Type 2 collagen

The collagen-coupled agarose gels (AG-Col) were prepared as previously described^{52 (link)}. Briefly, the type II collagen (Sigma) was suspended at 1.2 mg/ml in acetic acid (0.1 M), and was mixed with a 10-fold molar excess of the N-Sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (Sulfo-SANPAH; Thermo Fisher Scientific) in PBS at room temperature in the dark for 4 h. The 4% (w/v) agarose solution was prepared by dissolving low melting temperature agarose (Lonza) in sterile PBS. Three parts 4% agarose solution was then mixed with one part collagen/Sulfo-SANPAH solution to yield the mixture containing 3% agarose and 0.3 mg/ml collagen. The mixture was exposed under UV light for 20 min to allow the photocrosslinking reaction and conjugation of the collagen to the agarose. After conjugation, the agarose mixture was cooled down and washed with sterile PBS for 4 days to remove the unbound collagen and unreacted Sulfo-SANPAH. For control gels, AG/Col was prepared as stated previously without the addition of the Sulfo-SANPAH, and AG was prepared without the addition of the collagen and Sulfo-SANPAH.

Human IVD samples were harvested from lumbar spine surgery patients in compliance with the requirements of Qilu Hospital, Shandong University. NP cells were isolated and cultured as previously reported 34 (link). Briefly, after washing with sterile phosphate-buffered saline (PBS) 3 times, endplate cartilage and annulus fibrosus (AF) were meticulously eliminated from the human IVD tissues. IVD tissues were carefully cut into fragments approximately 1 mm³ in volume. Then, 0.25% trypsin (HyClone, Logan, USA) was used to digest the NP tissue for 30 minutes, followed by 0.2% type II collagen (Sigma-Aldrich, St. Louis, USA) for 4 hours. Then, NP cells were synchronously cultured (95% air, 5% CO2, 37°C) in DMEM/F-12 medium (HyClone, Thermo Co.) supplemented with 10% foetal bovine serum (FBS, Gibco, USA), 1% 100 U/mL penicillin and 100 mg/mL streptomycin (HyClone, USA) at pH 7.2. The culture medium was replaced every 3 days, and the cells were passaged when they reached 80-90% confluence. Second- or third-generation cells were used for the indicated experiments.

All experiments were performed using 8-week-old female Sprague Dawley rats (body weight 180~200g, purchased from Beijing Vital River Laboratory Animal Technology). A total of 140 rats were purchased, maintained and used according to institutional guidelines. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Inner Mongolia Medical University. Animals were housed 2 per cage and kept in the institutional animal facility at a constant temperature and humidity. Food pellets and water were provided ad libitum.
Rheumatoid arthritis was generated by type II collagen (Sigma) immunization as described previously (14). Briefly, type II collagen was dissolved in 0.01mol/L acetic acid overnight at 4°C. Collagen was then emulsified by complete Freund adjuvant (Sigma) and intradermally injected at the tail tip (0.2ml, 1mg/ml) for primary immunization. Two weeks later, type II collagen solution emulsified by incomplete Freund adjuvant (Sigma) was intradermally injected to the inguinal region at the same dose. A total of 100 rats were immunized to generate rheumatoid arthritis model.
Osteoarthritis was established by intra-knee joint cavity injection of L-cysteine papain (20 µl) in 20 rats [15 (link)]. 20 control animals received two intradermal injections of 0.9% saline.

CIA is a well-established mouse model for human RA [13 ]. Arthritic mice develop swollen joints, chronic inflammation, and joint destruction. CIA was induced by injecting a type II collagen at 2 mg/mL (Chondrex, Washington, USA) and complete Freud’s adjuvant (Sigma-Aldrich, Mannheim, Germany) 1:1 emulsion (200 μL) at the base of the tail in 10-week-old male WT and E2f2^−/− mice. A type II collagen and incomplete Freud’s adjuvant (Sigma-Aldrich) 1:1 emulsion was injected (200 μL) at the base of the tail in 14-week-old mice. Mice were monitored once per day for symptoms of arthritis.

For induction of CIA, we used the immunization protocol for C57BL/6 strain (H-2b)57 (link). Briefly, mice aged between 8 and 10 weeks of age were injected intradermally at several sites into the base of the tail and back with type II collagen (Sigma-Aldrich, Cat. no. C9301) emulsified in complete Freund adjuvant: incomplete Freund adjuvant (GIBCO), heat-killed mycobacterium tuberculosis (Difco Laboratories) on day −21 and the same injection was repeated on day 0 (Fig. 1a and Supplementary Fig. 2). Arthritis development in each paw was scored by macroscopic evaluation58 as follows: (0) no change, (1) erythema and mild swelling confined to the ankle, (2) erythema and mild swelling from the ankle to midfoot, (3) moderate swelling and (4) severe swelling. The maximum score per mouse is 16. The investigators (M.M., K.S., S.N. and J.S.H.) were blinded to the genotypes. Ten to twenty mice/genotype were used (Fig. 1 legend and Supplementary Fig. 2 legend). Mice were dissected 2 weeks after the second immunization to evaluate the draining lymph nodes (popliteal, inguinal, axillary and brachial).

DBA/1 mice were immunized with chicken type II collagen (Chondrex, Redmond, WA, USA) emulsified in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA) by subcutaneous injection in the tail (100 μL). After three weeks, mice received a booster immunization with type II collagen emulsified in incomplete Freund’s adjuvant (Sigma-Aldrich) (100 μL) [25 (link)]. Mice were anesthetized in 3–4% sevoflurane in 65% N₂ and 35% O₂ during both immunizations. Four weeks after initial immunization, signs of arthritis in the paws developed. Each paw was scored three times a week on a scale from 0 to 4. The following criteria were used: 0, normal paw; 1, one toe inflamed and swollen; 2, more than one toe, but not entire paw, inflamed and swollen or mild swelling of entire paw; 3, entire paw inflamed and swollen and 4, very inflamed and swollen paw or ankylosed paw. A mean score of 2.5 for all paws was considered maximum per animal.

Arthritis was induced through the standard procedure using Type II collagen obtained from the chicken tracheal cartilage purchased from Sigma-Aldrich (USA). In this method, the healthy, uninfected, acclimatized albino rats were immunized with II collagen, dissolved in 0.1 M acetic acid (2 mg/ml) at 4°C overnight, and emulsified with an equal volume of Complete Freund's adjuvant (CFA) (Sigma-Aldrich, USA) [20 (link)]. This was followed by each rat (n = 15) being injected intradermally with 4 mg/kg of collagen suspension at multiple areas around ankle joint and at the base of the tail. A booster dose injection was given after a week following the primary inoculation.
The development of rheumatic symptoms was observed following the second injection, paw edema being the first and most prominent visible signs of arthritic changes. The first set of readings for various evaluation parameters including paw edema was taken on the 15^th day. The second booster dose was administered 15 days after the first booster injection, i.e., on 22^nd day of experimentation. Following the protocol, the 1^st reading was taken on the 15^th day after immunization, and the subsequent readings were taken on the 30^thday and the 45^th day of experimentation.