Effects Of Hypoxia Microenvironment On Tenogenic Differentiation Of Adipose Derived Mesenchymal Stem Cells

Objective: In order to investigate the effect of microenvironment on tenogenic differentiation of adipose derived mesenchymal stem cells and its possible mechanism.Methods: Mesenchymal stem cells(MSCs)were detached from subcutaneous inguinal adipose tissue of SD rat according to our method.ADSCs were cultured and survived in vitro.The third generation was used for later experiments.Flow cytometry was used to detect the surface markers of ADSCs.At the same time,adipogenic and osteogenic induced differentiation experiment was performed to analyze the differentiation potential of the ADSCs.The P3 ADSCs were seeded on 96-well and 6-well plates,randomly divided into 2 groups: hypoxia group and normal group and evaluated their proliferation and migration status by CCK8 assay and scratch assay.Then the concentration gradient experiment was used to detect the optimal blocking concentration.The seeded on 6-well plates ADSCs were randomly divides into4 groups: hypoxia 0?M 2-MeOE2(hypoxia,0?M 2-MeOE2,HM0),hypoxia5?M 2-MeOE2(hypoxia,5?M 2-MeOE2,HM5),hypoxia 10?M 2-MeOE2(hypoxia,10?M 2-MeOE2,HM10),hypoxia 15?M 2-MeOE2(hypoxia,15?M 2-MeOE2,HM15).The cells were treated for 24 h in hypoxic three gas incubator.The Q-PCR assay was used to detect the expression of HIF-1? gene.The P3 ADSCs were seeded on 6-well plates and added 2-MeOE2 with 0?M and the optimal blocking concentration respectively.The treated cells wereplaced in hypoxia and normoxic incubator for 24 h.The expression of SCX mRNA and TNMD mRNA was analyzed by Q-PCRassay.The expressions of HIF-1? and SCX were detected by immunofluorescence staining or Q-PCR.Results: MSCs derived from subcutaneous inguinal adipose tissue were detached and cultured in vitro successfully.The CD29 and CD90 were highly expressed in ADSCs and the CD31 and CD45 were expressed exceedingly low.In addition,The induced differentiation experiment showed that the ADSCs have multiple differentiation potential and can be induced into adipocytes and osteocytes.The CCK8 and scratch assay exhibited that the ADSCs in hypoxia group grew and migrated slower than normxia group(P<0.05).Immunofluorescence and Q-PCR showed the expression of HIF-1? in hypoxia group was significantly higher than normoxia group.After induced 24 h in hypoxia microenvironment,the concentration gradient experiment detected the optimal blocking concentration was 10?M(P<0.05).In normxia group,the ADSCs expressed SCX and TNMD gene have no significant difference between normxia 0?M 2-MeOE2 group(NM0)and normxia 10?M 2-MeOE2group(NM10)(P>0.05).Under hypoxia condition,the expression level of SCX and TNMD of hypoxia 10?M 2-MeOE2 group(HM0)was obviously higher than normxia 10?M 2-MeOE2 group(HM10)group(P<0.05).In 0?M2-MeOE2 group,the mRNA of SCX and TNMD expression was much more than hypoxia group compared with normxia group(P<0.05).Conclusion:ADSCs highly expressed mesenchymal stem cell markers,and has the ability of multidirectional differentiation.ADSCs has a lower ability of proliferation andmigration,but has higher tenogenic differentiation potential under hypoxia microenvironment(5%O2).And ADSCs differentiation into tenocyte may play a role by regulating HIF-1? in hypoxia.Therefore,the hypoxia microenvironment conducive to ADSCs tenogenic differentiation and provide more application prospects broad of ADSCs in tendon tissue engineering.