Gene-programmed micro-nano metabolic engine drives coupled osteogenic-angiogenic regeneration

Effective bone healing is accompanied by coupled osteogenic-angiogenic regeneration, a process that critically depends on metabolic communication between bone marrow mesenchymal stem cells (BMSCs) and endothelial cells through an endogenous metabolic axis in which endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine into nitric oxide (NO). However, within hypoxic microenvironments such as those present in large bone defects, the restricted expression of eNOS and the depletion of its substrate synergistically suppress NO biosynthesis, thereby impairing osteogenic-angiogenic signaling coupling and subsequent tissue regeneration. Herein, we developed a gene-programmed micro-nano metabolic engine (GP-MNME) to actively reconstruct NO metabolic homeostasis. GP-MNME features a spatially hierarchical dual-module architecture: the nano-module is fabricated via a nanoprecipitation strategy and consists of amorphous calcium phosphate nanoparticles modified with l-arginine for efficient BMP-2 mRNA loading, while the micro-module is formed by microfluidic-fabricated GelMA microgels covalently conjugated with E7 peptides for BMSC targeting and encapsulation of the nano-modules. In a bone defect, the micro-module enriches BMSCs and releases the nano-modules. Upon cellular uptake, these nanoparticles concurrently upregulate eNOS expression and supply l-arginine to synergistically increase NO production (>3.1-fold). The re-established NO homeostasis serves as the central output of the metabolic engine, subsequently driving osteogenic mineralization and angiogenic sprouting (both > 3-fold), and ultimately promoting coupled osteogenic-angiogenic regeneration in rat bone defect (new bone formation >2.4-fold). Overall, this study presents a proactive metabolic modulation strategy for bone regeneration via reconstructing intracellular NO biosynthesis in situ.