Packed for Ossification: High-Density Bioprinting of hPDC Spheroids in HAMA for Endochondral Ossification

Long bone fractures are primarily repaired through endochondral ossification, a process in which a soft cartilage template forms at the injury site and is gradually replaced by bone. While bone has an innate self-healing capacity, this process can be disrupted in cases of large or complex defects, where regeneration fails, and clinical intervention is required. This study aimed at the development of a tissue engineering approach using human periosteum-derived cell (hPDC) spheroids encapsulated or bioprinted at high density within hyaluronic acid methacrylate (HAMA) hydrogels to support hypertrophic cartilage formation as a template for endochondral bone regeneration. We first compared different encapsulation time points (days 1, 7, and 14), finding that early encapsulation (day 1) enhanced spheroid fusion, increased DNA content, and promoted hypertrophic cartilage formation, as indicated by greater glycosaminoglycan (GAG) and collagen deposition along with lacunae formation. Next, HAMA-encapsulated spheroids were compared to spheroids formed using a standardized microwell platform, demonstrating that encapsulation promoted a more mature cartilage-like matrix with thicker collagen fibers and enhanced hypertrophic differentiation. Gene expression and immunostaining confirmed progression toward hypertrophic and osteogenic phenotypes. Finally, extrusion-based bioprinting of HAMA bioinks comprising a high-density of hPDC spheroids demonstrated scalability, improved spheroid alignment, and maintained robust cell viability and hypertrophic differentiation. HA’s bioactivity and regulatory advantages support clinical translation, although achieving spatial control remains an area for further optimization.