Development of a visible light, cross-linked GelMA hydrogel containing decellularized human amniotic particles as a soft tissue replacement for oral mucosa repair

Abstract

Early effective treatment of oral mucosal defects is the key to ensuring defect healing and functional recovery. The application of human amniotic membrane (HAM) in promoting wound healing has been shown to be safe and effective. However, amniotic membrane is thin, easy to tear and difficult to handle. Combined with the natural forces at play in the oral cavity, this has restricted the clinical applications of HAM for healing of mucosal defects. Methacrylated gelatin (GelMA) has good mechanical strength and adhesion, and can be used as a bionic repair film to attach to the damaged surface of oral mucosa, but GelMA lacks bioactive substances and cannot promote the rapid repair of oral mucosal defects. The aim of this study was to design a type of composite GelMA hydrogel mixed with decellularized human amniotic particles (dHAP) as an oral mucosa substitute, to promote regeneration of defective mucosa by stimulating rapid angiogenesis. The composite substitute GelMA-dHAP was easy to synthesize and store, and easy to operate for repair of oral mucosal defects. We show the angiogenic potential of GelMA-dHAP on chick chorioallontoic membrane and the curative effect of GelMA-dHAP as a treatment in the rabbit oral mucosa defect model. In conclusion, this study confirms the effectiveness of GelMA-dHAP as an ideal soft tissue substitute for the repair of oral mucosal defects, overcoming the shortcomings of using HAM or GelMA alone.

Fig. 1. Schematic preparation of the 3D porous structure scaffold, angiogenic potential of the GelMA–dHAP on CAM and efficacy of GelMA–dHAP as a wound treatment on the rabbit oral mucosa defect model. HAM: human amniotic membrane; dHAM: decellularized human amniotic membrane; dHAP: decellularized human amniotic particles; CAM: chick chorioallontoic membrane.

Fig. 2. (A) HAM before decellularization. (B) HAM after decellularization, H&E staining showed that there was no blue stain on the acellular matrix surface. (C) Under scanning electron microscope, acellular matrix showed no residual cells, while fibrous structures are observed.

Fig. 3. (A) SEM image of pore size distribution in the composite GelMA–dHAP scaffold. The scaffold has a 3D porous network structure in which the dHAP are adhered and wrapped. (B) The distribution of pore size in the GelMA–dHAP scaffold.

Fig. 4. The infrared spectra of GelMA, dHAM and GelMA–dHAP. No significant absorption peaks of new groups appeared in the GelMA–dHAP infrared spectra.

Fig. 5. (A) Stress–strain curves of dHAM, GelMA, and GelMA–dHAP. (B) Comparison of the maximum load value for dHAM, GelMA, and GelMA–dHAP (*P < 0.05, **P < 0.01).

Fig. 6. Cytotoxicity: compared with the negative control group (a: P < 0.05) and GelMA–dHAP showed no cytotoxicity.

Fig. 7. Evaluation of the angiogenic properties of GelMA–dHAP in the chick chorioallantoic membrane (CAM) assay. (A) Micrograph stereoscopy images of the control, GelMA and GelMA–dHAP groups taken on day 8 of embryonic development. (B) The change in total angiogenesis area of control, GelMA and GelMA–dHAP group calculated from stereoscopy images (*P < 0.05). Scale bars: 0.5 mm. (C) Representative images from each group demonstrating almost no inflammation in the CAM of the GelMA–dHAP group, whereas a mild inflammatory response, together with infiltration of inflammatory cells around the blood vessels, was observed consistently in the control group and GelMA group. Scale bars: 100 μm. The solid triangles indicate the presence of inflammatory cells in the blood vessels, and the hollow triangles indicate no inflammatory cell infiltration in blood vessels or less inflammatory cells.

Fig. 8. Histology of oral mucosa healing. H&E staining photographs of oral mucosa wound on days 3, 5, 7, and 14. The oral mucosal defects were covered with oiled gauze in group A; GelMA in group B; and GelMA–dHAP in group C.