| Objective: Rich platelet fibrin widely used in the oral tissue regeneration,but its mechanism is not yet clear.The aim of this study was to investigate the cytobiological effects of injectable-platelet rich fibrin(i-PRF)on stem cells from human bone mesenchymal(BMSCs)and the function and mechanism of this cell in the process of osteogenic differentiation in vitro.Methods: The whole bone marrow adherent method was used to obtain mesenchymal stem cells according to their characteristics,and then the cells were identified by flow cytometry to detect cell surface molecules and the multi-differentiation ability of stem cells.The human elbow forearm venous blood was obtained by centrifugation at room temperature 700 rpm for 3 minutes to obtain i-PRF.SEM and human growth factor array membrane were used to detect the microstructure and molecular composition of the specimen.After placing the obtained i-PRF in complete medium,10%,20% and 40% i-PRF were obtained.The control group and each experimental group were stimulated to determine the optimal concentration of subsequent osteogenic differentiation through CCK-8,Cell migration test and live/dead Cell staining.The i-PRF containing 10% and 20% was cultured with BMSCs in vitro with or without induction solution,and was detected by ALP activity,Alizarin red staining,and RT-q PCR.In this study,RT-q PCR and Western Bolt were used to explore the molecular mechanism of i-PRF in promoting osteogenic differentiation of BMSCs.Results: The positive rates of CD90?CD44?CD34 and CD45 on BMSCs were97.65%?96.37%?3.57% and 2.35%.The induction of osteogenesis was positive after21 days of osteogenic differentiation and 28 days of adipogenic induction.Observed under SEM,i-PRF for high density of 3D mesh structure,containing a large number of white blood cells and platelets embedded in its complex fibrous protein structure,and through the growth factor semi-quantitative detection array membrane i-PRF contains growth factors category,which is not only a large number of containing EGF,IGF,PDGF and other known factors,as well as M-CSF-R,and ?-NGF plays has not been reported.CCK-8 results showed that the 3rd day,differences appeared among the groups,until the 7th day,the proliferation of cells containing 10% and 20% i-PRF was higher than that of the blank group.However,in all the experimental groups,20%i-PRF had the most significant effect on promoting cell proliferation at 3,5 and 7 days.And i-PRF showed good biocompatibility by living/dead cell staining.There were no dead cells in all the experimental groups,and the proliferation effect of cells in the10% and 20% i-PRF groups was the most obvious by staining.Moreover,the cell migration experiment further confirmed that 10% and 20% concentration of i-PRF group had the best effect in promoting cell migration.In the process of osteogenesis induction,ALP activity and alizarin red staining showed that 20% i-PRF could induce osteogenic differentiation of BMSCs and significantly promote the differentiation.RT-q PCR results showed that after 7 days of culture,10% and 20% i-PRF significantly promoted the expression of RUNX2 and COL1,but in the expression of OCN and OPN,20% i-PRF in the experimental group could promote their expression,but there was no significant difference.Finally,RT-q PCR and Western Bolt were used to investigate the mechanism of osteogenic differentiation.The i-PRF and U0126 inhibitor stimulated cells showed that the expression of p-ERK1/2 and p-PLC?1increased by 47.2% and 66%,respectively.Compared with the control group,the expression of osteogenic related protein Runx2 was increased by 72.7% after i-PRF stimulation.The expression of Runx2,P-ERK1/2,and P-PLC?1 was inhibited when compared with the corresponding control group by adding the inhibitor U0126.The expression of genes ERK1/2,Runx2 and PLC?1 was increased after i-PRF stimulation.Compared with the groups of stimulated by U0126,these genes were inhibited.Conclusion: i-PRF can induce osteogenic differentiation of BMSCs partially through ERK1/2 pathway and it can promote the expression of PLC?1. |
PDF Full Text Request