Chi, A. K. | bioRxiv Science

bioRxiv2026

Hyaluronic Acid-Alginate Hydrazone Crosslinked Hydrogels Support the Generation and Maturation of V2a Interneurons

Injury to the central nervous system (CNS) causes inflammation, cell death, and glial scar formation that inhibits tissue repair. Injectable hydrogels modified with extracellular matrix (ECM)-derived peptides can provide biochemical cues to promote neural tissue repair and serve as a vehicle to deliver therapeutics across the blood-spinal/blood-brain barrier in a minimally invasive manner. We developed an injectable hydrazone crosslinked hyaluronic acid-alginate (HA-Alg) hydrogel for neural tissue repair. We fabricated hydrogels with a range of polymer concentrations and evaluated their physicochemical properties to identify formulations that mimic the stiffness and viscoelastic properties of the CNS tissue environment. Hyaluronic acid was further modified with ECM-derived, cell-adhesive peptides (RGD and IKVAV) to enhance neuronal adhesion and viability. To evaluate the therapeutic potential of our hydrogel platform, we embedded mouse embryonic stem cell aggregates and differentiated them toward mature V2a interneurons. These interneurons are critical for relaying motor signals and represent a promising therapeutic cell population for treating spinal cord injuries. We demonstrated successful enrichment for V2a interneurons in HA-Alg hydrogels containing ECM-derived peptides. Interestingly, both our newly described HA-Alg and established crosslinked HA-HA hydrogels containing IKVAV peptides demonstrated significantly increased neurite length in interneuron enriched cultures compared to hydrogels without peptides. This study demonstrates that the addition of ECM-derived peptides is essential to support the neuronal adhesion and viability required for functional CNS tissue repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/729352v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@666b1aorg.highwire.dtl.DTLVardef@17d4borg.highwire.dtl.DTLVardef@1c8ece2org.highwire.dtl.DTLVardef@37fdd5_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceDevelopment of injectable, peptide-modified hyaluronic acid alginate hydrogels to support neuronal maturation for central nervous system tissue repair.

bioengineering↗

bioRxiv2025

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

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.

bioengineering↗