An Evaluation of the Angiogenic Properties of Dehydrated Human Amnion Chorion Grafts

Abstract Body: The goal of this study was to characterize angiogenic proteins found within a commercially available dehydrated amnion chorion membrane graft (dACM) evaluate its angiogenic effects in vivo.   Proteomic analysis of dACM was conducted independently using a high-throughput 1000 target array (RayBiotech, Norcross GA) on 10 human donors. Proteins identified in the array were then annotated with manually selected Gene Ontology Terms based on UniProt Gene Ontology tags.  The in vivo assessment was conducted by implanting gelatin sponges (+/- media conditioned (CM) from dACM) subcutaneously into Sprague Dawley rats (protocol approved by Bridge PTS IACUC).  CM was generated by incubating dACM grafts in basal media for 5 days at 4°C at a concentration of 1 cm2 per mL.  Control groups consisted of implants adsorbed with basal media alone.  Implants were retrieved at 7- and 14-days post implantation and were evaluated for the presence of blood vessels via histology and gene expression via PCR.   Proteomic analysis identified 640 proteins present above the limit of detection; of these, 43 were related to angiogenesis with 72% of proteins tagged as pro-angiogenic, 19% were anti-angiogenic, and 9% were classified as playing a dual role in angiogenesis. Functionally, the majority of factors identified were pro-regulators of endothelial cell proliferation, migration, and sprouting angiogenesis and negative regulators of apoptosis. In vivo, implants with dACM CM showed significant increases in several pro-angiogenic genes at 7 days compared to controls including the genes FN1, EFNA1, TGFB3, VEGFC, TYMP, THBS1, and SERPINE1.  Histological analysis of the retrieved implants showed  increased angiogenesis as determined by CD31 and _SMA staining  in dACM CM treated groups compared to controls.   The results presented here further characterize the dACM proteome and native functions of the angiogenic proteins. In vivo, released factors from dACM result in pro-angiogenic response in vivo.