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

Objective: Placenta-derived extracellular matrix (dECM) has emerged as a promising raw material source, owing to its rich content of collagen, growth factors, and cytokines that support cell attachment and proliferation (2). However, traditional decellularization and sterilization methods often compromise ECM bioactivity and leave harmful chemical residues (3), limiting its therapeutic potential. To overcome these limitations, we have developed a new protocol which combines decellularization, drying, and sterilization using supercritical carbon dioxide (scCO2) to enhance ECM bioactivity retention but also to create a scalable and efficient process. The preserved bioactive properties of placenta dECM could make it an exceptional candidate for addressing critical healing challenges, such as those observed in chronic wounds. These represent a significant burden to global healthcare systems, affecting 1-2% of the population in developed countries (1), and are often the result of underlying conditions such as diabetes, vascular insufficiency, and aging, all of which compromise tissue repair and regeneration (1). Current treatments, including passive dressings and bioactive wound therapies, often fail to restore ECM function, leading to prolonged healing times and frequent infection (1). Consequently, there is a pressing need for advanced biomaterials which can restore the wound environment and accelerate healing. Methods: In this study, placenta-derived ECM was processed using scCO2 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 scanning electron microscopy (SEM), and the stability was assessed via zeta potential analysis. Additionally, the preservation of key ECM components—elastin, glycosaminoglycans (GAGs), and lipids—was quantified. Fourier Transform Infrared Spectroscopy (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 scCO2-treated dECM not only achieved improved stability and minimized agglomeration, but also 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. Decellularized ECM 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 represents a novel application of scCO2 for the decellularization and stabilization of placenta- derived ECM. The method preserves essential bioactive components while enhancing the material’s structural properties, making it a promising candidate for chronic wound treatment. Ongoing in vitro studies with human dermal fibroblasts and keratinocytes aim to confirm the regenerative potential and anti-inflammatory properties of scCO2-treated decellularized ECM in promoting wound healing. By optimizing production techniques, this approach holds the potential to provide a safe human-derived bioactive complex, suitable for advancing therapies for patients with non-healing wounds.