Abstract
Substrate stiffness critically regulates osteogenic differentiation, yet systematic identification of optimal mechanical conditions in three-dimensional culture remains limited. This study investigated how hydroxyapatite (HAp)-mediated mechanical modulation of gelatin methacryloyl (GelMA) hydrogels influences osteogenic differentiation of encapsulated SAOS-2 spheroids. GelMA hydrogels with HAp at 5, 10, and 15 μg/mL were characterized for mechanical properties and used to encapsulate pre-formed spheroids under osteogenic conditions. GelMA+HAp5 achieved the highest compressive modulus, while higher HAp concentrations reduced crosslinking efficiency. All formulations maintained comparable viability and metabolic activity. Notably, GelMA+HAp10 produced the highest alkaline phosphatase activity at Days 7 and 14, despite lower stiffness than GelMA+HAp5, demonstrating a non-linear relationship between substrate mechanics and osteogenic response. These results establish that optimizing rather than maximizing mechanical properties represents a more effective scaffold design strategy for bone tissue engineering.