Characterization and Preclinical Evaluation of Dehydrated Human Amnion Chorion Grafts

Placental membranes are known to be an abundant source of growth factors, cytokines, and extracellular matrix proteins. The purpose of this study is to characterize the composition of a commercially available dehydrated amnion chorion membrane (dACM), and to evaluate how cells important to the wound microenvironment respond to released growth factors and cytokines from dACM using in vitro and in vivo models.

dACM grafts from 10 independent donors were evaluated using a high-throughput 1000 target array to evaluate their content (Kiloplex Array, RayBioTech, Norcross GA). Human dermal fibroblasts, keratinocytes, and endothelial cells obtained from commercially available sources were used to evaluate the effects of released factors from dACM (conditioned media “CM”) on proliferation, migration, and gene expression. To evaluate the angiogenic responses in vivo, gelatin sponges soaked with or without conditioned media (CM) from dACM were implanted subcutaneously into Sprague Dawley rats, retrieved at seven and 14 days, and then evaluated via histology and gene expression.

Proteomic evaluation of dACM grafts identified 640 regulatory proteins present. In vitro, culture of fibroblasts, keratinocytes, and endothelial cells with dACM CM resulted in significant increases in proliferation and migration (fibroblasts and endothelial cells only). Significant changes were observed in the gene expression of all three types of cells exposed to CM. In response to CM, expression of KGF, IL-6, and IGF-1 was elevated in fibroblasts; HB-EGF and IL-ra was elevated in keratinocytes; and GM-CSF, TGFB3, and HGF was elevated in endothelial cells. In vivo, gelfoam sponges containing dACM CM had significant increases in the gene expression of several pro-angiogenic targets and increased αSMA and CD31 staining compared to controls.

In sum, these results elucidate the proteomic composition of dACM grafts and the effect of releasate from dACM on cells found within the wound microenvironment in vitro and on angiogenic responses in vivo.