Recombinant Spider Silk Enhances Engineered Cartilage Formation

Articular cartilage is characterized by its avascular, aneural, and alymphatic nature, which confers a limited intrinsic capacity for self-repair. Current regenerative strategies primarily focus on alleviating pain, mitigating symptoms, and restoring joint function. However, their long-term efficacy remains uncertain. Cartilage tissue engineering has emerged as a promising alternative to conventional therapies, offering innovative solutions for articular cartilage regeneration. Central to this approach is the development of functional biomaterials capable of supporting chondrogenic cell adhesion, proliferation, and differentiation, thereby facilitating effective cartilage repair. In this study, we introduce a novel protein-based recombinant spider silk (RSS) as a potential biomaterial for modulating chondrocyte behavior and enabling engineered cartilage formation both in vitro and in vivo. RSS was generated through molecular cloning and processed into silk fibers using biomimetic spinning and acidic coagulation techniques. In micromass cultures of murine chondrocytes, RSS significantly promoted cell aggregation, resulting in increased cell density. Alcian blue and Oil Red O staining demonstrated that RSS-treated cultures produced abundant glycosaminoglycans, a hallmark of chondrogenic activity, while exhibiting minimal lipid accumulation. These findings suggest that RSS supports chondrogenic differentiation and suppresses adipogenic lineage commitment. Real-time PCR analysis revealed upregulation of the chondrogenesis-related gene Sox9 and downregulation of the adipogenic marker PPARγ and the hypertrophic marker Runx2 in RSS-treated micromass cultures. RNA sequencing further corroborated these observations, underscoring the role of RSS in modulating extracellular matrix (ECM) remodeling in chondrocytes. In a subcutaneous transplantation model using severe combined immunodeficiency (SCID) mice, chondrocytes encapsulated in three-dimensional hydrogel scaffolds containing RSS exhibited significantly enhanced ECM accumulation compared to RSS-free controls, indicating that RSS supports the maintenance of the chondrocyte phenotype and promotes cartilage formation in vivo, and underscoring its promising potential as a component of hydrogel composite systems. These findings highlight the potential of RSS as a functional biomaterial to preserve chondrocyte functionality and advance engineered cartilage formation, presenting a promising avenue for cartilage tissue engineering and regeneration.