The Effects And Mechanisms Of CGF On Osteogenesis And Angiogenesis Of Co-cultured HBMSC And HUVEC

Objective:The effects of concentrated growth factors(CGF)on the proliferation,osteogenic and angiogenic differentiation of human bone marrow derived mesenchymal stem cells(h BMSC)and human umbilical vein endothelial cells(HUVEC)were studied by in vitro cell co-culture experiment.The mechanisms of CGF on osteogenesis of co-cultured h BMSC and HUVEC was preliminarily explored.Methods:(1)Platelet-rich plasma(PRP),platelet-rich fibrin(PRF)and concentrated growth factors(CGF)were prepared by extracting blood from healthy volunteers.Enzyme linked immunosorbent assay(ELISA)was used to detect the concentrations of PDGF-BB,TGF-β1 and VEGF in different platelet-rich hemoderivatives.(2)The specific surface markers of h BMSC were detected by flow cytometry.The differentiation ability of osteogenesis and adipogenesis was detected by staining after inducing osteogenic and adipogenic differentiation of h BMSC.(3)Flow cytometry analysis was used to detect the effects of different concentrations of CGF(2%,5%,10%,20%)on the proliferation of the co-culture of h BMSC and HUVEC.(4)Alkaline phosphatase(ALP)staining and quantitative real-time polymerase chain reaction(q RT-PCR)were used to detect the effects of 2%,5%,10% and 20% CGF on the expression of osteogenic genes(ALP,OCN,COL-1,RUNX2),angiogenic genes(CD31,ANG2,v WF,KDR,VEGF)and CX43 of directly co-cultured h BMSC and HUVEC.The presence of CX43 between h BMSC and HUVEC was verified by immunofluorescence staining.(5)The differences of osteogenic genes and CX43 between the direct and indirect co-culture of h BMSC and HUVEC were analyzed by the indirect co-culture of h BMSC and HUVEC in Transwell chamber.(6)TGF-β1 signaling pathway was blocked by TGF-β1 pathway inhibitor,and exogenous recombinant TGF-β1 was used to act on the direct co-culture of h BMSC and HUVEC.The osteogenic differentiation of co-cultured h BMSC and HUVEC were detected by q RT-PCR to explore the effect of TGF-β1 in CGF in osteogenic differentiation of directly co-cultured h BMSC and HUVEC.Results:(1)PRP,PRF and CGF were successfully obtained.More PDGF-BB,TGF-β1and VEGF were detected in CGF.(2)Morphological characteristics of cells purchased were consistent with the h BMSC,and had good abilities of osteogenic and adipogenic differentiation.The flow cytometry analysis showed the positive expression of CD44 and CD105,and negative expression of CD45 and CD31 on the cell surface,which could be used for subsequent experiments.(3)The number of h BMSC and HUVEC co-cultured directly at a ratio of 1:2was approximately the same on the day 7,10 and 14.When h BMSC and HUVEC were co-cultured directly at a ratio of 1:2 under different concentrations of CGF,the number of h BMSC increased with the increase of CGF concentration on the day 7.When the concentration of CGF was less than 5%,the number of HUVEC increased with the increase of CGF concentration,while the concentration of CGF was more than 5%,the proliferation level of HUVEC decreased with the increase of CGF concentration compared with the number of HUVEC in the direct co-culture group without CGF on the day 7.(4)ALP staining was the most obvious in the direct co-culture of h BMSC and HUVEC under the treatment of 10% CGF.The expressions of ALP,COL-1,RUNX2,OCN and CX43 in the direct co-culture group containing 10% CGF were up-regulated than those in the direct co-culture group without CGF,the difference was statistically significant(P<0.05).Immunofluorescence staining showed the expression of CX43 protein on h BMSC,HUVEC respectively,and on both.In addition,the expressions of CD31,v WF and KDR in the direct co-culture groups with different concentrations of CGF had no significant difference compared with the direct co-culture group without CGF.(5)The expression of ALP in the direct co-culture group containing 10% CGF and direct co-culture group without CGF was significantly higher than that in the indirect co-culture group containing 10% CGF and indirect co-culture group without CGF,the difference was statistically significant(P<0.05).The expression of OPG,OCN and CX43 in the direct co-culture group containing 10% CGF and direct co-culture group without CGF were up-regulated than that in the indirect co-culture group containing 10% CGF and indirect co-culture group without CGF.Whether direct co-culture or indirect co-culture,10% CGF could promote the up-regulation of ALP,OPG,OCN and CX43 compared with the group without CGF.The expressions of ALP,OPG,OCN and CX43 were the highest in the direct co-culture group containing 10% CGF.(6)The expressions of ALP,OPG,OCN and CX43 were all down-regulated in the direct co-culture group containing 10% CGF after TGF-β1 inhibitor was used,and the down-regulated expression levels were similar to those in the direct co-culture group without CGF.The expressions of ALP and OCN in the direct co-culture group containing exogenous TGF-β1 were higher than those in the direct co-culture group containing 10% CGF,and the difference was statistically significant(P<0.05).Conclusions:The direct co-culture of h BMSC and HUVEC changed the effect of CGF on the biological behavior of single-cell of h BMSC and HUVEC.10% CGF can significantly promote osteogenesis of h BMSC directly cocultured with HUVEC.The intercellular communication between the direct co-culture of h BMSC and HUVEC constructed by CX43 may be one of the main regulatory mechanisms.TGF-β1,a major growth factor in CGF,may be involved in regulating the effects of CGF on osteogenesis through the extracellular paracrine signaling pathway.