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Background: Decellularized tissues have been used successfully in reconstructive and regenerative medicine.Conventional decellularization involves multiple chemical processing steps to ensure completion of cell removal to minimize unwanted host tissue immune responses.To mitigate the inflammation risk of chemical decellularization,supercritical carbon dioxide(SCCO2)extraction technology was investigated for its effectiveness in cartilage tissue decellularization.Methods and Results: Porcine nasal cartilages were obtained from 6-month old pigs.The cartilaginous tissues were first incubated in NaOH and low concentration hydrogen peroxide,followed by SCCO2 extraction to complete tissue decellularization.The decellularized cartilage(DC)was evaluated histologically by hematoxylin-eosin(H&E),4,6-diamidino-2-phenylindole(DAPI),and Alcian blue stains.The microstructures of DC were examined by scanning electron microscopy(SEM).Biocompatibility of DC was assessed in 3T3 cell cultures.Results of histology showed significant reduction in the number of cells and nuclei as well as decreased amount of non-collagen proteins,such as glycosaminoglycan(GAG).At the microstructural level,the SCCO2-processed tissues appeared similar to the unprocessed tissue.3T3 cell viability was excellent as cells were able to migrate into the decellularized scaffold and proliferate in 7-day and 14-day cultures.Conclusions: The combination of mild chemical treatments and SCCO2 extraction were able to remove majority of cells from the cartilaginous scaffolds.The use of SCCO2 extraction in the terminal step has the additional advantage of eliminating chemical residues that remained in the prior process steps.SEM results showed no microstructural changes that could compromise biomechanical properties.The DC produced by this simple process can potentially be an ideal osteochondral scaffold for cartilage regeneration.