Hydrogel Delivery of Demineralized Bone Matrix Augmented with ROS-Triggered Biomineralization and Trb3 Activation for Enhanced Bone Regeneration

Abstract

Cranial bone defects remain a significant clinical challenge due to limited intrinsic regenerative capacity and an adverse oxidative microenvironment that impairs osteogenesis. Demineralized bone matrix (DBM), a clinically used bone graft substitute, exhibits osteoinductive potential but suffers from inconsistent performance and poor retention at defect sites. Here, we report a DBM-loaded injectable dynamic hydrogel that enhances bone regeneration through coordinated redox modulation, reactive oxygen species (ROS)-triggered biomineralization, and Tribbles homolog 3 (Trb3)-mediated osteogenic signaling. Black phosphorus (BP) nanosheets were functionalized with nuclear localization signal (NLS) peptides through a branched NLS–PEG construct formed by conjugating NLS to multi-armed PEG, followed by electrostatic assembly onto BP nanosheets and subsequent incorporation into a self-healing hydrogel network via dynamic Schiff base crosslinking. The hydrogel effectively scavenges excessive ROS and restores redox balance, while BP degradation releases phosphate to induce ROS-triggered biomineralization and promote a pro-osteogenic microenvironment. In parallel, the BP/NLS system enables gene delivery and nuclear localization of Trb3 plasmid DNA, leading to enhanced Trb3 expression and promotion of BMP/Smad-mediated osteogenic signaling. These combined effects significantly improve the osteoinductive capacity of DBM and promote enhanced bone regeneration in cranial defects. This study provides a strategy to enhance the therapeutic performance of clinically relevant bone graft materials through integrated microenvironment regulation and gene activation.