Introduction: Placental allografts have a long-documented use in wound care. Maintenance of structure, key components, and functionality of placental allografts may be significantly impaired due to harsh processing techniques. Here, a gentle process designed to maintain all key characteristics, hypothermically stored amniotic membrane (HSAM*) was compared to an unprocessed, fresh amniotic membrane (uAM) and evaluated for retention of ECM composition, degradation kinetics, and scaffold functionality.
Methods: Amniotic membranes were processed into either HSAM, which was stored at 1-10°C for up to 42 days before use, or uAM, which was used within 24 hours. Structural assessments were completed using scanning electron microscopy (SEM) and histological staining. Durability was evaluated by subjecting membranes to simulated wound fluid (SWF–) or to SWF plus collagenases type I and II (50U or 25U each; SWF+) to model a harsh, chronic environment. Degradation kinetics were assessed through imaging and changes in collagen content. Functionality of scaffolds was evaluated by assessing fibroblast attachment and proliferation for up to 14 days.
Results: HSAM and uAM have similar architectural characteristics, and HSAM retained expression of ECM components and key proteins. When exposed to SWF–, both had comparable degradation kinetics over 17 days and imaging revealed similar structural changes over time. Initial characterization of a SWF+ model with HSAM revealed an increased rate of degradation with increasing collagenases concentrations, as expected. To further characterize responses in the SWF+ environment, HSAM was subjected to 25U of collagenases over 17 days. Most degradation occurred between days 1 and 7 before plateauing. Histological analysis revealed changes in ECM structure over time. There was a corresponding reduction in collagen content from day 0 to 7, with no substantial changes in collagen levels from days 10 to 17. When evaluating scaffold functionality, fibroblasts seeded onto HSAM readily attached and proliferated robustly over 14 days compared to non-seeded controls, with significance at all timepoints. Immunofluorescent staining highlighted fibroblast proliferation and SEM revealed collagen deposition by the seeded fibroblasts.
Discussion: Results demonstrate that HSAM maintains native characteristics of unprocessed tissues, resists rapid enzymatic degradation, and functions as a scaffold to support fibroblast attachment and proliferation.
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