Study approval
Animal studies were approved by the Institutional Animal Care and Use Committees and Institutional Biosafety Committees of Central South University, China (CSU; 2013-05). Male C57BL/6 mice were purchased from the Animal Centre of Central South University (Changsha, Hunan, China). At the start of the experimental protocols, mice were 6 weeks old. Collection of human OA tissues from discarded knee replacement surgeries were approved by the Queensland University of Technology (QUT) and the St Vincent Private Hospital Ethics Committees (Human ethics number: #1400001024). Informed consent was obtained from all human subjects.
Human cartilage specimen preparation
Human cartilage was obtained from patients with OA (Kellgren-Lawrence grade 4) undergoing total knee replacement. Osteochondral plugs (including cartilage and subchondral bone) were taken from the tibial plateau region. All samples were prepared for histology and immunohistochemical analysis by washing in Phosphate-Buffered Saline (PBS) and immediately fixed in 4% paraformaldehyde (PFA). The samples were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH = 7.2-7.4). The specimens were embedded in paraffin, and 5 µm-thick sections were obtained as described in our previous studies [22].
Cell culture and bestatin treatment in vitro
Human chondrocyte cell line C28I2 was seeded into 6-well plates at a density of 1×106 cells/well and cultured in DMEM containing 10% foetal bovine serum (FBS) and 1% of penicillin and streptomycin (PS). At 90% confluence, cells were serum-starved for 24 h and then were incubated in the presence or absence of 10 ng/ml interleukin-1β (IL-1β) or 50 nM bestatin with culture medium for 24 h.
Cell viability test
Human chondrocyte cell line C28I2 was seeded into 96-well plates at a density of 5000 cells/well with culture medium. After 24 h, cells were serum-starved for 24 h and then were incubated in 0, 10, 50, 100, and 200 nM of bestatin separately. Cell viability test and cell count were performed according to our previous publication [23].
Preparation of bestatin
Bestatin was purchased from Cayman Chemical, 2-hydroxypropyl-β-cyclodextrin (HPCD) and polyethylene glycol 400 (PEG-400) was obtained from Sigma Aldrich. The oral formulation for bestatin was prepared as previously described [13]. Briefly, 100 µl PEG-400 and 100 mg of bestatin were gently mixed before dissolve in a 20% (w/v) aqueous solution of HPCD. Then, the solution was mixed at room temperature to obtain a homogeneous mixture at 2 mg/ml concentration.
Induction of OA in mice and bestatin treatment
Mice were group-housed (5 animals/cage) in ventilated cages in a temperature-controlled room on 12-h light/dark cycles with ad libitum access to standard mouse food and water and routine veterinary assessment. C57BL/6 mice (Animal Centre of Central South University, Changsha, Hunan, China) were randomly assigned and underwent destabilization of the medial meniscus (DMM) surgery to induce OA as previously described [22]. Briefly, OA was induced by dissecting the medial meniscus ligament to destabilize the medial meniscus in the knee joint of the hind limb on the right side. Mice in the sham group were surgically treated in which the ligament was visualized but not dissected. One week after experimental OA induction, mice were treated orally with 30 mg/kg bestatin [24] or vehicle (PEG-400 and HPCD). All experimental mice were then received twice-weekly oral bestatin after DMM surgery at a dose of 30 mg/kg for a total of 8 weeks. Mice were sacrificed 8 weeks after surgery for histological analysis.
Histologic evaluation of knee joint in mice
After euthanasia, the knee joints of hind limbs were fixed in 4% PFA, decalcified in 10% EDTA, embedded in paraffin and sectioned for histological and immunohistochemical analysis. Sections were stained with Safranin-O (Saf-O)/Fast green to evaluate cartilage degradation and synovitis. The severity of OA was assessed in the medial compartment of the knee using a modified Mankin scoring system (0-14) as previously described [22]. The degree of synovitis was scored using a 0-6 scoring system that measured the thickness of the synovial lining cell layer on a scale (0=1–2 cells, 1 = 2–4 cells, 2=4–9 cells and 3 = 10 or more cells) and cellular density in the synovial stroma on a scale of 0–3 (0 = normal cellularity, 1 = slightly increased cellularity, 2 = moderately increased cellularity and 3 = greatly increased cellularity). The sum of both parameters was used for analysis. Synovitis scores obtained from all four quadrants (lateral femur, lateral tibia, medial femur, and medial tibia).
Immunohistochemistry
Immunohistochemistry was performed according to our previously published methods [25, 26]. Sections were incubated overnight at 4°C with antibodies against LTA4H (Abcam, Melbourne, VIC, Australia; dilution 1:250) and leukotriene B4 receptor (BLT1, LSBio, Sapphire Bioscience, Redfern, NSW, Australia; dilution 1: 50) followed by incubation with anti-rabbit or anti-rat secondary antibody (1:5000 dilution; LI-COR Biosciences, Nebraska, USA) and visualization with 3,3-diaminobenzidine chromogen. The percentage of positive cells was determined by counting the number of immunostained cells and dividing by the total number of cells visualized by a hematoxylin counterstain using ImageJ (National Institute of Health, Bethesda, BA, USA).
Quantitative Real-time PCR
Total RNA was extracted with TRIzol, and cDNA was synthesized using the SensiFAST cDNA Synthesis Kit according to the manufacturer's protocol. SYBR Green detection chemistry was used on the QuantStudio Real-Time PCR system (Applied Biosystems, Thermo Scientific, Scoresby, VIC, Australia). Quantitative measurements of all primers used in this study were determined using (2−ΔΔCt) method, and GAPDH expression were used as the internal control, as described previously by our group [25, 27–29].
Statistical analysis
Statistical difference of the results was tested using the unpaired Student’s t-test comparison of two variables. One-way ANOVA followed by post-hoc test was used for comparison of more than two variables. All analyses were performed using GraphPad Prism 7, and p < 0.05 is significant. All data are presented as mean ± SD.