Abstract: Objective: To investigate the effects of cartilage extracellular matrix-derived oriented scaffolds on attachment,proliferation,distribution and alignment of chondrocytes seeded in the scaffolds cultured in vitro. Methods: Oriented cartilage extracellular matrix-derived scaffolds were fabricated with freeze-dried method using unidirectional solidification as a freezing process.After labeled with PKH26,canine chondrocytes were seeded onto the scaffolds and incubated for 1,3,5 and 7 days,respectively.Attachment,proliferation and viability of the cells were measured by mitochondrial dehydrogenase activity（CCK-8 assay）.Morphology,distribution and alignment of chondrocytes in the scaffolds were analyzed by invert microscopy,fluorescent microscopy,scanning electron microscopy and H＆E staining.Chondrocytes seeded into non-oriented cartilage extracellular matrix-derived scaffolds were used as controls. Results: Chondrocytes seeded in both scaffolds showed good attachment and viability from day 1 to day 7,most of the cells in both scaffolds were spheric in shape.However,proliferation of chondrocytes seeded in oriented scaffolds（OS） was higher than that of chondrocytes seeded in nonoriented scaffolds（NOS）.Moreover,chondrocytes seeded in the OS,were widely distributed within the scaffolds and aligned themselves along the oriented pore,mimicking a columnar arrangement whereas chondrocytes seeded in the NOS were randomly distributed within the scaffolds and less in number. Conclusion: Cartilage extracellular matrix-derived oriented scaffolds can support chondrocytes to accommodate and guide their growth into cartilagous tissue,and therefore,can be used as a three dimensional scaffold in cartilage tissue engineering.