Bone Morphogenetic Protein 2,6,and 9 Regulate The Osteogenic Differentiation Of Immortalized Preodontoblasts

Objective: This study was designed to observe the osteogenic differentiation efficacies of immortalized rat-derived preodontoblasts(iPDBs)transfected with recombinant adenovirus expression vectorsmediated bone morphogenetic protein 2,6,and 9(Ad-BMP2,6,9).Materials and Methods:In vitro studies: We immortalized PDBs by introducing a reversible SV40 T antigen-based immortalization system.The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay was examined to compare cell proliferation capability of iPDBs and PDBs.The effects of Ad-BMP2,6,and 9 on the osteogenic differentiation of immortalized preodontoblasts(iPDBs)were measured by alkaline phosphatase(ALP)activity assays,ALP and alizarin red S staining.The expression of osteogenic markers was evaluated by semi-quantitative reverse transcription and polymerase chain reaction analysis(Semi-QRT-PCR).In vivo studies: To further assess the ectopic bone formation,rat-derived iPDBs were transfected in culture with adenoviral vectors designated BMP2,6,and 9 and subcutaneously or intramuscularly transplanted into mice.Then the ectopic mineralized tissues were evaluated by micro-CT and histological staining.Results: There were several BMPs maintaining endogenous expression in iPDBs,such as BMP2 and BMP9.The overexpression of BMP2,6 and 9could regulate the m RNA expression of those proteins associated with mineralized tissue.Both ALP activity and the formation of mineralized nodule were significantly increased in the Ad-BMP9-transfected group as compared to the Ad-BMP2-transfected group,while the Ad-BMP2-transfected group was slightly stronger than the Ad-BMP6-transfected group.In addition,the most significant formation of hard tissue was also observed in the Ad-BMP9-transfected group.There were more bone-like trabeculae and osteoid matrices formed in the Ad-BMP2-transfected group when compared with the Ad-BMP6-transduced group.Conclusion: The results suggested that BMP2,6 and 9 played a role in the osteogenic differentiation of iPDBs.BMP9 could be treated as an efficacious accelerant for the osteogenic differentiation of iPDBs.In comparison with BMP6,BMP2 overexpression could induce the formation of more mature and dense osteoid tissues.