A Triple-Layer Amniotic Membrane Dressing Drives Robust Wound Healing: In-Depth Protein Profiling and <i>In Vivo</i> Validation in Rat and Human Subjects

In chronic ulcers, early intervention to obtain wound closure is a critical requirement for optimal reparative success. Human dried amniotic membrane (HDAM) has been widely used as a wound covering due to three characteristics: its content of highly enriched extracellular matrix (ECM) and bioactive cytokines, its long shelf life, and its suitable biomechanical properties adequate for handling in the dried state. The ECM composition can be enhanced by the creation of a sandwich composed of three distinct layers of the amniotic membrane without creating any detrimental manufacturing shortcomings or clinical procedural complications. In this study, we investigate the mechanisms of action that contribute to wound healing using the histological analysis of various proteins, which exist in tri-layer HDAM. In addition, extensive proteomic analysis of 80 cytokines and growth factors is compartmentalized and quantified based on their functions in wound healing. More importantly, the reciprocal interactions between ECM components and keratinocyte proliferation are investigated. Using an animal wound model, <i>in vivo</i> efficacy of the tri-layer HDAM is assessed based on wound closure, tissue granulation, and re-epithelialization rates and is compared to a no-graft group. Lastly, the therapeutic effects of tri-layer HDAM and possible adverse effects on patients are carefully recorded through conducting a clinical study with a 37-patient population. The collective results suggest that the tri-layer HDAM positively affects various critical pathways in chronic wound healing and shows no adverse effects in human clinical studies.