Application of supercritical fluids for complete decellularization of porcine cartilage

Abstract

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.