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

PURPOSE

In 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.

METHODS

Here, we describe the fabrication of a tissue-engineered construct consisting of a scaffold of aligned poly(ε-caprolactone) (PCL) microfibers coated in a dynamic covalent hydrazone crosslinked hyaluronic acid (HA) hydrogel to support myoblast attachment, alignment, and differentiation. 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 scaffolds with fiber diameters of 10 μm, 20 μm, and 30 μm of non-coated, HA-coated, and HA-RGD-coated MEW scaffolds through immunocytochemistry and creatine kinase activity assays.

RESULTS

HA-coated and HA-RGD-coated scaffolds showed increased cellular attachment of C2C12 mouse skeletal 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-RGD-coated scaffolds as seen by myosin heavy chain (MHC) staining. Highest creatine kinase (CK) activity on day 5 signified the successful differentiation of C2C12 cells into mature myotubes.

CONCLUSION

This 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.