In Vitro Cytocompatibility Assessment of Gentamicin-Impregnated Cryopreserved Amniotic Membrane Using 3T3-L1 Fibroblast Cell Line

Abstract

Background: Human amniotic membrane (HAM) has emerged as a promising biological scaffold in regenerative medicine owing to its anti-inflammatory, antimicrobial, and wound healing properties. Antibiotic impregnation of cryopreserved amniotic membrane may further enhance its therapeutic utility in infected chronic wounds such as diabetic foot ulcers. However, evaluation of its cytocompatibility is essential before clinical application. Aim: To evaluate the in vitro cytocompatibility and morphological effects of gentamicin-impregnated cryopreserved amniotic membrane on mouse fibroblast cell line (3T3-L1) using MTT assay and phase-contrast microscopy. Materials and Methods: Mouse fibroblast cell line (3T3-L1) was cultured in RPMI medium supplemented with 10% fetal bovine serum and antibiotics under standard conditions. Cells were exposed to gentamicin-impregnated cryopreserved amniotic membrane preparations containing three different gentamicin concentrations (40, 80, and 120 µg/mL), designated as 40 AM 276, 80 AM 125, and 120 AM 166, respectively. Cell viability was assessed at 24, 48, and 72 hours using the MTT assay. Morphological alterations were evaluated using inverted phase-contrast microscopy. Statistical analysis was performed using one-way ANOVA, and p<0.05 was considered statistically significant. Results: All experimental groups demonstrated high fibroblast viability throughout the study duration. Mean cell viability remained above 90% in all groups at 24, 48, and 72 hours, indicating no significant cytotoxicity. The 40 AM 276 group demonstrated significantly higher viability at 48 and 72 hours compared with other groups (p<0.05). Morphological evaluation revealed preserved fibroblast architecture with spindle-shaped morphology and intact cellular adherence across all groups without evidence of cellular degeneration or membrane damage. Conclusion: Gentamicin-impregnated cryopreserved amniotic membrane demonstrated excellent in vitro cytocompatibility with preserved fibroblast viability and morphology. These findings support its potential utility as a biologically compatible wound dressing material for regenerative wound management applications.