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mineralization [73, 75]. Moreover, by decreasing the amount of chondrocytes required (≤ 20% of the total cell mixture), culture-expansion was no longer necessary, which allowed the use of freshly isolated primary chondrocytes leading to improved cartilage formation. [76] Unfortunately, co-culture research has mainly focused on articular cartilage repair. The effect of non-articular chondrocytes in co-culture, such as ear [77-79] or nasal chondrocytes (NCs) [80], are sadly underexposed, although they seem essential for cell-based cartilage repair in the head and neck area. In depth understanding of the cellular interaction pathways between MSCs and chondrocytes is still under debate in literature: It is thought that the co-culture effect is either credited to (1) chondrocyte-drivenMSC-differentiation or ascribed to (2) chondrocytes, whose cartilage-forming capacity and proliferation activity are enhanced in the presence of MSCs. [81] In recent years, the trophic and paracrine functions of MSCs appeared most critical in this process, rather than the simple chondrogenic differentiation of MSCs alone. However, little is known as to whether their trophic function is a general characteristic of MSCs or dependent on the origin of the MSC source. Scaffolds For successful cartilage regeneration, the properties of the 3D matrix are of equivalent importance: (1) it must provide temporary or permanent cell-support while maintaining size and shape when subjected to the forces of the implanted environment; and (2) it needs to mimic the natural microenvironment to provide specific structural, mechanical and biological cues to cells, which guide tissue remodelling. [82] Currently, several 3D-scaffolds have been developed and investigated for their use in cartilage tissue engineering. [83] They can be roughly classified into synthetic and natural scaffolds. Synthetic scaffolds that are most intensely studied in the field of cartilage tissue engineering are the biodegradable polymers, such as polylactic acid (PLA), polyglycolic acid (PGA), and their co-polymers. [84] Their main benefit is that they can be fabricated in large quantities under controlled conditions and have predictable and reproducible physical properties. Although these materials are advantageous to work with, they are prone to induce a foreign body reaction which can inhibit cartilage regeneration and lead to tissue extrusion. [83] Next to synthetic materials, natural scaffolds have been introduced, such as hydrogels (e.g. alginate, chitosan, collagen, gelatine, hyaluronic acid, fibrin), bacterial nanocellulose, and decellularized ECM. [85] Unlike synthetic scaffolds, natural polymers are distinguished by low risk of toxicity and a reduced foreign body reaction. [85] On the contrary, their properties are less reproducible and more heterogeneous. Also, purification issues relevant to clinical use, represent a major challenge. Unfortunately, to date, no ideal scaffold has emerged as a promising scaffold for future clinical application for cell- based cartilage repair in the head and neck area. This thesis focuses on natural scaffolds. In particular, this thesis focusses on the quality and suitability of alginate, bacterial nanocellulose and decellularized ECM for tissue-engineering purposes in the head and neck area. Alginate is a hydrogel and formed from polysaccharides derived from brown algae. It consists of a mixture of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. [86] Both cell adhesion and hydrogel stiffness can be influenced by M to G ratio. [87] Alginate mechanical stiffness is however low and range from 1 to 1000 kPa. [88] Although alginate itself CHAPTER 1 18