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 0.05. Discussion: The processing technique of d-HAMs illustrates a high concentration of IL-4 and VEGF-A compared to inflammatory cytokines of IL-6 and IL-12p70. Higher levels of IL-4 and VEGF-A are associated with a predominantly anti-inflammatory host response and an M2 macrophage polarization. Moderate levels of GM-CSF expression can also assist with the controlled infiltration of macrophages to the site of repair. IL-4 is also a driver of IL-10 expression and M2 polarization, which is needed for the recruitment of satellite stem/progenitor cells in wound healing. Further studies are in progress to compare with hydrated HAMs and the benefits of other preservation techniques such as lyophilization and cryopreservation with the highlighted six factor panel illustrated in Figure 1. References:1. Koob, T. J., Lim, J. J., Zabek, N. & Massee, M., Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS 103B, 1133-1140 (2015). 2. McQuilling, J. P., Vines, J. B., Kimmerling, K. A. & Mowry, K. C., Proteomic Comparison of Amnion and Chorion and Evaluation of the Effects of Processing on Placental Membranes. Wounds 29 (6), E36-E40 (2017). 3. Kannaiyan, , Khare, S., Narayanan, S. & Mahuvawalla, F., Dehydrated Amniotic Allograft Seeded with Stem Cells: The Clinical Perspectives for Wound Healing. Journal of Stem Cell and Regenerative Biology 5 (1), 16-24 (2019). 4. Koob, T. J. et al., Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing. International Wound Journal, doi: 10.1111/iwj.12140 (2013). 5. Protzman, N. M. et al., Placental-Derived Biomaterials and Their Application to Wound Healing: A Review. Bioengineering 10 (829), https://doi.org/10.3390/bioengineering10070829 (2023). 6. Massee, M. et al., Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS 104B (7), 1495-1503 (2016).