15502-m-pleumeekers

where sGAG-production in the MNC/NC-seeded bilayer BNC scaffolds was significantly higher than the control condition (12-fold). The presence of cartilage matrix in the bilayer BNC scaffolds was also supported by biomechanical analysis. At 8 weeks post-implantation, a significantly higher initial matrix stiffness and improved relaxation kinetics (i.e. higher Ein and t 1/2 values) were observed in the MNC/NC-seeded scaffolds compared to the non-implanted and cell-free conditions. In fact, the effect size (i.e. ω and r > 0.5) obtained from the Ein and t 1/2 data represents a large effect by the MNC/NC condition. However, there was no significant difference in Eeq and σ max for the three conditions. Considering the improved relaxation kinetics in the MNC/NC-seeded constructs, we conclude that the ability of the MNC/NC-seeded scaffolds to attract and trap water was enhanced through the production and accumulation of proteoglycans and glycosaminoglycans in the bilayer BNC scaffolds. Nevertheless, since the intrinsic scaffold properties did not improve in the MNC/NC-seeded constructs compared to the no cell control (no difference in Eeq), it implies that collagen matrix was not effectively produced in the porous layer. To put the results from the biomechanical analysis in a clinical context, the values for instantaneous and equilibrium moduli measured from the MNC/NC-seeded constructs after 8 weeks of implantation were 8.4- and 17.4-fold lower, respectively, compared to human auricular cartilage (e.g. 6.4 ± 3.2 MPa for Ein and 3.3 ± 1.3 MPa for Eeq [329]). Therefore, the engineered cartilage as such would not be suitable for immediate ear cartilage replacement; rather modifications in cell concentration and perhaps a longer implantation period needs to be considered. The present study has certain limitations. Firstly, cell density plays a critical role when engineering functional and stable cartilage. Others have demonstrated that cell densities greater than 20 × 10 6 cells/ml are desirable, while low cell densities resulted in decreased cartilage formation [360]. During embryology of cartilage, densely packed and proliferative mesenchymal cells are responsible for depositing the vast amount of cartilage ECM. In cartilage TE, early phase of cartilage development needs to be simulated to generate functional and stable cartilage. Therefore, in order to enhance the outcome of tissue- engineered auricular cartilage, a higher cell density is needed to benefit from increased cell- cell contacts signaling chondrogenic ECM deposition and preventing the dedifferentiation process. Despite the limitations already stated, our findings support that bilayer BNC scaffolds in combination with alginate provide a suitable environment for MNCs and NCs to support the synthesis of neocartilage. Of equal importance, the use of freshly isolated human chondrocytes and mononuclear cells in the in vivo study gave us an indication of the potential of this strategy to advance the translation of cell-aided treatments to the clinic. ` Most auricular cartilage TE strategies have revolved around biodegradable scaffolds, where the hypothetical optimum has been the scaffold’s degradation orchestrated by the neo- tissue formation. It would be ideal if the scaffold could be degraded by the time the neocartilage has reached full mechanical strength. However, fine-tuning this intricate play has proven to be a challenge in TE. If the material degrades too rapidly, the neocartilage will collapse. Whereas if it degrades too late, it could induce a continuous inflammation that would affect the cartilage formation and when the material is finally degraded it would leave holes in the tissue, making it more prone to crack or collapse. Thus, we aim for a hybrid implant – 169 NOVEL BILAYER BNC: AN ECM-INSPIRED SCAFFOLD 8