Animal Experimental Study Of Yunnan Baiyao Influencing Odontoblast Differentiation On Human Dental Pulp Stem Cells

[Objective] Human dental pulp stem cells (hDPSCs) were cultured in vitro. The hDPSCs-HA/TCP composite was constructed in vitro. The composite was implanted subcutaneously in nude mouse. The aim of study was to investigate whether Yunnan Baiyao can induce hDPSCs into odontoblast in vivo.[Methods] The hDPSCs were isolated and cultured in vitro. Characterization of the phenotype of hDPSCs were detected by flow cytometry. Differentiation potential of hDPSCs was evaluated by osteogenic and adipogenic induction. The hDPSCs were seeded into HA/TCP in vitro, and these composites were divided into three groups to culture. The experimental group was exposed to an concentration of 10μg/ml Yunnan Baiyao solution. The positive group was exposed to 100ng/ml of recombinant human bone morphogenetic protein-2 (rhBMP-2). The control group was exposed to a-MEM containing 10% fetal bovine serum. These hDPSCs-HA/TCP composites were cultured in three groups for seven days. Then they were implanted subcutaneously in nude mouse. After eight weeks, the implants were taken out from nude mouse to detect changing of histology by HE staining, and to detect expression of OC and DSPP by immunohistochemistry (IHC) and qRT-PCR.[Results] 1. The surface antigen markers of hDPSCs were positive for STRO-1, CD 146, CD29, CD 105, and were negative for CD34, CD45 by flow cytometry. The hDPSCs in mineralized medium could formed mineralized nodules, in adipogenic medium could formed lipid droplets.2. HE staining showed that there were new-substrate and odontoblasts-like cells and capillaries in the surface and pore of HA/TCP. Yunnan Baiyao group and positive group was significantly higher than the control group. There was no pulp-dental complex. Some degradation of scaffold was seen.3. The result of IHC showed that OC and DSPP expression of Yunnan Baiyao group and positive group were positive and control group was negative. OC expression of Yunnan Baiyao group was lower than that of the positive group, the difference was statistically significant(P< 0.05). Yunnan Baiyao group and positive group were higher than that of control group, the difference was statistically significant (P< 0.05); DSPP expression of Yunnan Baiyao group and positive group were higher than that of control group, the difference was statistically significant(P< 0.05). There were no significant difference between Yunnan Baiyao group and positive group (P> 0.05).4. The result of qRT-PCR show that OC expression of Yunnan Baiyao group was higher than that of the control group and positive group, the difference was statistically significant (P<0.05); the DSPP expression of Yunnan Baiyao group and positive group were higher than that of control group, the difference was statistically significant (P<0.05), there were no significant difference between Yunnan Baiyao group and positive group (P> 0.05).[Conclusion] 1. The hDPSCs were obtained in vitro by cloning cells. These cells have ability of osteogenic and adipogenic differentiation.2. Yunnan Baiyao has the ability to induce hDPSCs to differentiate into odontoblast.