Polysaccharide hydrogel combined with mesenchymal stem cells promotes the healing of corneal alkali burn in rats

Abstract

Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection of mesenchymal stem cells (MSCs) have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid and mesenchymal stem cells (MSCs) were derived from Sprague-Dawley rats. Supplementation of the polysaccharide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs application on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduction in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-β), antiangiogenic cytokine (TSP-1) and decrease those promoting inflammation (TNF-α), chemotaxis (MIP-1α and MCP-1) and angiogenesis (VEGF and MMP-2). This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treatments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder.

Fig 1. Characterization of Hardy Orchid-derived polysaccharide and examination of its impacts on migration and proliferation of rat corneal epithelial cells (rCECs).

(A) Infrared spectrum analysis of Hardy Orchid-derived polysaccharide, (B) In vitro formed polysaccharide hydrogel composite, (C) Negative control for Cytokeratin-3 immunostaining of rCECs cultured with polysaccharide supplementation, (D) Cytokeratin-3 immunostaining of rCECs cultured with polysaccharide supplementation, (E) The migration rate of rCECs in control and polysaccharide treated groups 24h after wound. Polysaccharide treatment increased rCECs migration rate in a dose-dependent manner, (F) Proliferation analysis of rCECs in control and polysaccharide treated groups. At all three time points there were no significant differences between control and various polysaccharide dose groups. Data are presented as mean ± standard deviation (n = 5). One-way (E) or two-way (F) ANOVA analysis was performed to determine the significance of the difference between various treatment groups (* p<0.05, **p<0.01, ***p<0.001).

Fig 2. Re-epithelialization examination of alkali burn injured cornea.

(A-D) Fluorescence staining of corneal epithelium 3 days after injury in control, MSCs treatment, polysaccharide treatment and polysaccharide-MSCs combination (PM) groups, (E-H) Fluorescence staining of corneal epithelium 7 days after injury in various groups, (I) statistics of corneal defect area ratios in various groups on dya3 and day7 post-injury. Data are presented as mean ± standard deviation (n = 5). Two-way ANOVA analysis was performed to determine the significance of the difference between various treatment groups (* p<0.05, **p<0.01, ***p<0.001). At both time points, MSCs and polysaccharide treatments significantly enhanced the recovery of corneal epithelium. Polysaccharide-MSCs combination (PM) groups showed additive effects compared with single treatment groups.

Fig 3. Corneal opacity evaluation of alkali burn injured corneas.

(A-D) Corneal surface observation 3 days after injury in control, MSCs treatment, polysaccharide treatment and polysaccharide-MSCs combination (PM) groups, (E-H) Corneal surface observation 7 days after injury in various groups, (I-L) Corneal surface observation 14 days after injury in various groups, (M) statistics of corneal opacity grades in various groups on day3,7,14 post-injury. Data are presented as mean ± standard deviation (n = 5). Two-way ANOVA analysis was performed to determine the significance of the difference between various treatment groups (* p<0.05, **p<0.01, ***p<0.001). MSCs and polysaccharide treatments significantly decreased corneal opacity and Polysaccharide-MSCs combination (PM) groups showed additive effects compared with single treatment groups.

Fig 4. Analysis of corneal neovascularization after corneal alkali burn.

(A) Ratio of neovascularization area to corneal area in control, MSC, polysaccharide and PM groups at day7, 14, 28 days post-injury, (B-E) Ink perfusion detection of corneal neovascularization on day 28 post-injury in control, MSC, and PM groups. Statistics data are presented as mean ± standard deviation (n = 5). Two-way ANOVA analysis was performed to determine the significance of the difference between various treatment groups (* p<0.05, **p<0.01, ***p<0.001). MSCs and polysaccharide treatments significantly decreased corneal neovascularization and Polysaccharide-MSCs combination (PM) groups showed additive effects compared with single treatment groups.

Fig 5. Histological examination of cornea in the control, MSCs, polysaccharide and PM groups at day3 (A-D), and day14 post injury (E-H).

Fig 6. Quantitative Real-time PCR examination of inflammation, chemotaxis and neovascularization related genes.

The data are presented as mean ± SD from three assays. Two-way ANOVA analysis was performed to determine the significance of the difference between various treatment groups (* p<0.05, **p<0.01).

Fig 7. Evaluation of expression level of cytokines VEGF and TGF-β by ELISA.

The data are presented as mean ± SD from three assays. Two-way ANOVA analysis was performed to determine the significance of the difference between various treatment groups (* p<0.05, **p<0.01, ***p<0.001).