Protein was extracted from knee joints according to Nielsen et al (24 (link)) at week 0, 1, 2 and 4 after operation. Knee joints were isolated after euthanasia as described above, then immediately frozen in liquid nitrogen and stored at −80°C for use. The tibia and femur were cut 3 mm from the joint, producing samples weighing 500–700 mg. These samples were frozen in liquid nitrogen again, and transferred to a Bessman tissue pulverizer (Spectrum Chemical Manufacturing Corp.). The samples were crushed with 3 ml extraction buffer, consisting of 50 mM Tris-HCl buffer (pH 7.4), 0.1% Triton X-100, 0.1 M NaCl, 10 Mm (GM6001; Enzo Life Sciences, Inc.) and 1 tablet/10 ml buffer of Complete Mini EDTA-free protease inhibitor cocktail (Roche Diagnostics). Tissues were homogenized twice for 30 sec using an OMNI homogenizer (Omni International, Inc.) at speed level 4, then the samples were centrifuged for 10 min at 1,700 × g and 4°C, supernatants were obtained and centrifuged at 10,000 × g and 4°C. Finally, the supernatants were stored at −20°C until use.
Rat-specific commercially available ELISA kits (Elabscience®) were used to evaluate the levels of C-terminal telopeptide of type I collagen (CTX)-I (cat no. E-EL-R1456) and CTX–II (cat no. E-EL-R2554) in protein extracts collected from the knee, according to the manufacturer's instructions (24 (link)).
Rat-specific commercially available ELISA kits (Elabscience®) were used to evaluate the levels of C-terminal telopeptide of type I collagen (CTX)-I (cat no. E-EL-R1456) and CTX–II (cat no. E-EL-R2554) in protein extracts collected from the knee, according to the manufacturer's instructions (24 (link)).