A mitochondria-derived multicellular 3D-bioprinted gradient scaffold reprograms the bioenergetic microenvironment for functional tendon–bone interface regeneration

Healing failure after tendon–bone interface injury is associated with persistent inflammation-induced mitochondrial dysfunction and pathological metabolic reprogramming, resulting in bioenergetic imbalance, oxidative stress amplification, and loss of interface cell phenotypes. Here, we developed RGD-modified mitochondria-based nanoparticles cloaked with chondrocyte membranes (nMT@CM-RGD) to remodel the inflammatory bioenergetic microenvironment. Through homotypic membrane targeting and integrin-mediated recognition, nMT@CM-RGD enabled efficient cellular uptake, reduced lysosomal sequestration, and enhanced intracellular delivery of mitochondria-associated components. Integrated transcriptomic and metabolomic analyses showed that nMT@CM-RGD shifted inflamed cells from a glycolysis-dominant pathological state toward an oxidative phosphorylation-associated energy phenotype. Further analyses supported that nMT@CM-RGD attenuated inflammatory signaling and improved mitochondrial quality control, in association with modulation of the NF-κB/PKM2 axis and PINK1/Parkin-related pathways. At the multicellular level, nMT@CM-RGD preserved the chondrocyte phenotype, promoted osteogenic differentiation of bone marrow mesenchymal stem cells, and maintained the tenogenic phenotype of tenocytes, thereby supporting multiregional interface regeneration. We further incorporated nMT@CM-RGD into a tri-cellular 3D-bioprinted chondroitin sulfate (CS)/hyaluronic acid methacrylate (HAMA) gradient scaffold to construct MitoGel-Trio, a biomimetic regenerative platform, and evaluated it in a rat rotator cuff injury model. In vivo, MitoGel-Trio improved gait recovery, interface continuity, biomechanical performance, and fibrocartilage-like matrix regeneration. These findings identify inflammatory bioenergetic dysregulation as a potential therapeutic target in tendon–bone healing failure and support the combination of mitochondrial nanoengineering and multicellular 3D bioprinting as a materials-based strategy for functional interface regeneration. Graphical Abstract