# Hydroxyapatite-Mediated Mechanical Modulation of GelMA Hydrogels Influences Osteogenic Differentiation of 3D Spheroids

## TL;DR

This study shows that adding hydroxyapatite to hydrogels affects their stiffness and how well they support bone cell development in 3D cultures.

## Contribution

The study reveals a non-linear relationship between hydrogel stiffness and osteogenic differentiation, suggesting optimal rather than maximal stiffness is best.

## Key findings

- GelMA+HAp10 showed the highest alkaline phosphatase activity despite lower stiffness than GelMA+HAp5.
- Higher hydroxyapatite concentrations reduced crosslinking efficiency in GelMA hydrogels.
- Optimizing mechanical properties, not maximizing them, is more effective for bone tissue engineering.

## 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.