Effects Of MGF E Peptide Pretreatment On The Proliferation,Differentiation Of MSCs Under CoCl₂-induced Hypoxia

Mesenchymal stem cells?MSCs?have been widely utilized for the cell therapy and regenerative medicine due to its capacity of self-renewal and multi-lineage differentiation.Present studies and clinical detection confirmed that MSCs were involved and played an important role in bone repair process,especially in angiogenesis,immunoregulation and osteogenic differentiation.During bone regeneration,MSCs will migrate to the injured sites,proliferate and differentiate into chondrocytes and osteoblasts for endochondral and intramembranous ossification,respectively.Usually,MSCs always exist under a hypoxic environment?1%-7% pO2?and will endure much severer hypoxia during bone defect healing.As shown in previous studies,severe hypoxia will significantly inhibit the cell viability,proliferation and osteogenic differentiation,which is the currernt urgent needed to be solved.Kinds of growth factors are proved to have positive effect on the regulation of the cell motility,proliferation and osteogenic differentiation of MSCs.Mechano growth factor?MGF?E peptide?abbreviated as MGF in the following?exhibits a superior influence on protecting cells or tissues against hypoxia.Neverthless,whether MGF can regulate the proliferation and osteogenic differentiation of MSCs under severe hypoxia is still unknown.In the present study,we use CoCl₂ to mimic the severe hypoxic environment.Subsequently,we detect the effects of MGF pretreatment on cell proliferation and osteogenic differentiation of MSCs under severe hypoxic environmental condition.The main contents and results of this study are listed below:?1?The hypoxic environmental condition simulated by CoCl₂ and optimize selectionLarge numbers of studies had demonstrated that CoCl₂ could simulate hypoxic microenvironment effectively.The underlying mechanism is that Co2+ will displace Fe2+ in cells,and further prevent the degradation of hypoxia inducible factor 1a?HIF-1a?,by which Co2+ can simulate hypoxia indirectly.In this study,HIF-1a expression,cell survival,and cell activity of MSCs treated with different concentrations?0,100,200,500,800 and 1000 ?M?of CoCl₂ for 24 h were verified,respectively.The results indicated that HIF-1a expression could be increased by effectively with a gradully rising trend with the increase of CoCl₂ concentration and CoCl₂ could stimulate hypoxic environmental condition successfully.After 24 h,cell viabilities of MSCs were changed to 116.85±2.23%,117.89±0.81%,69.03±1.92%,28.46±6.46% and 11.55±1.99% by 0,100,200,500,800 and 1000 ?M CoCl₂ respectively.It was found that cell death obviously occured when the CoCl₂ concentration was greater than 500 ?M.Thus,500 ?M CoCl₂ was chosen as the optimal condition in the following experiments?adverse but non-lethal for MSCs?.What's more,cell activity was reduced to 92.83±2.58%,93.35±3.42%,92.55±5.35%,90.85±4.13%,91.25±5.63%,and 69.23±2.20% after culture for 1,2,4,8,12,and 24 h under 500 ?M CoCl₂.The concentration of 500 ?M CoCl₂ could significantly inhibit cell activity of MSCs but not induce apoptosis,so it was utilized to stimulate severe hypoxia.?2?The effect of MGF on the cell proliferation,apoptosis,morphology,adhesion and migration of MSCs under severe hypoxiaAs shown in the present study,MGF mRNA expression in MSCs was decreased ripidly by different concentrations?0,100,200,500,800,1000 ?M?of CoCl₂ treatment for 24 h,which suggested the potential possibility of MGF involved in the regulatory effect of hypoxia on the physiological and biochemical properties.Through detecting the effects of MGF pretreatment on cell morphology,proliferation,apoptosis,adhesion and migration of MSCs under hypoxia,it was found that cell area and roundness of MSCs were reduced by severe hypoxia.Meanwhile,cell adherence and transwell analysis showed that cell adhesion and migration were inhibited significantly,and cell kinetics was obviously insufficient.Fortunately,MGF pretreatment significantly improved cell activity of MSCs under severe hypoxia.Cell activities of MSCs under severe hypoxia were significantly increased by 53.07%,55.95%,71.23%,61.09% and 58.90% by 5,10,20,50 and 100 ng/ml MGF respectively,and showed similar levels with the normal group.In addition,MGF reduced HIF-1a expression and nuclear transfer in MSCs under severe hypoxic environmental condition,and improved cell roundness and area partly.What's more,MGF also promoted the cell adhesion and migration without any effect on the cell apoptosis of MSCs under severe hypoxia,that is MGF showed no cytotoxicity.?3?The effect of MGF on the osteogenic differentiation of MSCs under severe hypoxiaAlkaline phosphatase?ALP?activity and alizarin red s staining were used to detect the effect of MGF on the osteogenic differentiation of MSCs under severe hypoxic environmental condition.According to the previous studies,ALP activity was not only the marker of cell pluripotency,but also the marker of early stage of osteogenic differentiation of MSCs,and alizarin red s staining analysis could showed the calcium deposition in MSCs intuitively.The results of ALP activity and alizarin red s analyses showed that the osteogenic differentiation of MSCs under severe hypoxia was significantly inhibited,and ALP activity and calcium deposition in MSCs were obviously reduced.However,the osteogenic differentiation of MSCs could be significantly accelerated in MGF plus 500 ?M CoCl₂ group compared with the CoCl₂ alone group.Both ALP activity and calcium deposition were obviously increased.The expressions of osteogenic relative markers?ALP,runt-related transcription factor 2?Runx2?,and bone gamma-carboxyglutamic-acid-containing proteins?BGP??were detected after osteogenic differentiation culture for 0,7 and 14 days.The results showed that the expressions levels of ALP,Runx2 and BGP were decreased at 0,7 and 14 d,but could be recovered by MGF pretreatment at 7 and 14 d.Further,the effect of MGF on the osteogenic differentiation of MSCs with 100 ?M CoCl₂ supplement was detected as well,and the results showed the similar trend with above one,but showed a more obvious appearence.Interestingly,In this present study,it was found that MGF alone also enhanced the osteogenic differentiation of MSCs.Compared with the previous studies,MGF treatment manner in this study was different.Thus,MGF continuous stimulation or once stimulation before differentiation medium was performed in this study,and then osteogenic differentiation of MSCs was detected.The results showed that MGF once stimulation accerlerated the osteogenic differentiation capacity of MSCs,but MGF continuous stimulation inhibited,which was related to the MGF treatment manners.In conclusion,severe hypoxic environmental condition significantly inhibited cell activity,and changed cell morphology,and decreased cell proliferation,mobility and osteogenic differentiation of MSCs,which hindered bone fracture healing.After MGF pretreatment,cell activity was recovered effectively,and cell morphology,proliferation,migration and osteogenic differentiation of MSCs were improved.Besides,MGF pretreatment weakened the inhibitory effect of severe hypoxia on the mRNA expression levels of ALP,Runx2 and BGP.This study explained the regulatory effect of MGF on the cell properties of MSCs under severe hypoxia,and supplied theoretical basis for MGF clinical application of bone fracture healing.