The Combined Effects Of Mechanical Stretch And Recombinant Human Erythropoietin On Rat Bone Mesenchymal Stem Cells

Objectives1.To evaluate the osteogenic differentiation of recombinant human erythropoietin(rhEPO)with different concentrations on rat bone mesenchymal stem cells(BMSCs)and choose a concentration for further investigation with the highest ratio of osteogenic effectiveness to dosage.2.To investigate the combined effects of mechanical stretch and rhEPO on BMSCs.3.To understand whether or not extracellular signal-regulated kinase(ERK1/2)signaling pathway was involved in the enhancement of osteogenic differentiation by mechanical stretch and rhEPO on BMSCs.Methods1.BMSCs were isolated and cultured by whole bone marrow adherent method.After identification by flow cytometry,the third generation(P3)cells were selected for this study.L-DMEM containing 0.5%fetal bovine serum(FBS)was used as the culture medium,and the cells were treated with different concentrations of rhEPO(5 IU/ml,10 IU/ml,20 IU/ml and 40 IU/ml).Cells untreated with rhEPO defined as the control group.After 7day of culture,the proliferation of the cells was detected by CCK-8,and the osteogenic differentiation of the cells was detected by ALP activity.The optimal concentration of rhEPO(20 IU/ml)was used in the later study.2.The P3 cells were divided into four groups based on treatment methods:control group(CON),mechanical stretch(STR,1Hz,strain rate 10%,4h/time,2 times/d)group,the optimal concentration of rhEPO(E20)group and the mechanical stretch combined with rhEPO(STR+E20)group.After 7day treatment,CCK-8 was applied to detect the proliferation of the cells.The colony formation of the cells was detected by colony formation assay(CFA).The cell cycle of the cells was detected by flow cytometry.The migration of the cells was detected by Transwell assay.The osteogenic differentiation of the cells was detected by ALP staining and ALP activity.The gene expression of Ets-1,C-myc,Ccnd1,C-fos,ALP,OCN,COL and Runx2 was detected by qPCR.3.The P3 cells were divided into four groups based on treatment methods:CON,STR,E20 and STR+E20.After 7day treatment,the expression level of ERK1/2 and p-ERX1/2 was detected by Western blot,respectively.The P3 cells were divided into four groups based on treatment methods:CON,STR+E20,CON+blocking,STR+E20+blocking.Osteogenic induced medium was used to induce cells' osteogenesis while U0126(10 ?M)was used as ERK blocker.After 7day treatment,the osteogenic differentiation of the cells was detected by and ALP activity.Results1.The P3 cells were homogeneous and adhered to fibroblast-like adherents and the immunophenotyping of the cells was detected by flow cytometry.The results showed that the cells high express the CD79 and CD90,low express the CD11b and CD45.These results are compliance with the recognized BMSCs immunophenotyping.2.The OD values of CCK-8 were increased and the ALP staining became darker of the cells which subjected to different concentrations of rhEPO.Besides,when the concentration of rhEPO was 20 IU/ml,the OD value was the highest and ALP activity increased significantly.3.Considerable changes were revealed in the OD value,the number of colony formation,the number of cell migration,the staining of ALP,and the gene expression of the relatives,for cells subjected to mechanical stretch alone,rhEPO alone,and mechanical stretch combined with rhEPO,respectively.Nevertheless,the intervention of mechanical stretch combined with rhEPO yielded the largest effects.4.The relative expression of p-ERK1/2 increased after subjected to different treatments while mechanical stretch combined with rhEPO yielded the largest effect.After treated the cells with blocker alone with osteogenesis induction or STR+E20 treatment for 7d,the cells' ALP activity decreased significantly.Conclusions1.rhEPO promoted the proliferation and osteogenic differentiation of rat BMSCs in a concentration-respondent manner.A concentration of 20 IU/ml was therefore chosen for further investigation with the highest ratio of osteogenic effectiveness to dosage.2.Mechanical stretch combined with rhEPO promotes the proliferation,migration and osteogenic differentiation of rat BMSCs.3.ERK1/2 pathway was involved in the process of mechanical stretch+rhEPO induced osteogenic differentiation of rat BMSCs,and the osteogenic differentiation effect was weakened obviously when the pathway was blocked by the blockers.