Mitochondrial-targeted nano-enhancer driving a self-amplifying NAD+ feedback loop to reprogram MSC spheroids metabolism for accelerated bone regeneration.

Mitochondrial oxidative phosphorylation (OXPHOS) plays a crucial role in determining the functional fate of mesenchymal stem cells (MSCs). However, effective strategies to precisely modulate mitochondrial bioenergetics within three-dimensional (3D) MSC spheroids to improve bone repair remain limited. In this study, we have developed a mitochondria-targeted nano-enhancer (BP@PDA-TPP/Q) to enhance the therapeutic potential of MSC spheroids by reprogramming their energy metabolism for bone regeneration. The BP@PDA-TPP/Q nano-enhancer was constructed with black phosphorus (BP) nanosheets as a biodegradable core and polydopamine (PDA) as a stabilizing coating for ligand conjugation and PQQ loading; triphenylphosphonium (TPP) directed the nanocarrier to mitochondria, where the delivered pyrroloquinoline quinone (PQQ) served as an intramitochondrial redox cofactor to drive a self-amplifying NAD+ feedback loop. Within this cascade, the localized PQQ cofactor activates the SIRT3/PGC-1α axis to upregulate nicotinamide phosphoribosyltransferase (NAMPT) and the mitochondrial transporter SLC25A51, which cooperatively accelerate NAD+ salvage biosynthesis and mitochondrial import, thereby continuously sustaining and expanding the intramitochondrial NAD+ pool. Consequently, the metabolic preference of MSC spheroids shifts from glycolysis toward OXPHOS. This metabolic reprogramming significantly improves the osteogenic capacity of spheroids in vitro and leads to substantial bone regeneration in a rat calvarial defect model. Our study introduces a novel nano-therapeutic approach for bone regeneration and broadens the conceptual foundation for advancing stem cell therapy through targeted metabolic regulation.