Rapid Decellularization of Bovine Amniotic Membrane Using EDTA Pretreatment and Low Concentration SDS

Abstract

Objective: To identify a cost-effective protocol that achieves effective decellularization while preserving the key biological properties of bovine amniotic membrane.

Study Design: Experimental laboratory study
Place and Duration of Study: This was conducted at the Tropical Disease Center Laboratory, Universitas Airlangga, Surabaya, Indonesia; the Histology Laboratory, Faculty of Medicine, Universitas Airlangga, Surabaya,
Indonesia; and the Pathology Laboratory, R.T. Notopuro Regional General Hospital, Sidoarjo, Indonesia, between December 2025 and April 2026.

Methods: BAM samples from six healthy donor cows underwent 0.2% EDTA pretreatment followed by varying SDS concentrations and exposure durations. Decellularization efficacy was assessed by residual double stranded DNA (dsDNA) quantification and histological evaluation of residual nuclei. Collagen density, transforming growth factor beta 1 (TGFβ1), and BHK 21 fibroblast viability were evaluated to assess scaffold preservation and cytocompatibility.

Results: Decellularization significantly reduced residual dsDNA levels (p = 0.001). Treatment with 0.2% EDTA for 30 minutes followed by 0.1% SDS for 30 minutes reduced dsDNA below the accepted decellularization threshold
and eliminated detectable cellular nuclei. Increasing SDS concentration or exposure duration provided no additional decellularization benefit. Collagen density differed significantly among groups (p = 0.009), with higher SDS exposure associated with greater extracellular matrix disruption. TGFβ1 levels were not significantly different between groups (p = 0.157). All groups demonstrated cell viability above 70%, indicating acceptable cytocompatibility.

Conclusions: Pretreatment with 0.2% EDTA for 30 minutes followed by 0.1% SDS for 30 minutes provided effective decellularization while preserving extracellular matrix structure, TGFβ1 retention, and cytocompatibility.
This simple protocol may represent a practical approach for producing BAM derived scaffolds for regenerative medicine.