Effects of amniotic membrane suspension in human corneal wound healing in vitro

To investigate the biochemical mechanism of amniotic membrane (AM) suspension on corneal wound healing, particularly on epithelial proliferation and migration. Human corneal epithelial cells (HCECs) were cultured in media with different concentrations of AM suspension (5% and 30%), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (negative control), and serum containing Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (positive control). In an effort to evaluate the migratory potential of AM, migration assays were conducted via the manual scraping of HCECs and immunocytochemical staining of cell adhesion molecules (E-cadherin). The relative expression of matrix metallopeptidase 9 (MMP9) and adhesion molecules (E-cadherin, fibronectin) was determined via reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The proliferative potential of AM was evaluated via a proliferation assay using 5-Bromo-2-deoxyuridine (BrdU) incorporation and western blot analysis for proliferating cell nuclear antigen (PCNA). In addition, enzyme-linked immunosorbent assay (ELISA) was used to measure the protein concentrations of mitogenic growth factors (epidermal growth factor [EGF],keratinocyte growth factor [KGF], hepatocyte growth factor [HGF], and basic fibroblast growth factor [bFGF]) in AM suspensions. Migration assay rates were enhanced as AM concentrations increased, with statistically significant changes seen in 30% AM-treated and positive control cells, compared to negative control cells (p