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Category: Basic Sciences/Biologics; Other Introduction/Purpose: Placental tissue allografts have long been used as effective wound dressings for hard to heal wounds and are demonstrating increasing utility in the field of foot and ankle surgeries. Previous observations of the PURION-processed dehydrated amnion/chorion membrane (dHACM) demonstrated retention of regulatory proteins inherent to amniotic tissues and preservation of the bioactivity to stimulate cellular activities. Novel and patent-pending processing techniques have been developed to introduce a thicker and more robust allograft, as compared to dHACM. Lyophilized human amnion chorion membrane (LHACM) will confer versatility to clinicians with improved handling while maintaining the natural tissue composition and intrinsic biological activities. This study characterizes the biological properties of LHACM which may support the healing cascade. Methods: LHACM was processed using a modified PURION process which incorporates an optimized cleansing process, enabling retention of the intermediate layer, and lyophilization. Tissue architecture was visualized with hematoxylin and eosin staining. The hygroscopic contribution of the intermediate layer was appraised with absorbency measurements. Cleansing efficiency was determined through quantification of residual hemin remaining in the tissue. Regulatory proteins were quantified using a multiplex ELISA. The cellular response to LHACM treatment was assessed using multiple in vitro assays targeting cellular proliferation and analysis of signaling pathways. Adult dermal fibroblasts (HDFs) cultured over a 3-day period in LHACM eluates were assessed for cellular proliferation. In vitro models were developed to assess the regulation of relevant signaling pathways, including TGF-β1 and Wnt/β-catenin for fibrotic and proliferative processes, respectively. Cellular responses were evaluated through quantitative PCR and cell reporter lines. Results: Histological evaluation showed that processing maintains the tissue structure. Absorption testing demonstrated LHACM is more hygroscopic than dHACM. Hemin analysis demonstrated low levels of residual hemin which were comparable for LHACM and dHACM allografts. Screening for regulatory proteins indicate that LHACM contains a diverse array of regulatory proteins, inherent to amniotic membranes. In vitro treatment of primary cells with LHACM eluates promoted dermal fibroblast proliferation as well as regulation of elevated TGFβ- and Wnt/β-catenin signaling. Conclusion: The processing method of LHACM maintains the intrinsic properties of amniotic membranes with similar levels of biological activity as compared to dHACM, while providing an option for a thicker allograft with alternative handling characteristics. LHACM represents a novel product concept which increases versatility of amniotic membrane application in varied outpatient and surgical uses.