Synergistic Tuning of Pore Architecture and Mechanical Properties in a Biomimetic COL-HA-PVA Hydrogel Directs BMSC Chondrogenesis for Osteochondral Regeneration

Engineering hydrogels that simultaneously provide interconnected porosity for cell infiltration while delivering appropriate mechanical cues to direct stem cell fate remains a critical challenge in cartilage tissue engineering. Herein, we report a biomimetic COL-HA-PVA hydrogel scaffold with systematically tunable pore sizes (1.2, 1.4, and 1.6 mm) and concomitant mechanical properties, enabling the co-regulation of the structural and biomechanical microenvironment. The 1.2 mm scaffold exhibited the lowest and most stable compressive modulus across the physiologically relevant strain range, providing a compliant mechanical microenvironment that promoted BMSC chondrogenesis, coupled with enhanced surface hydrophilicity due to collagen functionalization. In vitro, this optimized pore architecture significantly promoted goat bone marrow mesenchymal stem cell (gBMSC) adhesion, spreading, and sustained proliferation over 14 days. More importantly, the 1.2 mm scaffold directed robust chondrogenic differentiation, as evidenced by markedly increased expression of the genes and proteins SOX9, COL-II, and aggrecan relative to larger-pore counterparts. Mechanistically, the scaffold activated the integrin β1-FAK-RhoA/ROCK mechanotransduction axis to enhance SOX9-mediated transcription while suppressing osteogenic markers RUNX2 and COL-I. In a caprine model of full-thickness osteochondral defects, the BMSC-laden 1.2 mm scaffold achieved seamless integration with host tissue, progressive subchondral bone regeneration, and formation of proteoglycan-rich hyaline-like cartilage, as confirmed by micro-CT, histological staining, and GAG quantification. This work demonstrates that synergistic tuning of pore architecture and mechanical properties represents a powerful design strategy for directing stem cell-based cartilage regeneration, offering mechanistic insights into scaffold-guided cell fate determination.