15502-m-pleumeekers

Elastin content Elastin content (EC: n =48) was measured using the Fastin Elastin Assay (Biocolor, Carrickfergus, UK) according to the manufacturer's instructions. Briefly, the sample was converted to water soluble α-elastin by three 12 hour heat extraction cycles at 100°C in 0.25 M oxalic acid before adding the kit's dye. Absorbance was measured at 513 nm on an Infinite F200 PRO (Tecan, Giessen, the Netherlands) plate reader. α-elastin from bovine neck ligament (from the manufacturer) was used as a standard. Elastin values were normalized to sample wet mass. Since NC samples contain no elastin [143], elastin was only quantified for EC samples. Histology EC samples from all ear regions and young and old adult age groups were embedded in OCT (Tissue-Teks O.C.T™ Compound, Sakura). Samples were cryosectioned at a 5 mm slice thickness and stained with Sirius red, Resorcin-Fuchsin and Safranin-O to visualize the collagen network, elastic fibers and sGAG, respectively. Statistical analysis A linear mixed-effect model, where donor was a random effect, and cartilage subtype, gender, age group and harvesting location were fixed effects, was used to analyze statistical differences between mechanical (Ein, σ max , Eeq, t 1/2 ) and biochemical (DNA, sGAG, hydroxyproline) results. Additionally bivariate, correlation analyses between mechanical and biochemical parameters were performed for EC and NC samples. All calculations were performed with SPSS (version 22.0, IBM Corp., New York, USA). All data are displayed as mean ± standard deviation, where ٭ indicates significance ( p <0.05). 32 CHAPTER 2