Bioengineered 3D Skeletal Muscle Model Reveals Complement 4b as a Cell‐Autonomous Mechanism of Impaired Regeneration with Aging

Abstract

A mechanistic understanding of cell-autonomous skeletal muscle changes after injury can lead to novel interventions to improve functional recovery in an aged population. However, major knowledge gaps persist owing to limitations of traditional biological aging models. 2D cell culture represents an artificial environment, while aging mammalian models are contaminated by influences from non-muscle cells and other organs. Here, a 3D muscle aging system is created to overcome the limitations of these traditional platforms. It is shown that old muscle constructs (OMC) manifest a sarcopenic phenotype, as evidenced by hypotrophic myotubes, reduced contractile function, and decreased regenerative capacity compared to young muscle constructs. OMC also phenocopy the regenerative responses of aged muscle to two interventions, pharmacological and biological. Interrogation of muscle cell-specific mechanisms that contribute to impaired regeneration over time further reveals that an aging-induced increase of complement component 4b (C4b) delays muscle progenitor cell amplification and impairs functional recovery. However, administration of complement factor I, a C4b inactivator, improves muscle regeneration in vitro and in vivo, indicating that C4b inhibition may be a novel approach to enhance aged muscle repair. Collectively, the model herein exhibits capabilities to study cell-autonomous changes in skeletal muscle during aging, regeneration, and intervention.

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

“…53 Previously, we found that, while young muscle constructs showed increased MPC amplification 1 day post injury (DPI), increases in MPC number were only detected in aged muscle constructs until 7 DPI. 53 More importantly, the aged muscle constructs recapitulated the regenerative responses of aged muscles to different interventions, including EVs. 53 Aged muscle constructs were prepared by culturing aged MPCs in a 3D hydrogel mixture of fibrin and Matrigel and maintained in a PDMS chamber containing two posts that provided physical anchors for attachment to induce the development of aligned myotubes (Figure 4A, S5A, S5B).…”

“…53 More importantly, the aged muscle constructs recapitulated the regenerative responses of aged muscles to different interventions, including EVs. 53 Aged muscle constructs were prepared by culturing aged MPCs in a 3D hydrogel mixture of fibrin and Matrigel and maintained in a PDMS chamber containing two posts that provided physical anchors for attachment to induce the development of aligned myotubes (Figure 4A, S5A, S5B). After 14 days of myotube induction, muscle constructs produced twitch and tetanic contractions upon 1 and 40 Hz electric stimulation, respectively (Figure S5C).…”

“…To evaluate muscle regeneration, we injured aged muscle constructs by treating the constructs with 0.4 μM CTX for 5 h, as we previously described. 53 With the goal of enhancing both MPC proliferation and differentiation to maximize muscle regenerative responses, we designed two strategies to treat injured aged muscle constructs: 1) administration of a mixture consisting of 1/2 fEVs and 1/2 nEVs ((f+n)EVs); and 2) sequential administration first of fEVs to promote proliferation followed by nEVs to induce cell differentiation ((f→n)EVs), as shown in Figure 4B. Injured muscle constructs without treatment were used as controls.…”

“…MPCs were isolated from young and aged mice based on a published protocol using a preplating technique. 53 Briefly, forelimb and hindlimb muscle tissues were minced and digested using collagenase II and Dispase to collect all populations of cells. The cells were then cultured in a collagen-coated T-flask.…”

“…The preparation of aged muscle constructs was conducted by following our previous study. 53 Briefly, 2 × 10 6 cells were suspended in 71.2 μL growth media followed by adding 0.8 μL Thrombin (Millipore Sigma, 100 U/mL in PBS containing 0.1% bovine serum albumin), 20 μL Matrigel (Corning), and 8 μL Fibrinogen (MP Biomedicals, Solon, OH, USA). 85 μL of the mixture of cells and hydrogels was then added to the sterilized PDMS frame and incubated for 25 min at 37 °C for gelation.…”