Osteoarthritis and Cartilage

Published by Elsevier
Online ISSN: 1522-9653
Publications
Article
To examine the chondrogenic activity of AG-041R and its mode of action in a bipotent chondroprogenitor cell line CL-1. Chondrogenic activity of AG-041R in CL-1 was examined by histology, alcian blue pH 1.0 intensity and mRNA expression of cartilage matrix proteins (collagen type II, aggrecan). Chondrogenic activities of other CCK2/gastrin receptor antagonists were also examined. Since TGF-beta1 induces dominant chondrogenesis and suppressed adipogenesis in CL-1, induction of TGF-beta by AG-041R was examined by enzyme linked immunosorbent assay. Involvement of MAP kinases in the chondrogenic effect of AG-041R in CL-1 was examined by Western blotting and MAP kinase inhibitors. AG-041R induced dominant chondrogenesis and marked suppression of adipogenesis in CL-1. Neither of the other CCK2/gastrin receptor antagonists tested showed chondrogenic activity in CL-1. AG-041R increased alcian blue pH 1.0 intensity and mRNA expression of collagen type II and aggrecan. TGF-beta1 and -beta2 proteins were increased by AG-041R. The chondrogenic activity of AG-041R in CL-1 was blocked by TGF-beta neutralizing antibody or inhibitors for activation of latent TGF-beta. AG-041R activated both Erk (p44/42) and p38 MAP kinases in CL-1. Inhibition of Erk (p44/42) by PD98059 canceled the adipogenesis suppression by AG-041R in CL-1. Inhibition of p38 by SB202190 completely canceled the chondrogenic activity of AG-041R in CL-1. AG-041R has chondrogenic activity in CL-1 not related to CCK2/gastrin receptor antagonism. It is suggested that TGF-beta induction and the activation of MAP kinases mediate the chondrogenic activity of AG-041R in CL-1.
 
Article
AG-041R, a novel indolin-2-one derivative, has recently been demonstrated to induce systemic hyaline cartilage hyperplasia in rats. The aim of this study was to characterize its anabolic actions on chondrocytes. Chondrocytes were isolated from knee joints of 5-week-old SD rats. Effects of AG-041R on cartilage matrix synthesis were examined by measuring [(35)S]sulfate incorporation into proteoglycans, Alcian blue staining, and Northern blotting of cartilage matrix genes. ALP activity, mineral deposition and the expression of markers for hypertrophic chondrocytes, were assessed for terminal differentiation of chondrocytes. Roles of endogenous TGF-beta/BMPs and MEK1/Erk signaling in the action of AG-041R were investigated using the neutralizing soluble receptors and the MEK1 inhibitor. AG-041R accelerated proteoglycan synthesis assessed by both [(35)S]sulfate incorporation and Alcian blue stainable extracellular matrix accumulation. It also up-regulated the gene expression of type II collagen and aggrecan, as well as tenascin, a marker for articular cartilage. In contrast, AG-041R suppressed ALP activity, mineralization, and the gene expression of type X collagen and Cbfa1, indicating that AG-041R prevents chondrocyte terminal differentiation. AG-041R increased in BMP-2 mRNA, and the neutralizing soluble receptor for BMPs reversed the stimulatory effects of AG-041R on cartilage matrix synthesis. Moreover, AG-041R activated MEK1/Erk pathway, which was revealed to prevent chondrocyte terminal differentiation. AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes, which is mediated at least in part by endogenous BMPs and Erk. The data demonstrates that AG-041R has a potential to be a useful therapeutic agent for articular cartilage disorders.
 
Article
To date semiquantitative whole-organ scoring of knee osteoarthritis (OA) relies on 1.5 Tesla (T) Magnetic resonance imaging (MRI) systems. Less costly 1.0 T extremity systems have been introduced that offer superior patient comfort, but may have limitations concerning field-of-view and image quality. The aim of this study was to compare semi-quantitative (SQ) scoring on a 1.0 T system using 1.5 T MRI as the standard of reference. The Multicenter Osteoarthritis Study (MOST) is a longitudinal study of individuals who have or are at high risk for knee OA. A sample of 53 knees was selected in which MRI was performed on a 1.0 T extremity system as well as on a 1.5 T scanner applying a comparable sequence protocol. MRIs were read according to the Whole Organ Magnetic Resonance Imaging Score (WORMS) score. Agreement was determined using weighted kappa statistics. Sensitivity, specificity and accuracy were assessed using the 1.5 T readings as the reference standard. In addition the number of non-readable features was assessed. Agreement (w-kappa) for seven main WORMS features (cartilage, bone marrow lesions (BMLs), osteophytes, meniscal damage and extrusion, synovitis, effusion) ranged between 0.54 (synovitis) and 0.75 (cartilage). Sensitivity ranged between 68.1% (meniscal damage) and 88.1% (effusion). Specificity ranged between 63.6% (effusion) and 96.4% (BMLs). Although the overall rate of non-readable features was very low, it was higher for the 1.0 T system (1.9% vs 0.2%). Semiquantitative whole organ scoring can be performed using a 1.0 T peripheral scanner with a moderate to high degree of agreement and accuracy compared to SQ assessment using a 1.5 T whole body scanner. Our results are comparable to the published inter- and intra observer exercises obtained from 1.5 T systems. Sensitivity to change of longitudinal scoring was not evaluated in this cross-sectional design and should be investigated in future validation studies.
 
Article
Quantitative magnetic resonance imaging (qMRI) of knee cartilage morphology is a powerful research tool but relies on expensive and often inaccessible 1.5 T whole-body equipment. Here we examine the reproducibility and accuracy of qMRI at 1.0 T by direct comparison with previously validated technology. Coronal images of the knee were obtained in six healthy and six osteoarthritic participants. Two data sets were acquired with a 1.5T whole-body magnetic resonance imaging (MRI) system and two with a 1.0 T peripheral MRI system, with repositioning between scans. Proprietary software was used to analyze surface area, volume, and thickness of femoral and tibial cartilage. At 1.0 T, precision errors for surface areas (root-mean-square (RMS) coefficient of variation (CV%)=1.7-2.6%) were higher than those at 1.5 T (1.0-2.1%). For volume and thickness, precision errors were 2.9-5.5% at 1.0 T compared to 1.6-3.4% at 1.5 T. High levels of agreement were found between the two scanners over all plates. With the exception of lateral femoral cartilage (volume and thickness), no statistically significant systematic bias was found between 1.0 T and 1.5 T. This is the first reported study to show that knee cartilage morphology can be determined with a reasonable degree of accuracy and precision using a 1.0 T peripheral scanner. Peripheral MRI is less costly, can be performed in clinical offices, and is associated with higher patient comfort and tolerance than 1.5 T whole-body MRI. Implementation of qMRI with peripheral systems may thus permit its more widespread use in clinical research and patient care.
 
Article
To compare articular cartilage signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and thickness measurements on a 1.5 T and a 3.0 T magnetic resonance (MR) scanner using three-dimensional spoiled gradient recalled echo (3D-SPGR) and two 3D steady-state free precession (SSFP) sequences. Both knees of five volunteers were scanned at 1.5 T and at 3.0 T using a transmit-receive quadrature extremity coil. Each examination consisted of a sagittal 3D-SPGR sequence, a sagittal fat suppressed 3D-SSFP (FS-SSFP) sequence, and a sagittal Dixon 3D-SSFP sequence. For quantitative analysis, we compared cartilage SNR and CNR efficiencies, as well as average cartilage thickness measurements. For 3D-SPGR, cartilage SNR efficiencies at 3.0 T increased compared to those at 1.5 T by a factor of 1.83 (range: 1.40-2.09). In comparison to 3D-SPGR, the SNR efficiency of FS-SSFP increased by a factor of 2.13 (range: 1.81-2.39) and for Dixon SSFP by a factor of 2.39 (range: 1.95-2.99). For 3D-SPGR, CNR efficiencies between cartilage and its surrounding tissue increased compared to those at 1.5 T by a factor of 2.12 (range: 1.75-2.47), for FS-SSFP by a factor 2.11 (range: 1.58-2.80) and for Dixon SSFP by a factor 2.39 (range 2.09-2.83). Average cartilage thicknesses of load bearing regions were not different at both field strengths or between sequences (P>0.05). Mean average cartilage thickness measured in all knees was 2.28 mm. Articular cartilage imaging of the knee on a 3.0 T MR scanner shows increased SNR and CNR efficiencies compared to a 1.5 T scanner, where SSFP-based techniques show the highest increase in SNR and CNR efficiency. There was no difference between average cartilage thickness measurements performed at the 1.5 T and 3.0 T scanners or between the three different sequences.
 
Article
The purpose of this study was to investigate the day-to-day reproducibility of the delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) measurement at different knee joint surfaces in healthy subjects at 1.5 Tesla (T). The dGEMRIC experiment was repeated for 10 asymptomatic volunteers three times with an average interval of 5 days between scans. The measurement was performed from a single sagittal slice through the center of the lateral femoral condyle and from the center of the patella in the axial plane. Cartilage was manually segmented into superficial, deep and full-thickness regions of interests (ROIs) at different topographical locations of the femur, tibia and patella. The reproducibility was evaluated separately for each ROI as well as for the entire bulk cartilage in the slice of each joint surface. The reproducibility at various ROIs expressed by root-mean-square average coefficient of variation (CV(RMS)) ranged between 4.7-12.9%. Thirty out of thirty-three ROIs showed a CV(RMS) less than 10%. Intraclass correlation coefficient (ICC) ranged between 0.45 and 0.98. The CV(RMS) and ICC for bulk dGEMRIC were 4.2% and 0.95 for femur, 5.5% and 0.87 for tibia, and 4.8% and 0.97 for patella. The dGEMRIC technique showed good day-to-day reproducibility, on the average 8% for small deep or superficial segments, 7% for full-thickness ROIs and 5% for bulk ROIs covering all visible cartilage in a single joint surface. We conclude that dGEMRIC imaging at field strength 1.5 T can be used as a reliable instrument for the assessment of articular cartilage when staff has been carefully trained.
 
Article
To assess the normal topographical variation of T2 relaxation time of articular cartilage in different compartments of the knee joint and at different tissue depths in young healthy adults. Twenty asymptomatic young adult volunteers (age range, 21-27 years; mean age, 22.5 years), were studied at 1.5T. Both axial and sagittal multi-slice multi-echo spin echo measurements were performed to determine the T2 relaxation time of cartilage in the femoral, tibial and patellar compartments. The cartilage surfaces were divided into 24 segments and each segment was divided into deep and superficial regions-of-interest (ROIs) of equal thickness. The reproducibility for ROI analysis was assessed for five patients by determining the interclass correlation coefficient (ICC) and the root-mean-square coefficient of variation (CV(RMS)). Cartilage T2 was significantly dependent on joint topography, compartment and tissue depth. For all joint surfaces, superficial T2 values were systematically higher as compared to deep tissue. The data showed a trend toward higher T2 values at the load bearing area of the femoral condyles. The interobserver error varied significantly among different locations and showed mostly good reproducibility with mean ICC of 0.70 and a CV(RMS) of 5.0%. The normal variation in cartilage T2 within a joint is significant and should be acknowledged when pathology-related T2 changes are investigated. The knowledge on normal variation can be used for power and sample size calculations in further studies, and the T2 values as control data in future patient studies.
 
Article
To use receiver operator characteristics (ROC) analysis for assessing the diagnostic performance of three cartilage-specific MR sequences at 1.5 and 3 T in detecting cartilage lesions created in porcine knees. Eighty-four cartilage lesions were created in 27 porcine knee specimens at the patella, the medial and lateral femoral and the medial and lateral tibial cartilage. MR imaging was performed using a fat saturated spoiled gradient echo (SPGR) sequence (in plane spatial resolution/slice thickness: 0.20 x 0.39 mm2/1.5 mm) and two fat saturated proton density weighted (PDw) sequences (low spatial resolution: 0.31 x 0.47 mm2/3 mm and high spatial resolution: 0.20 x 0.26 mm2/2 mm). The images were independently analyzed by three radiologists concerning the absence or presence of lesions using a five-level confidence scale. Significances of the differences for the individual sequences were calculated based on comparisons of areas under ROC curves (A(Z)). The highest A(Z)-values for all three radiologists were consistently obtained for the SPGR (A(Z) = 0.84) and the high-resolution (hr) PDw (A(Z) = 0.79) sequences at 3T. The corresponding A(Z)-values at 1.5 T were 0.77 and 0.69; the differences between 1.5 and 3 T were statistically significant (P < 0.05). A(Z)-values for the low-resolution PDw sequence were lower: 0.59 at 3 T and 0.55 at 1.5 T and the differences between 1.5 and 3T were not significant. With optimized hr MR sequences diagnostic performance in detecting cartilage lesions was improved at 3 T. For a standard, lower spatial resolution PDw sequence no significant differences, however, were found.
 
Article
To assess the effects of interference screws, which are commonly used to surgically fix an anterior cruciate ligament (ACL) graft in the ACL-deficient knee, and magnetic field strength on cartilage volume and thickness measurements with quantitative magnetic resonance imaging (qMRI). Five cadaver knees were imaged using a cartilage-sensitive sequence (T1-weighted water-excitation, three-dimensional (3D) fast low-angle shot) on 1.5T and 3T scanners with and without interference screws implanted. The tibiofemoral articular cartilage was segmented and reconstructed from the magnetic resonance images, and volume and thickness measurements were made on the resulting 3D models. Although several load-bearing regions showed significant differences in volume and thickness between magnet strengths, most showed no significant difference between screw conditions. The medial tibial cartilage showed a mean decrease in volume of 5.9% and 8.0% in the presence of interference screws at 3T and 1.5T, respectively. At 3T and 1.5T, the medial tibial cartilage showed a mean decrease in thickness of 7.0% and 12.0%, respectively, in the presence of interference screws. Caution should be used when interpreting thickness and volume of cartilage at 3T in the presence of interference screws, particularly in the medial tibial compartment. Additionally, 3T and 1.5T qMRI should not be used interchangeably to assess structural changes in tibiofemoral articular cartilage during longitudinal studies.
 
Article
To review the literature on modulation of chondrocyte activities in the osteoarthritic joint, and to discuss these changes in relation to established hard and soft tissue repair paradigms, with an emphasis on transforming growth factor beta (TGFβ1)-mediated signaling which can promote either a chondrogenic or fibrogenic phenotype. Papers addressing the close relationship between repair in general, and the specific post-injury response of joint tissues are summarized. Different interpretations of the role of TGFβ1 in the emergence of an "osteoarthritic" chondrocyte are compared and the phenotypic plasticity of "reparative" progenitor cells is examined. Lastly, emerging data on a central role for A-Disintegrin-And-Metalloproteinase-with-Thrombospondin-like-Sequences-5 (ADAMTS5) activity in modulating TGFβ1 signaling through activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 5 (ALK5) pathways is discussed. The review illustrates how a transition from ALK5-mediated fibrogenic signaling to ALK1-mediated chondrogenic signaling in joint cells represents the critical transition from a non-reparative to a reparative cell phenotype. Data from cell and in vivo studies illustrates the mechanism by which ablation of ADAMTS5 activity allows the transition to reparative chondrogenesis. Multiple large gene expression studies of normal and osteoarthritis (OA) human cartilages (CAs) also support an important role for TGFβ1-mediated pro-fibrogenic activities during disease progression. We conclude that progressive articular CA damage in post-injury OA results primarily from biomechanical, cell biologic and mediator changes that promote a fibroblastic phenotype in joint cells. Since ADAMTS5 and TGFβ1 appear to control this process, agents which interfere with their activities may not only enhance endogenous CA repair in vivo, but also improve the properties of tissue-engineered CA for implantation.
 
Article
Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects. HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells. Ten mM glucosamine and 5-10mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn(387) and Asn(440), abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells. Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.
 
Article
Whilst the characteristic pathologic feature of OA is the loss of hyaline cartilage, prior studies have demonstrated a poor relationship between severity of reported knee pain and degree of radiographic change. The aim of this study was to examine the association between knee symptoms and MRI cartilage volume. A cross-sectional study was performed to assess the association between knee symptoms and MRI cartilage volume in an unselected, community based population. The subjects were 133 postmenopausal females. The subjects had a T2-weighted fat saturated sagittal gradient-echo MRI performed of their right knee. Femoral, tibial and patella cartilage volumes were measured using three-dimensional (3D) Slicer, a software that facilitates semi-automatic segmentation, generation of 3D surface models and quantitative analysis. Qualitative data relating to symptoms, stiffness, pain, physical dysfunction and the quality of life using the WOMAC were recorded. The statistical analyses conducted to determine measures of association between knee pain/symptoms and cartilage volume were correlation, multiple regression and inter-quartile regression. Assessment of the association between patella cartilage volume and the WOMAC domains showed an inverse relationship between patella cartilage volume and pain, function and global score in a model including body mass index, physical activity and leg extensor power (all P=0.01). Inter-quartile regression comparing the lowest 25% with highest 25% patella cartilage volume demonstrated a stronger inverse relationship (P=0.005). This study suggests that alterations in patella volume are associated with pain, function and global scores of the WOMAC. In participants with more knee pain, there was an association with severity of patella cartilage reduction. Other MRI cartilage volume features were not strongly associated with WOMAC sub-scores.
 
Article
Calcification of hypertrophic chondrocytes is the final step in the differentiation of growth plates, although the precise mechanism is not known. We have established two growth plate-derived chondrocyte cell lines, MMR14 and MMR17, from p53-/- mice (Nakamata T, Aoyama T, Okamoto T, Hosaka T, Nishijo K, Nakayama T, et al. In vitro demonstration of cell-to-cell interaction in growth plate cartilage using chondrocytes established from p53-/- mice. J Bone Miner Res 2003;18:97-107). Prolonged in vitro culture produced calcified nodules in MMR14, but not in MMR17. Factors responsible for the difference in calcification between the two cell lines may also be involved in the physiological calcification in growth plate. Gene expression profiles of MMR14 and MMR17 were compared using a cDNA microarray to identify candidate genes involved in the calcification process. Forty-five genes were identified as upregulated in MMR14, including the cadherin-11 (Cdh-11) gene. The expression of Cdh-11 in MMR14 was detected in cell-cell junctions, while no expression was observed in MMR17. Primary cultured chondrocytes from growth plate (GC) also expressed the Cdh-11, and the staining of Cdh-11 was observed in the late hypertrophic zone of growth plate. Cell aggregation assays showed that chondrocytes required Ca2+ to form nodules, and knockdown of the Cdh-11 gene expression using short interfering RNA inhibited the formation of calcified nodules in MMR14. The introduction of Cdh-11 into MMR17 failed to produce calcified nodules indicating that Cdh-11 is one, but not the sole, factor responsible for the production of calcified nodules. Although the physiological role is still unclear, Cdh-11 is a discriminative factor between articular and growth plate chondrocytes.
 
Article
The goal of the study was to investigate the expression of cadherin-11 in synovial fibroblasts (SFs) under mechanical or inflammatory stimuli, and its potential relationship with PI3K/Akt signaling pathway. SFs separated from rat temporomandibular joint (TMJ) were treated with hydrostatic pressures (HP) of 30, 60, 90, and 120kPa, as well as TNF-α for 12, 24, 48, and 72 hrs. The location of cadherin-11 was observed by immunofluorecence microscopy, and its expression was detected by real-time PCR and Western blot. We also studied the activation of PI3K/Akt signaling pathway in SFs with HP or TNF-α stimulation. The results showed that increased expression of cadherin-11 could be found in the cell-cell contact site of SFs in response to HP and inflammatory stimulation. The mRNA and protein expression of cadherin-11 was positively correlated with the intensity of HP and the duration time of TNF-α treatment. Increased expression of vascular endothelial growth factor-D (VEGF-D) and activation of Akt were also found. Treatment with PI3K inhibitor LY294002 attenuated the pressure or inflammatory cytokine induction increases of cadherin-11, VEGF-D, and FGF-2 both in mRNA and protein levels. These findings suggest that cadherin-11 may play important roles in SFs following exposure to mechanical loading and inflammatory stimulation. In addition, PI3K/Akt pathway was associated with pressure or inflammation-induced cadherin-11 expression, which may involve in the pathogenesis of temporomandibular diseases.
 
The relative location of microsatellite markers on the 11q 12-13 chromosomal area used in our study and in the study of Chapman et al. 8. *Microsatellite markers that provided evidence for possible linkage disequilibrium in our study. 
Article
The aims of the present study were: (1) to evaluate the extent and mode of inheritance of hand osteoarthritis by using a large sample of ethnically homogeneous pedigrees of Caucasian origin; (2) to examine whether the synthetic measure of osteoarthritis according to Kellgren and Lawrence (K-L) and the more specific measure, namely, the extent of osteophytes development, have a similar putative genetic determination and pattern of biological inheritance and (3) to test the hypothesis that hand osteoarthritis dependent phenotypes are linked to the 11q 12-13 chromosomal region. The population of the present study comprised 1190 Chuvashians (Russian Federation) belonging to 295 nuclear families. Segregation analysis was carried out on a total sample. Sub-sample of 571 individuals was used to conduct Transmission/disequilibrium test (TDT) and model-based linkage analysis. ESULTS: Adjusted for age, sex and other covariates, both OA phenotypes showed significant familial aggregation. The model fitting analysis strongly supported the hypothesis of a major gene effect on study traits. The inferred major gene explained about 52% of the osteophyte score (OPS) and 49% of the K-L score variation adjusted for confounding variables. The series of model-based linkage analyses and TDTs provided inconclusive evidence on possible linkage of both phenotypes to the 11q 12-13 chromosomal region. We support the hypothesis of a major gene effect in heritability of hand osteoarthritis in both phenotypes. Despite the fact that some DNA markers showed statistically significant association to studied primary phenotypes, we find only weak evidence of linkage disequilibrium between hand osteoarthritis and the proximal part of the 11q 12-13 chromosomal segment (D11S1983 for K-L score and D11S1313 for OPS). The subject, however, a merit requires further investigation.
 
Article
Objective: Evaluation of the efficacy and safety of a single oral dose of a 1200 mg sachet of chondroitin 4&6 sulfate (CS 1200) vs three daily capsules of chondroitin 4&6 sulfate 400 mg (CS 3*400) (equivalence study) and vs placebo (superiority study) during 3 months, in patients with knee osteoarthritis (OA). Design: Comparative, double-blind, randomized, multicenter study, including 353 patients of both genders over 45 years with knee OA. Minimum inclusion criteria were a Lequesne index (LI) ≥ 7 and pain ≥ 40 mm on a visual analogue scale (VAS). LI and VAS were assessed at baseline and after 1-3 months. Equivalence between CS was tested using the per-protocol procedure and superiority of CS vs placebo was tested using an intent-to-treat procedure. Results: After 3 months of follow-up, no significant difference was demonstrated between the oral daily single dose of CS 1200 formulation and the three daily capsules of CS 400. Patients treated with CS 1200 or CS 3*400 were significantly improved compared to placebo after 3 months of follow-up in terms of LI (<0.001) and VAS (P < 0.01). No significant difference in terms of security and tolerability was observed between the three groups. Conclusion: This study suggests that a daily administration of an oral sachet of 1200 mg of chondroitin 4&6 sulfate allows a significant clinical improvement compared to a placebo, and a similar improvement when compared to a regimen of three daily capsules of 400 mg of the same active ingredient.
 
Article
To evaluate anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgical instability models of osteoarthritis (OA) in the 129/SvEv mouse knee joint. Micro-surgical techniques were used to perform ACLT or DMM under direct visualization. Histological scoring was performed on multiple sections to assess cartilage damage across the entire joint. The ACLT model gave severe OA, chondrogenesis of the joint capsule and, in some cases, severe subchondral erosion of the posterior tibial plateau. Surgical DMM was less invasive than the ACLT procedure and resulted in lesions primarily on the central weight-bearing region of the medial tibial plateau and medial femoral condyles. Lesions in the DMM model progressed from mild-to-moderate OA at 4 weeks, to moderate-to-severe OA at 8 weeks post-surgery. Destruction of the subchondral bone was never observed in the DMM model. ACLT is not recommended in the mouse due to the high surgical proficiency required and the development of severe OA that may involve subchondral bone erosion. The severity and location of lesions following DMM are consistent with lesions observed in aged spontaneous mouse models of OA. The DMM model has sufficient sensitivity to show disease modification, as observed with the ADAMTS-5 knock out (KO) mouse. The DMM model should be a first choice to challenge mice with gene deletions of potential targets in OA.
 
Article
We investigated the roles of CXC chemokine ligand 12a (CXCL12a), also known as stromal cell-derived factor-1α, in endochondral bone growth, which can give us important clues to understand the role of CXCL12a in OA. Primary chondrocytes and tibial explants from embryonic 15.5 day-old mice were cultured with recombinant mouse CXCL12a. To assess the role of CXCL12a in chondrogenic differentiation, we conducted mesenchymal cell micromass culture. In tibia organ cultures, CXCL12a increased total bone length in a dose-dependent manner through proportional effects on cartilage and bone. In accordance with increased length, CXCL12a increased the protein level of proliferation markers, such as cyclin D1 and PCNA, in primary chondrocytes as well as in tibia organ culture. In addition, CXCL12a increased the expression of Runx2, Col10 and MMP13 in primary chondrocytes and tibia organ culture system, implying a role of CXCL12a in chondrocyte maturation. Micromass cultures of limb-bud mesenchymal progenitor cells revealed that CXCL12a has a limited effect on early chondrogenesis, but significantly promoted maturation of chondrocytes. CXCL12a induced the phosphorylation of p38 and Erk1/2 MAP kinases and IκB. The increased expression of cyclin D1 by CXCL12a was significantly attenuated by inhibitors of MEK1 and NF-κB. On the other hand, p38 and Erk1/2 MAP kinase and NF-κB signaling were associated with CXCL12a-induced expression of Runx2 and MMP13, the marker of chondrocyte maturation. CXCL12a promoted the proliferation and maturation of chondrocytes, which strongly suggest that CXCL12a may have a negative effect on articular cartilage and contribute to OA progression. Copyright © 2015. Published by Elsevier Ltd.
 
Article
Objective: Growth plate chondrocytes up-regulate calcium-sensing receptor (CaR) expression as they mature to hypertrophy. In cells other than chondrocytes, extracellular calcium-sensing via the CaR functions partly to promote expression of parathyroid hormone-related protein (PTHrP), a critical regulator of endochondral development. Moreover, PTHrP is up-regulated in human osteoarthritis (OA) and surgically induced rabbit OA cartilages and may promote both chondrocyte proliferation and osteophyte formation therein. Hence, we examined chondrocyte CaR-mediated calcium-sensing in OA pathogenesis. Methods: We studied spontaneous knee OA in male Hartley guinea pigs. We also evaluated cultured bovine knee chondrocytes and immortalized human articular chondrocytes (CH-8 cells), employing the CaR calcimimetic agonist NPS R-467 or altering physiologic extracellular calcium (1.8 mM). Results: Immunohistochemistry revealed that CaR expression became up-regulated in the superficial zone at 4 months of age in the guinea pig medial tibial plateau cartilage as early OA developed. CaR expression later became up-regulated in the middle zone. PTHrP content, measured by immunoassay, was significantly increased in the medial tibial plateau cartilage as OA developed and progressed. In cultured chondrocytic cells, CaR-mediated extracellular calcium-sensing, stimulated by the calcimimetic NPS R-467, induced PTHrP and matrix metalloproteinase (MMP)-13 expression and suppressed expression of tissue inhibitor of metalloproteinase (TIMP)-3 dose-dependently, effects shared by elevated extracellular calcium (3 mM). Extracellular calcium-sensing appeared essential for PTHrP and interleukin (IL)-1 to induce MMP-13 and for PTHrP 1-34 to suppress TIMP-3 expression. Conclusions: Chondrocyte CaR expression becomes up-regulated early in the course of spontaneous guinea pig knee OA. Chondrocyte CaR-mediated extracellular calcium-sensing promotes PTHrP expression, modulates the effects of PTHrP and IL-1, and promotes MMP-13 expression and TIMP-3 depletion. Our results implicate up-regulated extracellular calcium-sensing via the CaR as a novel mediator of OA progression.
 
Article
To test the hypothesis that the spondyloepiphyseal dysplasia congenita (sedc) heterozygous (sedc/+) mouse, a COL2A1 mutant, is a model for the study of osteoarthritis (OA) in the absence of dwarfism and to investigate the presence of HtrA1, Ddr2, and Mmp-13 and their possible involvement in a universal mechanism leading to OA. Whole mount skeletons of adult animals were analyzed to determine whether sedc/+ mice exhibit dwarfism. To characterize progression of osteoarthritic degeneration over time, knee and temporomandibular joints from sedc/+ and wild-type mice were analyzed histologically, and severity of articular cartilage degradation was graded using the Osteoarthritis Research Society International (OARSI) scoring system. Immunohistochemistry was used to detect changes in expression of HtrA1, Ddr2, and Mmp-13 in articular cartilage of knees. As previously reported, the sedc/+ skeleton morphology was indistinguishable from wild type, and skeletal measurements revealed no significant differences. The sedc/+ mouse did, however, show significantly higher OARSI scores in knee (9, 12 and 18 months) and temporomandibular joints at all ages examined. Histological staining showed regions of proteoglycan degradation as early as 2 months in both temporomandibular and knee joints of the mutant. Cartilage fissuring and erosion were observed to begin between 2 and 6 months in temporomandibular joints and 9 months in knee joints from sedc/+ mice. Immunohistochemistry of mutant knee articular cartilage showed increased expression of HtrA1, Ddr2, and Mmp-13 compared to wild type, which upregulation preceded fibrillation and fissuring of the articular surfaces. With regard to skeletal morphology, the sedc/+ mouse appears phenotypically normal but develops premature OA as hypothesized. We conclude that the sedc/+ mouse is a useful model for the study of OA in individuals with overtly normal skeletal structure and a predisposition for articular cartilage degeneration.
 
Article
Objective: One of the proteoglycan families is the small leucine-rich proteoglycans (SLRPs) that are characterized by their association with collagen fibrils and/or some glycosaminoglycans. Opticin is a glycoprotein and class III member of the SLRP family, which was initially identified in the vitreous humour of the eye. In this study, we first investigated whether opticin is expressed and produced in normal and OA human articular tissues/cells. Further, we investigated the ability of the key metalloprotease involved in cartilage pathology, MMP-13, to cleave human cartilage opticin. Methods: Opticin gene expression was investigated in normal and OA human chondrocytes, synovial fibroblasts, and subchondral bone osteoblasts by reverse transcriptase-polymerase chain reaction (RT-PCR). Opticin protein production was determined in normal and OA synovial membrane and cartilage by immunohistochemistry. Opticin was isolated from human cartilage using guanidinium chloride extraction, and human MMP-13-induced opticin degradation analyzed by Western blotting. Finally, the opticin MMP-13 cleavage site was determined. Results: Opticin was expressed in human chondrocytes, synovial fibroblasts and subchondral osteoblasts, and the protein identified in synovial membrane and cartilage. At the protein level, OA cartilage showed a slightly higher level of opticin positive stained chondrocytes than normal cartilage; this did not reach statistical significance. However, in contrast with OA, normal cartilage demonstrated a high level of matrix staining in the superficial zone of the tissue, suggesting that in the OA cartilage matrix, opticin is degraded. Data also showed that cartilage opticin could be cleaved by MMP-13 after only 2h of incubation, indicating a preferential substrate compared to other SLRPs for this enzyme. Microsequencing revealed a major cleavage site at the G(104)/L(105)LAAP and a minor at P(109)/A(110)NHPG upon MMP-13 exposure. Conclusion: We demonstrated, for the first time, that opticin is expressed and produced in human articular tissues. Our data also showed that opticin in OA cartilage is degraded in a process that could be mediated by MMP-13. As opticin may contribute towards the structural stability of cartilage, its cleavage by MMP-13 may predispose cartilage to degeneration, particularly at the surface.
 
Article
Osteoarthritis (OA) is one of the most common diseases among the elderly. The main characteristic is the progressive destruction of articular cartilage. We lack quantitative and sensitive biomarkers for OA to detect changes in the joints in an early stage of the disease. In this study, we investigated whether a urinary metabolite profile could be found that could serve as a diagnostic biomarker for OA in humans. We also compared the profile we obtained previously in the guinea pig spontaneous OA model. Urine samples of 92 participants (47 non-OA controls and 45 individuals with radiographic OA of the knees or hips) were selected from the Johnston County Osteoarthritis Project (North Carolina, USA). Participants ranged in age from 60 to 84 years. Samples were measured by 1H nuclear magnetic resonance spectroscopy (NMR) with subsequent principal component discriminant analysis and partial least squares regression analysis. Differences were observed between urine NMR spectra of OA cases and controls (P<0.001 for both male and female subjects). A metabolite profile could be determined which was strongly associated with OA. This profile largely resembled the profile previously identified for guinea pigs with OA (approximately 40 out of the approximately 125 signals of the human profile were present in the guinea pig profile as well). A correlation was found between the metabolite profile and radiographic OA severity (R2 = 0.82 (male); R2 = 0.93 (female)). This study showed that a urine metabolite profile may serve as a novel discriminating biomarker of OA.
 
Article
In this study we investigated the effect of interleukin-13 (IL-13), an anti-inflammatory cytokine, for potential therapeutic use in osteoarthritis (OA). We examined the effect of IL-13 on the synthesis and expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-1 receptor antagonist (IL-1Ra) and stromelysin-1 on human OA synovial membrane in ex vivo cultures. In addition, we explored the effect of IL-13 on both the IL-1 receptor (IL-1R) and TNF-receptor (TNF-R) systems on OA synovial fibroblasts. This included determination of the levels of IL-1 beta and TNF-alpha receptor binding, IL-1Ra and TNF-soluble receptors 55 and 75 (TNF-sR55 and TNF-sR75). In OA synovial membrane treated with LPS, IL-13 inhibited the synthesis of IL-1 beta, TNF-alpha and stromelysin-1, but increased IL-1Ra production. In addition, IL-13 reduced the level of IL-1 beta mRNA and stimulated the level of IL-1Ra mRNA. In synovial fibroblasts, IL-13 decreased the level of IL-1 binding, an effect related to the increased production of IL-1Ra. Although IL-13 had no effect on the TNF-R level, this cytokine markedly decreased the shedding of TNF-R75. These experiments suggest that IL-13 is potentially useful in the therapeutic treatment of OA, as it could regulate the major pathological process of this disease by reducing the production of proinflammatory cytokines and metalloproteases, and favoring the production of IL-1Ra.
 
Article
Human osteoarthritic (OA) cartilage type-II collagen is preferentially cleaved by the proinflammatory cytokine-induced matrix metalloproteinases-13 (MMP-13). Interferon-gamma (IFN-gamma) potently inhibits interleukin-1 (IL-1)-induced MMP-13 expression in healthy chondrocytes. Our goal was to study the previously unknown impact of IFN-gamma on MMP-13 in OA and compare the levels and functional activity of IFN-gamma receptor (IFN-gammaR1) in healthy and OA chondrocytes. Chondrocytes were obtained from OA patients and non-arthritic control subjects and treated with IL-1+ or- IFN-gamma. MMP-13 mRNA and protein expression were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. IFN-gammaR1 expression was assessed by flow cytometry, immunoprecipitation and immunohistochemistry with fluorescein-labeled antibody. IFN-gammaR1 was neutralized with its antibody and signal transducer and activator of transcription 1 (STAT1) phosphorylation analyzed by Western blotting. OA chondrocytes were also transfected with control and IFN-gammaR1 expression vectors. OA chondrocytes displayed a drastically impaired MMP-13 suppression by IFN-gamma compared to control cells. IFN-gammaR1 levels were significantly decreased in OA chondrocytes as assessed by flow cytometry, immunoprecipitation and immunohistochemistry. Consequently, IFN-gamma-stimulated STAT1 phosphorylation mediated by IFN-gammaR1 was also considerably reduced in OA patient chondrocytes. IFN-gammaR1 overexpression in OA cells restored MMP-13 suppression by IFN-gamma. Ability of IFN-gamma to suppress IL-1-induced MMP-13 expression is diminished in OA chondrocytes due to decreased IFN-gammaR1 levels, activity and impaired downstream signal transduction. Therefore, IFN-gammaR1 modulation and weakened endogenous IFN-gamma response may be important mechanisms in OA pathogenesis and cartilage degradation.
 
Expression of hGITR and hGITRL on FLS and MH7A cells. (A) FLS or MH7A cells (10 6 cells/well) were prepared as described in the Methods section and stained with anti-hGITR Ab (2), or anti-hGITRL Ab (3) to detect hGITR [rat IgG: isotype control (1)] or hGITRL [rabbit IgG: isotype control (1)], respectively. The cells were further incubated with FITC-conjugated goat anti-rat IgG or FITC-conjugated goat anti-rabbit IgG for detection of hGITR and hGITRL, respectively, and analyzed by flow cytometry. (B) Total RNA was extracted from OA-FLS, RA-FLS, and MH7A cells (10 6 cells/well) and subjected to RT-PCR. Both cells contain transcript of hGITRL, but not hGITR. Similar results were obtained in three independent experiments.
Effect of direct contact with CD4 C T cells on MMP-13 induction by FLSs. (A) Confluent FLSs (10 5 cells/well) were cultured for 48 h either alone or in the presence of US CD4 C T cells or PMA/I(5/ 200 ng/ml)-stimulated (S) ones (2 ! 10 6 cells/well). (B) Human CD4 C T cells were stimulated with PMA/I or PHA (5 mg/ml). (C) PMA/I-stimulated CD4 C T cells were pre-incubated with anti-hGITR Ab (10 mg/ml) or rat IgG (10 mg/ml) for 8 h, and incubated with FLSs for 48 h. The CD4 C T cells were fixed as described in the Methods section; before coculture with FLS for 48 h. After incubation, medium samples of 50 ml each were concentrated by ultrafiltration (5-fold), separated on 10% SDS-PAGE gels and analyzed for MMP-13 by Western blotting. Similar results were obtained in three independent experiments.
Article
We tested the hypothesis that human glucocorticoid-induced tumor necrosis factor receptor (hGITR/TR11) expressed on the surface of activated CD4(+) T cells is responsible for up-regulating the production of matrix metalloproteinase (MMP)-13 by fibroblast-like synoviocytes (FLSs). The level of MMP-13 was measured by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR). Expressions of hGITR ligand (hGITRL) on the surface of FLSs and hGITR on the surface of human CD4(+) T cells were analyzed by flow cytometry and RT-PCR. Neutralizing antibodies (Abs) were used to block hGITRL and hGITR on the surface of FLSs and human CD4(+) T cells, respectively. Human CD4(+) T cells were cocultured with FLSs to facilitate interaction between hGITR on CD4(+) T cells and hGITRL on FLSs. Soluble hGITR (shGITR) stimulated FLSs to produce MMP-13, and blockade of hGITRL reduced this effect. Direct contact between activated CD4(+) T and FLSs also induced the production of MMP-13, and neutralization of hGITR on activated CD4(+) T cells during coculture decreased the amount of MMP-13 produced by FLSs. shGITR stimulated FLSs to produce MMP-13 via a signal through hGITRL. Direct contact between activated CD4(+) T cells and FLSs facilitated hGITR-hGITRL interaction, and resulted in inducing MMP-13. This effect may increase tissue destruction in chronic inflammation such as rheumatoid arthritis (RA).
 
Article
This study investigated the in vitro effects of hyaluronic acid (HA) of molecular weight (MW) 500-730 kDa on human articular chondrocytes cultivated for 48 h in the presence of interleukin-1beta (IL-1beta) with and without hydrostatic cyclical pressure. The effects of 10 and 100 microg/ml HA with and without IL-1beta were assessed in the culture medium of cells exposed to pressurization cycles in the form of sinusoidal waves (minimum pressure 1MPa, maximum pressure 5MPa) at a frequency of 0.25Hz for 3h, by the immunoenzymatic method on microplates for the quantitative measurement of human proteoglycans (PG) and by the Griess method for nitrites (NO). Morphological analyses were performed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The presence of IL-1beta determines a significant decrease in PG and a significant increase in NO concentrations measured in the culture medium. When the cells are cultured in the presence of IL-1beta and HA at the two concentrations, a statistically significant restoration of PG and a decrease in NO levels are observed. Under pressurization conditions, we observed that the PG concentration in the medium of cells presented a very significant increase in all the conditions used in the study, except for IL-1beta alone. NO production decreased very significantly in the presence of IL-1beta+HA 10 and IL-1beta+HA 100. The results of metabolic evaluation are confirmed by morphological findings obtained by TEM and SEM. These in vitro studies confirm both the protective role of HA (MW 500-730 kDa), which counteracts the IL-1beta-induced effects, and the importance of pressure on chondrocyte metabolism and morphology.
 
Article
Matrix metalloproteinase-13 (MMP-13) is an extracellular MMP that cleaves type II collagen, the major protein component of cartilage, with high specificity and has been implicated in the pathology of osteoarthritis. The present study aimed to characterize the binding and internalization kinetics of MMP-13 in normal rabbit chondrocytes and whether MMP-13 affected cell signalling. Rabbit chondrocytes were used in [125I]-MMP-13 binding assays to investigate the MMP-13 binding kinetics and Western analysis allowed for the assessment of intracellular signalling cascades. Rabbit chondrocytes were found to express the cartilage-specific genes aggrecan and type II collagen throughout their in vitro culture period. Appreciable specific cell-association of [125I]-MMP-13 was detected after 10 min of exposure to the ligand and equilibrium was obtained after 2 h. Binding of [125I]-MMP-13 to chondrocytes was specific and approached saturation at 75 nM. Internalization of MMP-13 was evident after 20 min, reached a maximum at 30 min and had returned to baseline by 90 min. Addition of receptor-associated protein (RAP) inhibited the internalization of MMP-13 indicating a likely role for low-density lipoprotein receptor-related protein-1 (LRP1) in this process. Interestingly the presence of MMP-13 induced phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2) protein showing that there is initiation of a signalling process in response to MMP-13 being bound and internalized by rabbit chondrocytes. However, this activation does not involve the MMP-13 internalization receptor LRP1. These studies demonstrate and characterize the MMP-13 binding and internalization system in rabbit chondrocytes and indicate that MMP-13 may regulate the phenotype of the chondrocytes through this receptor system.
 
Article
To detect and determine disease severity of osteoarthritis using a probe activated by matrix metalloproteinase-13 (MMP-13) in vivo in the murine destabilised medial meniscus (DMM) surgical model of osteoarthritis. We have previously described MMP12ap and MMP13ap, internally quenched fluorescent peptide substrate probes that are activated respectively by MMP-12 and MMP-13. Here we used these probes to follow enzyme activity in vivo in mice knees 4, 6 and 8 weeks following DMM surgery. After in vivo optical imaging, disease severity was determined through traditional histological analysis. The amount of probe activation was analysed for discrimination between DMM, contralateral and sham operated knees, as well as for congruence between activity and histological damage. There was no specific activation of MMP12ap at the time points observed between sham operated and DMM operated, or their respective contralateral joints. The activation of the MMP13ap in the DMM model was highest 6 weeks after surgery, but was only specific compared against sham surgery 8 weeks after surgery (1.5-fold increase). The activation of MMP13ap correlated with histological damage 6 and 8 weeks after surgery, with correlations of 0.484 (p = 0.0032) and 0.478 respectively (p = 0.0049). This correlation dropped to 0.218 (p = 0.011 ) if all data were considered. The current MMP-13 activity probe is suitable for the discrimination between DMM and sham or contralateral knees 8 weeks after surgery, when cartilage loss is typified by the appearance of small fissures up to the tidemark, but not earlier. This activity correlates with the histological damage observed.
 
Co-localization of RUNX2 and MMP-13 in clusters of chondrocytes in osteoarthritic (OA) cartilage. Adjacent sections from an OA lesion (B,D) from the femoral head of a 47-year-old male, and adjacent sections of control cartilage (A,C) from the femoral head of an 78-year-old female were stained with antibodies to RUNX2 (A,B) or MMP-13 (C,D). The immuno-reactive product is dark brown and the sections were counterstained with haematoxylin rendering the nuclei purple. Bar Z 100 m m. 
Increased expression of RUNX2 in OA cartilage. Articular cartilage was cut from the subchondral bone and full thickness sections were immunostained for RUNX2 and counterstained for nuclei (haematoxylin). Zones of the sections were assigned as the deep zone (within 275 m m of subchondral bone), the middle zone (between 275 and 550 m m) and the superficial zone (greater than 550 m m from bone) using a calibrated reticle. (A and B) The middle zone from the femoral head of 83-year-old female control (A) shows only a few RUNX2-expressing chondrocytes while the middle zone from the femoral head of a 38-year-old female OA patient (B) shows a majority of RUNX2 positive chondrocytes. Bar Z 20 m m. (C) Sections from OA patients were sub-categorized as severe OA or mild OA (for definitions, see Materials and methods) and the percentage of RUNX2-expressing chondrocytes in each zone were counted. The graph represents the combined data from five OA patients and three controls with at least three sections from each patient examined. Data represent mean G standard deviation (SD). *P ! 0.05 when compared with the control of the same zone. 
FGF2 up-regulates MMP-13 but not RUNX2 message levels. Bovine articular chondrocytes were cultured in medium with or without FGF2 (25 ng/ml) and total RNA samples were taken at days 1, 3 and 8. MMP-13 (A) and RUNX2 (B) mRNAs from at least three cultures were quantified by real-time RT-PCR and normalized against GAPDH. Data represent mean G SD (n Z 3 or 4). *P ! 0.05 when compared with the control of the same day.
Up-regulation of the MMP-13 promoter by RUNX2 and FGF2 is dependent on MEK/ERK signaling. Bovine articular chondrocytes were transfected with an MMP-13 promoter-luciferase construct (A,B) or a luciferase reporter driven by six tandem repeats of the RUNX2 binding site OSE2 (B,D). Cultures were co-transfected with a RUNX2 expression construct and/or treated with FGF2 as indicated. Luciferase activity was normalized against a control, co-transfected expression standard based on Renilla luciferase activity. Kinase inhibitors were added (C,D) 1 h prior to FGF2 treatment: CalC (Calphostin C, protein kinase C inhibitor) 0.5 m M; PD98059 (MEK/ERK inhibitor) 5, 30 and 100 m M; U0126 (MEK/ERK inhibitor) 0.1, 1.0, 10 m M. Data represent mean G SD ( n Z 3). *P ! 0.05 when compared with the control (Ctr) in panels A and B or between the two group means as indicated in panels C and D. 
Article
To understand the molecular mechanisms that lead to increased MMP-13 expression and cartilage degeneration during the progression of osteoarthritis (OA), we have investigated the expression of the transcription factor RUNX2 in OA cartilage and the regulation of MMP-13 expression by RUNX2 and FGF2 in articular chondrocytes. RUNX2 and MMP-13 expression in human OA and control cartilage was analyzed by immunohistochemistry. The effects of RUNX2 over-expression, with or without FGF2 treatment, on MMP-13 promoter activity and enzyme accumulation were measured in articular chondrocytes. Inhibitors of MEK/ERK were assayed for their ability to block FGF2 and RUNX2 up-regulation of the MMP-13 promoter. We analyzed RUNX2 phosphorylation in response to FGF2. Fibrillated OA cartilage exhibited increased RUNX2 immunoreactivity when compared to control cartilage. RUNX2 co-localized with MMP-13 in clusters of chondrocytes in fibrillated OA cartilage. RUNX2 over-expression in cultured chondrocytes increased their responsiveness to FGF2 treatment, which led to increased MMP-13 expression. Inhibitors of MEK/ERK signaling blocked up-regulation of the MMP-13 promoter by RUNX2 and FGF2, and also blocked the activation of RUNX2 by FGF2. FGF2 treatment of articular chondrocytes increased RUNX2 phosphorylation approximately 2-fold. Increased expression of RUNX2 in OA cartilage may contribute to increased expression of MMP-13. FGF2, which is present in OA synovial fluid, activated RUNX2 via the MEK/ERK pathway and increased MMP-13 expression. However, it is unlikely that RUNX2 is a substrate of ERK1/2. RUNX2 expression and activation may be a significant step in the progression of OA by promoting changes in gene expression and chondrocyte differentiation.
 
Article
Objectives: Glucosamine (GlcN), a natural amino monosaccharide, is a constituent of glycosaminoglycans (GAGs) found in hyaline cartilage. GlcN salts constitute a new class of nutraceutical components with putative chondroprotective activity, which may target chondrocytes as well as chondroprogenitors cells, such as mesenchymal stem cells (MSCs), during cartilage turnover and repair. In the present study, we examined the effects of GlcN on chondrogenesis of human MSCs (hMSCs) and the phenotype of normal and osteoarthritic human articular chondrocytes, using an in vitro pellet culture model maintained in a defined medium. Methods: hMSCs and normal and osteoarthritic human chondrocytes grown as pellet cultures, stimulated or not with interleukin-1beta (IL-1beta), were treated with varying doses of GlcN. Expression of cartilage matrix genes and cartilage degrading enzymes was determined by semiquantitative and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), and by histological staining of cartilage markers, as well as sulfated GAG (sGAG) analysis and Western blotting. Results: Chondrocytes grown in the presence of serum for 11 days showed decreased expression of the cartilage matrix genes, collagen type II (collagen II) and aggrecan, as early as day 3, which was reversed with GlcN treatment by day 11. Both hMSCs and chondrocytes grown as pellet cultures in defined medium and treated with 100 microM GlcN exhibited enhanced expression of collagen II and aggrecan as well as increased content of sGAG, when compared to control untreated pellets. However, high doses of GlcN (10-20mM) were inhibitory. GlcN treatment partially blocked IL-1beta mediated downregulation of collagen II and aggrecan expression and inhibited expression of the matrix degrading enzyme, matrix metalloproteinase 13 (MMP-13), in both chondrocytes and hMSCs undergoing chondrogenesis. Conclusions: These observations suggest that GlcN treatment enhances hMSC chondrogenesis and maintains cartilage matrix gene expression in chondrocytes, which may account for some of the reported chondroprotective properties of GlcN on cartilage.
 
Article
In idiopathic chondrocalcinosis and in osteoarthritis (OA), increased extracellular PP(i) (ecPP(i)) promotes calcification. In chromosome 5p-associated familial chondrocalcinotic degenerative arthropathy, certain mutations in the membrane protein ANK may chronically raise ecPP(i) via enhanced PP(i) channeling. Therefore, we assessed if dysregulated wild-type ANK expression could contribute to pathogenesis of idiopathic degenerative arthropathy through elevated ecPP(i). Using cells with genetic alterations in expression of ANK and the PP(i)-generating nucleotide pyrophosphatase phosphodiestrase (NPP) PC-1, we examined how increased ANK expression elevates ecPPI, testing for codependent effects with PC-1. We also evaluated the effects of ANK expression on chondrocyte growth, matrix synthesis, and MMP-13 expression and we immunohistochemically examined ANK expression in situ in human knee OA cartilages. Using cells expressing defective ANK, as well as PC-1 knockout cells, we demonstrated that ANK required PC-1 (and vice versa) to raise ecPP(i) and that the major ecPP(i) regulator TGFbeta required both ANK and PC-1 to elevate ecPP(i). Upregulation of wild-type ANK by transfection in normal chondrocytes not only raised ecPP(i) 5-fold to approximately 100nM but also directly stimulated matrix calcification and inhibited collagen and sulfated proteoglycans synthesis. In addition, upregulated ANK induced chondrocyte MMP-13, an effect that also was stimulated within 2h by treatment of chondrocytes with 100nM PP(i) alone. Finally, ANK expression was upregulated in situ in human knee OA cartilages. Elevation of ecPP(i) by ANK critically requires the fraction of cellular PP(i) generated by PC-1. The upregulation of ANK expression in OA cartilage and the capacity of increased ANK expression to induce MMP-13 and to promote matrix loss suggest that increased ANK expression and ecPP(i) exert noxious effects in degenerative arthropathies beyond stimulation of calcification.
 
Article
Objective: Abnormal maturation and ossification of the endplate chondrocytes play a central role in the pathogenesis of degenerative disorders of the cervical spine. It is widely held that insulin like growth factor-1 (IGF-1) stimulates chondrocyte proliferation and inhibits chondrocyte terminal differentiation both in vitro and in vivo. However, the mechanism underlying such regulation is not fully understood. The present study aimed to determine the role of IGF-1 on the mRNA expression of collagen type II, alpha 1 (Col2a1) and matrix metallopeptidase 13 (MMP-13) in rat endplate chondrocytes. The possible pathways that transduce IGF-1 effects such as phosphatidylinositol-3 (PI-3)-kinase (PI3K) and mitogen activated protein kinase (MAPK) were also investigated in these cells. Methods: Cultured endplate chondrocytes harvested from rat cervical spines were treated with IGF-1 (100ng/ml), and the changes in Col2a1 and MMP-13 mRNA were monitored with real-time polymerase chain reaction (PCR). MMP-13 activity was also assayed. Activation of signaling proteins was evaluated by western blot analysis. Cells were also treated with pharmacological agents that block PI3K and MAPK signaling pathways. Results: IGF-1 increased Col2a1 mRNA expression in rat endplate chondrocytes in a time- and dose-dependent manner. IGF-1 treatment resulted in a fourfold increase of Col2a1 mRNA with the effect maximizing at 24h. In contrast, IGF-1 treatment for 24h caused a roughly 50% reduction in MMP-13 mRNA. Similar effects were seen on the protein levels of type II collagen (col2) and MMP-13. Consistent with these results, IGF-1 also repressed MMP-13 activity. IGF-1 activated both the PI3K and the extracellular signal-regulated kinase (ERK) pathways as evidenced by phosphorylation of either Akt or ERK1/2 (respectively). The PI3K inhibitor Wartmannin significantly inhibited the IGF-1 effect on Col2a1 mRNA expression but did not affect IGF-1-induced repression of MMP-13 expression. In contrast, the ERK/MAPK inhibitor PD98059 significantly inhibited the effect of IGF-1 on MMP-13 mRNA repression and enhanced IGF-1-induced Col2a1 mRNA expression. Conclusions: In rat endplate chondrocytes the PI3K pathway mainly transduces IGF-1 effect on col2 expression while the ERK pathway mediates IGF-1 effect on MMP-13 expression.
 
Article
Diacerein has proved to be effective in the treatment of osteoarthritis (OA). However, the precise action mechanism of diacerein on OA is not yet fully understood. Therefore, we investigated the effects of rhein, an active metabolite of diacerein, on the production of promatrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase-1 (TIMP-1) in rabbit articular chondrocytes. Confluent rabbit chondrocytes were treated for 24 or 48 h with rhein, naproxen or dexamethasone in the presence of recombinant human IL-1alpha (rhIL-1alpha). ProMMP-9/progelatinase B in the culture medium was monitored by gelatin zymography, and proMMP-1/procollagenase 1, proMMP-3/prostromelysin 1, proMMP-13/procollagenase 3 and TIMP-1 were analysed by Western blot analysis. The steady-state levels of proMMP mRNAs were examined by Northern blot analysis. Total MMPs activity was also determined using FITC-labeled casein. Rhein suppressed the rhIL-1alpha-induced production of proMMPs-1, -3, -9 and -13 in a dose-dependent manner (0.1-30 microM). The suppressed production of proMMPs-1 and -3 was accompanied by a decrease in the steady-state levels of their mRNAs. Interestingly, rhein increased the production of TIMP-1. These observations were further supported by the fact that rhein decreased the apparent total activity of MMPs in the culture medium. We have demonstrated that rhein, an active metabolite of diacerein down-regulates the gene-expression and production of proMMPs and up-regulates the TIMP-1 production. The therapeutic effects of diacerein on OA may be due, at least in part, to the chondroprotective effect of rhein, its active metabolite.
 
Article
To investigate mitogen activated protein (MAP) kinase pathways for their ability to differentially regulate the expression of matrix metalloprotease (MMP)-1 and -13 in human chondrosarcoma cells using pathway-selective inhibitors. Human chondrosarcoma cell lines (SW1353 and JJ012) and human articular chondrocytes (HACs) were treated with cytokines (IL-1beta and TNFalpha) and the expression of MMP-1 and -13 was analyzed. The effects of MAP kinase inhibitors on cytokine-induced expression of MMP-1 and -13 were evaluated using ELISA and Western blot analyses. The possible involvement of the Runx2 pathway in mediating p38 effects on MMP-13 expression was analyzed using promoter-reporter assays, ELISA and immunoprecipitation analyses. IL-1beta and TNFalpha strongly induced the expression of MMP-1 and -13 in SW1353 cells and HACs, whereas only TNFalpha was found to induce the expression of these two MMPs in JJ012 cells. Cytokine treatment did not result in a significant increase in the activity of MMPs because of the excess production of endogenous tissue inhibitors of metalloproteases (TIMPs). Treatment with p38 kinase inhibitors (SB203580 and SB242235) strongly inhibited cytokine-induced MMP-13 expression in a dose-dependent fashion while having a somewhat weaker inhibitory effect on MMP-1 expression. In contrast, inhibitors of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways did not inhibit the expression of either MMP. Overexpression of Runx2 robustly stimulated the transcriptional activation of MMP-13 but had no effect on MMP-1 expression. Furthermore, IL-1beta induced the phosphorylation of Runx2, and this effect was blocked by a p38 kinase inhibitor. Our data suggest that Runx2 is likely to be a key downstream mediator of p38 effects in the differential regulation of IL-1beta induced MMP-13 expression. These studies demonstrate the differential inhibition of cytokine-induced MMP-1 and -13 expression by p38 kinase inhibitors in human chondrosarcoma cells. Our studies also suggest the involvement of Runx2, at least in part, in mediating the effects of p38 on MMP-13 expression.
 
Increased Ihh expression in OA cartilage . Ihh was analyzed with immunohistochemistry in knee joint cartilage from OA knees ( n 1⁄4 17) and resection specimens with normal cartilage ( n 1⁄4 6). The expression of Ihh is signi fi cantly increased in OA cartilage. Increased staining for Ihh protein is associated with increased severity of OA cartilage damage as demonstrated by Safranin-O staining. In contrast, Ihh staining was minimal in normal cartilage. 
Increased Ihh expression in OA synovial fl uid . (A) Radiographs con fi rmed cartilage damage and joint space narrowing in the OA patients and no joint changes in the normal controls. (B) Representative Western blot demonstrates a high level of Ihh in human OA synovial fl uid compared to normal control (54-year-old male healthy volunteer). Coomassie Blue staining was used to con fi rm equal loading. (C) Densi- tometry showed that the mean Ihh concentration was 137.2% higher in the OA group ( n 1⁄4 32) relative to the control group ( n 1⁄4 31) ( P 1⁄4 0.002). 
Increased expression of Ihh, type X collagen, and MMP-13 in OA cartilage . (A) Representative histologic sections from OA cartilage and adjacent relatively normal cartilage are shown. (B) Expression of Ihh, and typical molecular markers of chondrocyte hypertrophy: type X collagen and MMP-13, were determined by qPCR. Samples from the relatively normal are arbitrarily de fi ned as 1. Bar graphs show the averages with SD, N 1⁄4 3, * : P 1⁄4 0.0019. (C) Representative immunohistochemistry for type X collagen and MMP-13 in OA cartilage and cartilage from a control patient without OA. 
Ihh expression is associated with cartilage damage and chondrocyte morphological changes . (A) An increase in Ihh staining in OA cartilage correlates with OA grade and a larger cell size. Bar graphs show the averages of quanti fi ed data of Ihh intensity and cell size from 228 cells from nine patients. (B) There was a signi fi cant correlation ( P < 0.0001) between Ihh intensity of the chondrocytes and Mankin score ( R 2 1⁄4 0.89) (left) and between cell size and Mankin score ( R 2 1⁄4 0.80) (right). (C) Chondrocytes change in shape from 
Ihh induces chondrocyte hypertrophy and regulates type X collagen and MMPs in OA chondrocytes . Human OA chondrocytes were transfected with Ihh siRNA or treated with recombinant Ihh protein (5.0 m g/ml). The mRNA levels of Ihh, type X collagen, and MMPs were determined by qPCR. The endogenous Ihh was knocked down 70% by Ihh siRNA 
Article
The objectives of this study were to (1) determine the correlation between osteoarthritis (OA) and Indian hedgehog (Ihh) expression, and (2) establish the effects of Ihh on expression of markers of chondrocyte hypertrophy and matrix metalloprotease (MMP)-13 in human OA cartilage. OA cartilage and synovial fluid samples were obtained during total knee arthroplasty. Normal cartilage samples were obtained from intra-articular tumor resections, and normal synovial fluid samples were obtained from healthy volunteers and the contralateral uninjured knee of patients undergoing anterior cruciate ligament reconstruction. OA was graded using the Mankin score. Expression of Ihh in synovial fluid was determined by Western blot. Ihh, type X collagen and MMP-13 mRNA were determined by real time PCR. Protein expression of type X collagen and MMP-13 in cartilage samples was analyzed with immunohistochemistry. Chondrocyte size was measured using image analysis. Ihh expression was increased 2.6 fold in OA cartilage and 37% in OA synovial fluid when compared to normal control samples. Increased expression of Ihh was associated with the severity of OA and expression of markers of chondrocyte hypertrophy: type X collagen and MMP-13, and chondocyte size. Chondrocytes were more spherical with increasing severity of OA. There was a significant correlation between Mankin score and cell size (r(2) = 0.80) and Ihh intensity (r(2) = 0.89). Exogenous Ihh induced a 6.8 fold increase of type X collagen and 2.8 fold increase of MMP-13 mRNA expression in cultured chondrocytes. Conversely, knockdown of Ihh by siRNA and Hh inhibitor cyclopamine had the opposite effect. Ihh expression correlates with OA progression and changes in chondrocyte morphology and gene expression consistent with chondrocyte hypertrophy and cartilage degradation seen in OA cartilage. Thus, Ihh may be a potential therapeutic target to prevent OA progression.
 
Article
To investigate the mechanism of mechanical stress-induced expression and regulation of aggrecanases and examine the role of runt-related transcription factor 2 (RUNX-2) in chondrocyte-like cells. SW1353 cells were seeded onto stretch chambers at a concentration of 5×10⁴ cells/chamber, and a uni-axial cyclic tensile strain (CTS) (0.5 Hz, 10% stretch) was applied for 30 min. Total RNA was extracted, reverse transcribed, and analyzed by polymerase chain reaction (PCR) and real-time PCR. RUNX-2 overexpression and small interfering RNA (siRNA) targeting RUNX-2 were used to investigate the role of RUNX-2 in CTS-induced gene expression. The involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of RUNX-2, MMP-13 and ADAMTS-5 during CTS was examined by Western blotting. CTS induced expression of RUNX-2, MMP-13, ADAMTS-4, -5, and -9. Overexpression of RUNX-2 up-regulated expression of MMP-13 and ADAMTS-5, whereas RUNX-2 siRNA resulted in significant down-regulation of mechanically-induced MMP-13 and ADAMTS-5 expression. CTS induced activation of p38 MAPK, and CTS induction of RUNX-2, MMP-13 and ADAMTS-5 mRNA was down-regulated by the selective p38 MAPK inhibitor SB203580 but not by the p44/42 MAPK inhibitor U0126, or the JNK MAPK inhibitor JNK inhibitor II. RUNX-2 might have a role as a key downstream mediator of p38's ability to regulate mechanical stress-induced MMP-13 and ADAMTS-5 expression.
 
Article
The signaling protein p38 mitogen-activated protein kinase is required for inflammatory signaling in chondrocytes that regulates matrix metalloproteinase (MMP) production. We sought to determine the role of specific p38 isoforms in chondrocyte catabolic signaling in response to IL-1beta and fibronectin fragments (Fn-f). Human articular chondrocytes isolated from normal ankle cartilage from tissue donors or from osteoarthritic knee cartilage obtained during knee replacement were stimulated with IL-1beta or Fn-f, with or without pretreatment with p38 inhibitors (SB203580 or BIRB796) or growth factors (IGF-1 and OP-1). p38 isoform phosphorylation was measured by antibody array and immunoblotting. MMP-13 expression was measured by real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Chondrocytes were transfected with plasmids expressing constitutively active (CA) p38gamma or with adenovirus expressing dominant negative (DN) p38gamma. Stimulation of chondrocytes with either IL-1beta or Fn-f led to enhanced phosphorylation of p38alpha and p38gamma, with little phosphorylation of p38beta or p38delta isoforms. p38alpha localized to the nucleus and p38gamma to the cytosol. Inhibition of both p38alpha and p38gamma with BIRB796 resulted in less inhibition of MMP-13 production in response to IL-1beta or FN-f than did inhibition of only p38alpha with SB203580. Transfection with CA p38gamma resulted in decreased MMP-13 production while transduction with DN p38gamma resulted in increased MMP-13 production. IGF-1 and OP-1 pretreatment inhibited p38alpha phosphorylation but not p38gamma phosphorylation. p38gamma is activated by catabolic stimulation of human articular chondrocytes, but interestingly suppresses MMP-13 production. Treatments that increase p38gamma activation may be of therapeutic benefit in reducing chondrocyte production of MMP-13.
 
Article
To investigate the potential involvement of prostacyclin in basic calcium phosphate (BCP) crystal-induced responses in osteoarthritic synovial fibroblasts (OASF). OASF grown in culture were stimulated with BCP crystals. Prostacyclin production was measured by enzyme immunoassay. Expression of messenger RNA (mRNA) transcripts was assessed by real-time polymerase chain reaction (PCR). Expression of prostacyclin synthase (PGIS) and the prostacyclin (IP) receptor was measured. The effects of iloprost, a prostacyclin analogue, on expression of genes implicated in osteoarthritis such as microsomal prostaglandin E2 synthase 1 (mPGES1) and matrix metalloproteinases (MMPs) were also studied. FPT inhibitor II, a farnesyl transferase inhibitor, was used to antagonize iloprost-induced responses. BCP crystal stimulation led to a five-fold increase in prostacyclin production in OASF compared to untreated cells. This induction was attenuated by cyclooxygenase (COX)-2 and COX-1 inhibition at 4 and 32h, respectively. PGIS and IP receptor transcripts were constitutively expressed in OASF. BCP crystals upregulated IP receptor expression two-fold. While iloprost diminished BCP crystal-stimulated IP receptor upregulation, the inhibitory effect of iloprost was blocked by the farnesyl transferase inhibitor. In addition, iloprost upregulated mPGES1 and downregulated MMP-13 expression in BCP crystal-stimulated OASF, effects that were not influenced by the farnesyl transferase inhibitor. These data showed for the first time that BCP crystals can increase prostacyclin production and upregulate expression of the IP receptor in OASF. The potential of prostacyclin to influence BCP crystal-stimulated responses was supported by the effects of iloprost on the expression of the IP receptor, mPGES1 and MMP-13. These data demonstrate the potential involvement of prostacyclin in BCP crystal-associated osteoarthritis (OA) and suggest that inhibition of PG synthesis with non-steroidal anti-inflammatory drugs may have both deleterious and beneficial effects in BCP crystal-associated OA.
 
Article
To evaluate the efficacy and safety of the cyclooxygenase-inhibiting nitric-oxide donator, naproxcinod, compared with naproxen and placebo in patients with osteoarthritis (OA) of the knee. 918 eligible patients were randomly assigned to double-blind treatment with either naproxcinod 375 mg, naproxcinod 750 mg, naproxen 500 mg or placebo, twice daily for 13 weeks. The primary objective was to show superiority of naproxcinod compared to placebo. Main efficacy criteria were assessment of pain and physical function using the Western Ontario and MacMaster Universities Osteoarthritis Index (WOMAC) and patients' overall rating of disease status (Likert scale). The main secondary objectives were to show that naproxcinod was non-inferior to naproxen 500 mg and to evaluate overall safety. Both doses of naproxcinod were statistically and clinically superior to placebo in relieving signs and symptoms of OA of the knee after 13 weeks of treatment, as demonstrated by all three co-primary endpoints (P< or =0.0003). The evaluation of the other secondary efficacy measures was consistent with the primary endpoint results. Naproxcinod 750 mg was non-inferior to equimolar doses of naproxen 500 mg in the Intent-to-Treat (ITT) population. 24.5% of patients discontinued prematurely, with a higher incidence in the placebo group (18.6%) than the active groups (4.3-7.1%) discontinuing due to lack of efficacy. Both doses of naproxcinod were well-tolerated, with most adverse events being mild or moderate. Compared to placebo, naproxcinod 750 mg and 375 mg showed a similar blood pressure (BP) profile in contrast to naproxen which increased BP. These results demonstrated the clinical efficacy and safety of naproxcinod in the management of the signs and symptoms of OA. Naproxcinod was well-tolerated, with BP effects similar to placebo and different from naproxen. Clinical Trials.gov identifier: NCT00542555.
 
Article
To analyze the gene expression profile of human fetal cartilage by expressed sequence tags (ESTs). A human fetal cartilage (8-12 weeks) cDNA library was constructed using the lambda ZAP Express vector. ESTs were obtained by partial sequencing of cDNA clones. The basic local alignment search tool algorithm was used to compare all generated ESTs to known sequences. A total of 13,155 ESTs were analyzed, of which 8696 ESTs (66.1%) matched known genes, 53 ESTs (0.4%) were putatively novel (with no match) and the rest matched other ESTs, genomic DNA and repetitive sequences. Importantly, we identified 2448 unique known genes through non-redundancy analysis of the known gene matches, which were then functionally categorized. The tissue specificity of this library was reflected by its EST profile of the extracellular matrix (ECM) proteins. Collagens were the major transcripts, representing 68.5% of the ECM proteins. Proteoglycans were the second most abundant, constituting 9.5%. Collagen type II was the most abundant gene of all. Glypican 3, decorin and aggrecan were the major transcripts of proteoglycans. Many genes involved in cartilage development were identified, such as insulin-like growth factor-II, its receptor and binding proteins, connective tissue growth factor and fibroblast growth factors. Proteases and their regulatory factors were also identified, including matrix metalloprotease 2 and tissue inhibitor of metalloproteinase 1. The EST approach is an effective way of characterizing the genes expressed in cartilage. These data represent the most extensive molecular information on human fetal cartilage to date. The availability of this information will serve as a basis for further research to identify genes that are essential in cartilage development.
 
Article
The recent expert-consensus guidelines for the management of knee and hip osteoarthritis (OA) are very insightful and should serve as a powerful clinical tool for many providers 1 . Although minimal attention is given to the positive effects of manual physical therapy despite strong randomized control trials (RCT) in the literature 2,3 , the fact that the panel unanimously recommends evaluation and referral to a physical therapist is promising. These RCT’s evaluating manual physical therapy were included in the OsteoArthritis Research Society International (OARSI) panel’s literature review, however the emphasis of the actual recommendation appears to be focused more on the provision of assisted devices for ambulation, although none of the RCT’s referenced in that section include that as a studied intervention. As in many other disciplines, the specific intervention provided by a clinician can vary. When evaluating the efficacy of these interventions it is important for the medical provider to distinguish between current practice and best evidence-based practice when interpreting the results published in the literature. In this case, recommendations would be better served based on specific evidence-validated interventions provided by physical therapists. In addition, the statement that no RCT’s for management of hip OA by a physical therapist exist might imply a lack of thoroughness in the literature review for this portion. In a 2004 RCT in Arthritis and Rheumatism by Hoeksma et al., a 5-week manual therapy program was shown to be significantly superior to exercise therapy not only in general perceived improvement, but also in pain, hip function, walking speed, range of motion, and quality of life 4 .
 
Article
To investigate the relationships between serum and urinary molecular markers (MM) used to monitor osteoarthritis. Forty osteoarthritis patients had blood and urine collected at baseline and 1, 3, 6 and 12 months later. Specimens from 20 controls were obtained twice at a one month interval. The concentration of 14 different markers was determined at each time point and the data were analyzed by statistical methodology. The markers could be divided by the method of principal components analysis into five clusters of related markers: inflammation markers (C-reactive protein, tumor necrosis receptor type I and tumor necrosis receptor type II, interleukin 6, eosinophilic cationic protein), bone markers (bone sialoprotein, hydroxylysyl pyridinoline, lysyl pyridinoline), putative markers of cartilage anabolism (carboxypropeptide of type II procollagen, hyaluronan, epitope 846) and catabolism (keratan sulfate, cartilage oligomeric matrix protein), and transforming growth factor beta. Three markers (tumor necrosis factor receptor II, cartilage oligomeric matrix protein and epitope 846) from independent clusters discriminated osteoarthritis patients from controls. Inflammation was not a confounding factor in measurement, but a recognizable distinguishing factor in osteoarthritis. The markers separated into rational groups on the basis of their covariance, a finding with independent biochemical support. The covariance of markers from the same cluster suggests the use of a representative marker from the cluster to reflect changes in osteoarthritis. If multiple markers are being measured within a single cluster, then the use of a weighted cluster 'factor' may be preferable to the separate use of individual markers.
 
Article
The aim of this study was to investigate the pharmacokinetic and pharmacodynamic parameters of oral salmon calcitonin (oSCT) administered over 14 days to men and women presenting with osteoarthritis (OA). The study was a phase-I, 2-week, placebo-controlled, double-blind, double-dummy, randomized, gender-stratified study including 73 subjects aged 57-75 years. Patients had painful OA with a Kellgren and Lawrence index score of I-III. Treatment allocations were; 0.6 mg, 0.8 mg of oSCT, or placebo. Treatment was given twice daily for 14 days. The morning dose was administered between 07:00 and 08:00 at least 30 min before breakfast. The second dose was administered 30 min before evening dinner. On treatment day 1 and 14, the morning dose was followed by 5h of fasting, and blood samples and urine were collected immediately prior to dosing and according to the protocol. Study parameters were: plasma sCT levels, bone resorption by CTX-I (serum C-terminal telopeptide of collagen type I), bone formation by osteocalcin (serum OC), and cartilage degradation by CTX-II (urine C-terminal telopeptide of collagen type II) (clinicaltrials.gov identifier: NCT00486369). Doses of 0.8 mg compared with 0.6 mg produced significantly higher C(max) and AUC(0-4 hrs), of calcitonin, P=0.03. This resulted in significant reductions in CTX-I and CTX-II, [P<0.0001; P=0.007]. No differences were observed between baseline and follow-up at day 14 in pharmacokinetic and pharmacodynamic parameters. Gender had no observable influence on results. oSCT given twice daily with a pre-dinner and morning fasting dosing resulted in reductions in markers of bone resorption and cartilage degradation.
 
Article
The objective of this work was to compare the measurement properties of three categorical X-ray scoring methods for hip osteoarthritis (OA). In data obtained from trials and cohorts, radiographs were evaluated using the Kellgren and Lawrence (KL) grading system, the Osteoarthritis Research Society International (OARSI) joint space narrowing score, and quantitative measurement of joint space width (JSW), analysed as a categorical variable according to Croft and Lane's cutoffs (1.5, 2.5 and 3mm). Predictive validity was assessed through logistic regression to predict joint replacement in one database. Construct validity was assessed through logistic regression between pain and function and X-ray stages. Inter-observer and intra-observer reliability were assessed in 50 subjects by weighted kappa. Sensitivity to change was assessed in 50 patients over a 24-month interval, by standardized response mean (SRM). Radiographs were available from one trial and two cohorts (1404 X-rays). All three methods predicted joint replacement in the trial. Correlation with clinical parameters was low for the three scoring methods, except for the single community-based cohort. Interrater reliability was higher for categorical JSW (kappa, 0.71 vs 0.44 and 0.47 for KL and OARSI, respectively). Intrarater reliability was similar for the three methods (0.79 vs 0.69 and 0.81). Sensitivity to change was higher for categorical JSW than KL and OARSI (SRM, 0.77 vs 0.28 and 0.35). Categorical JSW has similar validity and higher sensitivity to change than the other categorical scoring techniques in hip OA. These results indicate categorical JSW may be the preferred method to evaluate structural severity in hip OA clinical trials.
 
Article
The objective of this study was to elucidate the role of the superficial region of articular cartilage in determining the dynamic properties of the tissue. It is hypothesised that removal of the superficial region will influence both the flow dependent and independent properties of articular cartilage, leading to a reduction in the dynamic modulus of the tissue. Osteochondral cores from the femoropatellar groove of three porcine knee joints were subjected to static and dynamic loading in confined or unconfined compression at increasing strain increments with and without their superficial regions. Equilibrium moduli and dynamic moduli were measured and the tissue permeability was estimated by fitting experimental data to a biphasic model. Biochemical analysis confirmed a zonal gradient in the tissue composition and organisation. Histological and PLM analysis demonstrated intense collagen staining in the superficial region of the tissue with alignment of the collagen fibres parallel to the articular surface. Mechanical testing revealed that the superficial region is less stiff than the remainder of the tissue in compression, however removal of this region from intact cores was found to significantly reduce the dynamic modulus of the remaining tissue, suggesting decreased fluid load support within the tissue during transient loading upon removal of the superficial region. Data fits to a biphasic model predict a significantly lower permeability in the superficial region compared to the remainder of the tissue. It is postulated that the observed decrease in the dynamic moduli is due at least in part to the superficial region acting as a low permeability barrier, where its removal decreases the tissue's ability to maintain fluid load support. This result emphasises the impact that degeneration of the superficial region has on the functionality of the remaining tissue.
 
Article
MiR146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) by impairing NF-κB activity and inhibiting the expression of target genes. Recent study suggests that histone deacetylases (HDACs) are involved in the regulation of microRNA expression. Therefore, we determined whether HDAC inhibitors can increase miR-146a expression, thereby inhibiting interleukin-1β (IL-1β)-induced signaling in osteoarthritis fibroblast-like synoviocytes (OA-FLS). MicroRNA expression was analyzed using real-time PCR. IL-1β-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA. Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays. IL-1β treatment of OA-FLS induced a mild (1.7-fold) increase in miR-146a expression that was unable to appropriately downregulate IRAK1 and TRAF6 expression. HDAC inhibitors, SAHA (vorinostat), and LBH589 (panobinostat) significantly (6.1- and 5.4-fold) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter, and negatively regulated IL-1β-induced IKK/IκB/p65 phosphorylation signaling and IL-6 secretion. The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or HDAC1 (class I HDAC), HDAC4 (class IIa HDAC), and HDAC6 (class IIb HDAC) overexpression, suggesting that they were due to inhibition of HDAC activity. Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1β-induced signaling and cytokine secretion. Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors.
 
Article
The PGD(2) metabolite 15-deoxy-delta12,14 PGJ(2) (15d-PGJ(2)), a potent peroxisome proliferator-activated receptor gamma (PPARgamma) activator, has recently received attention for its potential antiinflammatory effects, but its effect on the cyclooxygenase-2 (COX-2) production is still under debate. We investigated the effect of 15d-PGJ(2) on COX-2 and prostaglandin E(2) (PGE(2)) production in the absence or the presence of interleukin-1beta (IL-1beta) in human osteoarthritic chondrocytes.Data showed that, as expected, IL-1beta induced both COX-2 and PGE(2) production. The addition of 15d-PGJ(2) completely blocked (by 93%) the IL-1beta-induced PGE(2) synthesis, whereas COX-2 level was only partially reduced (by 72%). Interestingly in the absence of any COX-2 inducer, 15d-PGJ(2) up-regulated COX-2 expression without concomitant elevation of PGE(2) synthesis. This study showed that the PPARgamma agonist, 15d-PGJ(2), exerts a dual effect on COX-2 production. The mechanisms by which 15d-PGJ(2) favors COX-2 production will be discussed.
 
Top-cited authors
Sita Bierma-Zeinstra
Yves Henrotin
  • University of Liège
Francis Berenbaum
  • Sorbonne Université
Ali Guermazi
  • Boston University
Hiroshi Kawaguchi
  • Jhoban Hospital <Fukushima, Japan