Effects Of A-PRF On Proliferation And Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells In Vitro

Objective To investigate the effects of Advanced platelet-rich fibrin(A-PRF)on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells(HBMSCs)in vitro.Materials and methods Human bone marrow blood was collected under aseptic conditions in vitro and cultured according to the characteristics that stem cells were easily attached to plastic substrates.The third generation of HBMSCs were identified by flow cytometry to detect the expression of cell surface markers and cell differentiated into osteoblasts by means of alizarin red staining.Ten milliliter of blood was taken from volunteers.After centrifugation,the A-PRF fibrin clot was collected and placed in the complete medium for use.Cells cocultured with the different concentrations of A-PRF to detect the proliferative effects of A-PRF on HBMSCs and chose the best concentration with MTT.HBMSCs cocultured in the different conditioned medium containing 10% A-PRF to observe the osteogenic differentiation effects of A-PRF on the cells at different times by means of ALP,alizarin red staining.Gene expression of runt-related transcription factor 2(Runx2),alkaline phosphatase(ALP),bone morphogenetic protein 2(BMP-2)and collagen type I,alpha 1(Col1A1)were detected by quantitative real time polymerase chain reaction(qRT-PCR)to investigate the osteogenic differentiation effects.Results1.The cultured cells in vitro were identified as human bone marrow mesenchymal stem cells by immunophenotypic identification: the positive expression rates of CD44 and CD90 were 94.99% and 98.48%,respectively,and the negative expression rates of CD34 and CD45 were 0.41% and 0.31%,respectively,indicated that the cells were derived from bone marrow mesenchyme.After 21 days of osteogenic induction,a large number of calcium nodules were observed by alizarin red staining under inverted fluorescence microscope,indicated that the cells possess the osteogenic differentiation potential.2.MTT assay showed that different concentrations of A-PRF had no significant effects on cell proliferation on the 1st day,and there was no difference in the average absorbance values between the groups.On the 3rd day,the average absorbance values of the test groups were significantly higher than those of the control group(p<0.05).On the 5th and 7th day,there was a significant difference between the test groups and the control group(p<0.01).The proliferation-promoting effects of A-PRF on cells appeared obviously,especially with 10% A-PRF.The different concentrations of A-PRF stimulated the proliferation of HBMSCs in a dose-dependent way in vitro and 10% A-PRF was selected as the best concentration.3.ALP assay showed that the ALP activity value of the normal culture medium(10% A-PRF)group on the 7th day was significantly higher than that of the normal culture medium group(p<0.01),and the ALP activity value of the osteogenic-induced medium(10% A-PRF)than that of the osteogenic-induced culture medium group(p<0.01).The ALP activity value increased gradually over time.The ALP activity value of the test groups was significantly higher than that of the control groups(p<0.01)on the 14 th day.A-PRF promoted osteogenic differentiation of bone marrow mesenchymal stem cells in the early period.4.Alizarin red staining showed that the normal culture medium(10% A-PRF)group stained deeper than the normal culture medium group.Under the microscope,the shape with most of the cells in the former group were transformed into short fusiform and few cells underwent cell morphology transformation in the latter group,and the number of nodules was significantly higher than that of the latter group by means of semiquantitative analysis.The osteogenic-induced medium(10% A-PRF)group stained deeper than the osteogenic-induced medium group,and the above results confirmed with the counts of calcium nodules under the optical microscope.5.qRT-PCR results showed that the gene expression of Runx2,ALP,BMP2 and Col1A1 increased in normal culture medium(10% A-PRF)group compared with normal culture medium group(p<0.05).Gene expression of Runx2,ALP,BMP2 and Col1A1 were significantly upregulated in the osteogenic-induced(10% A-PRF)medium group compared with the osteogenic-induced medium group(p<0.05).Conclusions1.The whole bone marrow direct adherence method is a simple and effective method for cultivating high-purity human bone marrow mesenchymal stem cells.2.A-PRF can promote the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro.A-PRF combined with HBMSCs has great clinical application potential in promoting bone regeneration.