Alternative and complementary therapies in osteoarthritis and cartilage repair.

Osteoarthritis (OA) is the most common joint condition and, with a burgeoning ageing population, is due to increase in prevalence. Beyond conventional medical and surgical interventions, there are an increasing number of 'alternative' therapies. These alternative therapies may have a limited evidence base and, for this reason, are often only afforded brief reference (or completely excluded) from current OA guidelines. Thus, the aim of this review was to synthesize the current evidence regarding autologous chondrocyte implantation (ACI), mesenchymal stem cell (MSC) therapy, platelet-rich plasma (PRP), vitamin D and other alternative therapies. The majority of studies were in knee OA or chondral defects. Matrix-assisted ACI has demonstrated exceedingly limited, symptomatic improvements in the treatment of cartilage defects of the knee and is not supported for the treatment of knee OA. There is some evidence to suggest symptomatic improvement with MSC injection in knee OA, with the suggestion of minimal structural improvement demonstrated on MRI and there are positive signals that PRP may also lead to symptomatic improvement, though variation in preparation makes inter-study comparison difficult. There is variability in findings with vitamin D supplementation in OA, and the only recommendation which can be made, at this time, is for replacement when vitamin D is deplete. Other alternative therapies reviewed have some evidence (though from small, poor-quality studies) to support improvement in symptoms and again there is often a wide variation in dosage and regimens. For all these therapeutic modalities, although controlled studies have been undertaken to evaluate effectiveness in OA, these have often been of small size, limited statistical power, uncertain blindness and using various methodologies. These deficiencies must leave the question as to whether they have been validated as effective therapies in OA (or chondral defects). The conclusions of this review are that all alternative interventions definitely require clinical trials with robust methodology, to assess their efficacy and safety in the treatment of OA beyond contextual and placebo effects.

Meniscal extrusion and bone marrow lesions are associated with incident and progressive knee osteoarthritis.

OBJECTIVE: Whether meniscal extrusion and bone marrow lesions (BMLs) are independently associated with the risk of knee osteoarthritis (OA) is unknown. METHODS: Data was extracted from the Osteoarthritis Initiative (OAI) cohort. Participants were grouped according to the absence (Kellgren-Lawrence (KL) grade ≤ 1, n = 2120) or presence (KL ≥ 2, n = 2249) of radiographic OA (ROA). Baseline meniscal extrusion and tibial BMLs were assessed. Tibial plateau cartilage volume was assessed at baseline and 72 months, while radiographic disease was assessed at baseline and 48 months. Total knee replacement (TKR) was assessed at 72 months. RESULTS: In those with ROA, the presence of a baseline meniscal extrusion (independent of BMLs) was associated with accelerated cartilage volume loss (medial tibia: -2.1%/annum vs -1.5%; lateral: -2.6%/annum vs -1.6%; both P < 0.001), progressive ROA and TKR (Odds ratio (OR) range 1.4-1.8; 95% CI range 1.1-2.9). The presence of a baseline BML was associated with accelerated cartilage volume loss (medial tibia: -2.1%/annum vs -1.6%; lateral: -1.9%/annum vs -1.6%; P ≤ 0.02), progressive ROA and joint replacement (OR range 1.5-2.4; 95% CI range 1.1-3.4). In those with no ROA, a baseline medial meniscal extrusion was associated with accelerated cartilage volume loss (medial tibia: -2.1%/annum vs -1.2%, P < 0.001), and a baseline medial BML with incident ROA (OR 1.7, 95% CI 1.1 to 2.9). CONCLUSIONS: The presence of baseline meniscal extrusion and BMLs are associated with incident and progressive knee of each other (OA) and represent important structural targets for the treatment and prevention of knee OA.

What is the predictive value of MRI for the occurrence of knee replacement surgery in knee osteoarthritis?

Knee osteoarthritis is associated with structural changes in the joint. Despite its many drawbacks, radiography is the current standard for evaluating joint structure in trials of potential disease-modifying osteoarthritis drugs. MRI is a non-invasive alternative that provides comprehensive imaging of the whole joint. Frequently used MRI measurements in knee osteoarthritis are cartilage volume and thickness; others include synovitis, synovial fluid effusions, bone marrow lesions (BML) and meniscal damage. Joint replacement is considered a clinically relevant outcome in knee osteoarthritis; however, its utility in clinical trials is limited. An alternative is virtual knee replacement on the basis of symptoms and structural damage. MRI may prove to be a good alternative to radiography in definitions of knee replacement. One of the MRI parameters that predicts knee replacement is medial compartment cartilage volume/thickness, which correlates with radiographic joint space width, is sensitive to change, and predicts outcomes in a continuous manner. Other MRI parameters include BML and meniscal lesions. MRI appears to be a viable alternative to radiography for the evaluation of structural changes in knee osteoarthritis and prediction of joint replacement.

The BMP antagonists follistatin and gremlin in normal and early osteoarthritic cartilage: an immunohistochemical study.

OBJECTIVE: Bone morphogenic protein (BMP) activities are controlled in part by antagonists. In human osteoarthritic (OA) cartilage, the BMP antagonists follistatin and gremlin are increased but differentially regulated. Using the OA dog model, we determined if these BMP antagonists were produced at different stages during the disease process by comparing their in situ temporal and spatial distribution. METHODS: Dogs were sacrificed at 4, 8, 10 and 12 weeks after surgery; normal dogs served as control. Cartilage was removed, differentiating fibrillated and non-fibrillated areas. Immunohistochemistry and morphometric analyses were performed for follistatin, gremlin, BMP-2/4 and IL-1beta. Growth factor-induced gremlin expression was assessed in dog chondrocytes. RESULTS: Follistatin and gremlin production were very low in normal cartilage. Gremlin was significantly up-regulated in both non-fibrillated and fibrillated areas at 4 weeks, and only slightly increased with disease progression. Follistatin showed a time-dependent increased level in the non-fibrillated areas with significance reached at 8-12 weeks; in the fibrillated areas significant high levels were seen as early as 4 weeks. In the whole cartilage, follistatin and IL-1beta temporal production showed similar patterns; this was also true for gremlin and BMP-2/4, though BMP-2/4 production was already high in the normal dogs. Interestingly, data revealed that basic fibroblast growth factor (bFGF) could be another factor increasing gremlin expression early in the disease process. Comparison between superficial and deep zones revealed similar patterns for follistatin and IL-1beta in the superficial zone only; gremlin and BMP-2/4 had similar patterns in both zones. CONCLUSION: Data show, for the first time, different spatial and temporal production of gremlin and follistatin in cartilage during OA progression. These findings may reflect different roles for each antagonist in this disease.

Hsp90{beta} and p130(cas): novel regulatory factors of MMP-13 expression in human osteoarthritic chondrocytes.

BACKGROUND: Human osteoarthritic (OA) chondrocytes were previously classified into L (low)- and H (high)-OA according to matrix metalloproteinase-13 (MMP-13) basal levels and interleukin 1beta (IL1beta) inducibility. In H-OA chondrocytes, the regulatory proteins p130(cas) and nuclear matrix protein 4 (NMP4) acting on the MMP-13 promoter were identified. OBJECTIVE: To identify regulators of MMP-13 expression/production in human L-OA chondrocytes, to determine their effect on the expression of other MMPs and the effect of IL1beta on these molecules. METHODS: The identification of the L-OA chondrocyte proteins interacting specifically with the AGRE site of the MMP-13 promoter was performed by mass spectrometry. Heat shock protein 90beta (Hsp90beta), p130(cas) and NMP4 small interfering RNAs (siRNAs) were transfected into L-OA chondrocytes and incubated with or without IL1beta. Gene expression was determined by real-time PCR, MMP-1 and MMP-13 production by ELISA, and signalling pathway activation by western blotting and ELISA. RESULTS: Hsp90beta was identified as a protein of the L-OA/AGRE-specific complex. Silencing p130(cas) and Hsp90beta significantly increased MMP-13 expression (about four- and twofold, respectively) and production. sip130(cas) affected to a lesser extent MMP-1 expression (twofold) and production. siNMP4 showed no effect. Expression of MMP-2, -3, -9 and -14 was unaffected. Silencing both Hsp90beta and p130(cas) had a significant additive effect on MMP-13, but not on MMP-1 expression, the level of which was similar to that with sip130(cas) alone. IL1beta decreased p130(cas) and Hsp90beta expression/production, indicating another pathway by which this cytokine upregulates MMP expression. The IL1beta-triggered signalling pathways responsible for MMP upregulation were unaffected in the silenced cells. CONCLUSION: This study illustrates the complex regulation of MMP-13 by showing the inhibitory effect of the two cytoplasmic molecules, p130(cas) and Hsp90beta, in L-OA chondrocytes.

The differential expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in human osteoarthritic subchondral bone osteoblasts is an indicator of the metabolic state of these disease cells.

OBJECTIVE: We previously reported that human OA subchondral bone osteoblasts could be discriminated into two subpopulations identified by their levels of endogenous production (low [L] or high [H]) of PGE(2). Here, we investigated the OPG and RANKL expression levels, the histologic analysis of the subchondral bone as well as the osteoclast differentiation effect of osteoblasts on normal and both OA subpopulations (L and H), and further examined on the L OA osteoblasts the modulation of bone remodelling factors on the OPG and RANKL levels, as well as on the resorption activity. METHODS: Gene expression was determined using real-time PCR, PGE2 and OPG levels by specific ELISA, and membranous RANKL by flow cytometry. Histological observation of the subchondral bone was performed on human knee specimens. Osteoclast differentiation and formation was assayed by using the pre-osteoclastic cell line RAW 264.7. OPG and RANKL modulation on L OA osteoblasts was monitored following treatment with osteotropic factors, and the resorption activity was studied by the co-culture of differentiated PBMC/osteoblasts. RESULTS: Human OA subchondral bone osteoblasts expressed less OPG than normal. Compared to normal, RANKL gene expression levels were increased in L OA and decreased in H OA cells. The OPG/RANKL mRNA ratio was significantly diminished in L OA compared to normal or H OA (p<0.02, p<0.03), and markedly increased in H OA compared to normal. Inhibition of endogenous PGE(2) levels by indomethacin markedly decreased the ratio of OPG/RANKL on the H OA. In contrast to H OA osteoblasts, L OA cells induced a significantly higher level of osteoclast differentiation and formation (p<0.05). Histological analysis showed a reduced subchondral bone on the L OA and an increased bone mass on the H OA compared to normal. Treatment of L OA osteoblasts with osteotropic factors revealed that the OPG/RANKL mRNA expression ratio was significantly reduced by vitamin D(3) and significantly increased by TNF-alpha, PTH and PGE(2), while IL-1Beta demonstrated no effect. OPG protein levels showed similar profiles. No true effect was noted on membranous RANKL upon treatment with IL-1Beta, PGE(2) and PTH, but a significant increase was observed with vitamin D3 and TNF-alpha. The resorption activity of the L OA cells was significantly inhibited by all treatments except IL-1Beta, with maximum effect observed with vitamin D(3) and PGE(2). CONCLUSION: OPG and RANKL levels, and consequently the OPG/RANKL ratio, differed according to human OA subchondral bone osteoblast classification; it is decreased in L and increased in H OA. These findings, in addition to those showing that L OA osteoblasts have a reduced subchondral bone mass and induce a higher level of osteoclast differentiation, strongly suggest that the metabolic state of the L OA osteoblasts favours bone resorption.

Temporal assessment of bone marrow lesions on magnetic resonance imaging in a canine model of knee osteoarthritis: impact of sequence selection.

OBJECTIVE: To assess the evolution of bone marrow lesions (BMLs) in a canine model of knee osteoarthritis (OA) using three different magnetic resonance imaging (MRI) sequences. DESIGN: Three MRI sequences [coronal, T1-weighted three-dimensional fast gradient recalled echo (T1-GRE), sagittal fat-suppressed 3D spoiled gradient echo at a steady state (SPGR), and sagittal T2-weighted fast spin echo with fat saturation (T2-FS)] were performed at baseline, and at week 4, 8 and 26 in five dogs following transection of the anterior cruciate ligament. The same reader scored (0-3) subchondral BMLs twice, in blinded conditions, according to their extent in nine joint subregions, for all imaging sessions, and independently on the three MRI sequences. Correlation coefficients and Bland-Altman plots evaluated intra-reader repeatability. Readings scores were averaged and the nine subregions were summed to generate global BML scores. RESULTS: BMLs were most prevalent in the central and medial portions of the tibial plateau. Intra-reader repeatability was good to excellent for each sequence (r(s)=0.87-0.97; P<0.001). Maximal intra-reader variability (24%) was reached on T2-FS and was associated to higher scores (P<0.05). Global BML scores increased similarly on all three sequences until week 8 (P<0.05). At week 26, score on T2-FS was decreased, being lower when compared to T1-GRE and SPGR (P<0.05). CONCLUSION: In this canine OA model, the extent of BMLs varies in time on different MRI sequences. Until the complex nature of these lesions is fully resolved, it is suggested that to accurately assess the size and extent of BMLs, a combination of different sequences should be used.

Histone deacetylase inhibitors suppress interleukin-1beta-induced nitric oxide and prostaglandin E2 production in human chondrocytes.

OBJECTIVE: Overproduction of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) plays an important role in the pathogenesis of osteoarthritis (OA). In the present study, we determined the effect of trichostatin A (TSA) and butyric acid (BA), two histone deacetylase (HDAC) inhibitors, on NO and PGE(2) synthesis, inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 expression, and nuclear factor (NF)-kappaB DNA-binding activity, in interleukin-1beta (IL-1)-stimulated human OA chondrocytes, and on IL-1-induced proteoglycan degradation in cartilage explants. METHODS: Chondrocytes were stimulated with IL-1 in the absence or presence of increasing concentrations of TSA or BA. The production of NO and PGE(2) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of iNOS and COX-2 proteins and mRNAs was evaluated using Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Proteoglycan degradation was measured with dimethymethylene blue assay. Electrophoretic mobility shift assay (EMSA) was utilized to analyze the DNA-binding activity of NF-kappaB. RESULTS: HDAC inhibition with TSA or BA resulted in a dose-dependent inhibition of IL-1-induced NO and PGE(2) production. IL-17- and tumor necrosis factor-alpha (TNF-alpha)-induced NO and PGE(2) production was also inhibited by TSA and BA. This inhibition correlated with the suppression of iNOS and COX-2 protein and mRNA expression. TSA and BA also prevented IL-1-induced proteoglycan release from cartilage explants. Finally, we demonstrate that the DNA-binding activity of NF-kappaB, was induced by IL-1, but was not affected by treatment with HDAC inhibitors. CONCLUSIONS: These data indicate that HDAC inhibitors suppressed IL-1-induced NO and PGE(2) synthesis, iNOS and COX-2 expression, as well as proteoglycan degradation. The suppressive effect of HDAC inhibitors is not due to impaired DNA-binding activity of NF-kappaB. These findings also suggest that HDAC inhibitors may be of potential therapeutic value in the treatment of OA.

Correlation between bone lesion changes and cartilage volume loss in patients with osteoarthritis of the knee as assessed by quantitative magnetic resonance imaging over a 24-month period.

OBJECTIVE: To evaluate in patients with knee osteoarthritis (OA) the size changes in bone oedema and cysts over 24 months, and to contrast these changes with cartilage volume loss using quantitative magnetic resonance imaging. METHODS: 107 patients with knee OA, selected from a large trial evaluating the effect of a bisphosphonate, were analysed by magnetic resonance imaging at baseline and 24 months. Assessments of subchondral bone oedema and cysts, and cartilage volume were done. RESULTS: At baseline, 86 patients showed the presence of at least one type of bone lesion: 71 oedema, 61 cysts and 51 both. At 24 months, although not statistically significant, the oedema total size change increased by 2.09 (SD 15.03) mm, and the cyst by 1.09 (8.13) mm; mean size change for the oedema was +0.38 (2.18) mm and -0.10 (4.36) mm for the cyst. When analysed according to subregions, an increase was found for the cyst size in the trochlea (+0.67 (2.74) mm, p = 0.02) and in the lateral tibial plateau (+0.15 (0.83) mm, p = 0.09), and for the oedema size in the medial tibial plateau (+1.73 (8.11) mm, p = 0.05). At 24 months, significant correlations were seen between the loss of cartilage volume and oedema size change in the medial condyle (-0.40, p = 0.0001) and the medial tibial plateau (-0.23, p = 0.03), and the changes in cyst size in the medial condyle (-0.29, p = 0.01). A multivariate analysis showed that the oedema size change was strongly and independently associated with medial cartilage volume loss (-0.31, p = 0.0004). CONCLUSION: These data demonstrate that bone lesions are prevalent in knee OA. The correlation of the oedema and cyst size increase in the medial compartment over time with a greater loss of cartilage volume in this area underlines the importance of subchondral bone lesions in OA pathophysiology.

Two-year prospective longitudinal study exploring the factors associated with change in femoral cartilage volume in a cohort largely without knee radiographic osteoarthritis.

OBJECTIVE: To identify factors associated with change in femoral cartilage volume over 2 years in a cohort largely without knee radiographic osteoarthritis. METHODS: A total of 252 subjects (mean 45 years, range 28-60) were used for this study. T1-weighted fat saturation magnetic resonance imaging was performed at baseline and approximately 2 years later. Knee femoral condyle cartilage volume, femoral cartilage defect (0-4 scale) and tibial bone size were determined. RESULTS: The total femoral cartilage volume loss was 6.3% for the 2.3-year period. Factors associated with this annual change were female gender (females vs males: -1.69%, P<0.01), age (over vs under 40 years: -0.96%, P=0.01), smoking (beta: -0.04% per pack-years, P<0.01), as well as lower limb muscle strength (r: +0.32, P<0.01) and its change (beta: +0.34% per quartile, P<0.05). Structural factors associated with change included baseline femoral cartilage volume (beta: -0.36% per ml, P<0.01), femoral cartilage defects (beta: +1.07% per grade, P<0.01), tibial bone area (beta: +0.13% per cm(2), P<0.05), lateral osteophytes (beta: -1.91% per grade, P<0.01) and change in femoral cartilage defects (beta: -0.8% per grade, P<0.001). CONCLUSIONS: This study provides evidence confirming that significant risk factors are associated with femoral cartilage loss and these include gender (female), age, smoking, and severity of lower limb muscle weakness. It also supports the hypothesis that femoral cartilage swelling reflected by an increased baseline cartilage volume could be a predictor of disease progression. Our findings also provide interesting clues to implement preventive measures that can possibly prevent or reduce knee cartilage loss.

Magnetic resonance imaging can accurately assess the long-term progression of knee structural changes in experimental dog osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) structural changes take place over decades in humans. MRI can provide precise and reliable information on the joint structure and changes over time. In this study, we investigated the reliability of quantitative MRI in assessing knee OA structural changes in the experimental anterior cruciate ligament (ACL) dog model of OA. METHODS: OA was surgically induced by transection of the ACL of the right knee in five dogs. High resolution three dimensional MRI using a 1.5 T magnet was performed at baseline, 4, 8 and 26 weeks post surgery. Cartilage volume/thickness, cartilage defects, trochlear osteophyte formation and subchondral bone lesion (hypersignal) were assessed on MRI images. Animals were killed 26 weeks post surgery and macroscopic evaluation was performed. RESULTS: There was a progressive and significant increase over time in the loss of knee cartilage volume, the cartilage defect and subchondral bone hypersignal. The trochlear osteophyte size also progressed over time. The greatest cartilage loss at 26 weeks was found on the tibial plateaus and in the medial compartment. There was a highly significant correlation between total knee cartilage volume loss or defect and subchondral bone hypersignal, and also a good correlation between the macroscopic and the MRI findings. CONCLUSION: This study demonstrated that MRI is a useful technology to provide a non-invasive and reliable assessment of the joint structural changes during the development of OA in the ACL dog model. The combination of this OA model with MRI evaluation provides a promising tool for the evaluation of new disease-modifying osteoarthritis drugs (DMOADs).

Meniscal tear and increased tibial plateau bone area in healthy post-menopausal women.

OBJECTIVE: Meniscal tears detected using magnetic resonance imaging (MRI) have been identified as a risk factor for the development and progression of Osteoarthritis, however the prevalence and significance of meniscal tears in healthy, asymptomatic adults remains to be studied. We investigated the prevalence of meniscal tears in a healthy pain free population of post-menopausal women and whether meniscal tears in this population are associated with changes in cartilage volume and defects and tibial plateau bone area over 2 years. METHODS: Fifty-seven post-menopausal women underwent MRI of their dominant knee at baseline line and approximately 2 years later to assess meniscal tears, cartilage volume, cartilage defects and tibial plateau bone area. RESULTS: Forty-six percent of women had a meniscal tear in either the medial and/or lateral compartment. Women who had a tear were older (P=0.01) and had more lateral cartilage defects (P=0.02). Medial meniscal tear was associated with 103 mm(2) greater tibial plateau bone area within the medial [95% confidence of interval (CI) 6.2, 200.3; P=0.04] and a lateral meniscal tear with a 120 mm(2) greater area within the lateral compartment (95% CI 45.5, 195.2; P=0.002). CONCLUSION: This study demonstrates that meniscal tears are common in asymptomatic post-menopausal women and that they become more common with age. Meniscal tears were also associated with greater tibial plateau bone area but not cartilage volume, providing support to the hypothesis that tibial plateau bone changes occur before significant pathological changes in cartilage. Whether increased tibial plateau bone area predisposes to an increased risk of degenerative meniscal tears or whether it is a consequence of altered biomechanical forces in relation to meniscal tear will need to be determined.

Differential regulation of the bone morphogenic protein antagonist chordin in human normal and osteoarthritic chondrocytes.

OBJECTIVE: To investigate the distribution of the bone morphogenic protein (BMP) antagonist chordin in normal and osteoarthritic cartilage and synovial membranes, and its regulation in chondrocytes and synovial fibroblasts by inflammatory and growth factors. METHODS: Localisation of chordin in tissues was undertaken by immunohistochemistry and gene regulation was determined by real time polymerase chain reaction. RESULTS: In normal cartilage, chordin was found at low levels (mean (SD), 7.6 (1.3)%), mainly in the very superficial layers. In osteoarthritis, chordin was also found in the superficial layers (8.9 (1.1)%), though at a significantly higher level (24.7 (1.5)%) in the last two thirds of the cartilage. In contrast to normal cells, chordin mRNA and protein levels were significantly downregulated (p<0.01) in osteoarthritic chondrocytes by all the growth factors tested. Interferon gamma stimulated chordin expression in normal but not in osteoarthritic chondrocytes (p<0.0002), while interleukin 1 beta and tumour necrosis factor alpha did not affect the expression level. However, no difference was found in either the distribution or regulation of chordin in normal and osteoarthritic synovial membranes or synovial fibroblasts. CONCLUSIONS: The differential distribution and regulation of chordin in normal and osteoarthritic cartilage and chondrocytes suggests an involvement of this antagonist in the osteoarthritic process.

PD-0200347, an alpha2delta ligand of the voltage gated calcium channel, inhibits in vivo activation of the Erk1/2 pathway in osteoarthritic chondrocytes: a PKCalpha dependent effect.

OBJECTIVE: To explore the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage gated Ca(2+) channels, on cell signalling in osteoarthritic (OA) chondrocytes from an experimental dog model, and examine the effect of PD-0200347 on the major signalling pathways involved in OA cartilage degradation. METHODS: OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were divided into three groups and treated orally with (a) placebo; (b) 15 mg/kg/day PD-0200347, or (c) 90 mg/kg/day PD-0200347. The animals were killed 12 weeks after surgery. Cartilage specimens from femoral condyles and tibial plateaus were processed for immunohistochemistry. Specific antibodies against the phosphorylated form of PKCalpha, Ras, c-Raf, the MAP kinases Erk1/2, p38, JNK, and the transcription factors, CREB and Elk-1, were used. RESULTS: Levels of all the tested signalling mediators were increased in the placebo treated (OA) group compared with the normal group. PD-0200347 treatment significantly reduced the levels of the active forms of PKCalpha, c-Raf, Erk1/2, and Elk-1; however, the levels of the active forms of Ras, p38, JNK, and CREB were not affected by the PD-0200347 treatment. CONCLUSION: The action of PD-0200347 on OA chondrocytes is probably mediated through the inhibition of Erk1/2 activation via a Ras independent mechanism. This effect is associated with reduction of the activation of transcription factors such as Elk-1, which leads to the inhibition of the induction of the major catabolic factors involved in the degradation process of OA cartilage.

Subchondral and trabecular bone metabolism regulation in canine experimental knee osteoarthritis.

OBJECTIVE: To determine trabecular and subchondral bone metabolic changes in experimental canine osteoarthritis (OA). METHODS: OA was induced in 19 dogs by transection of the anterior cruciate ligament (ACL) of the right knee through a stab wound. Dogs were sacrificed at 8 (n=7) and 12 weeks (n=12) after surgery. Non-operated normal dogs (n=6) were used as controls. After sacrifice, samples were obtained from the weight-bearing area of medial tibial plateaus. Explants and cell cultures were prepared from subchondral and trabecular bone. Osteocalcin (Oc), cellular alkaline phosphatase (ALPase), urokinase plasminogen-activator (uPA), prostaglandin E2 (PGE2), metalloproteinase (MMP) and nitric oxide (NO) were measured using standard procedures. RESULTS: ALPase production was significantly increased only at week 12 in subchondral and trabecular bone, while an increase in Oc was noted at week 8. uPA and MMP activity were increased significantly at week 12 in subchondral bone, while PGE2 levels were significantly higher in subchondral and trabecular bone at week 12 compared to normal. A decrease in NO production appeared late at week 12 in trabecular bone, whereas NO levels from subchondral bone were significantly increased compared to normal at week 8. DISCUSSION: Intense bone remodeling takes place in both subchondral and trabecular bone in the knee following ACL transection. This process seems to occur around week 12, although Oc and NO appeared to be involved earlier at 8 weeks. These results suggest that not only subchondral but also trabecular bone metabolism is altered in this OA model.

Superiority of the gastroduodenal safety profile of licofelone over rofecoxib, a COX-2 selective inhibitor, in dogs.

This study assessed the gastroduodenal safety profile of licofelone, a new nonsteroidal anti-inflammatory drug with dual inhibitory activity against 5-lipoxygenase and cyclo-oxygenase (COX), by using endoscopic evaluations and by comparing licofelone to rofecoxib, a selective COX-2 inhibitor. Twenty-one dogs underwent blinded gastroduodenoscopies, during which the mucosa of the gastroduodenal tract was assessed and scored. Blood analyses were monitored on days 0 (baseline), 14, 28, 42, and 56. Examinations to detect fecal occult blood were performed daily. Dogs were randomly assigned to three groups that received either a placebo, licofelone at a dose of 2.5 mg/kg twice daily, or rofecoxib at a dose of 0.5 mg/kg daily, respectively. Significant differences between the groups in gastric (P = 0.003), duodenal (P = 0.009), and gastroduodenal (P = 0.002) endoscopic lesion scores were observed at day 56. Rofecoxib-treated dogs had more lesions in all areas when compared with placebo-treated dogs, more duodenal lesions when compared with licofelone-treated dogs and more lesions than they had at baseline. In contrast to licofelone, rofecoxib was found to induce significant gastric and gastroduodenal lesions in dogs that lacked pre-existing lesions at baseline. Blood analyses and fecal examinations did not reveal abnormalities in any of the experimental groups. Treatment with licofelone was well tolerated and was shown to be safer than rofecoxib in terms of upper gastrointestinal damage. In this way, this study demonstrates the gastroduodenal safety profile of licofelone for chronic treatment.

Treatment with licofelone prevents abnormal subchondral bone cell metabolism in experimental dog osteoarthritis.

OBJECTIVES: To determine if treatment with licofelone, a combined 5-lipoxygenase and cyclo-oxygenase inhibitor, in vivo in experimental dog osteoarthritis can modify bone cell metabolism in long term in vitro subchondral osteoblast cell cultures (Ob). METHODS: Group 1 received sectioning of the anterior cruciate ligament (ACL) of the right knee with no active treatment (placebo group). Groups 2 and 3 received sectioning of the ACL of the right knee, and were given licofelone (2.5 or 5.0 mg/kg daily by mouth, respectively) for eight weeks beginning the day after surgery. Primary Ob were prepared from the subchondral bone plate. Levels of phenotypic markers (alkaline phosphatase activity, osteocalcin release), and urokinase plasminogen activator (uPA) and insulin-like growth factor-1 (IGF-I) levels, were evaluated in each group. Lastly, prostaglandin E(2) (PGE(2)) and leucotriene B(4) levels were evaluated. RESULTS: No significant differences in alkaline phosphatase activity or osteocalcin release from Ob between the three groups, under either basal or 1,25(OH)(2)D(3) induction were seen. In contrast, treatment with licofelone reduced uPA and IGF-I levels in Ob. PGE(2) levels, which were still raised in the placebo group, were decreased sharply by licofelone. A relationship was found between licofelone treatment and either the reduction in the size of lesions on tibial plateaus or the levels of uPA, IGF-I, or PGE(2). CONCLUSIONS: Licofelone treatment prevents and/or delays the abnormal metabolism of subchondral osteoblasts in this model. Licofelone reduced PGE(2) levels after long term Ob, suggesting that the reduction in uPA and IGF-I levels is linked, at least in part, to this reduction.

Identification and differential expression of human collagenase-3 mRNA species derived from internal deletion, alternative splicing, and different polyadenylation and transcription initiation sites.

OBJECTIVE: Collagenase-3 is a metalloprotease that plays a role in tissue remodeling and pathological processes including arthritis. The human gene is transcribed into major (3.0 and 2.5 kb) and minor (2.2/2.0 kb) transcripts, as seen in Northern blot assays. We investigated the possibility that other transcripts, not detectable by Northern blot, were synthesized as either coding or regulatory RNAs that would modulate collagenase-3 expression and function/activity. DESIGN: We screened a cDNA library and total RNA from human chondrocytes by plaque hybridization and RT-PCR, and expressed the transcripts in a cellular environment. The levels of expression of each transcript in normal and osteoarthritic joint cells and cartilage were monitored by RT-PCR. RESULTS: We identified five different collagenase-3 RNA species derived from alternative polyadenylation sites (COL3-APS), internal deletion (COL3-DEL), alternative splicing (COL3-9B/COL3-9B-2), and different transcription initiation site (COL3-ATS and COL3-ATS-INT). Each transcript could be translated in a cellular environment. Interestingly, the proteins translated from the COL3-DEL and COL3-9B-2 transcripts had a modified hemopexin-like domain, suggesting altered collagenolytic activities. The transcript types COL3-APS, COL3-9B-2, and COL3-ATS were up-regulated in the osteoarthritic samples and expressed in the chondrosarcoma cell line SW1353. CONCLUSION: Our data show that the human collagenase-3 gene is subjected to different levels of regulation and constitutes a more complex system than was originally thought.

Diacerein reduces the level of cartilage chondrocyte DNA fragmentation and death in experimental dog osteoarthritic cartilage at the same time that it inhibits caspase-3 and inducible nitric oxide synthase.

OBJECTIVE: The primary objective of this study was to evaluate the ex vivo therapeutic efficacy of diacerein and its active metabolite, rhein, on osteoarthritic (OA) cartilage chondrocyte DNA fragmentation and death in the experimental canine model of OA. The study also aimed to explore the effect of the drug on the level of important factors involved in this phenomenon, i.e., caspase-3 and inducible nitric oxide synthase (iNOS). METHODS: OA knee cartilage was obtained from dogs that had received surgical sectioning of the anterior cruciate ligament (ACL) and were sacrificed 12 weeks after surgery. Cartilage explants were cultured in the presence or absence of therapeutic concentrations of diacerein (20 micrograms/ml) or rhein (20 micrograms/ml). Cartilage specimens were stained for TUNEL reaction and immunostained using specific antibodies for active caspase-3 and iNOS. Morphometric analyses were also performed. RESULTS: In OA cartilage specimens, a large number of chondrocytes in the superficial layers stained positive for TUNEL reaction. Treatment with therapeutic concentrations of diacerein (20 micrograms/ml) or rhein (20 micrograms/ml) significantly reduced the level of chondrocyte DNA fragmentation to about the same extent in both treatment groups (P < 0.006, P < 0.002, respectively). The levels of caspase-3 and iNOS in cartilage explants were also significantly decreased (caspase-3, diacerein P < 0.04; caspase-3, rhein P < 0.0003; and iNOS, rhein P < 0.009, respectively) when compared to the control group. CONCLUSIONS: This study shows that diacerein/rhein can effectively reduce the level of OA chondrocyte DNA fragmentation and death under the present experimental conditions. This effect is mediated by a decrease in the level of caspase-3 expression, which could possibly be related in part to the reduced level of iNOS and secondarily to NO production. These findings provide additional new information about the mechanisms of action of diacerein on the progression of OA.

Modulation of IL-1beta, IL-6, TNF-alpha and PGE(2) by pharmacological agents in explants of membranes from failed total hip replacement.

Introduction and goal Proinflammatory cytokines and prostaglandin E(2) (PGE(2)) play an important role in the pathophysiology of osteolysis and implant loosening. The aim of this study was to evaluate the role of pharmacological agents in the inhibition of Interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) and PGE(2) in explants of interface membranes from failed total hip replacements (fTHR). Material and methods Membranes from fTHR were retrieved (N=20) and explants were incubated for 72h in the absence or presence of tenidap at three different concentrations (5, 20 or 50 microg/ml) or diclofenac (125 microg/l). IL-1beta, IL-6, TNF-alpha, and PGE(2) levels were measured in the culture medium using ELISA Capture or EIA kits. Statistical analysis was done using the Mann-Whitney U-test. Results A statistically significant inhibition in IL-1beta synthesis was found at tenidap concentrations of 20 microg/ml (71.3%, P< 0.05) and 50 microg/ml (79.3%,P< 0.02). Tenidap reduced IL-6 levels by 90.4% at 20 microg/ml (P< 0.005) and 96.0% (P< 0.05) at 50 microg/ml. Tenidap also reduced the synthesis of TNF-alpha by 66.9% (P< 0.05) and 77.4% at concentrations of 20 microg/ml and 50 microg/ml. Tenidap had a marked suppressive effect of over 90% (P< 0.0001) on PGE(2) synthesis in all three concentrations. Diclofenac (125 microg/l) decreased PGE(2) production by 95% (P< 0.0001), but had no significant effect in IL-1beta, IL-6, and TNF-alpha levels in the culture medium. Conclusion The ability to simultaneously suppress the release of proinflammatory cytokines and PGE(2) may help control osteolysis and prevent aseptic loosening of THR. This effect could increase implant longevity and lead the way to the pharmacological treatment of this pathology.