| Objectives:The purpose of this study is to investigate the effect of Yunnan Baiyao on the proliferation and osteogenic differentiation of A-PRF on human periodontal ligament stem Cells(HPDLSCs)and the proliferation and migration of A-PRF on Human gingival fibroblasts(HGFs).Methods:1.Seven healthy volunteers were involved in this study.Ten ml venous blood was extracted from each participant before and after continuous oral Yunnan Baiyao administration for 3 days,according to the recommended doses(500mg/time,4 times/day),then the blood samples were centrifuged at 1300 rpm for 8min to prepare A-PRF.The concentration gradient conditioned mediums containing 1.25%,2.5%,5%,10%and 20%A-PRF were prepared for subsequent cell intervention experiments.2.The primary cells of HPDLSCs and HGFs were obtained by the improved tissue block adherent method.The phenotypes of HPDLSCs were identified by flow cytometry and multidirectional differentiation.The cell phenotypes of HGFs were identified by immunocytochemistry.3.Experimental group:A-PRF(YNB)group(group A);A-PRF group(group B);and blank control group(group C).The proliferation activities of HPDLSCs and HGFs in each group were detected by CCK-8 method,and the optimal concentration of HPDLSCs and HGFs in each group was selected based on the results.ALP kit,alizarin red staining and qRT-PCR were used to detect the ability of osteogenic differentiation of HPDLSCs in each group.And the cell migration ability of HGFs in each group was analyzed by scratch test.4.Statistical methods:All results was analyzed by one-way anova through SPSS 26.0.Results:1.Cell culture and identification:(1)Identification of HPDLSCs:The flow cytometry results were negative for CD34/CD45 and positive for CD105/CD90,indicating that HPDLSCs used in the experiment came from mesenchymal,not hematopoietic.The experimental cells showed fibroblast-like adherent growth,which was consistent with the growth characteristics of HPDLSCs.Alizarin red staining was performed on P3 HPDLSCs after osteogenesis induction for 21 days,and oil red O staining was performed after lipid induction for 28 days.Mineralized nodules and lipid droplets were observed under microscope in the induction group,indicating that HPDLSCs used in the experiment had the ability of multidirectional differentiation.(2)Identification of HGFs:The immunohistochemical identification results of P3 generation HGFs showing typical fibroblast-like adherent growth were positive for anti-vimentin staining and negative for anti-keratin staining,indicating that the experimental cells were mesoderm source without epithelial cell contamination,which was consistent with the development source and growth characteristics of HGFs.2.Effects of Yunnan Baiyao on the cell functions of A-PRF induced HPDLSCs:(1)The results of CCK-8:According to the difference in proliferation activity of HPDLSCs promoted by different concentrations,2.5%concentration of A-PRF(YNB)group and 10%concentration of A-PRF group were selected for subsequent experiments.On day 1,there was no statistical difference among three groups(P>0.05).On day 3,compared with the blank control group,both A-PRF(YNB)group and A-PRF group could significantly promote the proliferation of HPDLSCs(P<0.05),but there was no difference between the two groups.On day 5,compared with the other two groups,A-PRF(YNB)group had a more obvious effect on the proliferation of HPDLSCs(P<0.05).(2)Alizarin red staining results:Compared with the blank control group and the A-PRF group,after 14 days’ bone induction the number of calcium nodules formed by A-PRF(YNB)group increased significantly(P<0.05).(3)ALP activity:On day 3,7 and 14,compared with the blank control group,both A-PRF(YNB)group and A-PRF group increased ALP activity(P<0.05).However,the ALP activity of A-PRF(YNB)group was higher than that of A-PRF group only on day 7.(4)qRT-PCR results:Compared with the blank control group,both A-PRF(YNB)group and A-PRF group could promote the expression of OCN and OPN,and YNB+A-PRF group was significantly better than A-PRF group(P<0.05).There was no significant difference in Runx2 expression among the groups(P>0.05).And the expression of ALP was strongest in the blank control group(P<0.05).The results showed that A-PRF(YNB)group could promote the osteogenic differentiation of HPDLSCs best.3.Effects of Yunnan Baiyao on the cell functions of A-PRF induced HGFs:(1)The results of CCK-8:According to the difference in the proliferation activity of HGFs promoted by each concentration,both the 5%concentration of A-PRF(YNB)group and A-PRF group were selected for subsequent experiments.Compared with the blank control group,both A-PRF(YNB)group and A-PRF group promoted the proliferation of HGFs on day 1,3 and 5(P<0.05).Moreover,on day 3 and 5,A-PRF(YNB)group had better proliferation-promoting effect than A-PRF group(P<0.05).(2)The results of scratch experiment:At 12h,there was no difference in cell migration among three groups(P>0.05).At 24h,compared to the blank control group,both A-PRF(YNB)group and A-PRF group could promote the migration of HGFs(P<0.05),but there was no statistical difference between the two groups(P>0.05).At 48h.compared with the other two groups,A-PRF(YNB)group significantly promoted HGFs’ migration(P<0.05).Conclusions:A-PRF can promote the proliferation and osteogenic differentiation of HPDLSCs,as well as the proliferation and migration of HGFs.Moreover,the A-PRF prepared after taking Yunnan Baiyao further enhanced these effects. |
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