Development and characterization of small extracellular vesicles-loaded genipin cross- linked gelatine hydrogel for vocal fold fibroblasts regeneration in glottic insufficiency patients

Abstract

Glottic insufficiency occur due to the injury on laryngeal nerve, which affects the quality of voice and difficulty in breathing. Currently, injection augmentation using hyaluronic acid is done as treatment, but it requires re-injection every 3-6 months. The aim of this study is to incorporate small extracellular vesicles (sEV) derived from Wharton’s Jelly mesenchymal stem cells (WJMSCs) into genipin-crosslinked gelatin hydrogels (GCGH) and observe its therapeutic potential on vocal fold fibroblasts (VFF) regeneration in vitro. WJMSCs were isolated from umbilical cord, characterized, and expand for isolation of sEV by using tangential flow filtration (TFF) system. A total of number of sEV particles were detected using bicinchoninic assay (BCA). Then sEV were further characterized with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot. Afterwards, 6 different concentrations of sEV were assayed to observe its cytotoxicity and proliferative efficacy on VFF. From the results, 75 μg/mL of sEV was chosen because it promotes the highest VFF proliferation and cell viability up until day 7 when tested with MTT assay and LIVE/DEAD assay. Subsequent test incorporates GCGH with WJMSCs, WJMSCs with basic fibroblast growth factor (WJMSC-bFGF), or sEV, respectively. Physicochemical results showed that all the groups were able to be extruded from syringe in under 20 minutes and took average 42 days to fully degrade. In terms of biocompatibility with VFF, GCGH-MSC provide better cell viability than GCGH-sEV, GCGH-MSC-bFGF, and GCGH. The same result can be seen in collagen gel contraction assay, where foldchange collagen contraction of GCGH-MSC is higher than others. In addition, this study also found that all GCGH groups do not induce immune response when co-cultured with peripheral mononuclear blood cells (PBMC) for 7 days. In conclusion, GCGH was found suitable for delivery of WJMSC, bFGF, and sEV to VFF and has the potential in promoting regeneration of vocal fold tissues, based on the enhanced VFF proliferation and biocompatibility without any immune response. Further in vivo studies of the above combinations are necessary to uncover their full potential in true environment.