Enhanced decellularization of human amniotic membrane: optimizing dna clearance and protein retention for clinical applications.

BACKGROUND

The clinical efficacy of decellularized human amniotic membrane (dHAM) as a bioactive scaffold depends on the thorough removal of immunogenic components while maintaining the functional integrity of the extracellular matrix (ECM). This study aims to evaluate the impact of chemical and hypotonic decellularization protocols, specifically comparing 1 h and 24 h exposures on DNA clearance, protein retention, and growth factor accessibility.

METHODS AND

RESULTS

hAM samples were subjected to decellularization using 0.5% Triton X-100, 0.02% EDTA, and hypotonic saline solutions (0.2%, 0.3%, and 0.4%), followed by sonication (30 s) and gentle mechanical scraping. Decellularization efficiency was evaluated by genomic DNA quantification, whereas extracellular matrix preservation was assessed by total protein determination, SDS-PAGE analysis of retained protein profiles, histological staining (H&E, PAS, and Masson's Trichrome), and growth factor analysis (bFGF, EGF, KGF, HGF, and TGF-β1) using ELISA. Quantitative analyses demonstrated that EDTA-based hypotonic protocols, particularly the EDTA + 0.3 H group, achieved efficient cellular removal while maintaining extracellular matrix integrity. Genomic DNA content decreased to values below the accepted decellularization threshold of 50 ng/mg after 24 h, whereas total protein concentrations remained unchanged, indicating minimal matrix loss. Histological and biochemical analyses further confirmed effective decellularization with preservation of collagen architecture and endogenous growth factors.

CONCLUSION

The optimized EDTA-based hypotonic decellularization protocols (24 h) achieved efficient cellular removal while preserving extracellular matrix integrity and endogenous growth factor content. Compared with shorter incubation, the 24 h protocol exhibited higher detectable levels of bFGF and EGF, suggesting improved preservation and/or extractability of matrix-associated bioactive molecules. Collectively, these findings indicate that optimized hypotonic decellularization generates a structurally preserved and biologically active dHAM scaffold with considerable potential for future tissue engineering and regenerative medicine applications.