Gelatin sponge loaded with hypoxic human umbilical cord mesenchymal stem cell exosomes promotes healing of enterocutaneous fistula.

Enterocutaneous fistula (ECF) traditionally relies on surgical treatment and lacks effective therapies to promote tissue regeneration. This study constructed a composite system based on hypoxia-preconditioned human umbilical cord mesenchymal stem cell-derived exosomes loaded onto a gelatin sponge (GS Hypo-Exos), aiming to explore its potential application in ECF treatment. First, systematic characterization of exosomes was performed: transmission electron microscopy, nanoparticle tracking analysis, western blot, and flow cytometry confirmed successful exosome extraction. Material evaluation indicated that the gelatin sponge exhibited high porosity and good biocompatibility. Scanning electron microscopy confirmed successful loading of exosomes onto the sponge surface, and in vitro release experiments showed that GS Hypo-Exos enabled sustained release of VEGF and IGF-1, with release behavior superior to the normoxic control group. Small-animal in vivo imaging further demonstrated that exosome-loaded gelatin sponge could be retained locally at the fistula site for up to 21 days post-surgery. In the Sprague-Dawley rat ECF model, the GS Hypo-Exos treatment group (group C) showed superior healing effects compared with the normoxic exosome group: fistula area and circumference were significantly reduced, and animal weight recovery was the fastest. Serological analysis revealed that this group had the lowest level of inflammatory factor TNF-α and the highest level of anti-inflammatory factor IL-4. Local tissue levels of VEGF and TGF-β1 were significantly increased. Histopathological examination indicated reduced inflammatory cell infiltration and increased collagen fiber proliferation in group C. Regarding molecular mechanisms, real-time quantitative polymerase chain reaction and western blot results consistently showed significantly upregulated expression of the cell proliferation marker Ki67 and markedly downregulated expression of the apoptosis-related protein Caspase-3 in group C. In summary, GS Hypo-Exos effectively promotes structural and functional repair of ECF through synergistic sustained-release delivery and immunomodulation, providing experimental evidence for the development of novel ECF repair strategies.