The Effect Of Yunnan BaiYao On The Osteo/Odontogenic Differentiation Of Human Dental Pulp Stem Cells In Vitro

[Objective] To evaluate the effect of Yunnan Baiyao on the osteo/odontogenic differentiation of human dental pulp stem cells (hDPSCs), to investigate formation of mineral nodules and expression of alkaline phosphatase (ALP), dentin sialopho-sphoprotein (DSPP), osteocalcin (OCN) and dentin matrix protein 1 (DMP1).[Methods] The hDPSCs from human dental pulp tissues were isolated and cultured by the tissue block enzymolytic method, and the limiting dilution was used to obtain the relative homogeneous hDPSCs. Flow cytometry was used to analyze the surface markers of hDPSCs, inducing differentiations of odontoblast lineages and adipocyte lineages. The fourth generation of hDPSCs were used, a-MEM of 10% FBS was added into the control group, Yunan Baiyao of six different concentrations (1.25μg/ml, 2.5μg/ml,5μg/ml,10μg/ml,20μg/ml and 40μg/ml) and a-MEM of 10% FBS were added into the YunnanBaiyao groups. MTT assay was used to detect the proliferation of hDPSCs. At the same time, to test the formation of the mineral nodules of hDPSCs stimulated by Yunan Baiyao of six different concentrations (1.25μg/ml,2.5μg/ml, 5μg/ml, 10μg/ml,20μg/ml and 40μg/ml) on the 3rd and 7th day. Choosing Yunnan Baiyao at the concentration of 10μg/ml according to the previous results to conduct further experiments. Osteogenesis medium was added into the blank control group, osteogenesis medium and Yunnan Baiyao of 10μg/ml was added into the Yunnan Baiyao group. Observing the mineral nodules stained with alizarin red and detecting the expression of ALP, DSPP, OCN and DMP1 by real time PCR on the 3rd,7th and 14th day.[Results] The cells showed more proliferative at the 3 to 5 days of primary culture, obtaining the relative homogeneous hDPSCs by the limiting dilution. Flow cytometry results showed that CD29, CD90, CD 105, CD 146 and STRO-1 expressed positively, but CD34 and CD45 expressed negatively. Mineral nodules were heavily stained with alizarin red, and lipid droplets were stained red following the Oil red O staining. Compared with the control group,10μg/ml of Yunnan Baiyao promoted the proliferation (P<0.05). Alizarin red staining showed that 10μg/ml of Yunnan Baiyao promoted the formation of mineral nodules (P<0.05), and the expression of ALP, DSPP, OCN and DMP1 was up-regulated in different levels by real time PCR (P<0.05). [Conclusions]1.10μg/ml Yunnan Baiyao promoted the proliferation of hDPSCs;2.10μg/ml Yunnan Baiyao promoted the formation of mineral nodules;3.10μg/ml Yunnan Baiyao up-regulated the expression of ALP, DSPP, OCN and DMP1.