Assessment of Immunomodulatory Factors/Cytokines in Dual Layered Dehydrated Human Amniotic Membrane (d-HAM) Allografts

Introduction: Human amniotic membrane (HAM) allografts have numerous clinical applications to treat chronic wounds. Dual layered HAMs provide robust thickness for treating larger and deeper wounds, and the processing approaches for such HAMs directly impact the preservation of innate immunomodulatory factors [ 1]. Numerous studies have shown that the native composition of dehydrated human amniotic membrane (d-HAM) allografts possess key growth factors/cytokines [ 1, 2, 3, 4]. Passive drying methods for d-HAM allografts can provide efficiency for preservation of such growth factors/cytokines by removing excess water content in the amniotic tissue and extending shelf stability for the tissue grafts [ 4, 5, 6]. Through bead-based antibody multiplexing, this study aims to assess the preserved concentration of six pertinent immunomodulatory factors in dual layered d-HAMs (i.e., GM-CSF, IL-4, IL-6, IL-10, IL-12p70, VEGF-A) [Fig. 1], while giving insight to assessment techniques for further immunomodulatory factor/cytokine profiles. This will also provide a blueprint assessment for immunomodulatory factor/cytokine content of d-HAMs through the common preservation techniques of dehydration, lyophilization, and cryopreservation [ 5]. Methods: Donated amniotic tissue was processed and dried to develop a dual layered d-HAM. A multiplex antibody bead panel (Millipore; # HYCTA-60K) was utilized to assess relative quantification (RQ) concentrations of GM-CSF, IL-4, IL-6, IL-10, IL-12p70, and VEGF-A in Guanidine Hydrochloride digested d-HAM (RQ Values are referenced to GM-CSF). Results were statistically compared to internal controls and each individual test group. Results: Image not being displayed. Bar graph with IL-4/VEGF-A > 4X to GM-CSF/IL-6 and > > 200X to IL-10/IL-12p70. Caption below. Figure 1. Relative quantification levels of a six pertinent immunomodulatory factor/cytokine panel in dual layered dehydrated human amniotic membrane (d-HAM) [RQ Values referenced to GM-CSF]. Statistical significance between groups is p