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essential for cell migration, partial or even complete depletion of sGAGs could be beneficial to realize cartilage revitalization, as it has been shown that chondrogenic progenitor cells possess the capacity to migrate through degraded cartilage and repair ECM. [309] This indicates that optimization of cell migration could lead to matrix synthesis and restored biomechanical properties of the revitalized cartilage. Recovery of biomechanical properties due to matrix deposited by cells was previously seen by Reiffel et al. [160], who reported de novo cartilage deposition and a 30-fold increase in Eeq 3 months after in vivo implantation of a collagen type I hydrogel. This showed that the biomechanical properties returned to the native situation. Finally, the decellularized h EC scaffolds and whole human ear preserved their size and shape after decellularization. Also, approximately 25% more sGAGs were retained than in the decellularized b EC. The maturity of the h EC matrix might cause better retention of sGAG. In human ears, the ECM components and especially elastic fibers structurally change over the years. [164] When stained for elastin, the elastic fibers in our b EC are mainly directly located as a band around the lacunae whereas in h EC, this network extends more into the ECM, confirming what is shown previously by Ito et al. [164] This difference in elastic fibers in adult cartilage, could have protected the ECM from degradation during decellularization. Importantly, this retention was also reflected in the Eeq of the h EC scaffolds, which was not reduce compared to that of native h EC (3.3 ± 1.3 MPa for Eeq) measured in our previous work. [155] This shows that the decellularization process can also be translated to human tissue and provides the possibility to use decellularized ear cartilage as an improved reconstruction strategy. In conclusion, decellularization can provide scaffolds made of natural materials, even allogeneic or xenogeneic, for reconstruction of defects in cartilaginous structures. We have prepared decellularized ear cartilage scaffolds with an architecture and matrix composition that closely resembles native cartilage and that have the capacity to support chondrogenic differentiation of BMSCs. Furthermore, the translation of the decellularization method to whole human ear cartilage shows the possibility to use decellularization as an improved reconstruction strategy for large cartilage defects that hold complex shapes. In order to implement the method as a clinical treatment, long term in vivo studies should be conducted to assess the scaffold functionality and characteristics after implantation. Acknowledgements The authors would like to thank Prof. dr. Gert-Jan Kleinrensink (dept. of Neurosciences and Erasmus MC SkillsLab) and Cornelia Schneider (LBI/Red Cross Blood Transfusion Service of Upper Austria) for providing human cartilage. Thanks to Marcel Vermeij (dept. of Pathology, Erasmus MC) for his help with the RF stain, dr. Gert-Jan Kremers (Optical Imaging Centre, Erasmus MC) for his help with fluorescent imaging, Prof. dr. Heinz Redl (LBI for Experimental and Clinical Traumatology) for his comments on the manuscript. And finally, the authors would like to thank Mairéad Cleary (dept. of Orthopaedics, Erasmus MC/University College Dublin) for thoroughly reading the manuscript and providing constructive comments. This study was partially supported by the FFG-Bridge grant CartiScaff (842455). 144 CHAPTER 7