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

Long bone fractures are primarily repaired through endochondral ossification, where 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 fail in cases of large or complex defects, requiring clinical intervention. We developed 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 relevant to endochondral bone regeneration. Encapsulation at different timepoints (days 1, 7, and 14) showed that early encapsulation enhanced spheroid interaction, increased DNA content, and promoted hypertrophic cartilage formation, with greater glycosaminoglycan (GAG) and collagen deposition, and lacunae formation. HAMA-encapsulated spheroids were compared to spheroids formed using a standardized microwell platform, showing that encapsulation supported cartilage-like matrix formation with collagen deposition, Safranin O-positive extracellular matrix and lacunae, while maintaining progression toward a hypertrophic phenotype. Gene expression and immunostaining confirmed progression toward hypertrophic and osteogenic phenotypes. Finally, extrusion-based bioprinting of HAMA containing a high density of hPDC spheroids demonstrated scalability while maintaining cell viability and hypertrophic differentiation, although precise spatial control over spheroid positioning remains to be optimized.