| Objective The aim of this study is to compare the effects of different concentrations of CGF and PRF extracts on the proliferation of dental pulp stem cells and select the appropriate concentration,investigating the effects of this concentration of two extracts on odontoblastic differentiation and expression of related factors of human dental pulp stem cells.Methods(1)Human dental pulp cells were collected and cultured by tissue block combined with trypsin-digested culture technique.Human dental pulp stem cells were obtained,and their characteristic antigen profiles were identified by flow cytometry.Stem cell properties of h DPSCs were determined under osteogenic and adipogenic induction.(2)The extracts of CGF gel and PRF gel were prepared by freeze-thaw method and concentrations of TGF-β1 and PDGF-AA in both extracts were measured by enzymelinked immunosorbent assay(ELISA).Cells were co-cultured with different concentrations of CGF extracts and PRF extracts then CCK-8 assay was used to detect cell proliferation and select the appropriate concentration.(3)The wound healing assay was performed to observe the effects of 10%FBS,20% CGF and 20% PRF on cell migration ability.(4)Alizarin red staining and ALP staining kit were used to analyze the effects of 10%FBS,20%CGF and 20%PRF on osteogenic differentiation of h DPSCs.(5)q RT-PCR was performed to investigate the effects of 10%FBS,20%CGF and 20%PRF on expression of related specific factors of h DPSCs during odontoblastic induction.Results(1)Human dental pulp stem cells were successfully isolated and obtained by tissue block combined with trypsin-digested culture technique under our lab condition,presenting better proliferation and differentiation ability.(2)The amount of TGF-β1and PDGF-AA in 100%CGF extracts and 100%PRF extracts showed no obvious significance.Different concentrations of CGF and PRF extracts were demonstrated to have different capacity in enhancing the proliferation of h DPSCs,20% of these two extracts were presenting the highest proliferation ability(P<0.05)and 20%CGF group was superior than 20%PRF group(P<0.05).(3)The wound healing assay showed that 20%PRF group and 20%CGF group inhibited h DPSCs migration when compared to 10%FBS group,20% PRF group presented stronger inhibition ability.(4)Alizarin red staining and ALP staining results showed a trend that osteogenic differentiation ability of h DPSCs from top to bottom is 20%PRF group,20%CGF group and 10% FBS group.ALP activity staining showed 20%PRF group and 20%CGF group enhanced osteogenic differentiation of h DPSCs,which is significant compared with the control group(P<0.05).(5)q RT-PCR detection results showed that DSPP expression was the highest in 20%CGF group compared with 20%PRF and 10%FBS group(P<0.05)on day 3.The expression of BMP-2 and TGF-β1 were the highest in 20%PRF group(P<0.05)on day 3.No statistical difference between groups in the expression of DMP-1 on day 3.On day 7,expression of TGF-β1 and DMP-1 were the highest in 20%PRF group and there was no statistical difference among three groups in expression of DSPP and BMP-2.A significant difference in expression of DSPP,BMP-2,TGF-β1 and DMP-1 were confirmed in 20%CGF group and 20%PRF group when compared to FBS group(P<0.05)on day 14.The expression of DSPP and BMP-2 was the highest in 20%CGF group(P<0.05)and 20% PRF had the highest expression of TGF-β1 and DMP-1(P<0.05).Conclusions(1)Human dental pulp stem cells could be isolated and cultured by CD enzyme digestion technique under our lab condition.(2)10%CGF,20%CGF,30%CGF,40%CGF,10%PRF,20%PRF,30%PRF and 40%PRF extracts promoted the proliferation of h DPSCs.20%CGF extracts had the highest stimulating ability in cell proliferation.(3)20%PRF group and 20%CGF group inhibited h DPSCs migration when compared to 10%FBS group,while 20% PRF group presented stronger inhibition ability.(4)20%PRF group presented stronger ability in osteogenic differentiation of h DPSCs than 20%CGF group.(5)20%CGF and 20%PRF extracts could enhance odontoblastic differentiation of h DPSCs.20%PRF had the highest expression of TGF-β1 and DMP-1 while 20%CGF group had the highest in the expression of DSPP and BMP-2. |
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