Evaluating the Mechanical Properties of Silk Fibroin Scaffolds through Tensile Testing for Use As an in Vitro Culture Platform

One goal for engineered in vitro skeletal muscle models is to answer fundamental questions¹ about rare diseases, especially when animal models fail to recapitulate human disease.² One successful method by which simple skeletal muscle cell-based tissues are engineered is via in vitro addition of human myoblasts into an anisotropic biomaterial scaffold.³ To apply these methods to study the progression of rare muscular dystrophies, our scaffold material must withstand over 8 weeks of mechanical stimulation, to enable time for muscle myoblast growth and maturation (4 weeks) and time for a hypothesis driven experiment. To explore the feasibility of a long-term in vitro culture platform, we quantified tensile mechanical properties of anisotropic silk fibroin biomaterial scaffolds⁴ as a function of fabrication parameters via static and dynamic tensile testing. We evaluated polymer concentration (3% or 5% wt/v silk), freezing rate (50% or 100% ethanol and dry ice⁴), and crystallinity (water annealing or autoclaving). We performed analyses of static and fatigue tests as well as frequency and amplitude sweeps. Young’s modulus and ultimate tensile strength (UTS) was determined by hydrated static testing through stretching different scaffold formations from rest to 100% strain. The different fabrication parameters resulted in tunable Young’s modulus values of 600 kPa to 2800 kPa, relevant to skeletal muscle. Stress-strain curves show that the elastic and plastic response differ between autoclaved and water annealed samples. The fatigue tests were performed via continuous mechanical stimulation at 10% strain and 1 Hz for over 6000 minutes, and results show minimal change in the storage and loss modulus. Lastly, dynamic frequency and amplitude sweeps were performed. Frequency sweeps analyze the impact of the rate of reoccurring 10% strain on the material, a requirement for our in vitro model. The results of the frequency sweeps show that for all formulations of the anisotropic silk scaffold is not frequency dependent below 2 Hz, which is critical for use of these materials for skeletal muscle tissue engineering. Amplitude sweeps analyze the impact of stretching the material from a strain from 0% to complete failure while maintaining frequency value at 1 Hz. The results of the amplitude sweep show that these materials are highly amplitude dependent above 5% strain. Future work focuses on assessing human skeletal muscle myoblasts (HSMMs) within the 3% anisotropic silk scaffolds, 100% ethanol freezing solution, and water annealing, with the addition of decellularized extracellular matrix to improve adhesion and aid in cell maturation.