Promoting oral mucosal wound healing using a DCS-RuB2A2 hydrogel based on a photoreactive antibacterial and sustained release of BMSCs

Abstract

The high occurrence rate and difficulties in symptom control are listed as the major problems of oral mucosal disease by medical professionals. Following the development of oral mucosal lesions, the oral microenvironment changes, immunity declines, and continuous bacterial stimulation causes wound infection. Traditional antibacterial drugs are ineffective for oral mucosal lesions. To overcome this problem, a light-responsive antibacterial hydrogel containing sustained-release BMSCs was inspired by the trauma environment in the oral cavity, which is different from that on the body surface since it mostly remains under dark conditions. In the absence of light, the hydrogel seals the wound to form a barrier, exerts a natural bacteriostatic effect, and prevents invasion by foreign bacteria. Simultaneously, mesenchymal stem cells are presented, and the released growth factors and other substances have excellent anti-inflammatory and angiogenic effects, which result in rapid repair of the damaged site. Under light conditions, after photo-induced shedding of the hydrogel, RuB₂A exerts an antibacterial effect accompanied by degradation of the hydrogel. Results in a rat oral mucosal repair model demonstrate that DCS-RuB₂A₂-BMSCs could rapidly repair the oral mucosa within 4 days. Sequencing data provide ideas for further analysis of the intrinsic molecular mechanisms and signaling pathways. Taken together, our results suggest that this light-responsive antibacterial hydrogel loaded with BMSCs can be used for rapid wound repair and may advance the development of therapeutic strategies for the treatment of clinical oral mucosal defects.

Keywords: BMSCs; DCS; Hydrogel; Light-responsive; Oral mucosa.

Fig. 1

Schematic showing the preparation and application of the DCS-RuB₂A₂-BMSCs hydrogel.

Fig. 2

Performance characterization of DCS-Ru₂BA₂and exploration of the crosslinking mechanism. SEM images of CS (A, B) and DCS (C, D); scale bars 10 μm and 5 μm. (E) FTIR spectra of CS and DCS. (F) FTIR spectra of DCS with differing degrees of substitution. (G) Photographs of CS and DCS. (H) Photographs of DCS-RuB₂A₂. (I) UV–Vis spectral evolution of RuB₂A₂ (100 μmol L⁻¹) in deionized water with increasing irradiation time (λex = 450 nm, 14 mW cm⁻²). (J) The wavelength corresponding to the UV absorption peak of RuB₂A₂ varies with illumination time. Pink represents the first stage and blue represents the second stage. (K) HPLC analysis of the photodegradation of crosslinkers under dark conditions (blue) and after illumination (orange). (L, M) SEM images of DCS-RuB₂A₂; scale bars 10 μm and 1 μm. (N) FTIR spectra of DCS-RuB₂A₂. (O-Q) Mapping images of C, O, and Ru elements of DCS-RuB₂A₂ in (L).

Fig. 3

Coagulation and hemostatic properties of DCS. (A) Photographs of blood loss after tail docking in mice. (B) In vitro coagulation profiles following treatment with CS and DCS with differing degrees of substitution. (C) Histogram of blood loss in mice. (D) Histogram of hemostatic time in mice. (E) Statistical histogram of blood clotting time. (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4

In vitro antibacterial performance. (A) SEM images of E. coli and S. aureus treated with the control and RuB₂A₂. (B) Growth of E. coli and S. aureus with different concentrations of RuB₂A₂ added to the agar plates. Colony numbers of E. coli (C) and S. aureus (D). (E) ZOI method to test the effect of light on the antibacterial properties of the hydrogel and its components. The areas of the inhibition zone for E. coli (F) and S. aureus (G) in each group were counted.

Fig. 5

Cytocompatibility of DCS-RuB₂A₂. The CCK-8 method detected the cytotoxicity of CS and DCS with differing degrees of substitution (A, B) and RuB₂A₂ at different concentrations (C, D). (E, F) CCK-8 assays for the biocompatibility of DCS-RuB₂A₂ under light and dark conditions. (G) (PI/Hoechst) live and dead cell staining images and statistical analysis of HEK 293T cells cultured for 24 h, scale bars 50 μm (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 6

Paracrine effects of BMSCs. (A) Schematic diagram of the co-culture of BMSCs and HEK 293T cells. (B) CCK-8 assay to detect cell viability after co-culture of HEK 293T cells and BMSCs. (C) Cell scratch assay to detect the cell migration ability of HEK 293T cells after co-culture with BMSCs. (D) Statistical histogram of the cell migration ability after BMSCs co-culture. (E–I) Growth factor expression levels in HEK 293T cells after co-culture with BMSCs (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 7

Application of DCS-RuB₂A₂-BMSCs to promote mucosal defect healing in a rat model. (A) Schematic diagram of the construction of the rat oral mucosal defect model. (B) Photographs of oral mucosal defects in rats. (C) The wound area in (B) was measured. (D) Schematic diagram of the local bacterial culture of the extracted affected area. (E) OD values for the bacterial cultures at 600 nm after 8 h (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 8

Histological evaluation of the oral mucosal trauma healing process. (A) Images of H&E and Masson's trichrome staining, in addition to immunochemical staining of the inflammatory factors IL-6 and TNF-α and angiogenesis factors at the wound site in rats treated with different materials for 4 days. (B–D) Quantitation of IL-6, TNF-α, and angiogenesis factor levels in (A) (n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 9

Global assessment of the wound microenvironment using RNA‐seq after treatment with the photoresponse material. (A) Heat map of the upregulated and downregulated genes in the wound microenvironment after treatment with the photoresponse material (fold change ≥2 and p < 0.05). (B) Reactome enrichment analysis of the DEGs. (C)Gene ontology (GO) enrichment analysis of the DEGs. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the DEGs.