Mesenchymal stem cells in corneal neovascularization: Comparison of different application routes

Abstract

The purpose of the present study is to investigate the effect of mesenchymal stem cells in corneal neovascularization and wound healing, and to compare the effectiveness of two possible application routes, subconjunctival injection and amniotic membrane transplantation. Chemical injury was induced by application of sodium hydroxide to the rats' corneas. After 7 days, the animals were divided into three groups. Different treatment methods were used for each group as follows: i) Group 1, injection of bone marrow‑derived mesenchymal stem cells (BMSCs) under the conjunctiva; ii) group 2, transplantation of amniotic membranes, previously seeded with BMSCs; and iii) group 3, the untreated control group. The eyes were examined using a slit lamp on a weekly basis. After 4 weeks, the animals were sacrificed and corneas were removed for further examination. Corneal flat mounts were made following ink perfusion for improved vessel visualization, image capturing and quantitative evaluation. enzyme‑linked immunosorbent assay was performed to detect the levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP‑9). Reverse transcription‑quantitative polymerase chain reaction was used for detection of VEGF‑A, MMP‑9, Toll‑like receptor (TLR)2 and TLR4 gene expression levels. Cryosections were used for histological examination and immunostaining. Statistical analysis (Welch's one‑way analysis of variance) demonstrated a significant difference between the groups [P≤0.05, confidence interval (CI) 95%]. The level of injury in group 1 was significantly different from groups 2 and 3. Measurement of the vessel area and VEGF gene expression levels had a similar difference among the groups (P≤0.05, CI 95%), however the differences for TLR2 and TLR4 were not statistically significant. BMSCs were previously transduced with the green fluorescent protein gene by lentivirus to track the movement of the cells following transplantation. The transplanted cells enhanced corneal wound healing by trophic factor production and immune‑regulatory effect, rather than by direct transdifferentiation into corneal cells. The results of the current study demonstrated that BMSCs enhance corneal wound healing and decrease the area of neovascularization. Furthermore, the comparison of two application routes indicated that single subconjunctival injection appeared more effective than transplantation with amniotic membrane.

Figures

Figure 1

Bone marrow-derived mesenchymal stem cells under fluorescence microscopy (magnification, ×20). (A) Cells are attached to the bottom of the culture dish exhibit a spindle shape. (B) Flow cytometry results, cells express CD90 and CD44 and are negative for CD45 and CD11b. (C) Green fluorescent protein positive cells are observable under fluorescence microscopy. CD, cluster of differentiation.

Figure 2

Amniotic membrane with cultured stem cells under confocal microscope (magnification, ×40). (A) Normal amniotic membrane with epithelial layer, (B) epithelial cell nuclei stained with DAPI demonstrated regular arrangement; (C) denuded amniotic membrane with (D) no visible DAPI stained nuclei; (E) amniotic membrane with cultured stem cells; (F) nuclei of stem cells stained with DAPI demonstrated a irregular arrangement; (G) stem cells expressing GFP protein; (H) GFP-positive cells with DAPI stained nuclei. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein.

Figure 3

Corneal surface evaluation prior to and 4 weeks after treatments. (A–H) Images of rat corneas. (A) Cornea on the day of injury, (E) total epithelial defect stained with fluorescein dye; (B and F) Group 1, clear, smooth cornea without epithelial defects or vessels following 4 weeks of subconjunctival injection; (C and G) Group 2, cornea with irregular surface and new vessels 4 weeks following amniotic membrane transplantation; (D and H) Group 3 (control), small epithelial defect stained with fluorescein dye and new vessels visible.

Figure 4

Corneal flat mounts under light microscope (magnification, ×10). (A) Subconjunctival injection group (group 1), vessels are short and only in one quadrant; (B) amniotic membrane transplantation group (group 2), vessels are in more than half of the cornea and reach to the center; (C) control group (group 3), vessels are over the entire cornea; (D) VEGF protein level detected by ELISA; (E) length of the vessels measured by Image-Pro Plus 6.0; (F) VEGF, (G) MMP-9, (H) TLR2 and (I) TLR4 gene expression relative to actin. *P≤0.05, 95% confidence interval. VEGF, vascular endothelial growth factor.

Figure 5

Cryosections stained with hematoxylin and eosin under phase-contrast microscopy (magnification, ×20). (A) Group 1, epithelium is fully recovered and the stroma is compact; (B) group 2, epithelium is recovered, however, new vessels are observed and stroma is swollen; (C) group 3, epithelium is not fully recovered and severe stromal edema is observed.