Nanotopographical Cues Tune the Therapeutic Potential of Extracellular Vesicles for the Treatment of Aged Skeletal Muscle Injuries

Abstract

Skeletal muscle regeneration relies on the tightly temporally regulated lineage progression of muscle stem/progenitor cells (MPCs) from activation to proliferation and, finally, differentiation. However, with aging, MPC lineage progression is disrupted and delayed, ultimately causing impaired muscle regeneration. Extracellular vesicles (EVs) have attracted broad attention as next-generation therapeutics for promoting tissue regeneration. As a next step toward clinical translation, strategies to manipulate EV effects on downstream cellular targets are needed. Here, we developed an engineering strategy to tune the therapeutic potential of EVs using nanotopographical cues. We found that EVs released by young MPCs cultured on flat substrates (fEVs) promoted the proliferation of aged MPCs while EVs released by MPCs cultured on nanogratings (nEVs) promoted myogenic differentiation. We then employed a bioengineered 3D muscle aging model to optimize the administration protocol and test the therapeutic potential of fEVs and nEVs in a high-throughput manner. We found that the sequential administration first of fEVs during the phase of MPC proliferative expansion (i.e., 1 day after injury) followed by nEV administration at the stage of MPC differentiation (i.e., 3 days after injury) enhanced aged muscle regeneration to a significantly greater extent than fEVs and nEVs delivered either in isolation or mixed. The beneficial effects of the sequential EV treatment strategy were further validated in vivo, as evidenced by increased myofiber size and improved functional recovery. Collectively, our study demonstrates the ability of topographical cues to tune EV therapeutic potential and highlights the importance of optimizing the EV administration strategy to accelerate aged skeletal muscle regeneration.

Isolation of small extracellular vesicles from regenerating muscle tissue using Tangential Flow Filtration and Size Exclusion Chromatography

“…17 Once the muscle is pellet down remove PBS carefully with a glass fine pipette connected to the aspirator to avoid muscle loss for aspiration. 18 Rapidly resuspend explants with 10 ml of pre-warmed 37C°C FBS EVs-free. 19 Using a 1000 pipette connected to a tip, spot drops of this mixture on the surface of the wet dishes (Fig.…”

“…Particularly, there is an overwhelming evidence for the therapeutic use of EVs in muscle related pathologies [14][15][16][17][18][19][20][21]. In line with this, Wnt proteins, a conserved family of secreted glycoproteins that govern essential developmental, growth and regenerative stem cell processes, are a representative example of long-distance signaling and myoregenerative potential [22].…”