Use of supercritical carbon dioxide for decellularization of porcine trachea in an attempt to generate a tissue-engineered tracheal substitute

Purpose: Patients with tracheal lesions that exceed half of the trachea’s total length require a tracheal substitute. Tissue engineering, using either synthetic materials or decellularized tracheal tissue, opens up new possibilities for generating tracheal substitutes. Decellularization, a procedure in which the tissue's immunogenic cellular material is removed while the extracellular matrix (ECM) is preserved, seems the most promising approach. The majority of tracheal decellularization methods, however, are reliant on harsh chemicals and require lengthy wash procedures, resulting in damage to the ECM. To address these issues, a supercritical carbon dioxide (scCO2) decellularization approach has been suggested as an alternative solution due to its ability to both decellularize and sterilize tissues while leaving no toxic residues and requiring less treatment time. Therefore, the aim of this thesis was to compare scCO2 treatment with a chemical decellularization method, which is the current gold standard reported in literature, for creating a decellularized porcine tracheal scaffold with good cytocompatibility. Methods: A total of five different protocols were tested that varied in decellularization and sterilization methods used. Decellularization efficiency was evaluated in terms of DNA content, histological appearance, and retention of ECM components. Additionally, mechanical tensile testing and scanning electron microscopy were used to assess the effects of the different decellularization protocols. Further, decellularized scaffolds were recellularized with fibrin-encapsulated porcine adipose derived stem cells to assess the cytocompatibility of the scaffolds. Results: The highest reduction in DNA content was observed when samples were subjected to the detergent-enzymatic protocol, followed by sterilization with gamma irradiation, and when samples were subjected to scCO2 treatment, followed by washing with sodium hydroxide. The latter proto
Biomedical Engineering