660 nm LED preconditioning programs mitochondrial signaling in BMSCs for functional tracheal cartilage regeneration

Stem-cell-based cartilage regeneration is limited not only by cell delivery, but also by the difficulty of establishing a stable and functionally competent regenerative cell state before implantation. Here, we report a photonic mitochondrial programming strategy in which 660 nm light-emitting diode (LED) irradiation preconditions bone marrow-derived mesenchymal stem cells (BMSCs) to enhance functional tracheal cartilage regeneration. BMSCs were seeded onto egg white-derived ring-shaped porous scaffolds and exposed to defined 660 nm LED irradiation before implantation. Among the tested irradiation conditions, 15 J/cm² optimally promoted BMSC proliferation, chondrogenic differentiation and cartilage-like extracellular matrix deposition, while suppressing fibrotic, hypertrophic and matrix-degrading phenotypes. Mechanistically, 660 nm LED irradiation enhanced cytochrome c oxidase (CCO) activity, mitochondrial membrane potential, adenosine triphosphate (ATP) production and respiratory function, accompanied by moderate reactive oxygen species (ROS) signaling. This mitochondrial bioenergetic-redox response activated the PI3K/Akt/mTOR/GSK3β-SOX9 axis, thereby promoting type II collagen, aggrecan and glycosaminoglycan-rich matrix formation. Pharmacological inhibition of ROS signaling, mitochondrial activity, PI3K or mTOR attenuated the LED-induced chondrogenic program. In a rabbit tracheal cartilage ring defect model, 15 J/cm²-preconditioned BMSC-scaffold constructs improved cartilage-like tissue formation, airway epithelial repair, lumen patency and mechanical support after 30 days of observation. These findings support 660 nm LED irradiation as a controllable mitochondrial preconditioning strategy for stem-cell-based functional cartilage regeneration.