Lei Han1, Xiao-ling Zhang, Xuan Li, Guo-hua Tang. 1. Department of Orthodontics, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China. pinedream@163.com
Abstract
PURPOSE: The purpose of this study was to evaluate the effects of Hedgehog signaling pathway on osteoblast proliferation and differentiation. METHODS: Primary osteoblasts harvested from newborn rat calvarial bone were cultured for 21 days. The temporary expression of Shh and Ihh was detected by RT-PCR. The culture medium was then treated with either a recombinant Shh N-terminals protein (N-Shh) or cyclopamine (cy), a Hedgehog inhibitor. The proliferation of osteoblasts was quantified by MTT and FCM. The osteoblast differentiation and matrix mineralization were evaluated by ALP and Alizarin-Red staining. The expression of Ptch and Smo was quantified by real-time PCR.SAS 8.0 software package was used for statistical analysis. RESULTS: Osteoblast expressed both Shh and Ihh. The expression level of Shh decreased with culture time while the amount of Ihh expression increased. N-Shh treatment increased the cell population and the number of cells in S phase (P<0.05. N-Shh also promoted ALP activities (P<0.05) and bone matrix calcification. Moreover, N-Shh application led to an increase in Ptch and Smo mRNA level (P<0.05). Treatment with cy, on the other side, inhibited osteoblast proliferation and differentiation(P<0.05). CONCLUSION: Heghehog signaling pathway is actively involved in regulating osteoblast proliferation and differentiation.
PURPOSE: The purpose of this study was to evaluate the effects of Hedgehog signaling pathway on osteoblast proliferation and differentiation. METHODS: Primary osteoblasts harvested from newborn rat calvarial bone were cultured for 21 days. The temporary expression of Shh and Ihh was detected by RT-PCR. The culture medium was then treated with either a recombinant Shh N-terminals protein (N-Shh) or cyclopamine (cy), a Hedgehog inhibitor. The proliferation of osteoblasts was quantified by MTT and FCM. The osteoblast differentiation and matrix mineralization were evaluated by ALP and Alizarin-Red staining. The expression of Ptch and Smo was quantified by real-time PCR.SAS 8.0 software package was used for statistical analysis. RESULTS: Osteoblast expressed both Shh and Ihh. The expression level of Shh decreased with culture time while the amount of Ihh expression increased. N-Shh treatment increased the cell population and the number of cells in S phase (P<0.05. N-Shh also promoted ALP activities (P<0.05) and bone matrix calcification. Moreover, N-Shh application led to an increase in Ptch and Smo mRNA level (P<0.05). Treatment with cy, on the other side, inhibited osteoblast proliferation and differentiation(P<0.05). CONCLUSION: Heghehog signaling pathway is actively involved in regulating osteoblast proliferation and differentiation.