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Effects of adenovirus expressing bone morphogenetic protein-4 on different cell types of osteoblastic differentiation

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EAO-2018

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Abstract

Background Bone morphogenetic proteins(BMPs) play a pivotal role in inducing undifferentiated cells into osteoblastic cells that induce bone formation. Aim/Hypothesis In the present study, we investigated the effect of gene delivery of adenovirus expressing BMP-4 on osteoblastic differentiation of different cell types. Materials and methods An adenovirus expressing BMP-4(Ad5BMP4) was constructed by in vivo homologous recombination and transduced MC3T3-E1(mouse osteoblastic cell line), C2C12(mouse myoblastic cell line), NHDF(normal human dermal fibroblast-adult) to induce cell transformation. We assessed BMP-4 expression by ELISA for evaluation of gene expression activity of Ad5BMP4. MTT assay was performed at 3, 7, 15days to assess proliferation of cells that transduced with Ad5BMP4 and alkaline phosphatase activity was measured at 3, 7, 15days to assess osteoblastic differentiation of cells. The effect of Ad5BMP4 on mineralization was determined using alizarin red S staining after 14 days . Results At 3days, all three different cell types that transduced with Ad5BMP4 were observed BMP-4 expression and increased in proportion to the increase of concentration of Ad5BMP4 . Up to 14days, level of BMP-4 was maintained on MC3T3-E1, but remarkably decreased on C2C12 and NHDF compared with 7days. When transduced with Ad5BMP4, the cell proliferation was not inhibited on MC3T3-E1, but significantly decreased on C2C12 compared with not transduced cells at 14days. Cell proliferation of NHDF that transduced with 6.2 pfu/cell concentration of Ad5BMP4 was similar to that of not transduced NHDF, but decreased above the concentration after 7days. To observe the osteogenic abilities of cells that transduced with Ad5BMP4, we investigated alkaline phosphatase(ALPase) activity and mineralization formation. ALPase activities were significantly increased after 7days on MC3T3-E1 and C2C12 and after 14days on NHDF. Mineralization formation was observed on MC3T3-E1, but the other were not observed at 14days. Conclusionand clinical implication Present study demonstrated that gene delivery using adenovirus expressing BMP-4 facilitated osteoblastic differentiation of MC3T3-E1, C2C12, NHDF cells. Also we confirmed the possibility of ex vivo BMP-4 gene delivery that be able to apply to effective technique for regeneration of bone defects.

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