Study On The Induction System Of Differentiating Human Adipose-derived Mesenchymal Stem Cells Into Cartilage In Vitro

Background Treating articular cartilage injury still is a challenge in the field of articular surgery.Cartilage tissue engineering has provided a new strategy for the treatment of osteochondral lesions.Adipose-derived stem cell can be easily harvested and has multidifferentiation potential,which is an important seed cell for cartilage tissue engineering.How to efficiently induce adipose-derived stem cells into chondrocytes is a subject that needs further study.FGF signaling pathway plays an important role in the matrix metabolism of chondrocytes.In this study,Pellet culture was used to investigate the effect of FGF-18 on chondrogenesis of h ADSCs and the induction potential of chondrogenic differentiation on h ADSCs by FGF-18 and TGF-?3 two factor system.Meanwhile,we compare the effects of two different culture modes,scaffold-free pellet culture syestem and fibrin gel scaffold on the chondrocytic differentiation of ADSCs and the synthesis of cartilage extracellular matrix.From the two aspects of induction factor and culture mode,we summarize a culture system that can efficiently induce ADSCs to differentiate into cartilage,and provide the theoretical basis for the further construction of tissue engineering cartilage in vitro.Methods 1?Human adipose tissues were used to isolate h ADSCs that grew in high-glucose DMEM medium containing 10% FBS.After culturing the h ADSCs to the third generation,they were induced into cartilage by different cytokines using Pellet culture system,each pellet contains ADSCs 5×105.Experimental grouping:Blank control group,TGF-?3 group,FGF-18 group and two-factor group.Surface molecules was identified by cell flow analysis.After 5w,cells were observed by hematoxylin-eosin staining,alcian blue staining,safranine O staining and sirius red staining.The total GAG and DNA content of each pellet was determined by DMMB method at week1,week2,week3 and week4.The expression of cartilage related genes was assessed by real-time PCR at week3 and week5.2?After culturing the human adipose-derived stem cells to the third generation,they were induced into cartilage by two kinds of culture mode respectively.After 21 d,cells were observed by hematoxylin-eosin staining and safranine O staining.The GAG content in extracellular matrix was determined by DMMB method.The expression of type?collagen gene was assessed by real-time PCR.Results 1 ? Flow cytometry analysis showed that CD14-CD34-CD73-CD90+CD105+ CD166+,indicating that the cells we isolated expressed stem cell marker molecules.Pathology staining and GAG content detection shows FGF group can effectively induce differentiation of h ADSCs into cartilage,which is similar to the TGF-?3 positive control group.Moreover,synergistic chondrogenic-induction effect can be easily observed in two-factor group,while the synthesis of chondrocyte extracellular matrix was significantly increased.Real-time PCR results showed that FGF-18 group and TGF-?3 group both can promote the expression of chondrocyte marker genes,e.g.Aggrecan,Col2a1,Sox-9 and COMP.The two factor group can synergistically promote the expression of those genes.Col10 was the marker of cartilage hypertrophy,we found that the expression of Col10 was enhanced by FGF-18 and TGF-?3,while the expression of Col10 was the highest in double factor group,indicating that ADSCs tend to differentiate into fibrocartilage in the pellet model.2?Hematoxylin-eosin staining and safranine-O staining showed that the extracellular matrix of chondrocytes was strongly secreted in the fibrin gel group with TGF?3 and fused into a sheet,indicating that the fibrin gel scaffold could better promote cartilage differentiation of stem cells and maintain chondrocyte type.The results of GAG/DNA showed that the ability to secrete GAG in the fibrin gel group with TGF?3 was better than that of the other groups(P<0.05),and the expression of type?collagen m RNA was the strongest(P<0.05).Conclusion We have successfully established a h ADSCs isolation and culture system,found that the effect of FGF-18 on the chondrogenesis of h ADSCs was similar to that of TGF-?3,and FGF-18/TGF?3 two-factor system could significantly promote the differentiation of h ADSCs into cartilage,which was induced by Pellet's three-dimensional culture system.In the modified fibrin gel scaffold,the addition of TGF-?3 inducible factor can successfully induce the differentiation of human ADSCs into cartilage,construct cartilage-like tissue,and the ability to induce human adipose-derived stem cells to differentiate into cartilage by fibrin gel scaffold is superior to that of Pellet culture,the neo-cartilage tissue is closer to natural cartilage in function and structure.Our study are of great value for the application of h ADSCs in cartilage tissue engineering and cartilage repair.