Collagen extraction, ELISA and Western Blot

Pellets (n = 1 per donor and group) were digested with pepsin solution [2.5 mg pepsin/ml pepsin buffer (0.5 M acetic acid, 0.2 M NaCl)] for at least 16 hours. The pH was then adjusted to neutral pH 7 with 1 M Tris Base prior to extraction of the collagens with 4.5 M NaCl (overnight at 4 °C, both Roth, Karlsruhe, Germany). After centrifugation, the pellets were resuspended in 400 μl precipitation-buffer (0.1 M Tris Base, 0.4 M NaCl) and the collagens precipitated for 4 hours at −20 °C with 100% ethanol. After centrifugation, the pellets were resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100). The collagen type II content was measured by native type II collagen detection ELISA kit (Chondrex, Redmond, USA) according to manufacturer’s instructions.

For collagen type II and X Western blotting, pellet lysates were separated by denaturing SDS-PAGE and proteins blotted onto a nitrocellulose membrane. The lower part of the membrane was incubated with mouse anti-human collagen type X antibody (Quartett) and the upper part with mouse anti-human collagen type II antibody (ICN Biomedicals). Bands were visualized with peroxidase-coupled goat anti-mouse antibody using the ECL detection system (Roche).