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of chondrocytes. For example, human primary articular, auricular and nasoseptal chondrocytes cultured in macroporous BNC scaffolds in vitro have been shown to adhere, migrate, proliferate and maintain their chondrogenic phenotype - as confirmed by the synthesis of cartilage-specific ECM [322, 343, 344]. Engineering stable and functional auricular cartilage tissue also depends on the cell source used. Pleumeekers et al. showed that human auricular and nasoseptal chondrocytes possess a high chondrogenic capacity in vivo , making them attractive cell sources for auricular cartilage repair [345]. The use of cells in cartilage repair is an attractive strategy as it may result in regeneration of the lost tissue. However, the clinical application of a cell-aided treatment does feature challenges – a limited supply of autologous chondrocytes with the proper phenotype being the most stringent one. To cancel out cell culture, including the concomitant laboratory logistics and the double surgery, autologous cells should be isolated within the operating room and applied directly. In addition, the combination of chondrocytes with a less limited source of autologous cells, such as bone marrowmononuclear cells (MNCs), can overcome the challenge of having too few cells and may even increase the treatment’s performance [346, 347]. By resuspending the cells in alginate, also the factor of cell loss after scaffold seeding can be diminished whilst simultaneously providing the cells with a 3D environment to suppress dedifferentiation [348]. Several studies that have evaluated BNC as a scaffold material for auricular cartilage TE [316, 322, 329, 343] have contributed to the design and development of BNC scaffolds with a two-layer (bilayer) architecture. This study investigates the in vitro and in vivo performance of bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, designed to be mechanically stable and maintain a long-term structural integrity while providing a porous architecture that supports cell ingrowth and neocartilage formation. Moreover, this study explores the application of a clinically relevant strategy by seeding a low number of freshly isolated (uncultured) human chondrocytes combined with freshly isolated human MNCs, in order to test the translation of this auricular cartilage TE technology to the clinic. 150 CHAPTER 8