Hypoxic niches established via endogenous oxygen production in scaffold under anoxia for enhanced bone regeneration

Anoxia remains a challenging problem to effective graft implantation in bone tissue engineering for managing large-size bone defects. One promising strategy is to provide immediate oxygen required for cell viability and graft maturation by introducing oxygen-generating biomaterials. In this study, we present a novel composite oxygen-generating scaffold by integrating oxygen-generating microspheres (OMs) comprised of emulsified calcium peroxides (CPOs) encapsulated in poly (lactic-co-glycolic acid; PLGA) into the gelatin methacryloyl (GelMA) hydrogel. The <i>in vitro</i> results reveal that the scaffold encapsulating 2% (w/v) OMs (OM@GelMA) mildly sustained oxygen production for approximately 16 days, and hence, established hypoxic niches with low oxygen tension (10-46 mmHg) under anoxic culture condition (0.2% oxygen) for the viability of bone marrow-derived mesenchymal stem cells (BMSCs) and their enhanced osteogenic differentiation, which may be induced by activation of HIF-1/β-catenin signaling pathway by the compatibly hypoxic level as one of the underlying molecular mechanisms verified via transcriptome sequencing, western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) tests on <i>in vitro</i> samples. Moreover, the oxygen-generating hydrogel could enhance angiogenesis of human umbilical vein endothelial cells (HUVECs) under anoxia by preserving cell viability, accelerating cell migration, promoting tube formation and activating angiogenic genes and proteins expression. <i>In vivo</i> studies using rat cranial critical-size defect models demonstrated that OM@GelMA significantly enhanced bone regeneration, effectively promoting bone defect repair. In summary, the OM@GelMA, as a novel endogenously oxygen-generating scaffold, holds great potential to facilitate bone tissue regeneration subject to oxygen-deprived scenarios. This study provides a new insight for future research and clinical applications in bone tissue engineering, particularly for large bone defect repair.