Fabrication of Skeletal Muscle Fibers Within Hollow Silk Matrices

The controlled flow and flexibility of coaxial needles allows multi-layered gels in the form of gel fiber scaffolds to be formed, with important utility in the field of tissue engineering. Coaxial needles can generate outer and inner layered hollow structures, allowing for the fabrication of macroscale crosslinked hydrogel fibers that are promising as scaffolds for muscle growth. To develop <i>in vitro</i> muscle fiber formation, the inner layer of the hydrogels support 3D growth and cell spreading of encapsulated myogenic cells. Silk fibroin (SF) from the <i>Bombyx mori</i> silkworm functions as a scaffold that allows cells to distribute throughout the core of the hydrogel. Our objective was to develop a core-shell muscle cell hydrogel system using SF as the shell and the inner channel as the cell-supporting component, to generate environments for the 3D growth and maturation of mouse skeletal muscle myoblasts (C2C12). We hypothesized that the addition of SF and extracellular matrix (ECM) materials would promote the long-term growth and survival of the cells for muscle-related outcomes.<br/>Our methods first include culturing C2C12 cells in standard growth media. While passing confluent cells, two syringes were placed in a syringe pump and then attached to the inner (core) and outer (shell) ports of a coaxial needle using medical tubing. The core solution syringe was filled with the C2C12 cell suspension, collagen type 1 (Col1), SF, horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂), all suspended in standard growth media. The shell solution syringe was filled with sodium alginate (Na-Alg). A calcium chloride (CaCl₂) bath was used to crosslink the Na-Alg in order to form fiber-shaped gels. Fibers of desired length were cultured for up to 21 days in standard growth media. Fibers without SF/ECM components served as controls. Live-dead staining and light microscopy were used to assess cell viability following encapsulation.<br/>The coaxial needle system generated mechanically stable core-shell fibers with distinct inner and outer layers with control over the contents of the layers and the length of the fibers. Na-Alg formed the outer shell (1.5mm diameter), while the inner conduit (0.8mm diameter) was filled with the C2C12 cell suspension. Rapid crosslinking due to the enzymatic reaction generated a structure that encompassed the core materials upon extrusion and held shape for 21 days with minimal leakage. The cross linked hydrogel fibers were sufficiently stable to generate 3 meter-long structures. Over time, the integrity of the outer layer’s crosslinked hydrogel slowly decreased, but the addition of CaCl₂ in the growth media ameliorated this effect. At the same time, the cells suspended in SF rather than in just growth media resisted calcium toxicity under the processing conditions used. One day after extrusion, fluorescence images showed viable C2C12s that were distributed evenly throughout the core of the hydrogel fibers. The C2C12s proliferated over the 21-day period, measured through microscopy, with culture media changed every 4 days. After 5 days in culture, microscopy images showed cell alignment and developing matrix material, when compared to fibers without SF that showed more disordered cells.<br/>This method of fiber preparation supports the survival and alignment of skeletal muscle cells in developing core-shell hydrogel myofibers for muscle tissue engineering applications. Future improvements of the method include better approximating the mechanical stimuli cells experience during <i>in vivo </i>growth.