In vitro and in vivo effects of the overexpression of osteopontin on osteoblast differentiation using a recombinant adenoviral vector

Osteopontin (OPN) is a highly acidic secreted phosphoprotein that binds to cells via an RGD (arginine-glycine-aspartic acid) cell adhesion sequence that recognizes the alphaVbeta3 integrin. OPN may regulate the formation and remodeling of bone. To elucidate the function of OPN in bone tissue, we examined the overexpression of OPN in osteoblasts in vitro and in vivo using an adenoviral vector carrying an OPN cDNA (Adv-OPN). Rat bone marrow-derived osteoblasts infected with Adv-OPN were examined by Western blotting, immunofluorescence, nodule formation measurements, assay of alkaline phosphatase (ALP) activity, and Northern blotting. The results suggested that not only osteoblast differentiation markers such as osteocalcin and ALP, but nodule formation and ALP activity are markedly enhanced by OPN overexpression in the case of viral infection. On the contrary, when Adv-OPN and uninfected osteoblasts were implanted into subcutaneous sites with a porous ceramic scaffold, the ALP activity and calcium content of the OPN-infected composite were higher than in uninfected composites, however, the differences were smaller than expected from the in vitro experiments. We speculate that the difference in the result of in vitro and in vivo experiments originates from the inhibitory effect of secreted OPN on the crystal growth of apatite in vivo, which competes with the induced activity of osteoblasts.     

Recombinant human bone morphogenetic protein-2 promotes Indian hedgehog-mediated osteo-chondrogenic differentiation of a human chondrocytic cell line in vivo and in vitro

We examined osteo-chondrogenic differentiation of a human chondrocytic cell line (USAC) by rhBMP-2 in vivo and in vitro. USAC was established from a transplanted tumor to athymic mouse derived from an osteogenic sarcoma of the mandible. USAC usually shows chondrocytic phenotypes in vivo and in vitro. rhBMP-2 up-regulated not only the mRNA expression of types II and X collagen, but also the mRNA expression of osteocalcin and Cbfa1 in USAC cells in vitro. In vivo experimental cartilaginous tissue formation was prominent in the chamber with rhBMP-2 when compared with the chamber without rhBMP-2. USAC cells implanted with rhBMP-2 often formed osteoid-like tissues surrounded by osteoblastic cells positive for type I collagen. rhBMP up-regulated Ihh, and the expression of Ihh was well correlated with osteo-chondrogenic cell differentiation. These results suggest that rhBMP-2 promotes chondrogenesis and also induces osteogenic differentiation of USAC cells in vivo and in vitro through up-regulation of Ihh.     

Cbfa1/Runx2与成骨细胞分化调控

成骨细胞是由间充质干细胞经骨原细胞和前成骨细胞分化而来的。近年来已鉴定转录因子Cbfal(core binding factor α1)是成骨细胞分化和骨形成的关键调控因子。在成骨细胞分化的过程中,Cbfal通过调控成骨细胞特异性细胞外基质蛋白基因的表达和成骨细胞周期参与成骨细胞的分化过程。新近发现Cbfal能通过自身的PST序列区域与Smads结合形成复合物共同参与成骨细胞的分化调控。