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Background: Osteoarthritis is a degenerative disease caused by damage or trauma to articular cartilage, leading to pain, brittleness, limitation of joint motions and swelling of the tissue. Cartilage damage is common in older people and also athletes. Chondrocytes dedifferentiate in monolayer culture. Tissue engineering involves the use of scaffold to maintain the differentiation of the cells. In this study, maintaining chondrogenic differentiation of the chondrocytes within the pure silk fibroin (SF) and silk fibroin- chondroitin sulfate- alginate (SF-CHS-SA) was compared. Methods: Pure SF and SF-CHS-SA scaffolds were prepared through lyophilization. The microstructures of the scaffolds were studied by scanning electron microscopy (SEM). Chondrocytes were isolated from the articular cartilage tissue of rabbit and were cultured within the prepared scaffolds for 14 days. The percentage of chondrocytes viability was measured using 3-(5,4-dimethythiazol-2-yl)-5,2-diphenyltetrazolium bromide (MTT) assay, using extract of the scaffolds. Glycosaminoglycan (GAG) secretion and gene expression of collagen II were studied using alcian blue staining and real time polymerase chain reaction (real time-PCR), respectively. Findings: SEM showed that the composite scaffold had higher interconnected pores and pure SF scaffold had mainly closed pores. Results of the MTT assay confirmed no cytotoxicity of the prepared scaffolds. GAG secretion and collagen II expression were significantly higher in the SF-CHS-SA scaffold than the pure SF scaffold (P < 0.05). Conclusion: The SF-CHS-SA scaffold is a much more suitable substrate for maintaining differentiation of the chondrocytes than pure SF scaffold or monolayer culture of chondrocytes.