Cell culture
Murine osteoclast precursors from 7-week-old male C57BL/6J mice were obtained from Nippon CLEA (Tokyo, Japan) and osteoclast-like cells were induced using cytokines, as described elsewhere [19]. In brief, murine bone marrow cells were cultured (6 × 105 cells/well in a 48-well plate) in alpha-minimal essential medium (α-MEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10 % fetal bovine serum (FBS; Invitrogen, Grand Island, NY, USA), and 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich), and 25 ng/ml macrophage colony-stimulating factor (M-CSF; R&D Systems, Minneapolis, MN, USA) and 50 ng/ml RANKL (Wako, Osaka, Japan) in the presence or absence of either 0.05 % dimethyl sulfoxide (DMSO; Sigma-Aldrich), a control peptide (50 μM/l), or OP3-4 (1, 5, or 50 μM/l). The cells were incubated for 4 days at 37 °C. The OP3-4 peptide and the control peptide (FCYISEVEDQCY) were both purchased from American Peptide Company (Sunnyvale, CA, USA) [14]. Tartrate-resistant acid phosphatase (TRAP) staining was performed after fixation, and the number of TRAP-positive multinucleated cells (n >2) was counted.
An in-vitro osteoblastogenesis assay was carried out as described previously [19]. Briefly, primary osteoblast-like cells isolated from 1 day-old mice calvariae were seeded (5 × 104 cells/well in a 24-well plate) and cultured in α-MEM (Sigma-Aldrich) with 10 % FBS (Hana-Nesco Bio, Brisbane, Australia) and 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich) containing 50 mg/ml ascorbic acid (Wako), 10 mM β-glycerophosphate (Sigma-Aldrich), and 10 nM dexamethasone (Sigma-Aldrich). Alkaline phosphatase (ALP) staining was performed on day 7 and von Kossa staining was performed on day 21 of culture, and the ALP-positive and von Kossa-positive areas were measured using an image analysis system (KS400; CarlZeiss, Jena, Germany), as described previously [19].
mRNA analyses
Primary osteoblast-like cells were cultured in the osteogenic medium as already described for the indicated days. Total RNA was isolated from culture in osteogenic medium using Trizol (Invitrogen) and was treated with DNase I (Invitrogen). cDNA was synthesized using PrimeScript II reverse transcriptase (Takara, Kyoto, Japan) according to the manufacturer’s instructions. Quantitative RT-PCR analysis was performed using SYBR Premix Ex Taq II (Takara) and a LightCycler 2.0 (Roche, Basel, Switzerland). For normalization, the expression of hypoxanthine guanine phosphoribosyl transferase (Hprt) was measured as an endogenous reference gene. The following primers specific for mouse were used: Hprt, sense 5′-CTT TGC TGA CCT GCT GGA TT-3′ and antisense 5′-TAT GTC CCC CGT TGA CTG AT-3′; Runx2, sense 5′-ACT GGC GGT GCA ACA AGA C-3′ and antisense 5′-CGG TAA CCA CAG TCC CAT CT-3′; Alp, sense 5′-GCA CCT GCC TTA CCA ACT CT-3′ and antisense 5′-TCA GGG CAT TTT TCA AGG TC-3′; and Bglap1/2 (ostocalcin), sense 5′-TAG TGA ACA GAC TCC GGC GCT ACC TT-3′ and antisense 5′-AGC TCG TCA CAA GCA GGG TTA AGC TC-3′. The relative levels of expression to cells maintained in α-MEM were calculated by the ΔΔCt method.
Induction of CIA
The induction and assessment of CIA were performed as described previously [20]. Briefly, male DBA/1J mice (7 weeks old, six mice per group; Charles River Laboratories Japan, Kanagawa, Japan) were injected intradermally at the base of the tail with 200 μg bovine type II collagen (Collagen Research Center, Tokyo, Japan) in 0.05 M acetic acid (Sigma-Aldrich) emulsified in complete Freund adjuvant (CFA; Difco, Detroit, MI, USA). Twenty-one days after the primary immunization, the mice were boosted in the same way. The day of the first immunization was designated day 0. The mice were provided food (MF; Oriental Yeast Company, Tokyo, Japan) and distilled water ad libitum, and were maintained under a 12-hour light/dark cycle. The experimental procedures were reviewed and approved by the Animal Care and Use Committee of Tokyo Medical and Dental University (Tokyo, Japan) (authorization number: 120217A, 130255A, and 14070A).
Treatment with the OP3-4 peptide
The OP3-4 peptide was dissolved in phosphate-buffered saline (PBS)-buffered 20 % DMSO (Sigma-Aldrich). Alzet osmotic minipumps (Model 2001 or 2002; Alza, Palo Alto, CA, USA) were prepared according to the manufacturer’s instructions. On day 28, the mice were anesthetized with injections of medetomidine hydrochloride (0.5 mg/kg; Meijiseika, Tokyo, Japan) and ketamine hydrochloride (50 mg/kg; Sankyo, Tokyo, Japan). A 1 cm incision was made in the skin, and the osmotic minipumps filled with 20 % DMSO (vehicle) or OP3-4 peptide (to deliver 9 mg/kg/day or 18 mg/kg/day) were subcutaneously implanted. The osmotic minipumps were replaced on day 35 and day 42, and infusions were continued until the mice were killed (on day 49). For measurement of the bone formation parameters, calcein (Sigma-Aldrich) was injected on days 42 and 47. The mice (six per group) were divided into four groups: nonimmunized mice receiving vehicle (20 % DMSO) (Normal-vehicle group); immunized mice receiving vehicle (20 % DMSO) (CIA-vehicle group); immunized mice receiving 9 mg/kg/day OP3-4 peptide (CIA-9 mg OP3-4 group); and immunized mice receiving 18 mg/kg/day OP3-4 peptide (CIA-18 mg OP3-4 group).
Clinical assessment
To determine the arthritis score, two independent observers examined the mice daily from the day of the second immunization. The day of arthritis onset was considered to be when erythema and/or swelling was first observed. The severity of arthritis was graded on a 0–4 scale [21]. Briefly, the criteria for the grading were as follows: 0 = no evidence of erythema or swelling, 1 = erythema and mild swelling confined to the tarsals or ankle joint, 2 = erythema and mild swelling extending from the ankle to the tarsals, 3 = erythema and moderate swelling extending from the ankle to metatarsal joints, and 4 = erythema and severe swelling encompassing the ankle, foot, and digits, or ankylosis of the limb. Each paw was graded, and the four scores were added together so that the maximum possible score was 16 per mouse.
Radiographic assessment of arthritis
At the end of the experiment (day 49), the mice were killed using domitor anesthesia, blood was collected from the orbital vein, and the hind paws were removed and fixed in phosphate-buffered glutaraldehyde (0.5 %)–formalin (4 %) fixative (pH 7.4) for 2 days, washed with PBS for 1 day and then used for the radiographic analyses. Three-dimensional reconstruction images and sagittal images of the knee joints were obtained by microfocal computed tomography (μCT) (Scan Xmate-E090; Comscan, Kanagawa, Japan) [22]. Microarchitectural changes were then measured using a three-dimensional bone structure analyzing system (TRI/3D-BON; RATOC System Engineering, Tokyo, Japan) [23]. To exclude the primary spongiosa, the region of interest (ROI) for the microstructural analyses of secondary spongiosa was set at 0.2–1.7 mm longitudinal length from the proximal end of epiphysis, and trabecular bones where bone separation was less than 60 μm (4 voxels) were excluded from the measurement [24]. For the analyses of epiphysis, the two-dimensional ROI (150 μm width) along the proximal end surface of tibiae was used. The BMD of the knee joints was measured by dual X-ray absorptiometry (DXA) (DCS-600R; Aloka, Tokyo, Japan) using the high-resolution scanning mode. The ROI (2.0 × 2.0 mm) for measuring the BMD at knee joints was determined by excluding the secondary spongiosa of the tibial and femoral metaphysis.
Biochemical markers
The matrix metalloproteinase (MMP)-3, C-telopeptide fragments of type I collagen (CTX), and osteocalcin levels in the blood serum were analyzed according to the manufacturers’ instructions: for MMP-3, Mouse ELISA Kit (MMP 300) from R&D Systems, Inc. (Minneapolis, MN, USA); for CTX, RatLaps ELISA Kit (DS-AC 06F1) from Immunodiagnostic Systems Ltd (Fountain Hills, AZ, USA); and for osteocalcin, Mouse Gla-Osteocalcin High Sensitive EIA kit (MK 127) from Takara-Bio Inc. (Otsu-city, Shiga, Japan).
Histological assessment of arthritis
The tibial bones were embedded in methyl methacrylate monomer (MMA), as described elsewhere [25]. In brief, polymerization was performed at 4 °C. Standard undecalcified sections (3 μm) were prepared using a fully automated rotary microtome Leica RM2265 device (Leica Biosystems, Nussloch GmbH, Germany). The direction of the cuts made in the bones was guided by μCT images of embedded samples before the sections were made. After removing MMA resin using 1-acetoxy-2-methoxyethane (Wako), the sections were then stained with TRAP and counterstained with toluidine blue. Histomorphometric analyses [25] were performed using the KS400 system (CarlZeiss). Some sections were stained according to the von Kossa method to detect calcified tissue, and were counterstained with modified van Gieson stain. Toluidine blue-stained sections were used to detect the pannus infiltration site at the articular cartilage of the tibial epiphysis.
Histomorphometric evaluation of the epiphysis
The extent of cartilage degradation at the proximal end of the tibia was calculated using undecalcified toluidine blue-stained sections. The formula used was as follows:
$$ \mathrm{Extent}\ \mathrm{of}\ \mathrm{cartilage}\ \mathrm{degradation}\ \left(\%\right) = \left(\mathrm{length}\ \mathrm{of}\ \mathrm{cartilage}\ \mathrm{degradation}\right)\ /\ \left(\mathrm{total}\ \mathrm{length}\ \mathrm{of}\ \mathrm{proximal}\ \mathrm{end}\ \mathrm{of}\ \mathrm{tibial}\ \mathrm{epiphysis},\ \mathrm{excluding}\ \mathrm{anterior}\ \mathrm{intercondylar}\ \mathrm{area}\right) \times 100. $$
The undecalcified sections that were stained with TRAP and counterstained with toluidine blue were used for the assessments. The calcified area/tissue area and osteoclast number/bone volume (N.Oc/BV) ratios in the epiphysis of the proximal tibia were measured using the KS400 image analyzing system, as described previously [20]. TRAP-positive multinucleated (n >2) cells that formed resorption lacunae on the surface of the trabeculae were designated osteoclasts. The labeled surface was also measured in the region of epiphyses.
Bone histomorphometry of the tibial metaphysis
To investigate the secondary effects of CIA on bone resorption, in addition to the resorption of periarticular bone, standard histomorphometric analysis in the tibial metaphysis was performed [20, 26] using the image analysis system already described.
The ROI for the histomorphometric analyses of the metaphysis was set at 0.6 mm distal from the center of the growth plate and the size of the ROI was 1.1 mm (longitudinal length) × 0.7 mm (width) at the center of diaphysis to exclude the primary spongiosa.
Statistical analysis
The Kruskal–Wallis test was performed to analyze the arthritis score. For comparison purposes of each group, the Mann–Whitney U test with Bonferroni correction was applied. The other data were analyzed by an analysis of variance. When an F test yielded significant results (p <0.05), the groups were compared using Fisher’s protected least significant difference post-hoc test. Tests were carried out using an Apple software program, StatView 4.1 (SAS Institute, Cary, NC, USA). Values of p <0.05 were considered significant.