Silk Sponges As in Vitro Skeletal Muscle Tissue Model

Introduction: Limb Girdle Muscular Dystrophies (LGMD) encompass over 30 genetic mutations leading to degenerative muscle conditions. Limited patient data hampers LGMD study and treatment. Silk sponges are a biomaterial that can stimulate muscle cell growth in vitro.^1,2 We hypothesize introducing mechanical stimulation to myoblasts during growth will aid functional muscle tissue development. This approach holds potential for enhanced fundamental understanding and treatment strategies for diseases like LGMD.

Materials & Methods: Aqueous silk fibroin from Bombyx mori cocoons was processed using existing protocols for boiling and solubilization.³ The silk solution, diluted to 3% and mixed with porcine muscle decellularized extracellular matrix (dECM), was frozen in an anisotropic manner using a dry ice and ethanol slurry.⁴ After lyophilization (Labconco) and water annealing, the sponges were imaged via SEM (ThermoFischer Scientific) and mechanically evaluated via a tensile test using a rheometer (Anton Parr) with custom immersion cup to maintain hydration of samples.⁵ Sterile sponges were seeded with healthy human skeletal muscle myoblasts (HSMMs, Lonza®), incubated in growth media (GM) at 37°C for 8 days and subsequently fixed for analysis. The sponges were wax embedded and sectioned prior to H&E staining and imaging (Keyence) to assess cell integration.

Results & Discussion: SEM images show the sponges exhibit uniform and aligned pores that resemble the structure of mature muscle tissue formed by natural mechanical forces. Tensile tests simulate these forces and strain rates, producing comparable results for sponges composed of only silk and made of silk and dECM in various media conditions. The Young’s modulus of each sample type showed no significant difference at low strains, indicating that the sponges’ viscoelastic properties for applications in skeletal muscle tissue engineering are unaffected by ingredients in GM. However, at high strain rates, the presence of salts within the media did impact elastic properties. Staining HSMM-seeded dECM sponges showed widespread distribution of cells within the sponges, indicating cell migration from the surface. Future work includes putting HSMM-seeded sponges on a bioreactor to mechanically stimulate the myoblasts and encourage their maturation. These results show promising progress toward generating a human primary cell-derived in vitro muscle tissue model that can be customized to accommodate diseased cells, thereby advancing knowledge and therapeutic strategies for rare muscle diseases such as LGMD.