Understanding the Impact of Preservation Methods on the Integrity and Functionality of Placental Allografts

Introduction: Human placental membranes (hPMs) have a long history in treating burns and wounds. The composition of hPMs includes structural matrix, growth factors, and neonatal cells, all of which contribute to their regenerative potential. However, most hPM products are devitalized after dehydration and irradiation. We compared the functionality of single-layer viable cryopreserved human amniotic membrane (vCHAM) with multilayer devitalized dehydrated human amnion/chorion membrane (dHACM) in wound-relevant models to determine the effect of different processing methods on hPMs. Methods: Viable cryopreserved human amniotic membrane and dHACM were compared with fresh hPM for structural integrity and viability. Viable cell persistence in vCHAM over time was evaluated in vitro and in vivo in a diabetic chronic wound mouse model. Proliferation of cells within fresh hPM and vCHAM was evaluated with bromodeoxyuridine and Ki-67 staining, and proliferation of isolated cells in culture was evaluated. Growth factor release over time and in vitro response to chronic wound stimuli (tumor necrosis factor α, lipopolysaccharide, and hypoxia) were used to compare the functionality of vCHAM and dHACM. Results: The structure and thickness of fresh hPM were retained in vCHAM but were compromised in dHACM. Similar to fresh hPM, vCHAM contained viable cells, whereas dHACM did not. Cells in vCHAM remained viable after 4 and 7 days in culture and in an in vitro chronic wound environment and after 4 and 8 days in vivo after application to a mouse chronic wound. Staining for bromodeoxyuridine and Ki-67 did not reveal proliferative cells within fresh hPM and vCHAM. However, isolated cells proliferated in culture. Viable cryopreserved human amniotic membrane increased platelet-derived growth factor BB, hepatocyte growth factor, and epidermal growth factor levels over time and responded to chronic wound stimuli in vitro by significantly increasing levels of vascular endothelial growth factor and prostaglandin E2. Dehydrated human amnion/chorion membrane showed no significant accumulation of growth factors and did not respond to chronic wound stimuli. Conclusions: These results indicate that vCHAM retains intact, native matrix, and viable, active cells and responds to chronic wound stimuli in vitro. The inclusion of multiple layers of hPM does not compensate for structural degradation and loss of viability caused by dehydration as evidenced by a lack of functional response by dHACM. The clinical significance of these results remains to be answered.

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