Objective: Full-thickness cartilage defect are usually accompanied by subchondral bone damage, which is difficult to self-repair once damaged due to the lack of vascularization and innervation. In this study, a biphasic composite scaffold was developed by combining vacuum freeze-drying and iterative freeze-thawing with gelatin, chitosan, silk fibroin, and hydroxyapatite as the basic materials to explore the feasibility of using them for the repair of total cartilage defects. Methods and Results: Six groups of SF-CS-Gel-nHap porous scaffolds (Hap-0%, Hap-1%, Hap- 2%, Hap-3%, Hap-4%, Hap-5%) were prepared by vacuum freeze-drying and chemical cross-linking using filipin protein (SF), gelatin (Gel), chitosan (CS) and hydroxyapatite (Hap) as the base materials. A series of characterization methods were used to systematically analyze and test the morphological features as well as physical and mechanical properties of the scaffolds. Then a novel bionic biphasic porous scaffold was developed by a combination of freeze-drying and freeze-thawing using the SF-CS-Gel as the cartilage phase and the SF-CS-Gel-2%Hap as the subchondral bone phase. Finally, it was co-cultured with chondrocytes to verify the biological properties of the SF-CS-Gel/SF-CS-Gel-2%Hap bionic biphasic porous composite scaffold in vitro. The results showed that the SF-CS-Gel/SF-CS-Gel-2%Hap biphasic scaffolds had a highly porous mesh structure, with an average pore size of 156.06 ± 42.36 μm in the cartilage phase and 214.38 ± 65.82 μm in the subchondral bone phase. Co-cultured with chondrocytes, the live and dead cells stained, cck-8 growth and proliferation curves showed that the bionic scaffolds had good biocompatibility and cytotoxicity. Cytoskeletal staining showed that the morphology of chondrocytes in the bionic scaffolds could maintain three-dimensional growth as in vivo. Conclusion: The results showed that SF-CS-Gel/SF-CS-Gel-2%Hap biphasic scaffolds have good biocompatibility, biodegradability, stability, appropriate mechanical properties and porosity, and are suitable for repairing articular cartilage and subchondral bone. It is expected to be used as a repair material for articular cartilage in clinical applications.
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