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

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.