15502-m-pleumeekers

Mechanical evaluation Cartilage samples (EC: n =183; NC: n =103) were placed in close-fitting stainless steel cylindrical wells, and tested with a materials testing machine (Zwick Z005, Ulm, Germany) equipped with a 10 N load cell, built-in displacement control, and a cylindrical, plane-ended, stainless steel indenter (0.35 mm). During testing samples were immersed in PBS supplemented with antibiotic/antimycotic, and stress relaxation indentation tests were performed at room temperature, as described previously. [155, 156] Briefly, a preload of 3 mN was applied to locate the sample surface and measure sample thickness, h, and held for 5 min. Five successive strain steps (5% of h per step) were applied, and specimens were left to relax for 20 min at each step. An in-house Matlab® script converted force and displacement data to stress and strain, and instantaneous modulus (Ein), maximum stress (σ max ), equilibrium modulus (Eeq), relaxation half life time (t 1/2 ) were determined. To estimate viscoelastic relaxation, t 1/2 is computed after the first strain application. It is defined as the time needed for stress to decrease from its maximum value halfway to its equilibrium value. [155] Biochemical evaluation Following mechanical testing, each sample was cut into two and frozen at -80°C until processing. Samples were defrosted, and wet weight of each half was measured. One half was digested overnight at 60°C with papain buffer (0.2 M NaH2PO4, 0.01 M EDTA, pH 6.0 and freshly added 250 μg/mL papain, and 5 mM L-cystein), and analyzed for DNA, sGAG, and hydroxyproline content. The second half was analyzed for elastin content. DNA content Amount of DNA (EC: n =223; NC: n =153) was determined by ethidium bromide (GibcoBR1), using calf thymus DNA as a standard. Samples were analyzed with a spectrofluorometer (Wallac 1420 Victor 2; Perkin-Elmer, Wellesley, USA), using an extinction (340 nm) and an emission (590 nm) filter. DNA content was normalized to sample wet mass. Glycosaminoglycan content Sulfated-glycosaminoglycan content (sGAG) (EC: n =223; NC: n =154) was quantified using the 1,9-Dimethylmethylene blue (DMMB) dye-binding assay, where metachromatic reaction was monitored using a spectrophotometer. Absorption ratios of 540 nm and 595 nm were used to determine sGAG content with chondroitin sulfate C (shark) as a standard. sGAG values were normalized to sample wet mass. Hydroxyproline content Hydroxyproline content was measured to estimate collagen quantity (EC: n =189; NC: n =140) using the Total Collagen Assay (QuickZyme Biosciences, Leiden, the Netherlands) according to the manufacturer's instructions. Briefly, papain digests were hydrolyzed with equal volumes of 12 M HCL at 95°C for 18–20 hours. Hydroxyproline content was measured using a modification of the Prockop and Udenfriend method [157], and normalized to sample wet mass. 31 EAR CARTILAGE CHARACTERISTICS 2