Objective To fabricate a novel biological scaffold using bone marrow stem cells (BMSCs)-derived extracellular matrix and investigate the feasibility of the scaffold used in cartilage tissue engineering.Methods Bone marrows were isolated from immature New Zealand rabbits to further harvest second-or third-generation BMSCs.ECM derived from BMSCs was collected after high-density culture and was fabricated into a three-dimensional porous scaffold using a freeze-dying technique.Physicochemical properties of the scaffolds were determined by histology,immunohistochemistry,scanning electron microscope,porosity and water absorption measurement,and biomechanical study.Third-generation chondrocytes harvested from knee joints of adult New Zealand rabbits were seeded at a density of 5.0 ×104 cells/ml with Dulbecco' s modified Eagle' s medium (DMEM) (control group),scaffold lixivium at volume fraction of 50％ (study group Ⅰ),and scaffold lixivium at volume fraction of 100％ (study group Ⅱ),respectively.At days 1,3,and 7 after co-culture,MTT test was done to assess the absorbance and cytotoxicity of the scaffold.Results Scaffolds appeared white/silver white/ivory color.HE staining and scanning electron microscopy demonstrated that the scaffolds were cross-linked and pore interconnected with pore size of 100-400 μm,porosity of (91.50 ±2.84) ％,water absorption rate of (2 014 ± 224) ％,and compression strength of (6.80 ± 1.50) kPa.MTT test showed that the chondrocytes were proliferated well in each group and that the absorbance value was increased with time.Besides,the absorbance value was significantly different within groups at each time point (P ＜ 0.05).Conclusion Novel three-dimensional porous scaffolds made from BMSCs-derived ECM are acellular,safe and non-toxic,exhibit favorable pore size and porosity and good biocompatibility,and hence can be good scaffolds for cartilage tissue engineering.