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Background Osteochondral lesion repair is a challenging area of orthopedic surgery.Here we aimed to develop an extraceliular matrix-derived,integrated,biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells (BMSCs) in the repair of a large,high-load-bearing,osteochondral defect in a canine model.Methods The biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT).Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold,then assessed by SEM for cell attachment.Osteochondral defects (4.2 mm (diameter) x 6 mm (depth)) were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold (control group).The repaired defects were evaluated for gross morphology and by histological,biochemical,biomechanical and micro-CT analyses at 3 and 6 months post-implantation.Results The osteochondral defects of the experimental group showed better repair than those of the control group.Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group,and that the scores for the experimental group at 6 months were significantly higher than those at 3 months.The cartilage stiffness in the experimental group (6 months) was (6.95±0.79)N/mm,70.77％ of normal cartilage; osteochondral bone stiffness in the experimental group was (158.16±94.30) N/mm,74.95％ of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was (218±21.6) μg/mg (dry weight),84.82％ of native cartilage.Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months,with no significant difference between the experimental and control groups.Conclusion The extracellular matrix-derived,integrated,biphasic scaffold shows potential for the repair of large,high-load-bearing osteochondral defects.