| Elucidation of novel cellular and molecular mechanisms in moderating osteoblast mineralization is of great significance in osteogenesis mechanism research and bone tissue engineering. During the process of osteoblast mineralization, matrix vesicles(MVs) function as an initial or primary nucleation site that may be the prerequisite to subsequent secondary mineralization. Aberrant functions of MVs have been observed in multiple mineralization-defective diseases. MVs are highly enriched in proteins and lipids. Until now, about 2,000 proteins have been identified to be located in MVs. However, only a small set of MVs proteins such as annexins, had been demonstrated to be involved in the osteoblast differentiation. The roles of most of the MVs proteins during mineralization remain unclear. Therefore, we hypothesized that these MVs-enriched proteins could be specific sources to identify novel regulators for physiological and pathological mineralization process.Tenascin C(TNC) is an extracellular matrix glycoprotein synthesized by osteoblasts during bone growth and morphogenesis. It has been established that TNC was implicated in osteoblastic differentiation and mineralization within bone. In mechanism, TNC may act as a mediator of TGF-β-induced new bone formation. Additionally, bone morphogenetic protein(BMP) and Wnt growth factors, or mechanical loading and stress can also increase TNC expression in osteoblasts, but via distinct signaling pathways. However, little is known about the role of TNC in the regulation of osteogenesis and mineralization. In a previous study, we identified TNC as a novel MVs protein, and which raised our speculation that TNC might influence the mineralization process during osteoblast differentiation. Therefore, in this study, we investigated the effect of TNC on mineralization formation and MVs activity in an osteosarcoma-derived osteoblast-like cell line Saos-2.Objective: We aimed to examine the effects of TNC on mineralization in cell and MVs levels in Saos-2 cells and the possible mechanism.Methods:(1) Using mineralization inducing medium containing Ascorbic acid and β-glycerophosphate to promote Saos-2 cell osteo-differentiation and mineralization and establish the cell model of mineralization.(2) Saos-2 cells were divided into four groups: normal control group, inducted group(contained 3 groups). The normal control group were cultured in basis medium(McCoy’s 5A+15% fetal bovine serum+50 U/m L penicillin, and 50 μg/m L streptomycin), osteo-induced group were cultured in mineralization inducing medium(basis medium containing 50 μg/mL Ascorbic acid and 7.5mM β-glycerophosphate). When Saos-2 cells were cultured for 3, 6 and 9 days, the quantities and quality were detected by Alizarin Red.(3) Saos-2 cells were divided into three groups: negative group, RNAi groups(contained 2 groups). The negative control group was cultured in inducing medium and siRNA signed for negative control, while the RNAi groups were cultured with siRNA signed for down-regulation of TNC. When Saos-2 cells were cultured for 3days, the osteoblasts maker genes: ALP, COL1A1 were detected.(4) ALP activity of MVs was performed using p-NPP. Then the activity and ability to forming mineralization nodules were detected by Ca2+.(5) The expression of annexins located in MVs was detected by western blot.Results:(1) Quantitative analysis of mineralized nodules showed that mineralized nodules were six times more than that of control groups. That revealed the successes of establishment of cell model.(2)The expression of TNC was increased with mineralization, and the increase was slightly in earlier stage while the significant increase was found in later stage.(3) The results of RT-PCR and western blot revealed that the expression of TNC was decreased after adding siRNA fragments.(4) The expression of mineralization marker genes: ALP and COL1A1 of Saos-2 cells of RNAi group were obviously lower than that of negative group. Quantitative analysis of mineralized nodules and ALP activity showed that compared with negative control group, mineralized nodules formation in RNAi groups were significantly decreased respectively.(5) ALP activity analysis showed that ALP activity of MVs of RNAi group was lower than that of negative control group. After incubation, mineralization nodules of MVs of RNAi group were also lower than that of negative control group.Conclusions: Tenascin C could regulate the progress of mineralization in osteoblasts, and that may was achieved by regulating the activity and ability to forming mineralization nodules of MVs. |
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