| Objective: Pulp necrosis and periapical periodontitis are common diseases in young permanent teeth which were developing in clinic.As the root of young permanent teeth was not developed,if not treated in time,it would lead to the root resorption,loosening and even falling off.At present,most of the commonly used treatments were apical induction and apical barrier surgery,but this treatment would lead to root under development and incongruity of crown and root ratio.In recent years,some researches have shown that SCAP played an important role in the formation of dentin and the regeneration of root,and it was an ideal seed cell for repairing the defect of apical tissue.Baicalin has been proved to have good anti-inflammatory,anti-swelling and promoting cell proliferation and osteogenic differentiation.The aim of this study was to discuss the probabilities of different concentration of baicalin combined with immature tooth of pulp necrosis and periapical periodontitis.Methods : 1.SCAP were isolated from apical papilla by enzyme digestion and purified by monoclonal method.Flow cytometry was used to detect the expression of the third generation SCAP surface antigen STRO-1,CD146,CD90 and CD45.The cells which were induced in vitro on 14 and 21 days were stained with alkaline phosphatase(ALP),alizarin red and oil red to determine whether the cells had the potential of multidirectional differentiation.2.The third generation SCAP with vigorous growth activity were selected and inoculated in 96 holes plate after passage,then exchanged the culture of DMEM with different concentration of baicalin with 0,10,20,50,100μmol/L in another day.The effect of baicalin on SCAP proliferation were detected by MTT assay in 1,3,5,7 and 9 days.3.The third generation SCAP with high growth activity were divided into two groups:normal medium group(DMEM)and osteoblast induction medium group(ODM).According to the different concentrations of baicalin,the experimental group was divided into five concentration subgroups.The order was0,10,20,50,100μmol/L.The effects of baicalin at different concentrations on the early osteogenic differentiation of SCAP cultured for 7and14 days were studied by ALP staining and ALP quantitative detection.The effects of baicalin at different concentrations on osteogenesis and mineralization after 21 days of SCAP culture were studied by alizarin red staining and quantitative analysis.Results:1.After five days in the culture,the cells were removed from the edge of the tissue block under inverted microscope,and the cells were observed to be long fusiform.The expression of STRO-1、CD90 and CD146 was detected by flow cytometry of SCAP,the rates of expression of STRO-1、CD90 and CD146 were 14.73%,65.45% and 61.63%,respectively.The expression rate of CD45 was 0.19% and negative.After 14 days of induction of the SCAP by the osteogenic induction fluid,the ALP staining showed cytoplasm of cytoplasm in blue-purple,and 21 days after the induction of differentiation,alizarin red staining was observed in cytoplasm of mineralized nodules.The SCAP was induced 21 days after the lipid-induced fluid was induced,and the oil red-o staining was observed in the formation of red lipid droplets in the cytoplasm.2.MTT assay showed that there was no significant difference in OD values measured by the concentration cells in the control group and the experimental group at day 1(p> 0.05).On the 3th day,the OD values of experimental groups with baicalin concentration of 50 and 100μmol/L measured were significantly lower than that of control group(p< 0.05),indicating that contains 50 and 100 μmol/L baicalin concentration of SCAP plays a role of inhibiting proliferation activity.On the 7th day,compared with the control group,the concentration of baicalin 20 μmol/l was significantly higher in the experimental group than that in the control group(p< 0.05),indicating that the baicalin concentration of 20 μmol/l could promote the proliferation of SCAP.3.ALP quantitative results showed that the ALP activity of the group was significantly higher than that in the control group(p<0.05),and the ALP activity in the concentration group was significantly lower than that in the control group(p<0.05).After 14 days of induction of DMEM group,the ALP activity of the 20 μmol /L concentration group was significantly higher than that in the control group(p<0.05).4.Alizarin red quantitatively showed that the OD value of the experimental group of 100 μmol/L in the ODM group was significantly lower than that in the control group(p<0.05).Conclusions:1.Human apical papilla stem cells were successfully isolated by modified enzyme digestion,and osteogenesis in vitro could be proved by monoclonal method,flow cytometry,osteogenic and lipogenic test.SCAP had the potential of multiple differentiation.2.Baicalin could inhibit the proliferation of SCAP in the high concentration group,and could promote the proliferation of SCAP in the low concentration group,of which 20 μmol/L baicalin concentration group was most obvious.3.The baicalin concentration of 20 μmol/L could promote the early osteogenic differentiation of SCAP and 100 μmol/L could inhibit the differentiation of it in both early and late stages. |
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