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Previous studies have shown that novel scaffold-free self-assembled constructs can be an ideal alternative to cartilage tissue engineered based on scaffolds,which has many limitations.However,many questions remain,including the choice of seeding cells and the role of growth differentiation factor 5 in constructing self-assembled engineered cartilages.Moreover,whether the optimum construct is effective in human chondral defect repair is still unknown.In this study,we generated self-assembled constructs of human MSCs (hMSCs) using four different approaches: direct self-assembly of hMSCs with or without GDF-5 and predifferentiated hMSC self-assembly with or without GDF-5.Histological,immunohistochemical,and biochemistry analyses indicated that the constructs generated from predifferentiated hMSCs induced by GDF-5 (Group D2) exhibited up-regulated glycosaminoglycan and type Ⅱ collagen expression and contained higher amounts of GAG and total collagen than any other group.After 3-weeks of in vitro culturing of the contructs in a chondral defects explant culture system,the contructs from Group D2 exhibited stable adhesion to the surface of the cartilage matrix.Moreover,the repair tissue was stained positively with Toluidine blue,Safranin O,and type Ⅱ collagen.These data demonstrated that the generation of self-assembled tissue-engineered cartilage from chondrogenically differentiated hMSCs induced by GDF-5 is a promising therapeutic strategy for cartilage repair.