Engineering bioinspired, high-density collagen microgels with tunable intrafibrillar mineralization for accelerated osteogenesis in vitro and bone regeneration in vivo

The development of biomaterials that mimic native bone remains a major challenge in regenerative medicine. Here, we present a bioinspired platform using high-density collagen hydrogels with tunable mineral content. These engineered microenvironments promote rapid osteogenesis in vitro without osteogenic supplements and accelerate bone regeneration in vivo in critical-sized defects. By modulating mineralization, we demonstrate that early mechanosensitive signaling in human mesenchymal stem cells is linked to matrix stiffness and biochemical composition. Within two hours, focal adhesion formation decreased with increasing mineral content, and fully mineralized scaffolds significantly increased nuclear YAP1 localization. By 24 h, RUNX2 expression was markedly increased in fully mineralized scaffolds, with 40.7 ± 3.9% RUNX2⁺ nuclei (p < 0.0001), and this trend persisted at the gene expression level at 3 days. In a rat calvarial defect model, fully mineralized microgels significantly increased bone volume in males at 12 weeks (18.99 ± 2.66 mm³) compared to empty defects (11.60 ± 2.12 mm³, p = 0.0242), whereas females showed no added benefit of full mineralization. Two-way ANOVA confirmed significant effects of sex (p = 0.0006), treatment (p < 0.0001), and their interaction (p = 0.0158). Histological analyses confirmed osteoinductive behavior across all microgel groups and highlighted reduced scaffold degradation and limited cellular infiltration in mineralized conditions. Together, these results demonstrate that tunable intrafibrillar mineralization modulates early stem cell mechanosensing and osteogenic priming in vitro and drives sex-dependent regenerative outcomes in vivo, emphasizing the need to balance scaffold mechanics and degradation to suit the biological context and improve clinical outcomes. STATEMENT OF SIGNIFICANCE: This study introduces a strategy to fine-tune the properties of implantable materials for bone repair using microscale scaffolds with controlled mineral content. By adjusting composition at the nanoscale, our work identifies how early cellular responses can be directed to influence long-term healing. Importantly, the findings reveal that regenerative outcomes vary by sex, emphasizing the need to consider biological differences in biomaterial design. This work offers new insight into how tailored physical environments can guide tissue repair and highlights the potential for precision approaches in bone graft development.