Detailed Immunohistochemical and Proteomic Characterization of a Dehydrated Amnion Chorion Graft

Abstract Body: Placental membranes are known to contain a wide variety of growth factors, cytokines and extracellular matrix (ECM) proteins. The goal of this study was to conduct an in-depth characterization of a commercially available dehydrated amnion chorion membrane (dACM)º, by quantifying the proteins found within the tissue and using immunohistochemistry to identify location of key growth factors, cytokines and extracellular matrix proteins within the tissue.   Collagen, sulfated glycosaminoglycans, hyaluronic acid, and elastin were quantified within dACM samples using commercially available colorimetric kits (Biocolor, Carrickfergus, UK).  Proteomic analysis of dACM grafts (n=10) was independently conducted using a high-throughput 1000 target array (RayBioTech, Norcross GA).  For the histological analysis of dACM grafts, tissue was fixed and paraffin embedded.  Standard staining techniques were utilized including hematoxylin and eosin, Alcian Blue, and Verhoeff–Van Gieson.  Localization of collagen I, collagen III, fibronectin, laminin, hyaluronic acid, TIMP-1, VEGF, bFGF, TGF-_1, IL-1Ra, HGF, and IGF-1 were evaluated via immunohistochemistry.   Quantification of ECM proteins within dACM indicated that collagen and elastin were found at the highest concentrations (386.65 and 137.07 ug/cm2, respectively).  Proteomic evaluation of dACM grafts identified 640 regulatory proteins present; utilizing Uniprot keywords, we found that receptors, hydrolases, proteases, and cytokines were the most commonly tagged molecular function terms. Immunohistochemical analysis of dACM found that TIMP-1, VEGF, collagen I, collagen III, and fibronectin were highly concentrated in the chorion layer; while IL-1ra and IGF-1 were located primarily in the amnion and were concentrated in the spongy layer of the amnion.  Additionally, Hyaluronic acid, TGF-_1, and HGF were found in high concentrations in the spongy layer.    In sum, we have presented the proteomic composition of dACM grafts including categories of native molecular functions represented. Further, we have highlighted the importance of various layers of the tissue by identifying the location of key proteins within the graft.