Abstract
Glottic insufficiency, often caused by laryngeal nerve injury, impairs voice quality and breathing. Current treatments, such as hyaluronic acid injection, require frequent reapplication every 3-6 months. This study aimed to investigate the therapeutic potential of small extracellular vesicles (sEVs) derived from Wharton's Jelly mesenchymal stem cells (WJMSCs) incorporated into genipin-crosslinked gelatin hydrogels (GCGHs) for promoting vocal fold fibroblast (VFFs) regeneration in vitro. WJMSCs were isolated from umbilical cords, expanded to passage 4, and used for sEV isolation via tangential flow filtration (TFF). The sEVs (585.89 ± 298.93 µg/mL) were characterized using bicinchoninic acid assay (BCA), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot. Seven concentrations of sEVs were tested on VFFs to evaluate cytotoxicity and proliferation, identifying 75 µg/mL as the optimal dose. GCGHs were then combined with WJMSCs and sEVs and evaluated for physicochemical properties, degradation, biocompatibility, and immune response. The hydrogels were injectable within 20 min and degraded in approximately 42 ± 0.72 days. The optimal sEV concentration significantly enhanced VFFs proliferation (166.59% ± 28.11) and cell viability (86.16% ± 8.55, p < 0.05). GCGH-MSCs showed the highest VFFs viability (82.04% ± 10.51) and matrix contraction (85.98% ± 1.25) compared to other groups. All hydrogel variants demonstrated minimal immune response when co-cultured with peripheral blood mononuclear cells (PBMCs). GCGH is a promising scaffold for delivering WJMSCs and sEVs to support VFF regeneration, with demonstrated biocompatibility and regenerative potential. Further in vivo studies are warranted to validate these findings.