Bovine type 2 collagen

Bovine type II collagen (2 mg/mL in 0.05 M acetic acid; Chondrex, Inc., Redmond, WA, USA) was mixed with the same volume of Freund complete adjuvant (2 mg/mL Mycobacterium tuberculosis; Chondrex, Inc.). DBA/1 mice were given an intradermal injection of 100 μg of Bovine type II collagen on day 1 into the base of the tail. On day 21, mice received a booster intradermal injection of 100 μg of Bovine type II collagen emulsified in an equal volume of incomplete Freund adjuvant (Chondrex, Inc.). The mice were randomly divided into control, collagen-induced arthritis (CIA), SR9009 low-dose, and SR9009 high-dose groups. To evaluate the effect of SR9009 on the development of arthritis, mice were administered intraperitoneally 50 or 100 mg/kg body weight SR9009 (low and high doses, respectively) seven times per week for 4 weeks after the booster injection. At 48 days, the animals were anesthetized and euthanized.

The AIA mouse model was established through intradermal injection of 0.05 ml of CFA (Sigma, San Diego, CA, USA) into the right plantar of mice on day 0. For mice in the control group, 0.05 ml of 0.9% normal saline was injected into the right plantar. Bovine type II collagen emulsion was prepared by mixing 2 mg/ml of Bovine type II collagen (Chondrex, USA) with 1 mg/ml of complete Freund’s adjuvant. The solution was injected subcutaneously at the base of the DBA/1 mouse tail (100 μl per mouse) on day 0. At 21 days after the first immunization, the mice were injected with Bovine type II collagen emulsion subcutaneously as a booster immunization (100 μl/only)(11 (link)). Mice in the control group were injected with 0.9% saline solution 100 μl at the base of the DBA/1 mouse tail.

Bovine type II collagen (Chondrex, Redmond, WA, USA) was emulsified at a ratio 1:1 with compelet Freund’s adjuvant (Chondrex) containing 2 mg/mL heat-killed Mycobacterium tuberculosis. 6-week-old male DBA/1 mice (n = 5 per group; OrientBio, seongnam, Korea) received a primary immunization, followed by a boosting immunization on day 21 using the same concentration of Bovine type II collagen and incomplete Freund’s adjuvant (Chondrex). Injection was intradermally conducted at the base of tail. The severity was observed for 28 days after first injection. The severity of arthritis was monitored and scored as determined by hind paw swelling and clinical scoring [19 (link)]. To evaluate therapeutic efficiency, 1 × 10⁶ of AMMs, AMM/I and PBS were injected intraperitoneally twice a week (day 0 and day 7) when the arthritis score reached 3 or more.

The process for CIA induction has been described previously [34 (link)]. In brief, bovine type II collagen (Chondrex, Woodinville, WA, USA, Cat#20021) and complete Freund’s adjuvant (Chondrex, Cat#7001) were fully emulsified. Then, 0.1 mL of the emulsion was injected into the tail root intradermally. The day of the primary injection was defined as day 0. A booster injection of 0.1 mL emulsion of bovine type II collagen and incomplete Freund’s adjuvant (Chondrex, Cat#7002) was intradermally injected into the back of mice on day 21. Meanwhile, normal saline was injected, in parallel, into mice of the control group.

After nine administrations of bacteria or vehicle, mice (11 weeks old) were immunised with bovine type II collagen (Chondrex, Inc., Redmond, WA) emulsified in complete Freund’s adjuvant (Chondrex) at a final concentration of 0.5 mg/mL of heat-killed Mycobacterium tuberculosis H37 RA (non-viable) and 0.1 mg/mL of collagen. Injections were administered subcutaneously in 100 µL volume of the emulsion at the base of the tail. At 3 weeks after primary immunisation (14 weeks old), mice were administered a booster immunisation dose with bovine type II collagen in incomplete Freund’s adjuvant (Chondrex). The control groups (W and P) were injected with 100 µL PBS twice instead of the emulsion.
Arthritis was scored by a blinded examiner using a visual assessment scoring (VAS) system with a scale of 0–4 per limb, as described previously^{49 (link)}. Scoring was performed as follows: 0: no swelling or redness of paws or digits, 1: swelling and redness in one or two digits, 2: swelling and redness of the ankle or three or more digits or the midfoot, 3: swelling and redness of the ankle and midfoot or digits and midfoot, and 4: swelling and redness of the entire foot or ankylosis.

The Bovine type II collagen (Chondrex, Redmond, WA, USA) was emulsified using a complete Freund’s adjuvant (Chondrex) containing 2 mg/mL heat-killed Mycobacterium tuberculosis [44 (link)]. The six-week-old male DBA/1 mice (OrientBio) received a primary immunization, followed by a boosting immunization on day 21 using the same concentration of Bovine type II collagen and incomplete Freund’s adjuvant (Chondrex) [44 (link)]. The injection was intradermally conducted at the base of the tail. The severity was measured for 28 days after first injection. The severity of the arthritis was observed and scored as determined by the hind paw swelling and clinical scoring [44 (link)]; 1 × 10⁶ of AMMs or AMM/B were resuspended in 50 μL of PBS. When the arthritis score reached 3 or more, resuspended AMMs or AMM/B or 50μL of PBS were injected intraperitoneally twice a week.

12 DBA/1 mice (RA and RA+Th) were injected s.c. with 50 μg bovine type II collagen (Chondrex Inc., Redmond, USA) in Freund's complete adjuvant (Sigma Aldrich, St. Louis, USA) into the base of the tail. 14 days later, these mice were injected with 50 μg bovine type II collagen (Chondrex Inc., Redmond, USA) in Freund's incomplete adjuvant (Sigma Aldrich, St. Louis, USA). 6 control mice (Ctrl) were injected at the same time points with PBS with pH 7.2. Therapy was initiated in 6 mice (RA+Th) at day 1 after CIA induction. These animals were injected i.p. once per week with 1 mg/kg methotrexate (Sigma Aldrich, St. Louis, USA) and 4.2 mg/kg methylprednisolone acetate (Pfizer, La Jolla, USA). All animals received the opioide analgesic Buprenorphin (0.1 mg/kg) to prevent pain after CIA induction.