Cysteine

Crude peptides were all purchased from Beijing SciLight Biotechnology Ltd. Co.; glutathione (GSH), oxidized glutathione (GSSG), DTT, and cysteine were obtained from Gibco (Carlsbad, CA). All other reagents were analytically pure. All peptides were folded and purified as described previously [24 (link),25 (link)]. The amino acid sequences of MVIIA and its variants are listed in Scheme 1.

Fresh cartilage tissues were isolated from the knee joints of newborn mice and digested for 6 h at 37 °C in DMEM F12 medium (Hyclone, Logan, UT, USA), which contained 10% FBS (Gibco, New York, NY, USA), P/S (meilunbio, Dalian, China), cysteine and collagenase P (Sigma, St. Louis, MO, USA), and the digestion was terminated by adding the complete medium to the basal digest. The digest was then collected and centrifuged at 200 g for 15 min. The supernatant was removed, the precipitate was resuspended in 10 mL DMEM F12 + P/S + FBS, and the suspension was filtered through a 100 mm Falcon filter in a fresh 50 mL test tube. Next, the pellet was resuspended in DMEMF12 + P/S + FBS by centrifuging the tube at 200 g for 5 min, and the suspension was filtered through a 40 mm Falcon filter into the used 50 mL tube. The filter was flushed with 10 mL DMEMF12 + P/S + FBS, and the suspension was again filtered through the used filter. The centrifugation and suspension cycles were performed twice. Finally, chondrocytes were obtained and inoculated in 6‐well plates at 5 × 10⁵ cells·mL⁻¹. Chondrocytes were cultured in a humidified environment supplemented with 10% FBS, 0.1% P/S, 50 mg·mL⁻¹l‐ascorbic acid (Gibco, New York, NY, USA), and cysteine solution (35.1 mg·mL⁻¹) containing 5% carbon dioxide at 37 °C. The culture medium was changed every 2 days.