An "inside-out"-guided genetically engineered hydrogel for augmenting aged bone regeneration

Senescent bone repair faces significant obstacles due to reduced cellular activity and an unfavorable microenvironment, both of which hinder the osteogenic differentiation of bone marrow-derived stem cells (BMSCs) into osteoblasts (OBs) and subsequent bone formation. Current approaches primarily target senescent cell clearance (senolytics) or suppression of the senescence-associated secretory phenotype (senomorphics), neglecting the complex interactions between BMSCs and the osteogenic microenvironment. In this study, a genetically engineered hydrogel incorporating NAD-dependent deacetylase sirtuins 3 (SIRT3)-loaded nano-vectors and poly (glycerol sebacate)-co-poly (ethylene glycol)/polyacrylic acid (PEGS/PAA) was developed as an "inside-out" strategy for bone regeneration. At the intracellular level, BMSC function is restored, and osteogenesis is promoted through genetically enhanced SIRT3 expression. At the extracellular level, carboxyl functional groups chelate iron ions, simulating a hypoxic environment and promoting synergistic interactions between angiogenesis and osteogenesis. The therapeutic effects of the genetically engineered hydrogel in alleviating senescent damage and enhancing osteogenic differentiation were confirmed in both chemically and naturally induced senescence models <i>in vitro</i>. Local delivery of the hydrogel significantly increased newly formed bone in rat cranial defects. Mechanistically, the central role of SIRT3 in balancing senescence and osteogenesis, as well as its involvement in bone immune signaling pathways, was elucidated through CRISPR/Cas9-mediated editing in mice and transcriptome sequencing. This work presents a novel paradigm that integrates cellular and microenvironmental factors to enhance bone regeneration, offering new hope for treating age-related bone injuries.