15502-m-pleumeekers

MATERIALS AND METHODS Chemicals were obtained from Sigma-Aldrich, USA unless stated otherwise. Samples Cartilage samples were harvested from fresh frozen cadaveric donors ( n =8 male; n =2 female), average age 66.5 ± 6 years at UMCU (University Medical Center of Utrecht, the Netherlands) according to the ethical guidelines of the institution. From each donor 2 adjacent samples from the ear concha, medial nasoseptal cartilage and lateral alar cartilage were removed with a 4 mm biopsy puncher. The samples were shipped at -20°C to either the VUmc (Vrije Universiteit Medical Center, Amsterdam, the Netherlands) for biomechanical and microscopic evaluation or EMC (Erasmus Medical Center, Rotterdam, the Netherlands) for biochemical evaluation. Samples were thawed prior to experiments and remaining tissue and perichondrium were surgically removed. Indentation To determine mechanical properties indentation measurements were performed using a novel commercial nano-indenter (Piuma, Optics11, the Netherlands). The device utilizes a ferrule-top cantilever probe [197] to apply load and simultaneously measure indentation depth using a fiber optic based readout. (Figure 1A and C) In this set up a 78 µm diameter spherical probe was used capable of applying forces ranging from 0.1 µN to 7.5 mN at indentation depths ranging 1 to 17 µm. Cantilever bending calibrations were performed prior to each series of experiments by indenting a rigid surface and equating cantilever bending to probe displacement. Each sample was indented 10 times on the same anatomical location in a grid pattern with 100um distance between measurements. The resulting stress strain curves (Figure 1B) were analysed using the mathematic model derived by Oliver and Pharr for a spherical indenter to determine the effective Young´s modulus (E*). [198] The indentation protocol was carefully optimized to minimize viscoelastic effects from influencing the measurements. (Data not shown) Biochemical evaluation Prior to biochemical analysis, wet weight was determined of all cartilage samples which were then digested overnight at 60°C in a papain solution (0.2 M Na2H2PO4, 0.01 M EDTA.2H2O, 250 µg/mL papain, 5 mM L-cystein, pH 6.0). The amount of DNA measured in each papain- digested cartilage sample was determined by Ethidium bromide (GibcoBR1), using calf thymus DNA as a standard. Samples were analysed with a spectrofluorometer (Wallac 1420 Victor 2; Perkin-Elmer, Wellesley, USA), using an extinction filter (340 nm) and an emission filter (590 nm). A 1,9-Dimethylmethylene Blue (DMMB; pH 3.0) assay was performed to measure the sulphated-glycosaminoglycan (sGAG) content in each papain-digested cartilage sample. The metachromatic reaction of DMMB was monitored using a VersaMax spectrophotometer at 530 and 590 nm. Shark chondroitin sulphate C was used as a standard. 47 FACIAL CARTILAGE CHARACTERISTICS 3