Comprehensive evaluation of ethanol-preserved amniotic extracts: Exploring antioxidant properties, proliferation enhancement, protective efficacy and regeneration potential in wound healing

Abstract

Amniotic membrane extract (AME) has significant regenerative potential, which has been preserved using various methods over the years. This study explores ethanol (ETOH) as a preservative for AME in regenerative medicine. The study evaluated ETOH-preserved AME at 70 % (ETOH 70 %) and 50 % (ETOH 50 %) concentrations stored at room temperature (RT) and −80 °C. These were compared to phosphate buffer saline (PBS) and Dulbecco's Modified Eagle Medium/Glycerol (DMEM/GLY). In vitro assays included MTT for proliferation, apoptosis assays for protective effects, and scratch assays for migration using adipose stromal cells (ASCs) and fibroblasts. In vivo assessments involved subcutaneous injections in rats, with wound size measured at 4-, 8-, and 11-days post-injury. Histopathological parameters were analyzed after 11 days to evaluate tissue healing and regeneration.The results showed that ETOH preservation, particularly at 50 % and 70 %, exhibited significant antioxidant potential for AME, outperforming traditional methods. ETOH-preserved AME inhibited reactive oxygen species (ROS) and enhanced proliferation rates in ASCs compared to DMEM/GLY (p < 0.01). The ETOH 50 % variant at RT significantly reduced apoptosis and sustained cell proliferation. In vivo, ETOH-preserved AME at 50 % and 70 % accelerated wound closure significantly compared to the control group (p < 0.05). Histopathological analysis revealed improved healing, collagen deposition, and tissue regeneration in treated groups. ETOH preservation is a viable and cost-effective method for maintaining the regenerative potential of AME. This approach offers notable antioxidant, proliferative, and cytoprotective benefits, suggesting its promise for diverse biomedical applications.