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MATERIALS AND METHODS All chemicals were obtained from Sigma-Aldrich, St. Louis, USA unless stated otherwise. Preparation of decellularized cartilage scaffolds To obtain full thickness bovine ear cartilage ( b EC), macroscopically intact cartilage was harvested from calves ( n =3) less than 8 months old (T. Boer & Zn., Nieuwerkerk aan den IJssel, the Netherlands) and washed with phosphate buffered saline (PBS) after careful resection of the perichondrium. Bovine articular cartilage samples ( b AC) were harvested from the metacarpophalangeal joints ( n =3) and included as controls to compare decellularization outcomes. Samples were made using an 8 mm dermal biopsy punch (Spengler, Asnières sur Seine, France) and kept in PBS until decellularization. Human ear cartilage ( h EC) was obtained from post mortem donors ( n =2; M, 83 and 84 Y) who donated their bodies to medical science at Erasmus Medical Center (EMC; Rotterdam, the Netherlands). Dermal tissue was macroscopically removed, followed by careful removal of the perichondrium and samples were made using an 8 mm dermal biopsy punch. Untreated (i.e. native) cartilage samples were immediately stored dry at -80°C after harvest for biochemical analysis or in 4% formaldehyde for histological analysis and scanning electron microscopy (SEM). All human and bovine cartilage samples were decellularized according to the protocol of Kheir, et al . [101], which was further optimized to specifically decellularize ear cartilage. Briefly, the samples were subjected to 2 overnight dry freeze-thaw cycles followed by 2 overnight freeze-thaw cycles at -20°C in hypotonic buffer (10 mM Tris-HCl in Mili-Q water, pH 8.0) following a 24 hour incubation in hypotonic buffer at 45°C. Next, samples were treated for 24 hours with an ionic detergent consistent of 0.1% sodium dodecyl sulfate (SDS), 0.1% ethylenediaminetetraacetic acid (EDTA) and 10 KUI/mL aprotinin in Mili-Q water. Then, samples were washed twice for 30 minutes in wash solution (PBS with 10 KIU/mL aprotinin) before a 24 hour wash at 45°C in wash solution. Since the protocol of Kheir et al . was not sufficient to reduce or remove cellular remnants, an elastase solution was incorporated into the protocol to improve the removal of cellular remnants. Therefore, the samples were treated next with a low concentration elastase solution (0.2 M Tris-HCl in Mili-Q water, 10 KIU/mL aprotinin and 0.03 U/mL elastase, pH 8.6) for 24 hours at 37°C, as a high concentration elastase would completely damage the matrix structure due to the complete depletion of elastin and glycosaminoglycans (GAGs). (Supplementary figure 1) Next, samples were washed twice and incubated for 3 hours at 37°C in nuclease solution (50 mM Tris-HCl in Mili-Q water, 10 mM MgCl, 50 µg/mL bovine serum albumin (BSA), 50 U/mL DNAse and 2.5 U/mL RNAse, pH 7.5). Samples were washed again in wash solution and treated for 3 hours in decontamination solution (0.1% peracetic acid in PBS). All incubation and wash steps were performed with agitation. Finally, the samples were transferred to sterile tubes and washed twice for 30 minutes in sterile PBS before starting a 24 hour wash cycle in sterile PBS at 45°C. To assess the decrease in wet weight after decellularization, samples from one donor of both cartilage types were weighted directly after harvest and subjected to an individual decellularization treatment taking into account volume ratios of the used solutions. After the individual treatment, wet weight was determined again. Samples from the remaining donors 132 CHAPTER 7