(247i) Engineering a Long-Term Silk Fibroin Skeletal Muscle Tissue Platform for Modeling Disease Progression

Statement of Purpose: A continued need for engineered skeletal tissue models stems from challenges in skeletal muscle regeneration and our limited fundamental understanding of muscular degeneration in patients with muscular dystrophies. Many current methods for engineering skeletal muscle tissues show structural similarities of native tissue, but do not have the functional contractility of muscle over long time frames.¹ Mechanical stimulation can be applied during in vitro culture to increase the contractile forces of the engineered tissue, but the most successful materials for this kind of 3-dimentional (3D) culture are made of hydrogels, which deform and change volume overtime. In these models, 3D culture allows for mechanical strain on skeletal muscle cells to enhance their growth and differentiation through mechanotransduction pathways. Previously, work on engineered skeletal muscle tissue has shown that the time scale at which mechanical strain is applied directly affects myotube formation.² We utilize these successful stimulation regimens² to mature skeletal muscle myoblasts toward myotubes using uniaxial strain as it mimics the behavior of muscles during exercise, but we are specifically investigating long-term (>8 weeks) culture platforms to ensure skeletal muscle myoblast maturity can be reached within the material prior to the start of experimentation and investigation of muscle degeneration. Thus, our platform is made from anisotropic silk fibroin (aSF) scaffolds. Here, we show that these aSF scaffolds are an ideal material platform for long-term in vitro culture due to maintenance of constant shape, volume, and elasticity over long-term physiologically relevant mechanical regimes and ability to support and sustain human skeletal muscle myoblast phenotypes.

Methods: In this work, aligned silk scaffolds are generated at different formulations, by changing silk polymer concentration (3% or 5%), freezing time (50% vs 100% ethanol), and crystallization (autoclaving vs water annealing)³. Six different scaffold formulations were generated from these different fabrication parameters. Silk fibroin solution was isolated from the cocoons of Bombyx mori silkworms and alignment was induced through directional freezing with a slurry of dry ice and ethanol.³ Extensional uniaxial rheology (n=6) was used to calculate the modulus of the material. Dynamic oscillations were utilized to determine the scaffolds dependence on strain and frequency and the linear region for future mechanical testing. Fatigue tests and hysteresis testing were used to determine the influence of long-term cyclic stress on the silk scaffolds which was used to inform the mechanical stimulation during culture. After evaluation of the mechanical properties and scaffold formulations, the conditions which most closely resemble the native tissue were utilized for cell seeding experiments (3% silk, 100% ethanol, water annealing). Human skeletal muscle myoblasts (Lonza ®) (HSMM) are seeded at 5000 cells/cm² onto the silk scaffolds (20mm x 10mm x 2mm). Growth occurred over 7 days and differentiation for 5 days. Control samples were HSMMs cultured in 2D. Histological assays were performed to assess homogeneity, cell attachment, and myofiber formation. The histological assessments were performed over periods of no mechanical stimulation and 2 weeks of mechanical stimulation to ensure control of the engineered platform before long-term (>8 weeks) assessment via immunohistochemistry (structure), western blot (protein expression), and qPCR (gene expression).

Results: Dynamic tensile testing of anisotropic silk fibroin scaffolds has not been performed previously. We hypothesized that higher polymer concentration, higher crystallinity, and a faster freezing rate would produce scaffolds with a higher modulus and smaller pore sizes. The role of silk concentration was found to change the pore architecture, with higher silk concentrations generating more lamellar shaped architecture. The change in freezing rate generated different sizes of pores. The method of inducing crystallinity was found to significantly affect the mechanical strength and modulus of the scaffolds (Figure 1a). Silk fibroin scaffolds that were water annealed exhibited stress-strain curve behavior similar to elastomers while the scaffolds that were autoclaved behaved more like semi-crystalline materials. Young’s modulus was not significantly affected by changes in silk fibroin concentration or freezing rate. The extensional Young’s modulus for the range of fabrication parameters was found to be 600 kPa to 2800 kPa. Dynamic oscillatory testing showed the linear region of these materials to be below 15% strain amplitude and below 2 Hz frequency. The amplitude sweeps showed that the oscillatory scaffold performance is not dependent on silk fibroin concentration (Figure 1b). Hysteresis tests showed that the material reaches an equilibrium lost energy after around 20 loops of stimulation (Figure 1c). Fatigue tests showed minimal change in the storage and loss modulus of the material for more than 6000 minutes of continuous mechanical stimulation at 10% strain and 1 Hz (Figure 1d). This indicates that the cellularized silk fibroin scaffolds can be mechanically stimulated for 2 hours per day for 8 weeks.

H&E was used to confirm cellular infiltration and adhesion of myoblasts throughout the entire silk fibroin scaffolds (Figure 1e). Human skeletal muscle myoblasts were confirmed to grow and differentiate into myotubes within the anisotropic silk fibroin scaffolds as confirmed via fluorescent staining and western blot analysis. Myosin heavy chain was increased in the differentiated conditions to confirm myotube formation. Control samples were HSMMs grown in 2D conditions. Cell attachment in the silk fibroin scaffolds was increased through the addition of 0.2 mg/mL of decellularized extracellular matrix (dECM) which was included during formation of the scaffolds. The dECM was not shown to have any significant differences in the mechanical properties of the silk only scaffolds. The cellularized scaffolds that had 2 weeks of mechanical stimulation (10% strain, 1 Hz frequency, for 2 hours per day) showed larger area and longer myotube formation.

Future work will increase the length of the time of mechanical stimulation to at least 8 weeks as current work in engineered skeletal muscle tissue cannot perform for long-time periods. Confirmation of mature tissue will be assessed through contraction force of the engineered skeletal muscle tissue. This work is innovative in that it will improve upon current 3D cell culture techniques and create a novel long-term platform for engineered skeletal muscle tissue. This initial healthy construct will be used as foundation to explore mechanobiology in skeletal muscle cell lines and cells from rare muscular disorders.

References: [1] Somers SM. Tissue Eng. Part B Rev. 2017;23(4): 362-372. [2] Somers SM., Acta Biomaterialia, 2019;94: 232-242. [3] Stoppel WL., Biomed. Materials, 2015;10(3): 1-13. [4] Kuthe CD. J Appl Biomater Funct Mater 2016; 14(2): e154-e162.