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One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from a donor tissue, reducing its immunogenicity and the risk of rejection. Supercritical CO2 (scCO2)-assisted processing has been proposed to improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, the decellularization of heterogeneous 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, the type of co-solvent, values of pressure and temperature inside the scCO2 reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the effectiveness of decellularization approaches based on various combinations of these parameters. The protocols were based on the combinations of treatments in alkaline, ethanol or detergent solutions with scCO2-assisted processing at different modes. Histological analysis demonstrated favorable effects of the preconditioning in a detergent solution. Following processing in scCO2 medium provided a high decellularization degree, reduced cytotoxicity, and increased ultimate tensile strength and Young’s modulus of the aortic valve leaflets, while the integrity of the extracellular matrix was preserved.

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Porcine cartilage was ground by cryomill (Retch, Germany) at –196°C. A fraction of cartilage microparticles (CMP) of size 100-250 µm was isolated. CMP was decellularized at room temperature with periodic mixing in 3 shifts of 0.1% sodium dodecyl sulfate buffer solution, containing an increasing concentration (1, 2 and 3%) of Triton X100. CMP treatment in a supercritical CO2 (sc-CO2) atmosphere was carried out at a pressure of 300 bar, T = 35°C, with a flow rate of sc-CO2 of 2.5 ±0.5 ml/min for 8-24 hours using RESS-SAS equipment (Waters Corporation, USA). Ethanol (96%) at a concentration of 10% was used as a polarity modifier. The degree of decellularization of CMP was assessed by histological methods (stained by hematoxylin and eosin) and by detection of the residual amount of DNA in samples using DNA-binding fluorescent dye DAPI. In the case of treatment with the detergents only and detergents after sc-CO2, the required degree of decellularization of CMP was not achieved. Histological analysis of the samples has shown that only a partial release of chondrocytes occurs. CMP treatment by detergents followed by sc-CO2 was more effective. Complete removal of cells can be achieved if the cartilage is first treated with surfactant, and then CO2. When ethanol was added as a polarity modifier, histological studies confirm that non-disrupted cells were almost completely absent and study with the DAPI dye has shown that more than 90% of CMP samples were completely free of DNA or contained only single whole cells.To achieve the highest possible degree of decellularization, the treatment of cartilage microparticles should be carried out first with detergent solutions followed by exposure to sc-CO2. The introduction of a polarity modifier (ethanol) at a concentration of 10% has a positive effect on the degree of decellularization and in combination with lengthy treatment time allows to reach complete decellularization of cartilage tissue.

To obtain a supported heterogeneous catalyst, laser ablation of metallic palladium in supercritical carbon dioxide was performed in the presence of a carrier, microparticles of γ-alumina. The influence of the ablation process conditions—including supercritical fluid density, ablation, mixing time of the mixture, and laser wavelength—on the completeness and efficiency of the deposition of palladium particles on the surface of the carrier was studied. The obtained composites were investigated by scanning and transmission electron microscopy using energy dispersive spectroscopy. We found that palladium particles were nanosized and had a narrow size distribution (2–8 nm). The synthesized composites revealed high activity as catalysts in the liquid-phase hydrogenation of diphenylacetylene.

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