15502-m-pleumeekers

Macroscopic examination of the bilayer BNC scaffolds after the in vitro and in vivo studies revealed that the adhesion between the dense and porous layers remained stable, as there were no signs of adhesive failure. We postulate that the interfacial bonding between the layers is like a molecular welding process. As the BNC-EMIMAc solution partly dissolves both surfaces at the interface, this makes it possible for the long chains of BNC to diffuse into both layers. Once the dissolved BNC is precipitated in ethanol, the interface structure is locked, which results in a stable interfacial bonding. We have observed that when pulling the dense and porous layers apart, the scaffold breaks at the porous layer, similar to a structural failure. In contrast, a weak adhesion would have had resulted in an adhesive or cohesive failure at the interface. Based on this observation we speculate that the interfacial bonding between the layers is stronger than the structure of the porous layer, although in this study the interfacial strength of the bilayer BNC scaffolds was not measured. The compact BNC network structure of the dense layer provided a good mechanical stability, while the interconnected high porosity layer (75% porosity with mean pore size of 50 ± 25 µm) supported the ingrowth and homogeneous distribution of NCs throughout this layer. In agreement with our previous study, which evaluated BNC/alginate composite scaffolds in vitro [343], bilayer BNC scaffolds also supported the redifferentiation of NCs to a more chondrogenic phenotype, which led to the formation of neocartilage; as demonstrated by the increase in gene expression of chondrogenic marker genes ACAN and COL2A1 and homogeneous distribution of cartilage-specific ECM after 6 weeks of in vitro culture. Although, the expression of dedifferentiation marker VCAN remained constantly low, a strong expression of COL1A1 was observed during the in vitro culture. The presence of COL2A1 and COL1A1 indicates a subpopulation of NCs that did not switch to a chondrogenic phenotype during 3D culture in bilayer BNC scaffolds under chondrogenic medium conditions. After 6 weeks of in-vitro culture, a rich and homogenous distribution of cells and neocartilage was observed throughout the porous layer of the scaffold, even in the center, which is known to be a critical region in static 3D culture due to the limited supply of nutrients and oxygen. This outcome could have been accelerated by increasing the percentage of cells retained in the scaffolds after cell seeding, as there was a substantial loss of cells when these were seeded in medium. Embedding the cells in alginate significantly improved cell retention in the scaffolds after seeding. Alginate was chosen, since it has been successfully used to seed chondrocytes in a scaffold for in vivo implantation [358, 359] and this hydrogel is well known to maintain a chondrogenic phenotype of human chondrocytes and stimulate neocartilage formation [348]. In the in-vivo study we explored the application of a clinically relevant strategy, by seeding bilayer BNC scaffolds with a low number of freshly isolated human chondrocytes combined with freshly isolated human mononuclear cells, in order to test the translation of this auricular cartilage TE technology to the clinic. At 8 weeks post-implantation, deposition of cartilage matrix components such as proteoglycan and type II collagen were observed predominantly in MNC/NC-seeded constructs. The strong Safranin-O stain surrounding the cells showed the presence of proteoglycans in the newly synthesized ECM, while the presence of type II collagen was confirmed by immunohistochemistry. These results, showing the formation of neocartilage in the porous layer, were further confirmed by biochemical analysis; 168 CHAPTER 8