Osteoarthritis and Cartilage

MAPKs are essential upstream signaling pathways in proteolytic cartilage degradation – divergence in pathways leading to aggrecanase and MMP-mediated articular cartilage degradation

2009-12-04

Summary: Objectives: Matrix metalloproteinases (MMPs) and aggrecanases are essential players in cartilage degradation. However, the signaling pathways that results in MMP and/or aggrecanase synthesis and activation are not well understood. We investigated the molecular events leading to MMP- and aggrecanase-mediated cartilage degradation.Methods: Cartilage degradation was induced in bovine articular cartilage explants by oncostatin M (OSM) and tumor necrosis factor (TNF), in the presence or absence of specific inhibitors of the mitogen-activated protein kinases (MAPKs) P38, P44/42 and Src family. Toxicity was followed by the AlamarBlue colorimetric assay. MMP-activity was assessed using a fluorescent substrate assay and MMP-9 and -2 activities by gelatinase zymography. MMP-mediated collagen type II degradation and MMP as well as aggrecanase-mediated aggrecan degradation was investigated with specific ELISA and hydroxyproline release by standard methods. The findings were verified by immunohistochemistry and histology.Results: Stimulation of cartilage degradation by OSM+TNF resulted in 100-fold induction of CTX-II release (P<0.01). This was dose-dependently inhibited by MAPK P38 inhibitors and by the MAPK P44/42 inhibitors. MMP-activity and expression was significantly decreased, as evaluated by cleavage of fluorescence MMP-substrate and zymography. Immunohistochemistry confirmed these findings. Interestingly, only the P44/42 inhibitors abrogated aggrecanase-mediated aggrecan degradation.Conclusion: We found that inhibition of MAPK P38, P44/42 and Src family abrogated proteolytic cartilage degradation by blocking MMP synthesis and activity. However, only MAPK P44/42 was essential for aggrecanase-mediated aggrecan degradation. These data suggest that various aspects of cartilage degradation can be targeted independently by inhibiting specific upstream signaling pathway.

Symptomatic efficacy and safety of diacerein in the treatment of osteoarthritis: a meta-analysis of randomized placebo-controlled trials

E.M. Bartels, H. Bliddal, P.K. Schøndorff, R.D. Altman, W. Zhang, R. Christensen — 2009-11-02

Summary: Objective: To estimate the efficacy and safety of diacerein as a pain-reducing agent in the treatment of osteoarthritis (OA), using meta-analysis of published randomized placebo-controlled trials (RCTs).Methods: Systematic searches of the bibliographic databases Medline, Embase, Cinahl, Chemical Abstracts, Cochrane and Web of Science for RCTs concerning diacerein treatment of OA. Inclusion criteria: explicit statement about randomization to either diacerein or placebo, and co-primary outcomes being reduction in pain and improvement in function. Efficacy effect size (ES) was estimated using Hedges's standardized mean difference. Safety was measured via the risk ratio (RR) of patients having at least one episode of diarrhoea, or withdrawal due to adverse events. Trials were combined by using random-effects meta-analysis. Consistency was evaluated via the I-squared index.Results: Six trials (seven sub-studies; 1533 patients) contributed to the meta-analysis, revealing a large degree of inconsistency among the trials (I2=56%) in regard to pain reduction: the combined ES was −0.24 [95% confidence intervals (CI): −0.39 to −0.08, P=0.003], favouring diacerein. The statistically significant improvement in function (P=0.01) was based on a small amount of heterogeneity (I2=11%), but presented a questionable clinical effect size (ES=−0.14). Risk of publication bias could not be excluded, and trials with duration of more than 6 months did not favour diacerein. There was an increased risk of diarrhoea with diacerein (RR=3.51 [2.55–4.83], P<0.0001), and some withdrawal from therapy following adverse events (RR=1.58 [1.05–2.36], P=0.03).Conclusions: Diacerein may be an alternative therapy for OA for patients who cannot take paracetamol or non-steroidal anti-inflammatory drugs (NSAIDs) because of adverse effects or lack of benefit. However, it is associated with increased risk of diarrhoea, and the symptomatic benefit after 6 months remains unknown.

The human pharmacokinetics of oral ingestion of glucosamine and chondroitin sulfate taken separately or in combination

2009-11-26

Summary: Objective: As part of the National Institutes of Health (NIH)-sponsored Glucosamine/Chondroitin sulfate Arthritis Intervention Trial (GAIT) our objective here was to examine (1) the pharmacokinetics (PK) of glucosamine (GlcN) and chondroitin sulfate (CS) when taken separately or in combination as a single dose in normal individuals (n=29) and (2) the PK of GlcN and CS when taken as a single dose after 3 months daily dosing with GlcN, CS or GlcN+CS, in patients with symptomatic knee pain (n=28).Methods: The concentration of GlcN in the circulation was determined by established fluorophore-assisted carbohydrate electrophoresis (FACE) methods. The hydrodynamic size and disaccharide composition of CS chains in the circulation and dosage samples was determined by Superose 6 chromatography and FACE.Results: We show that circulating levels of CS in human plasma are about 20μg/ml. Most significantly, the endogenous concentration and CS disaccharide composition were not detectably altered by ingestion of CS, when the CS was taken alone or in combination with GlcN. On the other hand, the Cmax (single-dose study) and AUC values (multiple-dose study) for ingested GlcN were significantly reduced by combination dosing with CS, relative to GlcN dosing alone.Conclusions: We conclude that pain relief perceived following ingestion of CS probably does not depend on simultaneous or prior intake of GlcN. Further, such effects on joint pain, if present, probably do not result from ingested CS reaching the joint space but may result from changes in cellular activities in the gut lining or in the liver, where concentrations of ingested CS, or its breakdown products, could be substantially elevated following oral ingestion. Moreover, since combined dosing of GlcN with CS was found to reduce the plasma levels seen with GlcN dosing alone, any improved pain relief by combination dosing cannot be explained by higher circulating concentrations of GlcN.

Development of a population-based microsimulation model of osteoarthritis in Canada

2009-11-20

Summary: Objectives: The purpose of the study was to develop a population-based simulation model of osteoarthritis (OA) in Canada that can be used to quantify the future health and economic burden of OA under a range of scenarios for changes in the OA risk factors and treatments. In this article we describe the overall structure of the model, sources of data, derivation of key input parameters for the epidemiological component of the model, and preliminary validation studies.Design: We used the Population Health Model (POHEM) platform to develop a stochastic continuous-time microsimulation model of physician-diagnosed OA. Incidence rates were calibrated to agree with administrative data for the province of British Columbia, Canada. The effect of obesity on OA incidence and the impact of OA on health-related quality of life (HRQL) were modeled using Canadian national surveys.Results: Incidence rates of OA in the model increase approximately linearly with age in both sexes between the ages of 50 and 80 and plateau in the very old. In those aged 50+, the rates are substantially higher in women. At baseline, the prevalence of OA is 11.5%, 13.6% in women and 9.3% in men. The OA hazard ratios for obesity are 2.0 in women and 1.7 in men. The effect of OA diagnosis on HRQL, as measured by the Health Utilities Index Mark 3 (HUI3), is to reduce it by 0.10 in women and 0.14 in men.Conclusions: We describe the development of the first population-based microsimulation model of OA. Strengths of this model include the use of large population databases to derive the key parameters and the application of modern microsimulation technology. Limitations of the model reflect the limitations of administrative and survey data and gaps in the epidemiological and HRQL literature.

Hip osteoarthritis and risk factors in elderly Korean population

2009-11-30

Summary: Objective: To investigate the prevalence of hip osteoarthritis (OA) in a community-based elderly Korean population and to identify its risk factors.Design: Radiographs of hip and knee were evaluated in 288 men and 386 women (age≥65 years) that participated in the Korean Longitudinal Study on Health and Aging (KLoSHA). Minimum joint space widths (JSW), center-edge angles (CEA), and neck–shaft angles were measured on hip radiographs, and tibio–femoral angles on knee radiographs. Hip OA was defined as minimum JSW of ≤2mm or ≤2.5mm. The following potential risk factors of OA were examined; demographic data, acetabular dysplasia, large CEA (≥40°) and deformities of femoral neck and knee joint. Multivariate analysis with generalized estimating equation (GEE) model was performed to exclude confounding factors.Results: When hip OA was defined as JSW≤2mm, the overall prevalence of the disease was 2.1% (95% confidence interval [CI], 1.0–3.2%), and only older age (≥70 years) was identified as a significant risk factors with an odds ratio (OR) of 10.0. However, when hip OA was defined as a JSW of ≤2.5mm, the overall prevalence of the disease was 13.1% (95% CI, 10.5–15.6%), and older age (≥70 years), female, large CEA (≥40°), and acetabular dysplasia (CEA<20°) were identified as significant risk factors with ORs of 2.1, 2.1, 2.3, and 10.2, respectively.Conclusions: The prevalence of hip OA in elderly Korean was 2.1% (JSW≤2mm) in community-based population. Older age (≥70 years), female, large CEA (≥40°), and acetabular dysplasia (CEA<20°) appeared to be significant risk factors of hip OA.

Foot structure and function in older people with radiographic osteoarthritis of the medial midfoot

H.B. Menz, S.E. Munteanu, G.V. Zammit, K.B. Landorf — 2009-12-09

Summary: Objective: To investigate whether foot structure and dynamic foot function differ between older people with and without radiographically confirmed osteoarthritis (OA) of the talo-navicular joint (TNJ) and navicular-first cuneiform joint (N1stCJ).Method: Dorso-plantar and lateral weighbearing foot radiographs (right feet) were obtained from 205 older people aged 61–94 years, and the presence of OA in the TNJ and N1stCJ was determined using a standardized atlas. Foot structure was assessed using a clinical measure (the arch index [AI]) and two radiographic measures (calcaneal inclination angle [CIA] and calcaneal-first metatarsal angle [C1MA]). Dynamic plantar pressure assessment during walking was undertaken using the Tekscan MatScan® system.Results: Thirty-five participants exhibited radiographic OA in the TNJ and N1stCJ. There were no significant differences between the groups in relation to age, sex, weight or walking velocity. Compared to those without OA in these joints, those with OA had significantly flatter feet, as evidenced by larger AI (0.26±0.05 vs 0.25±0.05, P=0.02), smaller CIA (18.5±6.3 vs 21.3±5.4°, P<0.01) and larger C1MA (137.0±9.3 vs 132.4±8.0°, P<0.01), and exhibited significantly higher maximum forces in the midfoot (15.2±7.3 vs 11.2±7.0kg, P<0.01; 36% increase).Conclusion: Older people with radiographic OA of the TNJ and N1stCJ exhibit flatter feet and increased loading of the plantar midfoot when walking. Excessive loading of the midfoot may predispose to OA by increasing dorsal compressive forces, although prospective studies are required to confirm whether this relationship is causal.

Pre-radiographic MRI findings are associated with onset of knee symptoms: the most study

2009-11-30

Summary: Objective: Magnetic resonance imaging (MRI) has greater sensitivity to detect osteoarthritis (OA) damage than radiographs but it is uncertain which MRI findings in early OA are clinically important. We examined MRI abnormalities detected in knees without radiographic OA and their association with incident knee symptoms.Method: Participants from the Multicenter Osteoarthritis Study (MOST) without frequent knee symptoms (FKS) at baseline were eligible if they also lacked radiographic features of OA at baseline. At 15 months, knees that developed FKS were defined as cases while control knees were drawn from those that remained without FKS. Baseline MRIs were scored at each subregion for cartilage lesions (CARTs); osteophytes (OST); bone marrow lesions (BML) and cysts. We compared cases and controls using marginal logistic regression models, adjusting for age, gender, race, body mass index (BMI), previous injury and clinic site.Results: 36 case knees and 128 control knees were analyzed. MRI damage was common in both cases and controls. The presence of a severe CART (P=0.03), BML (P=0.02) or OST (P=0.02) in the whole knee joint was more common in cases while subchondral cysts did not differ significantly between cases and controls (P>0.1). Case status at 15 months was predicted by baseline damage at only two locations; a BML in the lateral patella (P=0.047) and at the tibial subspinous subregions (P=0.01).Conclusion: In knees without significant symptoms or radiographic features of OA, MRI lesions of OA in only a few specific locations preceded onset of clinical symptoms and suggest that changes in bone play a role in the early development of knee pain. Confirmation of these findings in other prospective studies of knee OA is warranted.

Osteoarthritis may not be a one-way-road of cartilage loss – comparison of spatial patterns of cartilage change between osteoarthritic and healthy knees

2009-12-14

Summary: Objective: To explore whether longitudinal change in cartilage thickness in femorotibial subregions of knees with radiographic osteoarthritis (ROA) differs from that in healthy knees.Methods: 3T coronal magnetic resonance (MR) images were acquired in 152 women at seven clinical centers at baseline (BL) and 24 months. Knees from 75 women with signs of ROA in either anterior–posterior or Lyon schuss radiographs were compared with those from 77 asymptomatic healthy controls without ROA to identify knees showing greater change in cartilage thickness than expected based on observations in healthy knees. The femorotibial cartilage thickness was determined in BL and follow-up MR images across five tibial and three femoral subregions in the medial/lateral compartment, respectively.Results: A substantial portion of knees with ROA were classified as having longitudinal cartilage thinning (28%) or thickening (20%) in at least one medial femorotibial subregion based on comparisons to longitudinal changes observed in healthy knees; only 5% showed both subregional thinning and thickening across (different) medial subregions at the same time. Whereas the estimated proportion of Kellgren Lawrence grade (KLG) 3 knees (n=28) with significant medial cartilage thinning (46%) was substantially greater than that with cartilage thickening (18%), the estimated percentages of KLG2 knees (n=30) with significant medial thinning (20%) and thickening (23%) were similar.Conclusion: This exploratory study indicates that OA may not be a one-way-road of cartilage loss. Subregional analysis suggests that, compared with healthy knees, cartilage changes in ROA may occur in both directions. Medial femorotibial cartilage thickening was observed as frequently as cartilage thinning in KLG2 knees.

The association of prevalent medial meniscal pathology with cartilage loss in the medial tibiofemoral compartment over a 2-year period

2009-11-30

Summary: Objective: To investigate the association of different types of magnetic resonance imaging (MRI)-detected medial meniscal pathology with subregional cartilage loss in the medial tibiofemoral compartment.Methods: A total of 152 women aged ≥40 years, with and without knee osteoarthritis (OA) were included in a longitudinal 24-month observational study. Spoiled gradient recalled acquisitions at steady state (SPGR) and T2-weighted fat-suppressed MRI sequences were acquired. Medial meniscal status of the anterior horn (AH), body, and posterior horn (PH) was graded at baseline: 0 (normal), 1 (intrasubstance meniscal signal changes), 2 (single tears), and 3 (complex tears/maceration). Cartilage segmentation was performed at baseline and 24-month follow-up in various tibiofemoral subregions using computation software. Multiple linear regression models were applied for the analysis with cartilage loss as the outcome. In a first model, the results were adjusted for age and body mass index (BMI). In a second model, the results were adjusted for age, BMI and medial meniscal extrusion.Results: After adjusting for age, BMI, and medial meniscal extrusion, cartilage loss in the total medial tibia (MT) (0.04mm, P=0.04) and the external medial tibia (eMT) (0.068mm, P=0.04) increased significantly for compartments with grade 3 lesions. Cartilage loss in the total central medial femoral condyle (cMF) (0.071mm, P=0.03) also increased significantly for compartments with grade 2 lesions. Cartilage loss at the eMT was significantly related to tears of the PH (0.074mm; P=0.03). Cartilage loss was not significantly increased for compartments with grade 1 lesions.Conclusion: The protective function of the meniscus appears to be preserved in the presence of intrasubstance meniscal signal changes. Prevalent single tears and meniscal maceration were found to be associated with increased cartilage loss in the same compartment, especially at the PH.

Semi-automated segmentation to assess the lateral meniscus in normal and osteoarthritic knees

2009-11-09

Summary: Objective: The goal of this study was to develop an algorithm to semi-automatically segment the meniscus in a series of magnetic resonance (MR) images to use for normal knees and those with moderate osteoarthritis (OA).Method: The segmentation method was developed then evaluated on 10 baseline MR images obtained from subjects with no evidence, symptoms, or risk factors of knee (OA), and 14 from subjects with established knee OA enrolled in the Osteoarthritis Initiative (OAI). After manually choosing a seed point within the meniscus, a threshold level was calculated through a Gaussian fit model. Under anatomical, intensity, and range constraints, a threshold operation was completed followed by conditional dilation and post-processing. The post-processing operation reevaluates the pixels included and excluded in the area surrounding the meniscus to improve accuracy. The developed method was evaluated for both normal and degenerative menisci by comparing the segmentation algorithm results with manual segmentations from five human readers.Results: The semi-automated segmentation method produces results similar to those of trained observers, with an average similarity index over 0.80 for normal participants and 0.75, 0.67, and 0.64 for participants with established knee OA with Osteoarthritis Research Society International (OARSI) joint space narrowing (JSN) scores of 0, one, and two respectively.Conclusion: The semi-automatic segmentation method produced accurate and consistent segmentations of the meniscus when compared to manual segmentations in the assessment of normal menisci in mild to moderate OA. Future studies will examine the change in volume, thickness, and intensity characteristics at different stages of OA.

Site specific osteoarthritis and the index to ring finger length ratio

B. Ferraro, F.V. Wilder, P.E. Leaverton — 2009-11-23

Summary: Objective: To quantify the relationship between the index to ring finger length ratio second digit:fourth digit(2D:4D) and radiographic osteoarthritis (OA) of the knee.Methods: Data from the Clearwater Osteoarthritis Study (COS) were analyzed. We selected a random sample of 236 subjects with knee OA (Kellgren–Lawrence scores ≥2) and compared their finger length ratio pattern with a random sample of 242 controls. Finger length measurements were recorded from digitized hand radiographs. Subjects were classified into three groups: type 1 (index finger longer than ring finger), type 2 (fingers of equal length) and type 3 (index finger shorter than ring finger). Using a case-control design, we calculated odds ratios (OR).Results: The type 3 finger pattern was significantly associated with knee OA (OR 2.59, 95% confidence interval (CI) 1.54–4.37). Women demonstrated a stronger association of visual type 3 finger pattern and knee OA (OR 4.40, 95% CI 2.62–7.38) compared to men (OR 2.59, 95% CI 1.34–5.00).Conclusions: The type 3 finger length pattern is associated, to a statistically significant degree, with OA of the knee. The type 3 finger length pattern (ring finger longer than index finger) appears to be an indicator of OA predisposition. Consideration of this pattern in clinical assessments may be an added aid as clinicians screen patients for OA risk.

Functional cartilage MRI T2 mapping: evaluating the effect of age and training on knee cartilage response to running

T.J. Mosher, Y. Liu, C.M. Torok — 2009-12-03

Summary: Objective: To characterize effects of age and physical activity level on cartilage thickness and T2 response immediately after running.Design: Institutional review board approval was obtained and all subjects provided informed consent prior to study participation. Cartilage thickness and magnetic resonance imaging (MRI) T2 values of 22 marathon runners and 15 sedentary controls were compared before and after 30min of running. Runner and control groups were stratified by age≤45 and ≥46 years. Multi-echo [(Time to Repetition (TR)/Time to Echo (TE) 1500ms/9–109ms)] MR images obtained using a 3.0T scanner were used to calculate thickness and T2 values from the central femoral and tibial cartilage. Baseline cartilage T2 values, and change in cartilage thickness and T2 values after running were compared between the four groups using one-way analysis of variance (ANOVA).Results: After running MRI T2 values decreased in superficial femoral (2ms–4ms) and tibial (1ms–3ms) cartilage along with a decrease in cartilage thickness: (femoral: 4%–8%, tibial: 0%–12%). Smaller decrease in cartilage T2 values were observed in the middle zone of cartilage, and no change was observed in the deepest layer. There was no difference cartilage deformation or T2 response to running as a function of age or level of physical activity.Conclusions: Running results in a measurable decrease in cartilage thickness and MRI T2 values of superficial cartilage consistent with greater compressibility of the superficial cartilage layer. Age and level of physical activity did not alter the T2 response to running.

An examination chair to measure internal rotation of the hip in routine settings: a validation study

S. Reichenbach, P. Jüni, E. Nüesch, F. Frey, R. Ganz, M. Leunig — 2009-10-28

Summary: Objective: To determine the performance of a newly developed examination chair as compared with the clinical standard of assessing internal rotation (IR) of the flexed hip with a goniometer.Methods: The examination chair allowed measurement of IR in a sitting position simultaneously in both hips, with hips and knees flexed 90°, lower legs hanging unsupported and a standardized load of 5kg applied to both ankles using a bilateral pulley system. Clinical assessment of IR was performed in supine position with hips and knees flexed 90° using a goniometer. Within the framework of a population-based inception cohort study, we calculated inter-observer agreement in two samples of 84 and 64 consecutive, unselected young asymptomatic males using intra-class correlation coefficients (ICC) and determined the correlation between IR assessed with examination chair and clinical assessment.Results: Inter-observer agreement was excellent for the examination chair (ICC right hip, 0.92, 95% confidence interval [CI] 0.89–0.95; ICC left hip, 0.90, 95% CI 0.86–0.94), and considerably higher than that seen with clinical assessment (ICC right hip, 0.65, 95% CI 0.49–0.77; ICC left hip, 0.69, 95% CI 0.54–0.80, P for difference in ICC between examination chair and clinical assessment ≤0.001). The average range of motion (ROM) obtained with examination chair and clinical assessment were similar (difference 1.1°, 95% CI – 0.7–2.8°, P=0.23), and the correlation was strong (Pearson's coefficient, 0.75, 95% CI 0.62–0.84).Conclusions: The use of the examination chair resulted in a precise assessment of hip IR in our population-based inception cohort study of young asymptomatic males. It was strongly correlated with standard clinical assessment of IR but was considerably more reliable.

Reliability, validity and responsiveness of the Portuguese version of the Knee injury and Osteoarthritis Outcome Score – Physical Function Short-form (KOOS-PS)

R.S. Gonçalves, J. Cabri, J.P. Pinheiro, P.L. Ferreira, J. Gil — 2009-11-26

Summary: Objective: To test the reliability, validity and responsiveness of the Portuguese version of the Knee injury and Osteoarthritis Outcome Score – Physical Function Short-form (KOOS-PS).Methods: The Portuguese full KOOS and Medical Outcomes Study – 36 item Short-Form (SF-36) questionnaires, and a form of individual characteristics of the patients were applied to 85 subjects with knee osteoarthritis (OA).Results: Cronbach's alpha coefficient was 0.89 and intraclass correlation coefficient (ICC) was 0.85, certifying that KOOS-PS reliability was acceptable. Construct validity was supported by the confirmation of the five predefined hypotheses involving expected correlations between KOOS-PS scale, KOOS subscales and SF-36 subscales. An additional predefined hypothesis was also confirmed with the subjects that need walking aids obtaining higher KOOS-PS scale scores (P=0.011). Responsiveness to 4 weeks of conventional physical therapy treatments and to a 6-week health education and exercise program was demonstrated with a standardized effect size of 0.88 and 0.50, and a standardized response mean of 1.21 and 0.73, respectively.Conclusion: The Portuguese KOOS-PS evidenced acceptable psychometric characteristics.

Cartilage degeneration in the goat knee caused by treating localized cartilage defects with metal implants

2009-11-12

Summary: Objective: The purpose of the current study was to investigate the feasibility of applying defect-size femoral implants for the treatment of localized cartilage defects in a 1-year follow-up model.Methods: In 13 goats, a medial femoral condyle defect was created in both knees. Defects were randomly treated by immediate placement of an oxidized zirconium (OxZr) (n=9) or cobalt–chromium (CoCr) implant (n=9) or left untreated (n=8). Six un-operated knee joints served as a control. Animals were sacrificed at 52 weeks. Joints were evaluated macroscopically. Cartilage quality was analyzed macroscopically and microscopically and cartilage repair of untreated defects was scored microscopically. Glycosaminoglycan (GAG) content, release and synthesis were measured in tissue and medium. Implant osseointegration was measured by automated histomorphometry.Results: Cartilage repair score of the defects was 13.3±3.0 out of 24 points (0=no repair, 24=maximal repair). Articular evaluation scores decreased (indicative of degeneration) in untreated defects and in defects treated with either implant (P<0.05). Macroscopical, microscopical and biochemical analysis showed that the presence of untreated defects and the implants caused considerable degeneration of medial tibial plateau, and to a lesser extent of the lateral compartment. Mean bone-implant contact was extensive and not different between materials (39.5±28.1% for OxZr and 42.3±31.5% for CoCr) (P=0.873).Conclusions: Considerable cartilage degeneration was induced in the articulating cartilage of the medial tibial plateau 1 year after creating an osteochondral defect in the medial femoral condyle. Treating this defect with a small metal implant, made of either OxZr or CoCr, could not prevent this degeneration. Further optimization of defect-size implants and their placement is required to make this the therapy of choice for the treatment of local cartilage defects.

Tumor risk by tissue engineering: cartilaginous differentiation of mesenchymal stem cells reduces tumor growth

2009-12-07

Summary: Objective: Implantation of autologous chondrocytes (AC) is a promising option for the treatment of cartilage defects, but problems with cell harvesting, dedifferentiation, or the donor age limit the clinical outcome. Mesenchymal stem cells (MSC) gain much interest because of their simple isolation and multipotential differentiation capacity along with their immunosuppressive properties. The latter might introduce tumor manifestation. The influence of undifferentiated and chondrogenically differentiated MSC or AC on tumor growth and metastasis formation was investigated in a murine melanoma model.Methods: Allogeneic melanoma cells and either syngeneic MSC (C3H10T1/2, transduced with enhanced green fluorescent protein gene) or AC were co-injected at a distance of 3cm into the contra lateral groins of five mice/group, and evaluated macroscopically and histologically after 4 weeks.Results: Undifferentiated MSC migrated to the tumor site and induced strong tumor growth and metastasis formation. Even avital MSC promoted tumor growth and spreading, but insignificantly without detectable MSC at the tumor site. Chondrogenically differentiated MSC did not migrate and had a significantly lower impact on tumor growth and spreading; AC had no measurable influence on melanoma cells.Conclusions: Our data suggest that differentiation of MSC reduces MSC-dependent promotion of latent tumors and that native AC do not introduce any increased risk of tumor growth. The question of how far MSC should be differentiated prior to clinical application should be addressed in further studies.

Collagen fibril disruption occurs early in primary guinea pig knee osteoarthritis

J.L. Huebner, J.M. Williams, M. Deberg, Y. Henrotin, V.B. Kraus — 2009-10-26

Summary: Objective: A major barrier inhibiting the discovery of structural modifying agents for osteoarthritis (OA) is an incomplete understanding of early disease events. Herein, we investigated the time course of collagen II cleavage and fibril disruption in the well-validated Hartley guinea pig model of spontaneous OA of the knee.Methods: Knee joints of 46 male Hartley guinea pigs were analyzed at 3 weeks, 2, 4, 7, 10, 12, and 18 months of age for histological severity of OA, cartilage collagen fibril disruption by semi-quantitative polarized light microscopy, and expression of type II collagen degradation biomarkers, 9A4 and Coll2-1, by immunohistochemistry. In addition, serum biomarkers specific for collagen II degradation, CTX-II, C2C, and Coll2-1 were quantified.Results: Collagen fibril disruption and expression of the collagenase-generated cleavage neoepitope, 9A4, were observed as early as 2 months of age, despite the appearance of histological OA at 4 months of age. Only serum Coll2-1 increased coincident with the early disruption of the collagen fibril between 3 weeks and 7 months, in contrast to serum C2C, which did not change significantly or correlate with histological severity. Inversely, CTX-II declined dramatically from 3 weeks to 4 months and remaining low thereafter, coincident with growth plate turnover.Conclusions: Collagenase cleavage and disruption of the type II collagen network are early OA disease events in this model, preceding histological evidence of proteoglycan loss. The markedly different serum profiles of collagen II-related biomarkers during the early stages of disease development suggest compartmental segregation and temporal regulation of collagen degrading enzymes.

Maturation of collagen fibril network structure in tibial and femoral cartilage of rabbits

P. Julkunen, J. Iivarinen, P.A. Brama, J. Arokoski, J.S. Jurvelin, H.J. Helminen — 2009-11-30

Summary: Objective: The structure and composition of articular cartilage change during development and growth, as well as in response to varying loading conditions. These changes modulate the functional properties of cartilage. We studied maturation-related changes in the collagen network organization of cartilage as a function of tissue depth.Design: Articular cartilage from the tibial medial plateaus and femoral medial condyles of female New Zealand white rabbits was collected from six age-groups: 4 weeks (n=30), 6 weeks (n=30), 3 months (n=24), 6 months (n=24), 9 months (n=27) and 18 months (n=19). Collagen fibril orientation, parallelism (anisotropy) and optical retardation were analyzed with polarized light microscopy. Differences in the development of depth-wise collagen organization in consecutive age-groups and the two joint locations were compared statistically.Results: The collagen fibril network of articular cartilage undergoes significant changes during maturation. The most prominent changes in collagen architecture, as assessed by orientation, parallelism and retardation were noticed between the ages of 4 and 6 weeks in tibial cartilage and between 6 weeks and 3 months in femoral cartilage, i.e., orientation became more perpendicular-to-surface, and parallelism and retardation increased with changes being most prominent in the deep zone. At the age of 6 weeks, tibial cartilage had a more perpendicular-to-surface orientation in the middle and deep zones than femoral cartilage (P<0.001) and higher parallelism throughout the tissue depth (P<0.001), while femoral cartilage exhibited more parallel-to-surface orientation (P<0.01) above the deep zone after maturation. Optical retardation of collagen was higher in tibial than in femoral cartilage at the ages of 4 and 6 weeks (P<0.001), while at older ages, retardation below the superficial zone in the femoral cartilage became higher than in the tibial cartilage.Conclusions: During maturation, there is a significant modulation of collagen organization in articular cartilage which occurs earlier in tibial than in femoral cartilage, and is most pronounced in the deep zone.

Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration

2009-10-23

Summary: Objective: Regardless of recent progress in the elucidation of intervertebral disc (IVD) degeneration, the basic molecular characteristics that define a healthy human IVD are largely unknown. Although work in different animal species revealed distinct molecules that might be used as characteristic markers for IVD or specifically nucleus pulposus (NP) cells, the validity of these markers for characterization of human IVD cells remains unknown.Design: Eleven potential marker molecules were characterized with respect to their occurrence in human IVD cells. Gene expression levels of NP were compared with annulus fibrosus (AF) and articular cartilage (AC) cells, and potential correlations with aging were assessed.Results: Higher mRNA levels of cytokeratin-19 (KRT19) and of neural cell adhesion molecule-1 were noted in NP compared to AF and AC cells. Compared to NP cytokeratin-18 expression was lower in AC, and alpha-2-macroglobulin and desmocollin-2 lower in AF. Cartilage oligomeric matrix protein (COMP) and glypican-3 expression was higher in AF, while COMP, matrix gla protein (MGP) and pleiotrophin expression was higher in AC cells. Furthermore, an age-related decrease in KRT19 and increase in MGP expression were observed in NP cells. The age-dependent expression pattern of KRT19 was confirmed by immunohistochemistry, showing the most prominent KRT19 immunoreaction in the notochordal-like cells in juvenile NP, whereas MGP immunoreactivity was not restricted to NP cells and was found in all age groups.Conclusions: The gene expression of KRT19 has the potential to characterize human NP cells, whereas MGP cannot serve as a characteristic marker. KRT19 protein expression was only detected in NP cells of donors younger than 54 years.

Mitochondrial DNA damage is involved in apoptosis caused by pro-inflammatory cytokines in human OA chondrocytes

J. Kim, M. Xu, R. Xo, A. Mates, G.L. Wilson, A.W. Pearsall, V. Grishko — 2009-10-26

Summary: Objective: Pro-inflammatory cytokines play a pivotal role in cartilage destruction during the progression of osteoarthritis (OA). Additionally, these cytokines are capable to generate reactive oxygen and nitrogen species within chondrocytes. Mitochondrion is a prime target of oxidative damage and an important player in aging and degenerative processes. The purpose of the present study was to investigate whether these cytokines will alter the mitochondrial DNA (mtDNA) integrity and mitochondrial function in both normal and osteoarthritic human chondrocytes.Design: Primary normal and osteoarthritic human chondrocyte cultures were exposed to various concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) for different time. Following exposure, chondrocytes were evaluated for mitochondrial DNA damage, ATP production, changes in mitochondrial transcription, and apoptosis. Adenoviral vectors were used to deliver DNA repair enzyme hOGG1 to mitochondria.Results: Pro-inflammatory cytokines IL-1β and TNF-α disturb mitochondrial function in human chondrocytes by inducing mitochondrial DNA damage, decreasing energy production and mitochondrial transcription, which correlated with the induction of apoptosis. Increased NO production was the key factor responsible for accumulation of mtDNA damage after cytokine exposure. Mitochondrial superoxide production was also enhanced following pro-inflammatory cytokine exposure. OA chondrocyte mitochondria were more susceptible to damage induced by pro-inflammatory cytokines then mitochondria from normal chondrocytes. Protection of human chondrocytes from mtDNA damage by the mitochondria-targeted DNA repair enzyme hOGG1 rescued mtDNA integrity, preserved ATP levels, reestablished mitochondrial transcription, and significantly diminished apoptosis following IL-1β and TNF-α exposure.Conclusion: Mitochondrion is an important target in pro-inflammatory cytokine toxicity, maintaining of mitochondrial DNA integrity is necessary to prevent chondrocytes from apoptosis induced by IL-1β and TNF-α.

Osmolarity effects on bovine articular chondrocytes during three-dimensional culture in alginate beads

X. Xu, J.P.G. Urban, U.K. Tirlapur, Z. Cui — 2009-11-04

Summary: Objective: With the development of engineered cartilage, the determination of the appropriate culture conditions is vital in order to maximize extracellular matrix synthesis. As osmolarity could affect the fate of chondrocytes, the purpose of this study was to determine the effects of osmolarity on chondrocytes during relatively long-term culture.Design: Bovine articular chondrocytes were cultured in alginate beads in a biocarbonate free system at 280, 380 and 550mOsm at pH 7.4 for up to 12 days, respectively. Cell volume, intracellular pH (pHi), cell number, glucosaminoglycan (GAG) and collagen retention were measured at day 5 and 12. Cell viability and volume were monitored over the 12 days of culture.Results: By day 5 and 12, compared to the cell volume at 380mOsm, around 20% (P<0.01) swelling and 15% (P<0.05) shrinkage were observed when the cells were cultured at 280 and 550mOsm. The pHi over the 12 days of culture varied with osmolarity of the culture medium. In comparison with fresh cells, pHi became slightly more acidic by 0.15pH units at 280mOsm at day 5. However, by day 12, an alkalization of pHi, by 0.2pH units, was noted. A higher proliferation rate was seen at 280mOsm than at other osmolarities while less GAG was produced.Conclusions: Chronic exposure to anisotonic conditions results in cell swelling at 280mOsm and shrinkage at 550mOsm. The osmolarity of 280mOsm appears to encourage proliferation of chondrocytes, but inhibits matrix production.

Proteome characterization of human articular chondrocytes leads to novel insights in the function of small heat-shock proteins in chondrocyte homeostasis

2009-11-11

Summary: Objective: In recent years, studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chondrocyte proteome remains limited. This study aimed to further explore the proteome expressed by human knee chondrocytes, and to study the functional aspects of heat-shock protein 27 (HSP27), a protein related to the previously described αBcrystallin, in chondrocyte biology.Methods: Chondrocytes isolated from human knee articular cartilage were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydrophobicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line two-dimensional (2-D) nanoliquid chromatography (nanoLC)-system coupled to a quadrupole time-of-flight (Qq-TOF) mass spectrometer. Differential expression analysis of HSP27 was performed by Western Blotting and quantitative polymerase chain reaction (QPCR). The effects of HSP27 on chondrocyte biology were explored by suppression of HSP27 expression induced by RNA interference (RNAi).Results: In this study, we identified proteins with unknown functions together with membrane proteins, transcription factors and other low abundant proteins, which have not yet been described in chondrocytes. Based on previous knowledge on the related protein αBcrystallin, we selected HSP27 from the chondrocyte proteome database. Differential expression analysis revealed a decreased expression of HSP27 in Osteoarthritic (OA) chondrocytes. RNAi experiments revealed that HSP27 is involved in interleukin-1β (IL-1β) induced IL-6 secretion.Conclusion: These findings highlight that small HSPs, especially HSP27, play a prominent role in the maintenance of human articular chondrocyte homeostasis.

Hyaluronan oligosaccharide treatment of chondrocytes stimulates expression of both HAS-2 and MMP-3, but by different signaling pathways

I. Schmitz, W. Ariyoshi, N. Takahashi, C.B. Knudson, W. Knudson — 2009-11-06

Summary: Objective: Small hyaluronan (HA) oligosaccharides displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and includes stimulation of genes involved in matrix degradation such as matrix metalloproteinases (MMPs) as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2 (HAS-2). The objective of this study was to determine whether stimulation of HAS-2 and MMP-3 by HA oligosaccharides is due to the activation of a single, cascading pathway or multiple signaling pathways.Method: Bovine articular chondrocytes were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in HA were monitored by real time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HAS-2 mRNA, HA ELISA and HA accumulation at the cell surface. A 1900 base pair sequence containing the proximal promoter of HAS-2 was inserted into a luciferase reporter construct, transfected into human immortalized chondrocytes and assayed in a similar fashion.Results: While our previous studies demonstrated that HA oligosaccharides stimulate MMP-13 activity via activation of p38 MAP kinase and NF-κB, inhibitors of these pathways did not affect the stimulation of HAS-2 mRNA expression. However, inhibiting the phosphatidylinositol-3-kinase pathway blocked HA oligosaccharide-mediated stimulation of HAS-2 yet had no effect on MMP-3. Wortmannin and LY294002 also blocked HA oligosaccharide-induced serine and threonine Akt phosphorylation. Treatment of transfected immortalized chondrocytes with HA oligosaccharides resulted in stimulation of HAS-2 mRNA, activation of Akt and enhanced luciferase activity—activity that was blocked by inhibitors of Akt phosphorylation.Conclusions: Changes in chondrocyte–matrix interactions by HA oligosaccharides induce altered matrix metabolism by the activation of least two distinct signaling pathways.

10mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin

D.R. McCulloch, J.D. Wylie, J.-M. Longpre, R. Leduc, S.S. Apte — 2009-11-23

Summary: Objective: 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.Design: 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.Results: 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, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.Conclusions: 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.

Cartilage shear dynamics during tibio-femoral articulation: effect of acute joint injury and tribosupplementation on synovial fluid lubrication

B.L. Wong, S.H. Chris Kim, J.M. Antonacci, C. Wayne McIlwraith, R.L. Sah — 2009-12-23

Summary: Objective: To determine the effects of acute injury and tribosupplementation by hyaluronan (HA) on synovial fluid (SF) modulation of cartilage shear during tibio-femoral articulation.Methods: Human osteochondral blocks from the lateral femoral condyle (LFC) and tibial plateau (LTP) were apposed, compressed 13%, and subjected to sliding under video microscopy. Tests were conducted with equine SF from normal joints (NL-SF), SF from acutely injured joints (AI-SF), and AI-SF to which HA was added (AI-SF+HA). Local and overall shear strain (Exz) and the lateral displacement (Δx) at which Exz reached 50% of peak values (Δx1/2) were determined.Results: During articulation, LFC and LTP cartilage Exz increased with Δx and peaked when surfaces slid, with peak Exz being maintained during sliding. With AI-SF as lubricant, surface and overall Δx1/2 were ∼40% and ∼20% higher, respectively, than values with NL-SF and AI-SF+HA as lubricant. Also, peak Exz was markedly higher with AI-SF as lubricant than with NL-SF as lubricant, both near the surface (∼80%) and overall (50–200%). Following HA supplementation to AI-SF, Exz was reduced from values with AI-SF alone by 30–50% near the surface and 20–30% overall. Magnitudes of surface and overall Exz were markedly (∼50 to 80%) higher in LTP cartilage than LFC cartilage for all lubricants.Conclusion: Acute injury impairs SF function, elevating cartilage Exz markedly during tibio-femoral articulation; such elevated Exz may contribute to post-injury associated cartilage degeneration. Since HA partially restores the function of AI-SF, as indicated by Exz, tribosupplements may be beneficial in modulating normal cartilage homeostasis.

Erratum to “Risk factors of knee osteoarthritis – excellent evidence but little has been done” [Osteoarthritis and Cartilage 18 (2010) 1–2]

W. Zhang — 2010-02-03

The full citation for reference 7 should be as follows: Blagojevic M, Jinks C, Jordan K. Risk factors for onset of osteoarthritis of the knee in oldest adults, a systematic review and meta-analysis. Osteoarthritis Cartilage 2009;18(1):24–33.