Single-cell transcriptomic-driven design of cascade-responsive hydrogel with bioinspired nanocellular pacemaker for aged bone regeneration.

Age-related bone defects remain poorly repaired due to a vicious cycle involving senescent bone marrow mesenchymal stem cells (BMSCs) and pro-inflammatory macrophages. We unveil circadian disruption as the underlying driver via single-cell transcriptomics and propose an "internal clock-repairing, external immunity-reprogramming" strategy. Inspired by cardiac pacemakers that sense signals and rectify rhythms, we engineer nanocellular pacemakers (AMC) with a "sense-release-regulate" paradigm for aged bone regeneration. AMC comprises melatonin-loaded, cobalt-based metal-organic frameworks (MOF) coated with membrane vesicles derived from the new generation of probiotic Akkermansia muciniphila. The cobalt-based MOF functions as an artificial sensor that specifically senses senescence-associated signals to release melatonin in the senescent microenvironment. The targeted delivery of melatonin repairs the internal clock and rejuvenates BMSCs by precisely regulating circadian rhythms to inhibit p53 and TNF signaling pathways. While probiotic membrane on AMC actively reprograms macrophages toward a pro-regenerative M2 phenotype in the senescent niche. To enable minimally invasive delivery, a dual-network hydrogel with reactive oxygen species-responsive boronic ester bonds is developed for spatiotemporal release of AMC. In aged mice, this cascade-responsive therapeutic system fully repairs bone defects within 4 weeks by breaking the senescence-inflammation vicious cycle, offering a novel "cellular pacing" paradigm for age-related tissue repair.