparative dentin formation as well as mineralization. It might regulate the process associated with MGP.Part two:The effects of HtrAl in odontoblastic differentiation of human dental pulp cellsExperiment four:Expression of HtrAl, BMP-2and MGP during the odontoblastic differentiation of human dental pulp cellsObjective:The purpose of the present study was to investigate the expression of HtrA1, BMP-2and MGP during the odontoblastic differentiation of human dental pulp cells (hDPCs).Methods:HDPCs were cultured in mineralization medium for28days. Mineralized nodules formation, alkaline phosphates (ALPase) activity and the expressions of the mineralization-related genes including ALP, collage I and DSP were evaluated to determine the odontoblastic differentiation. Simultaneously, the expressions of HtrAl, BMP-2and MGP also were detected.Results:Mineralized nodules formation, ALPase activity and the expressions of mineraliazation-related genes progressively increased in the process of odontoblastic differentiation of hDPCs. Meanwhile, the mRNA and protein levels of HtrAl, BMP-2and MGP also increased in this procedure.Conclusion:HtrA1, BMP-2and MGP were involved in the process of odontoblastic differentiation of hDPCs; they might interact with each other to modify the procedure of odontoblastic differentiation and dentin mineralization.Experiment five:The effects of HtrAl knockdown in odontoblastic differentiation of hDPCsObjective:The purpose of the present study was to determine the mineralized nodles formation, the expression of HtrA1, BMP-2, MGP and mineralization-related genes during the odontoblastic differentiation of hDPCs with HtrA1knockdown by RNA interference, exploring the role of HtrAl in dentin formation.Methods:Two lentiviral vectors of RNAi targeting human HtrA1gene were constructed for knocking down the expression of HtrA1in hDPCs. After packaging, the virus was used to infect hDPCs. Treated the infected hDPCs with odontoblastic induction medium for28days, then mineralized nodules formation, ALPase activity, the expressions of the mineralization-related genes were evaluated. Simultaneously, the expressions of HtrAl, MGP and BMP-2were detected.Results:The mineralized nodules formation decreased significantly in HtrA1knockdown group. In contrast to the scramble group, the ALPase activity as well as the expressions of mineralization-related gene including ALP, collenge I and DSP were downregulated significantly. Meanwhile, HtrA1knockdown led to decreased expression of BMP-2mRNA and increased the mRNA and protein levels of MGP during odontoblastic differentiation.Conclusion:HtrA1promotes odontoblastic differentiation and dentin mineralization; it might regulate the process through modifying the expression of BMP-2and MGP. |
PDF Full Text Request