Comparative Effects of Hypoxic vs. Normoxic Mesenchymal Stem Cell-Derived Extracellular Vesicles on Tissue Repair Following Volumetric Muscle Loss (VML)

Volumetric muscle loss (VML) is an irreversible muscle injury that results in chronic functional impairment. Mesenchymal stem 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 MSC metabolism, proliferation, and EV production. Hypoxic (3% O ₂ ) preconditioning of MSCs increased the yield of EVs (30-300 nm) by 1.5-fold but decreased the expression of characteristic 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 the retention of EVs at the VML injury site. VML was created by removing ∼20% of the gastrocnemius-soleus muscle's mass in mice using a 3 mm biopsy punch. EVs (4.48×10 ¹⁰ particles/mL) derived from MSCs cultured under hypoxic (Hypo-EV) or normoxic (Norm-EV) conditions were encapsulated within fibrin hydrogels and implanted at the VML injury site. Fibrin hydrogels containing PBS (PFG) were used as controls. On 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 groups 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. We speculate that hypoxic conditions could have caused MSCs to prioritize survival over repair-promoting activities, thereby producing EVs with less pro-regenerative signals. The increased quantity of EVs in response to hypoxia doesn't compensate for their diminished regenerative potential, highlighting the importance of quality over quantity when considering EVs for therapeutic applications.