Heparin Promotes The Induction Ability Of Transforming Growth Factorβ₃ On Rabbit Dental Pulp Stem Cells’ S Osteogenic Differentiation In Vitro

Objective: to investigate the induction ability of Transforming Growth Factor β₃（TGF-β₃） combined with heparin on rabbit dental pulp stem cells’ s osteogenic differentiation in vitro. Methods: Young New Zealand rabbit is choosen for the study,isolate the rabbit dental pulp stem cells with tissue enzymetic digestion method and continue routine culture,when the cells grow to 80-90%,then pass down,the third passage DPSCs is choosen and culture with a medium contain 0, 5u/mL, 10u/mL,20 u/m L heprin respectively,after 9 days of culture obtain the cell growth curve,and choose the most favourable concentration of heparin for later use. The osteogenic inductive study is devided into control group, TGF-β₃group,heparin group and TGF-β₃ + heparin group,the control group is cultured with total medium,the TGF-β₃ group is cultured with medium containing 20μg/L TGF-β₃,the heparin group is cultured with medium containing 5 u/m L heparin,the TGF-β₃+heparin group is cultured with medium containing 5 u/mL heparin and 20μg/L TGF-β₃.alkaline phosphate activity assay,immunocytochemistry assay,and Alizarin red staining is conducted,for heparin group we conducted RTQ- PCR to tese the mRNA expression of osteogenic related markers as RUNX-2,BSP. Result:the rabbit DPSCs isolated by tissue enzymetic digestion method grows well in the inducing system,the 9days of culture in different concentration Of heparin reveal that the concentration 10u/m L,20u/m L inhibit the growth of DPSCs,how ever the concentration 5u/m L heparin has no influence on the growth of heparin,indicating that this concentration has no damage on the DPSC.the ALP activity of TGF-β₃+heparin group on 7,14 is[（27.20±1.01）、（29.06±1.39）u/mg·pro]higher than the TGF-β₃ group’s [（12.69±1.03）、（9.44±1.05）u/mg·pro] and the control group’s[（5.96±3.68）、（6.10±3.66）u/mg·pro]（P<0.01）,TGF-β₃ group is higher than the control group（P<0.001）;on the day 7 of osteogenic induction RUNX-2 is positive in TGF-β₃ group,heparin group,and TGF-β₃+heparin group.OC is positive On day 14 th in the TGF-β₃ group,TGF-β₃+heparin group,wich is negative in heparin group and the control group,the 21 th day’s Alizarin red staining is positive in TGF-β₃ group,and TGF-β₃+heparin group,negative in control group and the heparin group.the RTQ-PCR results reveal that when comparing with control groups heparin group’s mRNA expression of RUNX-2 is rised,but the mRNA expression of BSP is not rised.Conclusions Heparin posess the role of Promoting The Induction Ability Of Transforming Growth Factor β₃ On Rabbit Dental Pulp Stem Cells’ s osteogenic differentiation In Vitro.