Modulation of Primary Chondrocyte Responses by Silk Fibroin Scaffold Architecture In Vitro

Articular cartilage is a highly specialized avascular tissue with limited intrinsic regenerative capacity, making the treatment of cartilage defects particularly challenging. Silk fibroin (SF) is a promising biomaterial for cartilage tissue engineering due to its biocompatibility, tunable degradation, and versatile processing into three-dimensional structures. In this study, two SF scaffolds obtained from the same aqueous fibroin solution were fabricated using freeze-drying (FD) or freeze–thawing (FT) to elucidate how processing-induced architectural differences regulate chondrocyte responses. Morphological analysis showed that FD scaffolds exhibited higher porosity and larger pore size, whereas FT scaffolds displayed a denser structure and significantly higher compressive modulus, highlighting distinct structure–property relationships. ATR-FTIR analysis further revealed a higher β-sheet fraction in FT scaffolds, indicating processing-dependent differences in fibroin molecular organization. These structural differences translated into distinct biological outcomes. FD scaffolds supported higher chondrocyte metabolic activity, consistent with their more open porous architecture. In contrast, FT scaffolds promoted stronger expression of cartilage-related markers, including COL2A1 and ACAN, indicating enhanced chondrogenic maturation. Overall, the results demonstrate that silk fibroin scaffold architecture modulates the balance between cellular metabolic activity and cartilage-specific matrix production. The ability to precisely tune scaffold architecture through processing techniques represents a key strategy for optimizing silk fibroin-based systems in cartilage tissue engineering.