Acute effects of hypoxic vs. normoxic mesenchymal stem cell-derived extracellular vesicle treatment on traumatized muscle

Volumetric muscle loss (VML) is an irreversible muscle injury leading to chronic functional impairment. Murine Mesenchymal Stromal Cell (MSC)-derived extracellular vesicles (EVs) can facilitate tissue repair through immunomodulatory, angiogenic, and anti-fibrotic effects. However, their low yield and poor on-site retention limit their therapeutic efficacy. Hypoxia can boost EV production from MSCs. Hypoxic (3% O 2 ) preconditioning of MSCs increased the yield of EVs by 1.5-fold but decreased the expression of EV markers (i.e., CD81, ICAM, and FLOT1). Fibrin hydrogels promote skeletal muscle regeneration and can sequester EVs via integrins or electrostatic interactions. We hypothesized that encapsulating EVs in fibrin hydrogels would further enhance regeneration and prolong their retention in the injured muscle. EVs derived from MSCs cultured under hypoxic (Hypo-EV) or normoxic (Norm-EV) conditions were encapsulated within fibrin hydrogels and implanted at VML injury created in gastrocnemius-soleus muscle of 129S1/SvlmJ female mice. Fibrin hydrogels containing PBS (PFG) served as controls. Day 14 post-injury, Norm-EV treatment resulted in increased muscle mass, angiogenesis, and myofiber regeneration relative to the Hypo-EV group. Both the Norm-EV and Hypo-EV treatment reduced macrophage infiltration at the injury site compared to the PFG. These findings highlight that while both Norm-EV and Hypo-EV exhibit immunomodulatory effects, they differ in their regenerative potential.