Developing a Fibronectin-Enhanced Elastomer Substrate for the Improved Visualization of 2D Muscle Fiber Morphology

In vitro tissue engineering allows for faster and more efficient studies of the morphology and function of biological systems compared to in vivo models. Skeletal muscle tissue models enable researchers to study electrical signaling and muscle cell growth and differentiation, providing insights into disease mechanisms. Studies have developed 2D methods of engineering muscle fibers and studying their morphology, chemical signaling, and contractility. Muscle alignment, length, and width are key characteristics related to muscle function, necessitating the ability to view and measure whole fibers. However, the common use of extracellular matrix (ECM)-mimicking hydrogels (e.g., fibrin) as substrates results in an uneven surface plane, compromising reproducibility and imaging capabilities. This study develops an alternative substrate for skeletal muscle cell growth using functionalized polydimethylsiloxane (PDMS) with the ECM protein fibronectin, demonstrated in previous studies with cardiac muscle. Various compositions of commercially available PDMS were mechanically tested to match the mechanical properties of fibrin. This blend was then spin-coated onto glass to achieve a thin, clear, uniform layer, and tested first with fibronectin adsorption, then functionalized using aminosilane and glutaraldehyde chemistry, forming stable covalent linkages to fibronectin. C2C12 mouse muscle myoblasts successfully adhered to the substrate and differentiated into measurable muscle fibers, however, they began to detach from the surface by Day 5 of differentiation, possibly due to spontaneous twitching of thickly-formed fibers. Thus, the contractile strength of skeletal muscle fibers may necessitate either a thicker substrate layer or altered attachment geometry.