A total of 40 children (29 girls and 11 boys, Table 1), admitted to the Division of Paediatric Rheumatology of University Hospital Centre Zagreb, between December 2008 and December 2010 with the diagnosis of JIA, were included in the study after obtaining approval from the regional Ethics Committee and informed consent from patients. oJIA was diagnosed in 18 and pJIA in 22 children, in accordance with the ILAR criteria 3 . Eight patients with oJIA and ten patients with pJIA did not receive any therapy at the time of sampling, and the others received non-steroid anti-inflammatory drugs (NSAID) (5 patients each with oJIA and pJIA); disease modifying anti-rheumatic drugs (DMARDs) - methotrexate (MTX) (5 oJIA, 4 pJIA); anti-TNF - etanercept (1 pJIA) or MTX + prednisone (2 pJIA).

Disease activity was followed by clinical examination and laboratory assessment (Table 1).

Healthy children without history of autoimmune or joint diseases, admitted to the Division due to non-inflammatory conditions during the same period were also included in the study as controls (n = 18, 12 girls and 6 boys, Table 1) after obtaining informed consent.

Peripheral blood was obtained from patients and controls during routine clinical assessment, followed by peripheral blood mononuclear cell (PBMC) separation using Histopaque (Sigma-Aldrich, St. Louis, MO, USA). All participants' samples were obtained in the morning after overnight fasting. In addition, SF samples were collected at the same time from children with JIA with indication for arthrocentesis (18 patients with oJIA and 9 patients with pJIA), and synovial cells were separated by centrifuging. Serum and SF samples were collected in aliquots of 500 μL and stored at -20°C until used. Routine laboratory tests were performed at the Department of Clinical Laboratory Diagnostics of the same Hospital Center.

SF-derived cells were cultured in a density of 0.75 × 10⁶ cells/cm² in 24-well plates in minimal essential medium-α (α MEM) supplemented with 10% fetal bovine serum (FBS). Osteoblast differentiation was induced on culture day 7 by the addition of 50 μg/mL ascorbic acid and 5 mM β-glycerophosphate, and assessed on culture day 21 by alkaline phosphatase (AP) histochemical staining using a commercially available kit (Sigma Aldrich, Milwaukee, MI, USA). Since colonies formed by SF-derived cells were poorly delineated and confluent, the area with the red staining was measured by image-analyzing custom made software for quantification of osteoblast differentiation. Total cellularity in each well was estimated by staining with methylene blue (MB), and quantified as the area of blue stain using the same software. Osteoblast differentiation was additionally assessed by measuring expression of osteoblast-specific genes on culture days 14 and 21 by real-time polymerase chain reaction (PCR). To expand SF-derived mesenchymal progenitors and remove inflammatory and hematopoietic cells from the culture, adherent cells were passaged three times, and osteoblastogenesis was induced only in the fourth passage (P4) cells by plating 0.5 × 10⁵ cells/cm² in α-MEM/10% FBS in 24-well plates and addition of 50 μg/mL ascorbic acid and 5 mM β-glycerophosphate on culture day 2. Assessment of osteoblast differentiation was performed on day 14 by AP histochemical staining using a commercially available kit (Sigma Aldrich), and expression of osteoblast genes on culture days 10 and 14 by real-time PCR. Total cellularity in each well was estimated by staining with MB. Osteoblast differentiation was quantified by AP activity colorimetric assay (Sigma Aldrich), reflecting the number of active osteoblasts per well.

The time points for gene expression analysis were determined in a preliminary set of experiments by multiple time point analysis of expression of differentiation genes in human primary and P4 SF-derived osteoblasts. Based on these data, we chose optimal time points which reflected immature and mature stage of osteoblast differentiation.

Normal hBM was obtained from a healthy donor after obtaining approval from the regional Ethics Committee and informed consent from the patient. The 2 × 10⁶ cells were plated in 75 cm² flasks and cultured in α-MEM medium supplemented with 10% FBS until reaching confluence. After two passages, 5 × 10³ cells/cm² were plated in a 24-well culture plate. After reaching confluence, control cells were grown in α-MEM/10% FBS, 50 μg/mL ascorbic acid and 5 mM β-glycerophosphate for 17 days. Additionally, cells were cultured with 10% SF from oJIA or pJIA patients. Osteoblastogenesis was assessed by AP activity colorimetric assay (Sigma Aldrich) and expression of osteoblast genes by real-time PCR.

For the AP activity assay, total cells from two wells plated in a density of 0.75 × 10⁶ cells/cm² for primary cultures, and 0.5 × 10⁵ cells/cm² for P4 cultures, were pooled for analysis. Upon removal of cell culture media, cells were washed 3 times with phosphate-buffered saline (PBS) and lysed with 200 μL lysing buffer (10 mM Tris, 0.1% Triton X-100, pH 7.5) per well. AP activity was measured in a 96-well plate. Five μL of the sample and 15 μL of lysing buffer were added in duplicates into each well, together with 180 μL p-nitrophenyl phosphate (pNP) solution (Sigma Aldrich) and incubated for 30 minutes at 37°C. Color development was measured at 405 nm using the enzyme-linked immunosorbent assay (ELISA) microplate reader (Bio-Rad, Hercules, CA, USA). Results were expressed as micromoles of pNP per hour.

Total RNA was extracted from peripheral blood cells and SF-derived cells using 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA, USA). For PCR amplification, 1 μg of total RNA was converted to complementary DNA (cDNA) by reverse transcriptase (Applied Biosystems). The amount of cDNA corresponding to 20 ng of reversely transcribed RNA was amplified by real-time PCR. Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), TNF-α, Fas, Fas ligand, IL-1β, IL-4, IL-6, IL-17, IL-18, CC chemokine ligand (CCL) 2, CCL3, and CCL4 was analyzed using commercially available TaqMan Assays (Applied Biosystems).

Real-time PCR was conducted using the ABI Prism 7500 Sequence Detection System (Applied Biosystems). Each reaction was performed in duplicate in a 25 μL reaction volume. The relative quantities were calculated using the standard curve designed from 6 serial dilutions of the calibrator sample (control PBMC, SF-derived cells or osteoblast culture cells). According to the standard curve, the relative amounts of messenger RNA for target genes were calculated as the ratio of the quantity of the target gene normalized to GAPDH as the endogenous control.

The concentration of IL-17 and TNF-α in SF was determined using a commercial kit (Quantikine, Human IL-17 Immunoassay and Quantikine High Sensitivity, Human TNF-α Immunoassay, R&D systems, Minneapolis, MN, USA). Briefly, samples were added to anti-IL-17 or anti-TNF-α monoclonal antibody-precoated plates and incubated for 2 or 3 hours respectively at room temperature, washed five times and incubated for the next 2 hours with horseradish peroxidase conjugated IL-17 or AP-conjugated TNF-α-specific Ab. After five further washes, the reaction was visualized with substrate solution (tetramethylbenzidine) for IL-17 or substrate and amplifier solutions (NADPH and amplifier enzymes) for TNF-α, and arrested with hydrochloric or sulfuric acid respectively. Optical density was determined within 15 minutes, on the microplate reader (Bio-Rad, Hercules, CA, USA) set to the excitation wavelength at 450 nm or 490 nm respectively.

Clinical and laboratory data for each type of arthritis were presented as mean ± standard deviation (SD) and compared using analysis of variance. AP activity, AP staining intensity, and gene expression values in JIA and control samples were expressed as median with interquartile range (IQR) and compared using the nonparametric Kruskal-Wallis test followed by the Mann-Whitney test and Bonferroni's correction for multiple testing. Osteoblast differentiation parameters were additionally correlated to inflammation markers using rank correlation and Spearman's coefficient rho (ρ) with its 95% confidence interval (CI). Statistical analysis was performed using the MedCalc software package (Mariakerke, Belgium). For all experiments, the α-level was set at 0.05.

SF-derived cells from oJIA formed more AP-positive, osteoblast-like colonies than SF-derived cells from patients with pJIA when plated at the same density (Figure 1A, B). At the same time, total cellularity assessed by MB staining was also higher in cultures of SF-derived cells from patients with oJIA in comparison to those with pJIA, (median 11.49, IQR 8.23 to 12.82 vs. median 5.23, IQR 0.43 to 6.47, arbitrary units/cm², P = 0.004, Mann-Whitney test). Similar findings were observed in osteoblastogenic cultures from adherent SF-derived cells after removal of inflammatory cells by cell passaging (P4-cells). As the difference in the histochemical staining intensity was not so prominent in P4 cultures, since P4-cells grew in homogenous monolayers (Figure 1C), we used AP activity colorimetric assay to quantitatively assess osteoblast differentiation. AP activity was significantly higher in osteoblastogenic cultures from patients with oJIA, than in those with pJIA (Figure 1D). When P4-cells from patients with oJIA were plated at equal densities, total cellularity (MB staining, Figure 1C) was higher than in patients with pJIA (median 11.09, IQR 6.52 to 12.10 vs. median 2.41, IQR 1.74 to 5.91, arbitrary units/cm², P = 0.04, Mann-Whitney test).

In addition, the second passage was efficient only in 4 out of 9 pJIA patients, significantly less in comparison to oJIA patients (16 out of 18, Fisher's exact test, P = 0.0235).

Inhibition of osteoblast differentiation was confirmed at the level of osteoblast gene expression as the early osteoblast marker Runx2, essential for osteoblast differentiation 23 , was significantly decreased in pJIA on day 14 of the primary SF-derived osteoblast culture (P = 0.02, Mann-Whitney test; Figure 2A, Table 2), and day 10 of P4-SF-derived cell culture (P = 0.02, Mann-Whitney test; Figure 2B). OPG gene expression, the marker of mature osteoblasts, was significantly decreased in pJIA on day 21 of the primary SF-derived osteoblast culture (P = 0.03, Mann-Whitney test; Figure 2A, Table 2), and day 14 of the P4-SF-derived cell culture (P = 0.01, Mann-Whitney test; Figure 2B, Table 2). Expression of RANKL was significantly lower in pJIA only on culture day 10 in P4-SF-derived osteoblast culture (P = 0.04 Mann-Whitney test; Figure 2B, Table 2).

Primary SF-derived cells from patients with oJIA and pJIA had similar expression of the early osteoblast marker Runx2, as well as the late osteoblast marker OPG gene (Figure 3A). Expression of RANKL was significantly higher in patients with pJIA than in patients with oJIA (P = 0.05, Mann-Whitney test; Figure 3A). PBMCs from patients with JIA expressed less OPG than healthy control patients (P = 0.05, Kruskal-Wallis test: Figure 3B), whereas there was no difference in the expression of RANKL and Runx2 between the patient and control groups (Figure 3B).

As estimated by the area of the red stain of AP-histochemically positive colonies, SF-derived osteoblast differentiation negatively correlated with erythrocyte sedimentation rate (ESR, P = 0.05, rank correlation; Figure 4A). The correlation was stronger for C-reactive protein (CRP, P < 0.01, rank correlation; Figure 4A). Furthermore, osteoblast differentiation negatively correlated with local synovial concentration of IL-17 (P = 0.01, rank correlation; Figure 4B), an inflammatory cytokine closely related to the development of bone erosions and produced by Th17 cells, which are enriched in joints of children with arthritis 24 25 . The inverse relation was also found between synovial concentrations of TNF-α and osteoblastogenesis from SF-derived progenitors, although the correlation was not statistically significant (P = 0.34, rank correlation; Figure 4B). Finally, osteoblast differentiation negatively correlated with expression of the CCL2 gene in SF-derived cells (P = 0.05, rank correlation; Figure 4B).

Mesenchymal lineage cells, as well as differentiating and mature osteoblasts, are known to have immunoregulatory properties, expressing various pro- or anti-inflammatory cytokines and chemokines 26 . We assessed gene expression of several candidate cytokines and chemokines which could be involved in the regulation of inflammation in the course of JIA.

The expression of pro-inflammatory CCL2 was significantly higher in synovia-derived immature osteoblasts from patients with pJIA in comparison those with oJIA (P = 0.04, Mann-Whitney test; Figure 5). The expression of CCL3 (macrophage inhibitory protein (MIP)1α) was significantly higher in mature SF-derived osteoblasts from patients with pJIA than those with oJIA (P = 0.03, Mann-Whitney test; Figure 5). Fas was significantly increased in mature SF-derived osteoblasts in patients with pJIA compared with oJIA (P = 0.03, Mann-Whitney test; Figure 5). Expression of TNF-α, IL-4, IL-6, IL-17 and IL-18 was not detected in SF-derived osteoblasts, and expression of CCL4, IL-1β and FasL was not different between the groups.

To assess whether soluble factors from SF of patients with JIA could inhibit differentiation of osteoblasts, we treated BM-derived osteoblasts obtained from a healthy donor with 10% of SF obtained from patients with JIA, and compared osteoblast differentiation with untreated BM osteoblast culture. Assessed by AP activity assay, SF from both oJIA and pJIA patients inhibited the differentiation of hBM-derived osteoblasts (P < 0.01, Kruskal-Wallis test; Figure 6A). The specificity of this effect was confirmed by inhibited expression of Runx2 in early osteoblastogenic culture (P = 0.04, Kruskal-Wallis test; Figure 6B).