Effects of amniotic membrane suspension in corneal epithelialization

Abstract

Abstract Purpose To define the biochemical role of amniotic membrane (AM) suspensions on corneal epithelial wound healing, particularly with regards to epithelial proliferation Methods ELISAs were used to measure the concentrations of mitogenic growth factors in AM suspensions compared to human serum. Human corneal epithelial cells (HCECs) were cultured in medium with AM suspension (5%, 30%), negative and positive control. Migration assays and immunocytochemical staining of cell adhesion molecules were conducted. The relative expression of MMP9 and adhesion molecules and the proliferation assay using BrdU incorporation and Western blot analysis for PCNA were determined. Results ELISAs showed that our AM suspension contained large amount of HGF, EGF, KGF, FGF, especially large amount of HGF, followed by EGF compared to human serum. On migration assay, migration rates enhanced as the concentrations of AM increased, with statistical significant changes seen in AM 30% and positive control compared to negative control (P<0.05). RT‐PCR and Western blot revealed that the expression of MMP9 was upregulated by AM, and the expressions of E‐cadherin and fibronectin were downregulated by AM. Upon immunocytochemistry, large quantities of E‐cadherin were detected near the wound edges in the AM‐treated groups. On the proliferation assay, the BrdU positive cell counts / total cell counts (labeling index) were augmented by AM to a statistically significant degree. On Western blot analysis, the expression of PCNA was increased gradually with increasing AM concentration. Conclusion The suspension form of AM contains large amount of growth factors and maintains its beneficial effect on corneal epithelial wound healing in vitro, especially in corneal epithelial migration and proliferation.