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DISCUSSION For successful cartilage regeneration 3D-scaffolds are crucial. We were able to obtain decellularized bovine and human scaffolds from whole full-thickness ear cartilage (EC) tissue. These scaffolds preserved their native collagen and elastin contents, as well as their major architecture and shape. Furthermore, these decellularized EC scaffolds were non-cytotoxic and have the capacity to allow chondrogenic differentiation of human BMSCs in vitro . To date, decellularized scaffolds are extensively used for the reconstruction of various tissues and organs. [293] In addition, several cartilaginous structures have been decellularized. These studies, however, mainly focus on hyaline (i.e. articular cartilage, nasal cartilage, tracheal cartilage) or fibrous (i.e. meniscal cartilage, annulus fibrosis) cartilaginous tissues. Other cartilage decellularization techniques described in literature are the fabrication of decellularized ECM-derived scaffolds, by either pulverizing cartilage tissue [290, 291] or stacking thin cartilage slices. [294] Although these seem effective methods to decellularize the tissue, its major drawback is that it completely disrupts the native tissue architecture and/or shape. In fact, no method to specifically decellularize full thickness EC has been described in literature yet. This study is the first to evaluate structural and functional properties of decellularized full-thickness EC scaffolds of both bovine ( b EC) and human ( h EC) origin. Various decellularization protocols are proposed for cartilaginous tissues, each aiming to maximize the decellularization effect, while reducing any adverse effect of the process on the structural composition and functionality of the remaining ECM. Therefore, decellularization outcome was evaluated based on: (1) the removal of cellular material and (2) matrix integrity which was characterized by its components, architecture and biomechanical properties. First, removal of native cellular material is highly imperative, as it reduces the possibility of an immune reaction in case of in vivo implantation. For this reason, one of the criteria for successful decellularization is to reduce the DNA content to less than 50 ng/mg tissue. [295] Unfortunately, most recently developed decellularization protocols for cartilage do not meet this requirement at all [100, 105]. Decellularized cartilage scaffolds still show distinct cell remnants on histological examination [94-96, 99-101, 104, 105, 296-299] or need multiple decellularization cycles to remove nuclear material, [101, 299] leading to further degradation of the ECM. To specifically decellularize EC, the samples were decellularized according to the protocol of Kheir et al. [101] which was further optimized to ensure the decellularization outcome was satisfactory for EC and cell remnants reduced. The incorporation of an additional 24 hour incubation with a low concentration of elastase (0.03 U/mL), enabled the removal of nuclear material and a reduction of cell remnants in decellularized b EC scaffolds and near-complete removal in decellularized full size human ear cartilage scaffolds. It should be noted though, that the 10 ng detection limit of the DNA assay, concerns a fraction of papain digest used in the DNA assay (50 µL). Because the DNA content was undetectable in that fraction of the decellularized b EC scaffolds, it is reasonable to assume that DNA was removed from the entire scaffolds after decellularization. Second, the balance between the removal of nuclear material and preserving the matrix integrity should be considered carefully. We showed that the decellularized EC scaffolds preserved their native collagen and elastin contents, as well as their major architecture and shape, while sGAG content significantly decreased during the process. 142 CHAPTER 7