Hyaluronic Acid-Coated Melt Electrowritten Scaffolds Promote Myoblast Attachment, Alignment, and Differentiation

Abstract

PurposeIn muscle tissues, anisotropic cell alignment is essential for optimal muscle fiber development and function. Biomaterials for muscle tissue engineering must guide cellular alignment while supporting cell proliferation and myogenic differentiation. MethodsHere, we describe the fabrication of a tissue-engineered construct consisting of a scaffold of aligned poly({varepsilon}-caprolactone) (PCL) microfibers coated in a dynamic covalent hydrazone crosslinked hyaluronic acid (HA) hydrogel to support myoblast attachment, myoblast alignment, and myotube formation. Norbornene modification of HA further enabled functionalization with fibronectin-derived arginine-glycine-aspartic acid (RGD) peptide. Scaffolds were fabricated using melt electrowriting (MEW), a three-dimensional (3D)-printing technique that uses stabilization of fluid columns to produce precisely aligned polymeric microfibers. We evaluated C2C12 mouse skeletal myoblasts cultured on non-coated, HA-coated, and HA-RGD-coated MEW scaffolds with fiber diameters of 10 {micro}m, 20 {micro}m, and 30 {micro}m using immunocytochemistry and creatine kinase activity assays. We further evaluated the mechanical properties of 20 {micro}m fiber scaffolds and their effect on myogenic gene expression and alpha-actinin protein expression of C2C12 myoblasts undergoing differentiation. ResultsHA-coated and HA-RGD-coated scaffolds increased attachment of C2C12 myoblasts on all fiber diameters compared to non-coated scaffolds, with HA-RGD-coated scaffolds demonstrating the highest cell attachment. All scaffolds supported cellular alignment along the fibers. Cells differentiated on scaffolds showed anisotropic alignment with increased myotube formation on HA-coated and HA-RGD-coated scaffolds as demonstrated by myosin heavy chain (MHC) staining and by the presence of striations on HA-coated scaffolds visualized with alpha-actinin staining. Increased creatine kinase activity and myogenic gene expression on day 5 further indicated myotube formation on all scaffolds, with HA-coated scaffolds significantly increasing the expression of several key myogenic markers. ConclusionThis unique combination of tunable biophysical and biochemical cues enables the creation of a biomimetic tissue engineered scaffold, providing a platform for new therapeutic approaches for muscle regeneration.