Osteoarthritis and Cartilage

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.