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.

Keywords: aging; cell-free therapy; exosomes; nanotopography; skeletal muscle repair.

Figure 1

Nanogratings promoted myogenic differentiation of young MPCs. (A) Scanning electron microscope micrographs of PDMS flat substrate (Flat) and nanogratings (NGs). Scale bars = 1 μm. (B) Morphology of MPCs and myotubes on flat and nanograting substrates. The red arrow indicates the nanograting direction. Scale bars = 100 μm. (C) Western blot analysis of MyoD and MyoG expression in MPCs cultured on flat or nanograting substrates. * p < 0.05, *** p < 0.001, two-tailed Student’s t test (n = 4–5). (D) Immunofluorescent images of myotubes stained for MHC (red), nuclei (DAPI; blue). The white arrow indicates the nanograting direction. Scale bars = 100 μm. (E) Quantification of the relative number, length, and fusion index of myotubes formed by MPCs on flat and nanograting substrates. The myotube fusion index is defined as the percentage of nuclei inside the myotubes. * p < 0.05, *** p < 0.001, two-tailed Student’s t test (n = 3). Data are presented as means ± SEM.

Figure 2

Substrate nanotopography altered the biochemical composition of MPC-derived EVs. (A) Representative fluorescence images of young MPCs cultured on flat or nanograting substrates without or with the treatment of GW4869 or Pitsop2. Magenta: MHC; blue: nuclei. CTRL: no treatment control. Scale bar: 100 μm. (B) Quantification of the number and (C) length of myotubes formed by MPCs with or without GW4869 or Pitstop2 treatments. * p < 0.05 (compared to flat CTRL); ## p < 0.01, ### p < 0.001 (compared to nanograting CTRL). One-way ANOVA. (n = 3–5) (D) Western blot analysis and (E, F) quantification of MyoD and MyoG expression in MPCs cultured on different substrates with or without GW4869 treatment. * p < 0.05, *** p < 0.001, One-way ANOVA. (n = 3) (G) Western blot analysis and (H, I) quantification of MyoD and MyoG expression in MPCs cultured on different substrates with or without Pitstop2 treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, One-way ANOVA. (n = 3) (J) Average size and (K) concentration of fEVs and nEVs. EV concentration was normalized to cell protein abundance. ns: no significance, two-tailed Student’s t test. (EV size: n = 6, EV concentration: n = 3). (L) Average spectra of fEVs and nEVs acquired from Raman analysis (spectra taken from n = 4/group). (M) Linear Discriminant Analysis of spectra acquired from fEVs and nEVs. ***p < 0.001, two-tailed Mann–Whitney test. (N) Subtraction spectrum of the differences between average spectra acquired from fEVs and nEVs. Data are presented as means ± SEM.

Figure 3

Cell substrates modulated the function of MPC-derived EVs. (A) EV uptake by aged MPCs. EVs were labeled by PKH26 in red. Cells were stained by phalloidin in green. Blue: nuclei. Scale bar: 50 μm. (B) Representative fluorescence images and (C) quantification of Ki-67 positive aged MPCs after EV treatment. CTRL: no treatment. Red: Ki-67; blue: nuclei. Scale bar: 50 μm. * p < 0.05, ** p < 0.01, One-way ANOVA. (n = 5–9). (D) Western blot analysis and (E) quantification of MHC expression in MPCs after EV treatment. * p < 0.05, One-way ANOVA. (n = 3). (F) Representative fluorescence images and (G) quantification of myotubes formed by age MPCs after EV treatment. The maturation index is defined as the percentage of myotubes containing more than five nuclei. Magenta: MHC; Blue: Nuclei. Scale bar: 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001, nonparametric ANOVA (myotube number) and one-way ANOVA (myotube length, fusion index, and maturation index) (n = 4). Data are presented as means ± SEM.

Figure 4

Optimization of EV treatment strategies using bioengineered aged muscle constructs. (A) Schematic illustration of 3D muscle construct preparation. (B) Schematic illustration of EV administration strategies and timeline. Aged muscle constructs were injured by CTX, followed by EV treatments for a total of 7 days. At 7 DPI, myotube regeneration and muscle construct force recovery were evaluated. In control group (CTRL), injured muscle constructs were cultured in EV-free differentiation media (DM). (C) Representative confocal images of injured muscle constructs (7 DPI) stained for sarcomeric α-actinin (SAA) to show myotube regeneration without or with EV treatment. Red: SAA; blue: nuclei. Scale bar = 100 μm. (D) Quantification of myotube number and (E) myotube diameter of injured muscle constructs without or with the EV treatment at 7 DPI. * p < 0.05, *** p < 0.001, one-way ANOVA. (n = 4) (F, G) Relative twitch and tetanic forces of injured muscle constructs (7 DPI) without or with EV treatment. Force data were normalized to the CTRL group. ** p < 0.01, *** p < 0.001, one-way ANOVA. (n = 4–10). Data are presented as means ± SEM.

Figure 5

Sequential administration of fEVs and nEVs enhanced aged muscle regeneration in vivo. (A) Schematic illustration of EV administration. (B) Representative cross-sectional images of injured TA muscles (12 DPI) with or without EV treatment. Young CTRL: injured TA muscles of young mice injected with PBS; Aged CTRL: injured TA muscles of aged mice injected with PBS; (f+n)EVs: injured TA muscles of aged mice injected with an EV mixture containing 5 × 10⁸ fEVs and 5 × 10⁸ nEVs at both 1 and 5 DPI; (f →n)EVs: injured TA muscles of aged mice injected with fEVs (1 × 10⁹) and nEVs (1 × 10⁹) at 1 and 5 DPI, respectively. Green: laminin; blue: nuclei. Scale bar = 100 μm. (C) Size distribution of myofibers of injured TA muscles with or without EV treatment (n = 5–6). (D) Relative twitch and (E) tetanic force of TA muscles with or without EV treatment (12 DPI). Force data were normalized to the aged CTRL group. * p < 0.05, *** p < 0.001, ns: no significance, one-way ANOVA (n = 5–12). Data are presented as means ± SEM.