Exploring supercritical CO2 processing to generate highly preserved decellularized placenta for wound healing and regeneration

Introduction/Objectives: Placenta-derived extracellular matrix (dECM) is a promising material source due to its collagen, growth factors, and cytokines that support cell attachment and proliferation (1). However, traditional decellularization and sterilization methods often compromise ECM bioactivity and leave harmful residues (2), limiting therapeutic potential. To address this, we developed a protocol combining decellularization, drying, and sterilization using supercritical carbon dioxide (scCO 2 ) to enhance bioactivity retention while ensuring scalability and efficiency. Preserved placenta dECM bioactivity could help tackle critical healing challenges, such as chronic wounds, which burden healthcare systems, affecting 1-2% of developed populations (3), and arise from conditions like diabetes, vascular insufficiency, and aging (3). Current treatments, including passive dressings and bioactive therapies, often fail to restore ECM function, leading to prolonged healing and frequent infections (3). Consequently, there is a pressing need for advanced biomaterials that restore wound environment and accelerate healing. Methods: In this study, placenta ECM was processed using scCO 2 combined with sucrose to minimize exposure time to decellularization agents (sodium dodecyl sulfate and sodium hydroxide) while preserving bioactive ECM components. To minimize batch to batch variability equal amounts of 6 human donor placentas were mixed. Decellularization efficiency was evaluated by DNA quantification. Structural integrity was verified through SEM, and the stability was assessed via zeta potential analysis. Additionally, the preservation of key ECM components—elastin, glycosaminoglycans (GAGs), and lipids—was quantified. FTIR analysis was performed to evaluate alterations in the chemical composition. Sterilization was confirmed through a 14-day turbidity test, followed by plating to detect microbial growth. Biocompatibility was assessed using direct contact assays with Human Dermal Fibroblasts (HDFs). Results: Our results revealed that scCO 2 -treated dECM achieved improved stability, minimized agglomeration, and ensured complete sterilization, while preserving vital macromolecules essential for tissue regeneration. The optimized decellularization protocol produced a high yield of sterile, dry material, ranging from 42%-52%, and was validated by DNA quantification. DECM spectra revealed reduced lipid and nucleic acid bands and collagen and GAGs preservation. Furthermore, initial in vitro studies with HDFs demonstrated the bioactivity of the preserved ECM components, with a notable increase in cellular metabolic activity, indicating strong potential for enhancing wound healing and tissue repair. Conclusions: This work introduces scCO 2 as a novel method for decellularizing and stabilizing placenta ECM, preserving bioactivity while enhancing structural properties for chronic wound treatment. In vitro studies with HDFs and keratinocytes aim to confirm the regenerative and anti-inflammatory potential of scCO 2 -treated ECM. Optimized production techniques could provide a safe, human-derived bioactive complex to advance therapies for non-healing wounds.