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to the septal cartilage and low in elastin content, demonstrated mechanical behaviour that is more comparable to ear cartilage. While similar in general appearance, we observed that chondron size seemed to be different between ala nasi and septum cartilage. Sample size was small however and no statistical evidence was gathered to support this side finding. Donor variability is large and general comparison of our data with literature is difficult as no research has previously been performed comparing these three cartilage types both mechanically, structurally and biochemically. Our data match the observations of Nimeskern and colleagues [201] that septal cartilage is stiffer and contains higher sGAG than auricular cartilage but lower DNA, indicative for lower cell concentration. Our findings also match the results of a study performed by Griffin and colleagues [28] who measured lower stiffness of the ala nasi cartilage compared to the septum. For tissue engineering purposes the scale at which the indentation experiments were performed gives a good reference for the appropriate scaffold stiffness on the cellular level. SHG proved a good tool to non-invasively depict the collagen and elastin bundle architecture in 3D. This information could be translated to serve as a structural template for 3D printing of scaffolds and together with the data on mechanics and biochemical content provide a new step towards scaffold optimisation for facial cartilage reconstruction. We used cartilage samples from donors at higher age (average 66.5 ± 6 years). Mechanical behaviour and histology might differ in younger patients due to calcifications and structural changes during aging. Ears for example continue to expand in volume throughout a lifetime which is attributed to alterations in the elastic fibers during aging. [164] For the septum however Richmon and colleagues [26] found no significant differences in mechanical properties between age or gender. Although samples were taken from the same anatomical location in all donors, minor variation might have occurred. This is a limitation, as several studies indicate that within the separate cartilage types there are regional differences in content. [28] Despite their localisation and comparable role as soft tissue support suggesting similar characteristics, the facial cartilages are in fact quite different from another. The specific function of cartilage tissue, for example compression for articular cartilage and flexibility for ear cartilage, may demand different mechanical testing regimes. We chose micro-indentation to explore the stiffness of the ECM, in regard of our findings perhaps a combination of mechanical tests is necessary to be able to elicit the different structural roles of the various cartilage components. In the future it might be interesting to also include macroscopic mechanical testing, as gross mechanical traits are also influenced by other factors such as the perichondrium and anatomical form. [29, 204] From a surgical perspective, it is interesting to note that tissue composition and mechanical behaviour are not always related as expected. We did not find an explanation for the lower stiffness of ala nasi cartilage. It does support the concept that tissue transplants from different origins can serve as structural surrogate in reconstructive surgery. The use of concha tissue for ala nasi reconstruction is an excellent example thereof. Other fields that are not covered in this paper but are important to consider are cellular interaction including proteomics and metabolism, as cell survival and behaviour are key to tissue engineering and long-term successful transplantation. The finding that the facial cartilage types not only structurally 54 CHAPTER 3