Osteoblastogenesis from synovial fluid-derived cells is related to the type and severity of juvenile idiopathic arthritis
© Lazić et al.; licensee BioMed Central Ltd. 2012
Received: 17 January 2012
Accepted: 12 June 2012
Published: 12 June 2012
Juvenile idiopathic arthritis (JIA) is characterized by synovial inflammation, followed by hyperplastic changes of the synovium, and destruction of articular cartilage along with underlying bone. This hyperplastic process is the result of inflammation-induced activation of NF-κB, which may be accompanied by decreased osteogenic differentiation of synovial mesenchymal progenitors and contribute to bone resorption. We aimed to explore osteoblast differentiation of synovial fluid (SF)-derived mesenchymal progenitors and correlate it with intensity of inflammation in patients with JIA.
Peripheral blood from 18 patients with oligoarticular (o)JIA, 22 patients with polyarticular (p)JIA and 18 controls was collected along with SF from 18 patients with oJIA and 9 patients with pJIA. SF-derived cells were cultured to assess osteoblastogenesis, using alkaline phosphatase histochemical staining and colorimetric activity assay. The expression of osteoblast-related genes, Runt-related transcription factor 2 (Runx2), Osteoprotegerin (OPG), Receptor activator of nuclear factor κB ligand (RANKL) and arthritis-related cytokine/chemokine genes, Tumor necrosis factor alpha (TNF-α, Fas, Fas ligand (FasL), Interleukin (IL)-1β, IL-4, IL-6, IL-17, IL-18, CC chemokine ligand (CCL)-2, CCL3, CCL4 was evaluated. Osteoblastogenesis was correlated with systemic and local inflammatory indicators. Expression of osteoblast genes was also analyzed in peripheral blood mononuclear cells (PBMC) and total SF-derived cells from patients with JIA. Additionally, we assessed the inhibitory effect of SF from patients with JIA on differentiation of human bone marrow (hBM)-derived osteoblasts.
Osteoblastogenesis from SF-derived progenitors was decreased in patients with pJIA compared to those with oJIA. Osteoblastogenesis from primary SF-derived cells negatively correlated with erythrocyte sedimentation rate (ρ = -0.391, P = 0.05), C-reactive protein concentration (ρ = -0.527, P<0.01) and synovial concentration of IL-17 (ρ = -0.552, P = 0.01). SF-derived osteoblasts from pJIA patients expressed more CCL2 and CCL3 genes than in oJIA (P = 0.04 and P = 0.03, respectively; Mann-Whitney test). Expression of Fas was significantly higher in osteoblasts from patients with pJIA than those with oJIA (P = 0.03, Mann-Whitney test). SF-derived cells from patients with pJIA expressed higher levels of RANKL than in oJIA (P = 0.05, Mann-Whitney test). PBMCs from patients with JIA expressed less OPG than healthy control patients (P = 0.05, Kruskal-Wallis test). SF from all tested JIA patients inhibited differentiation of hBM-derived osteoblasts (P = 0.04, Kruskal-Wallis test).
Osteoblast differentiation was decreased in patients with severe forms of JIA and accompanied by altered cytokine/chemokine expression pattern. Development of therapeutic interventions targeting synovial mesenchymal or osteoblast lineage cells in JIA would contribute to alleviating both bone destruction and inflammation in severe forms of the disease.
Juvenile idiopathic arthritis (JIA) is one of the leading causes of disability in children, characterized by synovial hyperplasia and formation of pannus, which cause destruction of articular cartilage and underlying bone [1, 2]. Clinically, JIA is defined as arthritis appearing before 16 years of age, with a minimum duration of six or more weeks and exclusion of other forms of childhood arthritis . According to the International League of Associations for Rheumatology (ILAR) JIA is classified into the following subtypes: systemic JIA, oligoarticular JIA (oJIA, affecting four or fewer joints), polyarticular JIA (pJIA, affecting five or more joints), psoriatic arthritis, enthesitis-related arthritis and undifferentiated arthritis . In general, oJIA is the most frequent disease form (56 to 60% of cases), followed by pJIA (28 to 30%). The course of disease is variable; patients with oJIA have the best outcome, while the course of pJIA is characterised by progressive and diffuse joint involvement and early radiographic changes.
The pathogenesis of bone loss in children with JIA involves inflammation, physical inactivity, medication intake and malnutrition . Inflammation-induced bone loss in JIA is driven by the interactions of the immune system and bone, which share a number of regulatory molecules . The stimulatory effects of inflammation on osteoclast-mediated bone resorption are well established but the influence of pro-inflammatory cytokines on osteoblast function in vivo requires further elucidation . It is known that tumor necrosis factor (TNF) and interleukin (IL)-1β may directly impair osteoblast differentiation [7–10]. Bone marrow-derived mesenchymal stem cells (MSC) from TNF transgenic mice, that develop chronic inflammatory arthritis, form fewer osteoblast colonies with decreased expression of osteoblast genes . In addition, pro-inflammatory factors are able to disrupt the Wnt signaling pathway, which normally induces the differentiation and maturation of osteoblasts [12, 13]. Among Wnt antagonists, murine models revealed that Dickkopf and secreted Frizzled-related proteins are upregulated in arthritic synovial tissues and may contribute to decreased osteoblast function . Inhibition of Dickkopf-1 was able to reverse bone destruction towards bone formation in the mouse model of rheumatoid arthritis (RA) . The involvement of Fas and Fas ligand (FasL) has also been proposed in osteoblast differentiation, and confirmed in animal models of Fas deficiency, which is found to protect animals from osteoporosis and joint destruction in arthritis [15–17].
Therapeutic treatment of joint destruction in JIA aims to attenuate inflammation and bone resorption, as well as to increase regeneration of subchondral bone and cartilage. Several therapeutic approaches involve MSC because of their immunomodulatory and regenerative capacity . MSC are multipotent cells with ability to differentiate into osteoblasts, chondroblasts, adipocytes, connective tissue fibroblasts and myoblasts, thus contributing to tissue regeneration . They are present in various tissues such as bone marrow, skin, adipose and connective tissues, as well as the synovia [19, 20].
In a murine model, synovial hyperplasia develops as a result of inflammation-induced activation of nuclear factor κB (NF-κB) in synovial fibroblasts, which promotes their proliferation and inhibits osteoblast/chondroblast lineage differentiation [21, 22]. This mechanism could also be relevant for human JIA, promoting inflammation and suppressing joint regeneration.
The overall aim of the present study was to assess the osteoblastogenesis from synovial fluid (SF)-derived cells in patients with JIA and its association with disease type and activity. We first assessed osteoblastogenesis and osteoblast gene expression in SF-derived cells from patients with JIA and correlated them with systemic and local inflammatory activity. We also assessed the systemic expression of osteoblast-related genes in patients with JIA and control patients, as well as cytokine and chemokine expression in osteoblasts from JIA patients. Finally, we investigated the effect of SF from patients with JIA on the differentiation of human bone marrow (hBM)-derived osteoblasts.
Materials and methods
Demographic and laboratory data for juvenile idiopathic arthritis (JIA) patients and controls*
Control (n = 18)
oJIA (n = 18)
pJIA (n = 22)
7.33 ± 5.22
7.13 ± 5.20
11.36 ± 4.50
ESR (1 h)
8.33 ± 4.27
16.44 ± 9.46
53.09 ± 41.03
4.39 ± 6.80
4.34 ± 6.36
47.31 ± 46.3
Leukocyte number (x109/L)
6.54 ± 1.65
10.03 ± 4.01
9.57 ± 4.62
124 ± 9.70
123.69 ± 11.36
117.4 ± 14.02
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.
Osteoblast differentiation from synovial fluid-derived cells
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.
Bone marrow osteoblast culture
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.
Alkaline phosphatase colorimetric assay
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.
Osteoblast differentiation from synovial fluid-derived cells in patients with JIA
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).
Osteoblast gene expression in primary and fourth passage (P4) SF-derived osteoblast cultures from patients with juvenile idiopathic arthritis*
oJIA (n = 12)
pJIA (n = 4)
P (vs. pJIA)
P (vs. day 21)
P (vs. pJIA)
P (vs. day 21)
P (vs. pJIA)
P (vs. day 21)
oJIA (n = 7)
pJIA (n = 4)
P (vs. pJIA)
P (vs. day14)
P (vs. pJIA)
P (vs. day14)
P (vs. pJIA)
P (vs. day14)
Expression of osteoblast genes in synovial fluid-derived cells and peripheral blood mononuclear cells from patients with JIA
Osteoblast differentiation negatively correlated with systemic and local inflammatory indicators
Expression of cytokines and chemokines in osteoblasts from patients with JIA
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 . We assessed gene expression of several candidate cytokines and chemokines which could be involved in the regulation of inflammation in the course of JIA.
Effect of synovial fluid from patients with JIA on differentiation of human bone marrow-derived osteoblasts
The results of our study clearly demonstrate that osteoblastogenesis from SF-derived progenitors was impaired in patients with pJIA in comparison to patients with oJIA. Osteoblastogenesis from SF-derived progenitors correlated with systemic and local inflammatory indicators, suggesting the impact of inflammation on bone formation.
Decreased osteoblast differentiation was confirmed by the decreased area of AP-positive osteoblast colonies in SF-derived osteoblastogenic cultures from JIA patients, as well as the decreased expression of Runx2, a transcription factor essential for the commitment of mesenchymal progenitors to the osteoblast lineage . We also assessed gene expression levels of RANKL and OPG, associated with osteoblast maturation and, at the same time, important for the regulation of osteoclastic bone resorption [27, 28]. Decreased expression of OPG in SF-derived osteoblasts from patients with pJIA, together with comparable expression of RANKL in both patient groups, resulted in the lower OPG/RANKL ratio in children with pJIA, which might contribute to the increase in osteoclastic bone resorption. On the other hand, expression of RANKL was higher in total SF-derived cells from patients with pJIA compared to those with oJIA, which may be explained by the fact that RANKL is produced not only by osteoblasts but also by activated T lymphocytes . Furthermore, both oJIA and pJIA patients expressed less OPG in PBMCs than the control group, which is consistent with the recent prospective cohort study reporting lower OPG serum levels, higher levels of RANKL and decreased OPG/RANKL ratio in children with JIA compared to healthy children .
Since oJIA had lower laboratory inflammation markers than pJIA, we expected that osteoblastogenesis would be negatively regulated by inflammatory processes. This was confirmed by the negative correlation of osteoblastogenesis with the levels of CRP and ESR, demonstrating an osteoblast-related mechanism of bone loss which accompanies autoimmune disorders .
Negative correlation of osteoblastogenesis with synovial IL-17 levels found in patients with JIA is consistent with IL-17 contribution to the cartilage and bone damage seen in the animal model of autoimmune arthritis . IL-17 participates in the arthritic process by affecting B- and T-lymphocytes, epithelial, myelomonocytic and BM stromal cells and synovial fibroblasts and chondrocytes, stimulating their production of various cytokines, chemokines and tissue destructive mediators [32, 33]. Soluble IL-17 and high numbers of IL-17-producing Th17 cells have been found in synovial tissue from adults and children with inflammatory arthritis, particularly in those with a more severe clinical course [33, 34]. Although we were unable to confirm statistically significant correlation between synovial TNF-α concentration and SF-derived osteoblasts differentiation, we observed an inverse relationship between these two variables. The lack of significance for this correlation is probably related to high variability of synovial TNF-α concentration in study patients (13.03 ± 11.08 pg/ml).
In addition, we assessed whether osteoblastogenesis could be altered by therapy, but we were unable to detect a significant difference either in osteoblastogenesis or in systemic (SE, CRP) and local (synovial TNF-α) inflammation parameters between patients receiving therapy and patients without therapy. This could be ascribed to the poor therapeutic response in treated patients or activation of disease in the untreated patient group. A more homogenous and larger group of patients according to the duration of the disease and the applied therapy is needed to address this issue with adequate power.
Mesenchymal cells are known to have immunoregulatory properties . Upon inflammatory stimuli, osteoblasts also may express various cytokines and chemokines that augment or suppress inflammation. Synovial inflammation in patients with RA and human TNF transgenic mice upregulates WNT5A in synovial fibroblasts . WNT5A is able to induce IL-1β, IL-6, CCL2, CCL5, CXCL1, and CXCL5 in osteoblasts, thus altering their regulatory properties . It has been previously shown that osteoblasts and osteocytes from RA patients immunohistochemically expressed CCL20, which was absent in osteoblasts from osteoarthritic patients . We demonstrated that the expression of CCL2 was higher in SF-derived immature osteoblasts from patients with pJIA, whereas CCL3 expression was increased in mature SF-derived osteoblasts derived from patients with pJIA. In the animal model of adjuvant arthritis, CCL2 and CCL3, up-regulated by TNF and IL-1β, are shown to contribute to the pathogenesis of inflammatory arthritis . Our finding of increased proinflammatory CCL2 and CCL3 in osteoblasts derived from pJIA patients suggests that osteoblastic cells in severe forms of JIA may themselves perpetuate joint inflammation via cytokine secretion. In addition, the expression of Fas, a TNF superfamily member characteristic for T lymphocytes but also expressed on osteoblasts and osteoclast lineage cells , was increased in mature SF-derived osteoblasts from pJIA patients. We have previously described that Fas expressed by mature murine osteoblasts is able to specifically inhibit their differentiation . This mechanism could also be effective in human JIA and lead to suppressed osteoblasts differentiation as a result of inflammatory process in JIA.
Our study also provides experimental evidence that SF from patients with both oJIA and pJIA may adversely influence the differentiation of hBM derived osteoblasts. Together with the in vitro finding of Caparbo, et al.  that serum from patients with active pJIA decreases differentiation and increases apoptosis in human osteoblasts, our study experimentally demonstrated that bone loss in JIA was associated with decreased osteoblastogenesis and results in impaired bone formation. This may be the cellular mechanism for lower levels of bone formation biochemical markers found in JIA patients [40, 41].
In conclusion, decreased osteoblast differentiation is accompanied by altered properties of SF-derived osteoblasts from patients with severe forms of JIA, which may promote osteoclastic bone resorption and perpetuate local and systemic inflammation. Development of therapeutic interventions targeting synovial mesenchymal and/or osteoblast lineage cells in JIA would contribute to alleviating both bone destruction and inflammation in severe forms of the disease.
CC chemokine ligand
disease modifying anti-rheumatic drug
enzyme-linked immunosorbent assay
erythrocyte sedimentation rate
fetal bovine serum
glyceraldehyde 3-phosphate dehydrogenase
human bone marrow
International League of Associations for Rheumatology
juvenile idiopathic arthritis
macrophage inhibitory protein
mesenchymal stem cells
nuclear factor κB
non-steroid anti-inflammatory drugs
oligoarticular form of juvenile idiopathic arthritis
peripheral blood mononuclear cell
polymerase chain reaction
polyarticular form of juvenile idiopathic arthritis
receptor activator of nuclear factor κB ligand
Spearman's coefficient rho
runt-related transcription factor 2
tumor necrosis factor.
We thank Mrs. Sanja Ivcevic and Mrs. Katerina Zrinski-Petrovic for their technical assistance.
This work was supported by grants from the Croatian Ministry of Science, Education and Sports (108-1080229-0140, 108-1080229-0142, and 108-1080229-0341).
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