| Objectives This study aims to investigate the roles and the mechanisms of injective platelet-rich fibrin(i-PRF)in bone marrow mesenchymal stem cells(BMSCs)proliferation,osteogenic differentiation and adipogenic differentiation.It will provide the basis for application of i-PRF in clinical oral implant repair and other aspects.Methods 1 In this study,rabbit primary BMSC were established by tissue mass method,and the cells were identified by morphological observation and immunofluorescence assay.2 The experiment was divided into the blank control group,PRF group and i-PRF group.Cell Counting Kit-8(CCK-8)was used to detect the proliferation ability of the three groups.3 The activity of ALPase protein in the three groups was detected by ALPase activity assay kit.4 Alizarin red staining was used to detect the formation of osteoblasts and mineralized nodules in the three groups,and oil red O staining was used to detect the formation of adipogenic lipid droplets in the three groups.5 RT-qPCR was used to detect the mRNA levels of osteogenic differentiation-related genes Runx2,BGP and OPN,and the mRNA level of adipogenic differentiation-related gene PPAR?.Results 1 Rabbit alveolar bone tissue-derived cells were successfully established by tissue block assay.The cells exhibited long spindle-shaped.Immunofluorescence assay showed that the cells were negative for hematopoietic stem cell marker CD34,while were positive for the mesenchymal stem cell marker CD44 and STRO-1.Therefore,the cells were identified as BMSCs.The proliferation activities of rabbit BMSCs in the i-PRF group and PRF group on the third,fourth,fifth and sixth days were all significantly higher than that in the blank group(all P<0.05);There was no significant difference of proliferation activity on the three,four and five days between the i-PRF group and PRF group(all P>0.05),whereas the proliferation acitvity of i-PRF group on the sixth day was significantly higher than that of PRF group(P<0.05).This indicated that both i-PRF and PRF have the ability to promote the proliferation of BMSCs,and i-PRF has a stronger ability to promote the proliferation of BMSCs than PRF.3 ALPase activity in BMSCs: On the third,fifth,seventh and ninth days,the ALPase activity of cells in i-PRF group and PRF group were markedly higher than that of the blank control group(all P<0.01),and there was no difference between i-PRF group and PRF group at each time point(all P> 0.05).This suggests that i-PRF has the same effect as PRF on promoting osteogenic differentiation of BMSCs.4 BMSCs calcified nodules: The formation of intracellular calcium nodules in i-PRF group and PRF group was significantly higher than that in blank group(all P<0.05),and there was no significant difference between i-PRF group and PRF group(P>0.05).This indicates that both i-PRF and PRF have the similar ability to promote bone mineralization in BMSCs.5 The mRNA levels of osteogenic genes: The mRNA levels of Runx2,OPN and BGP in i-PRF group and PRF group on the fifth and seventh day were significantly higher than that in the blank group(all P<0.05);There was no difference between the i-PRF group and the PRF group at each time point(all P>0.05).This indicates that i-PRF and PRF have the similar ability to promote the osteogenic differentiation of BMSCs.6 BMSCs lipid droplet formation: The intracellular lipid droplet formation abilities in i-PRF group and PRF group were significantly lower than that in the blank group(all P<0.05),there was no significant difference between i-PRF group and PRF group(P>0.05).This suggests that i-PRF and PRF have the similar ability to inhibit adipogenic differentiation of BMSCs.7 The mRNA level of lipogenic gene PPAR?: The mRNA lelvels of PPAR? in i-PRF group and PRF group were significantly lower than that in blank group on the fifth and seventh days(all P<0.05),while there was no difference between i-PRF group and PRF group at each time point(P>0.05).This indicates that i-PRF and PRF have the similar ability to inhibit the adipogenic differentiation of BMSCs.Conclusions 1 Rabbit alveolar bone derived cells cultured in vitro conform to the characteristics of BMSCs.2 i-PRF and PRF can promote BMSCs proliferation,and the effect of i-PRF on BMSCs proliferation is better than PRF.3 i-PRF and PRF have the similar effect on promoting osteogenic differentiation of BMSCs cells,as well as the mRNA expression of osteogenic genes.4 i-PRF and PRF have the similar effect on inhibiting adipogenic differentiation of BMSCs cells,as well as the mRNA expression of lipogenic gene.Figure 8,Table 4,Reference 78... |
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