Impact of crystalline domains on long-term stability and mechanical performance of anisotropic silk fibroin sponges

Sponge-like materials made from regenerated silk fibroin biopolymers are a tunable and advantageous platform for in vitro engineered tissue culture and in vivo tissue regeneration. Anisotropic, three-dimensional (3D) silk fibroin sponge-like scaffolds can mimic the architecture of contractile muscle. Herein, we use silk fibroin solution isolated from the cocoons of Bombyx mori silkworms to form aligned sponges via directional ice templating in a custom mold with a slurry of dry ice and ethanol. Hydrated tensile mechanical properties of these aligned sponges were evaluated as a function of silk polymer concentration (3% or 5%), freezing time (50% or 100% ethanol), and post-lyophilization method for inducing crystallinity (autoclaving, water annealing). Hydrated static tensile tests were used to determine Young's modulus and ultimate tensile strength across sponge formulations at two strain rates to evaluate rate dependence in the calculated parameters. Results aligned with previous reports in the literature for isotropic silk fibroin sponge-like scaffolds, where the method by which beta-sheets were formed and level of beta-sheet content (crystallinity) had the greatest impact on static parameters, while polymer concentration and freezing rate did not significantly impact static mechanical properties. We estimated the crystalline organization using molecular dynamics simulations to show that larger crystalline regions may be responsible for strength at low strain amplitudes and brittleness at high strain amplitudes in the autoclaved sponges. Within the parameters evaluated, extensional Young's modulus is tunable in the range of 600–2800 kPa. Dynamic tensile testing revealed the linear viscoelastic region to be between 0% and 10% strain amplitude and 0.2–2 Hz frequencies. Long-term stability was evaluated by hysteresis and fatigue tests. Fatigue tests showed minimal change in the storage and loss modulus of 5% silk fibroin sponges for more than 6000 min of continuous mechanical stimulation in the linear regime at 10% strain amplitude and 1 Hz frequency. Furthermore, we confirmed that these mechanical properties hold when decellularized extracellular matrix is added to the sponges and when the mechanical property assessments were performed in cell culture media. We also used nano-computed tomography (nano-CT) and simulations to explore pore interconnectivity and tortuosity. Overall, these results highlight the potential of anisotropic, sponge-like silk fibroin scaffolds for long-term (>6 weeks) contractile muscle culture with an in vitro bioreactor system that provides routine mechanical stimulation.