3D-printed biphasic iPSC-BMSC scaffold for osteochondral repair and chondrogenic stability

Repair of osteochondral defects remains a critical unmet clinical challenge in knee osteoarthritis management, with core bottlenecks of insufficient regenerative capacity at the cartilage-subchondral bone interface and the high propensity for hypertrophy and calcification of bone marrow mesenchymal stem cells (BMSCs) after chondrogenic differentiation. To address these hurdles, we developed a 3D-printed biphasic scaffold. The subchondral bone layer was a composite system of BMSC-laden hydrogel (composed of Alginate Methacryloyl, hyaluronic acid methacrylate (HAMA), polycaprolactone, and gelatin methacrylate (GelMA)) integrated with a polycaprolactone electrospun support skeleton, loaded with bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor 165, to promote osteogenic differentiation of BMSCs. The cartilage layer was an induced pluripotent stem cell (iPSC)-laden hydrogel consisting of decellularized porcine knee cartilage extracellular matrix, HAMA, chondroitin sulfate methacrylate, and GelMA, loaded with BMP-2 and transforming growth factor-β3. This formulation induced chondrogenic differentiation of iPSCs while maintaining their chondrogenic phenotype and reducing hypertrophy/calcification-associated gene expression. Importantly, transcriptomic analysis suggested that PI3K/Akt pathway suppression was most evident during the middle-to-late stage of chondrogenic induction (14-21 d), a window that may be associated with maintenance of the iPSC chondrogenic phenotype and suppression of hypertrophy and calcification. The scaffold exhibited native tissue-matched biomechanical properties, excellent biocompatibility, and sustained growth factor release capacity. In a rabbit knee osteochondral defect model, the scaffold achieved substantial osteochondral tissue regeneration at 16 weeks post-implantation, providing a potential strategy for integrated osteochondral defect repair.