Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes

Abstract

Vascular bio-scaffolds produced from decellularized tissue offer a promising material for treatment of several types of cardiovascular diseases. these materials have the potential to maintain the functional properties of the extracellular matrix (ecM), and allow for growth and remodeling in vivo. the most commonly used methods for decellularization are based on chemicals and enzymes combinations, which often damage the ECM and cause cytotoxic effects in vivo. Mild methods involving pressurized co 2 -ethanol (EtOH)-based fluids, in a supercritical or near supercritical state, have been studied for decellularization of cardiovascular tissue, but results are controversial. Moreover, data are lacking on the amount and type of lipids remaining in the tissue. Here we show that pressurized co 2 -etoH-H 2 O fluids (average molar composition, Χ CO2 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized co 2 -limonene fluids (Χ CO2 0.88) and neat supercritical CO 2 (scco 2 ) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized co 2 -limonene followed by enzyme treatment, resulted in efficient DNA removal. The structure of elastic fibers was preserved after pressurized treatment, regardless solvent composition. in conclusion, pressurized co 2 -ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO 2 -limonene fluids facilitate subsequent enzymatic removal of DNA.Cardiovascular diseases (CVDs) are responsible for 17.9 million deaths per year in the world (31% of total deaths) 1 . In 2015, the global prevalence of arterial hypertension (AHT), the most prevalent risk factor for CVD development, was estimated to be around 30-45% of the adult population, increasing up to 60% in people above 60 years of age 2 . Moreover, the prevalence of AHT is estimated to increase by 15-20% in 2025 2 . Pulmonary arterial hypertension (PAH), a sub-form of AHT, is characterized by breakdown of elastic fibers and alterations in the cross-linking of collagen, resulting in remodeling the extracellular matrix (ECM) in pulmonary arteries 3,4 . Hypertrophic remodeling of the media and endothelial cell dysfunction result in a high vascular resistance and thrombosis 4,5 , potentially leading to right ventricular failure and death in severely affected patients.Organ or tissue transplantation is the last option proposed for such CVDs-affected patients with a poor prognosis. However, the lack of compatible organs and tissues constitutes a major limitation. Even though the global rate of transplantation increased by 7.25% between 2015 and 2016, reaching a rate of 15.5 organs transplanted per hour 6 , less than 10% of the transplant needs are covered. Consequently, patients often have to wait long time for transplantation, resulting in worsening of their medical condition. Furthermore, those that are offered a transplantation require life-long immune therapy to redu...

Protocols for the Preparation and Characterization of Decellularized Tissue and Organ Scaffolds for Tissue Engineering

“…Transmission electron microscopy studies on scCO 2 -treated tissues revealed collagen fibrils displaying thickness and organization equivalent to the native tissue. Collagen fiber organization seemed intact, as confirmed by the density and parallel appearance of the collagen fibrils [15]. Our present investigation is concurrent with the previous report that scCO 2 -mediated production of tissue and organ scaffolds did not alter the elastic collagen fibers or ECM -or, more importantly, that of the native tissue -indicating that scCO 2 is the holy grail technology for the production of tissue and organ scaffold.…”

“…Tissue scaffold structure and the integrity of elastic fibers (as examined by histology) in scCO 2 -mediated production of tissue scaffold revealed an overall preserved morphology with parallel aligned continuous elastic fibers and without any differences between treated and untreated tissue [15]. In detergent-treated tissue samples, spacing between elastic fibers was more noticeable compared with the scCO 2treated tissues [15]. Transmission electron microscopy studies on scCO 2 -treated tissues revealed collagen fibrils displaying thickness and organization equivalent to the native tissue.…”

Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds

“…This approach yields a nearly intact decellularized tissue free of cells, lipids, and nucleic acids, proposing an alternative to traditional decellularization methods. Nevertheless, further in vitro and in vivo analyses need to be completed [125].…”

Decellularized Scaffolds for Skin Repair and Regeneration

“…Supercritical CO 2 has been used initially to gently sterilize natural materials and tissues [107]. In recent years, supercritical CO 2 decellularization was developed on vascular tissues [108], and most recently, processing of human skin and porcine skin has been reported.…”