Early Radial Extracorporeal Shockwave Stimulation on Proximal Tibial Circular Osteotomy Site Enhanced Heterotopic Skin Wound Healing via Small Extracellular Vesicles

Wound healing represents a complex biological process necessitating innovative therapeutic approaches, particularly for chronic wounds such as diabetic foot ulcers (DFUs). Inspired by the regenerative effects of tibial bone transport for DFUs, we explored whether radial extracorporeal shockwave therapy (rESWT) at the tibial circlularr osteotomy site (TOE) could enhance wound healing in rats. We found that TOE accelerated dorsal foot wound healing via small extracellular vesicles (sEV). This sEVs showed strong regenerative effects and modulated macrophage polarizations in vitro and in vivo. Single-cell sequencing revealed mesenchymal stem cells as the likely sEV source, with rESWT triggering sEV release through ATP/P2X7R/p38MAPK pathway. Proteomic analysis identified five differentially expressed proteins in sEVs, with Thbs1-enhanced sEVs enhancing wound healing by promoting IL4-induced M2 macrophage polarization. This innovative approach represents a promising alternative strategy for wound management, with potential for future DFU treatment. Overall design: Single-cell RNA sequencing (scRNA-seq) was performed on all nine samples from circular osteotomy tissues at the proximal tibia to elucidate the cellular origins of the pro-healing sEVs derived from TOE. We established a 3-mm circular osteotomy model at the proximal tibia (TO model) and a concurrent 4-mm full-thickness cutaneous wound on the ipsilateral dorsum of the foot in Sprague-Dawley rats (n=9, 6-8 weeks old). Shock wave therapy was administered to the osteotomy site on postoperative days 2 and 4. Tissue samples from the proximal tibial osteotomy site were collected at 4 and 24 hours after each shock wave session (TOE group), with non-treated osteotomy site samples (TO group) serving as controls. An additional baseline sample (TO_2d_0h) was obtained prior to shock wave intervention on postoperative day 2. Single-cell RNA sequencing (scRNA-seq) was subsequently performed on all nine samples.