Knee Osteoarthritis Cartilage Research Articles

Levels of serum biomarkers from a two-year multicentre trial are associated with treatment response on knee osteoarthritis cartilage loss as assessed by magnetic resonance imaging: an exploratory study

BackgroundThere is an obvious need to identify biomarkers that could predict patient response to an osteoarthritis (OA) treatment. This post hoc study explored in a 2-year randomized controlled trial in patients with knee OA, the likelihood of some serum biomarkers to be associated with a better response to chondroitin sulfate in reducing cartilage volume loss.MethodsEight biomarkers were studied: hyaluronic acid (HA), C reactive protein (CRP), adipsin, leptin, N-terminal propeptide of collagen IIα (PIIANP), C-terminal crosslinked telopeptide of type I collagen (CTX-1), matrix metalloproteinase-1 (MMP-1), and MMP-3. Patients were treated with chondroitin sulfate (1200 mg/day; n = 57) or celecoxib (200 mg/day; n = 62). Serum biomarkers were measured at baseline. The cartilage volume at baseline and its loss at 2 years were assessed by quantitative magnetic resonance imaging (MRI). Statistical analysis included analysis of covariance.ResultsAs data from the original MOSAIC trial showed no differences in cartilage volume and loss in the lateral compartment of the knee joint between the two treatment groups in any comparison, only the medial compartment and its subregions were studied. Stratification according to the median biomarker levels was used to discriminate treatment effect. In patients with levels of biomarkers of inflammation (HA, leptin and adipsin) lower than the median, those treated with chondroitin sulfate demonstrated less cartilage volume loss in the medial compartment, condyle, and plateau (p ≤ 0.047). In contrast, patients treated with chondroitin sulfate with higher levels of MMP-1 and MMP-3, biomarkers of cartilage catabolism, had less cartilage volume loss in the medial compartment, condyle, and plateau (p ≤ 0.050). Patients with higher levels of PIIANP and CTX-1, biomarkers related to collagen anabolism and bone catabolism, respectively, had reduced cartilage volume loss in the medial condyle (p ≤ 0.026) in the chondroitin sulfate group.ConclusionThis study is suggestive of a potentially greater response to chondroitin sulfate treatment on cartilage volume loss in patients with knee OA with low level of inflammation and/or greater level of cartilage catabolism.Trial registrationThis is a post hoc study. Original trial registration: ClinicalTrials.gov, NCT01354145. Registered on 13 May 2011.

Effects of TLR-2/NF-κB signaling pathway on the occurrence of degenerative knee osteoarthritis: an in vivo and in vitro study.

The study aims to explore the effects of TLR-2/NF-κB signaling pathway on the occurrence of degenerative knee osteoarthritis (OA). Degenerative knee OA and normal cartilage samples were collected from patients with degenerative knee OA receiving total knee arthroplasty and amputation. Expressions of TLR-2, NF-κB and MMP-13 were determined by qRT-PCR and immunochemistry. The chondrocytes were divided into control, IL-1β, IL-1β + anti-TLR-2 and IL-1β + PDTC groups. MTT assay and flow cytometry were performed to determine proliferation and apoptosis of the chondrocytes. Expressions of TLR-2, NF-κB and MMP-13 were measured by Western blotting. ELISA was conducted to detect the expressions of related inflammatory factors. The positive expressions of TLR, NF-κB and MMP13 were associated with body mass index (BMI), family history, exercise, and WOMAC scores of OA patients. Logistic regression analysis showed that OA influencing factors were TLR, NF-κB, MMP13, BMI, family history and exercise. Compared with normal chondrocytes, the expressions of TLR-2, NF-κB, MMP-13 and related inflammatory factors increased in degenerative knee OA. The chondrocytes in the IL-1β + anti-TLR-2 and IL-1β + PDTC groups showed lower apoptosis rates than those in the IL-1β group. Compared with the control group, increased expressions of TLR-2, NF-κB, phosphorylated-NF-κB (p-NF-κB), MMP-13, IL-1, IL-6 and TNF-α were found in the IL-1β group. In the IL-1β + anti-TLR-2 and IL-1β + PDTC groups, decreased expressions of NF-κB, p-NF-κB, MMP-13, IL-1, IL-6 and TNF-α were found compared with those in the IL-1β group. TLR-2/NF-κB signaling pathway contributes to the occurrence of degenerative knee OA.

Role of Fibulin 3 in Aging-Related Joint Changes and Osteoarthritis Pathogenesis in Human and Mouse Knee Cartilage.

The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA. Immunohistochemical analysis was performed on normal and OA knee cartilage samples from humans and mice. Experimental OA was induced in wild-type and fibulin 3-/- mice, and the severity of OA was evaluated by histologic scoring. To examine fibulin 3 function, human chondrocyte monolayer cultures were transfected with small interfering RNA (siRNA), followed by quantitative polymerase chain reaction and Western blot analyses. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with an EFEMP1 lentivirus and analyzed for markers of chondrogenesis. Fibulin 3 was specifically expressed in the SZ of normal knee joint cartilage from humans and mice, and the expression levels declined with aging. Both aging-related OA and experimental OA were significantly more severe in fibulin 3-/- mice compared with wild-type mice. Fibulin 3 expression was high in undifferentiated human BM-MSCs and decreased during chondrogenesis. Suppression of fibulin 3 by siRNA significantly increased the expression of SOX9, type II collagen, and aggrecan in human articular chondrocytes, while overexpression of fibulin 3 inhibited chondrogenesis in BM-MSCs. Fibulin 3 is specifically expressed in the SZ of articular cartilage and its expression is reduced in aging and OA. Fibulin 3 regulates differentiation of adult progenitor cells, and its aging-related decline is an early event in the pathogenesis of OA. Preventing aging-associated loss of fibulin 3 or restoring it to normal levels in SZ chondrocytes has the potential to delay or prevent the onset of OA.

Differences in type II collagen turnover of osteoarthritic human knee and ankle joints.

We analysed hyaline cartilage of human knee and ankle joints for collagen and proteoglycan turnover in order to find differences in the metabolism and biochemical content of the extracellular matrix that could explain the higher prevalence of osteoarthritis (OA) in the knee joint, compared to the ankle joint. Cartilage tissue from ankle and knee joints of OA patients were assessed for total collagen and proteoglycan content. For turnover, the aggrecan 846-epitope (CS 846), the type II collagen C-propeptide (CP2) and the collagenase-generated intrahelical cleavage neoepitope (C2C) were quantified. Molecular analyses showed that type II collagen turnover (CP2 and C2C) was significantly elevated in the ankle, whereas aggrecan turnover (CS 846), total proteoglycan and total collagen were comparable between both joints. Analysis of the inter-relationships in the components of cartilage matrix turnover showed a significant positive correlation of C2C vs CP2. The data suggest an increased type II collagen turnover in ankle vs knee OA cartilage but a comparable aggrecan turnover and comparable contents of type II collagen and proteoglycan. These findings point towards a focused attempt in advanced OA cartilage to structurally repair the collagen network that was more pronounced in the ankle joint and may explain in part the higher prevalence of OA in the knee as compared to the ankle joint.

Identification of DNA methylation changes associated with disease progression in subchondral bone with site-matched cartilage in knee osteoarthritis.

Subchondral bone plays a key role in the development of osteoarthritis, however, epigenetics of subchondral bone has not been extensively studied. In this study, we examined the genome-wide DNA methylation profiles of subchondral bone from three regions on tibial plateau representing disease progression using HumanMethylation450 BeadChip to identify progression associated DNA methylation alterations. Significant differential methylated probes (DMPs) and differential methylated genes (DMGs) were identified in the intermediate and late stages and during the transition from intermediate to late stage of OA in the subchondral bone. Over half of the DMPs were hyper-methylated. Genes associated with OA and bone remodeling were identified. DMGs were enriched in morphogenesis and development of skeletal system, and HOX transcription factors. Comparison of DMGs identified in subchondral bone and site-matched cartilage indicated that DNA methylation changes occurred earlier in subchondral bone and identified different methylation patterns at the late stage of OA. However, shared DMPs, DMGs and common pathways that implicated the tissue reparation were also identified. Methylation is one key mechanism to regulate the crosstalk between cartilage and subchondral bone.

MicroRNA-9 regulates the development of knee osteoarthritis through the NF-kappaB1 pathway in chondrocytes.

It has been suggested that microRNA-9 (miR-9) is associated with the development of knee osteoarthritis (OA). This study was aimed to investigate the association between the mechanism of miR-9 targeting nuclear factor kappa-B1 (NF-κB1) and the proliferation and apoptosis of knee OA chondrocytes.Cartilage samples were collected from 25 patients with knee OA and 10 traumatic amputees, and another 15 OA rat models, together with 15 rats without knee OA lesions were also established. MiR-9 expressions in both knee OA cartilage and normal cartilage samples were detected using quantitative real-time PCR. The expressions of related genes (NF-κB1, IL-6, and MMP-13) in the two groups were also detected. Dual luciferase reporter gene assay was employed to examine the effect of miR-9 on the luciferase activity of NF-κB1 3'UTR. Knee OA chondrocytes were transfected with miR-9 mimics, miR-9 inhibitor, and NF-κB1 siRNA, respectively, and changes in cellular proliferation and apoptosis were detected via MTT assay and flow cytometric analysis, respectively. Western blotting assay was used to detect the expressions of NF-κB1, interleukin-6 (IL-6), and matrix metalloproteinase-13 (MMP-13).According to results from human OA samples and rat OA models, miR-9 was significantly downregulated in knee OA cartilage tissues compared with normal cartilage tissues (P < 0.01). The expressions of NF-κB1, IL-6, and MMP-13 in knee OA cartilage tissues were significantly higher than those in normal cartilage tissues (P < 0.01). Dual luciferase reporter gene assay showed that miR-9 could bind to the 3'UTR of NF-κB1 and significantly inhibit the luciferase activity by 37% (P < 0.01). Upregulation of miR-9 or downregulation of NF-κB1 could promote cell proliferation and suppress cell apoptosis.Conclusively, downregulated miR-9 can facilitate proliferation and antiapoptosis of knee OA chondrocytes by directly binding to NF-kB1, implying that stimulating miR-9 expressions might assist in treatment of knee OA.

Effects of close-to-bone needling on expression of type-Ⅱ collagen and markers in cartilage in rabbits with osteoarthritis of knee and imaging observation

To observe the effects of close-to-bone needling (CBN) on the expressions of type-Ⅱcollagen, pro-collagen type Ⅱ C-terminal propeptide (PⅡCP) and C-telopeptide of type Ⅱ collagen (CTX-Ⅱ) in rabbits with knee osteoarthritis (KOA). Among 40 New Zealand rabbits, 10 rabbits were selected into a normal group; the remaining 30 rabbits were made into KOA model, and X-ray was used to evaluate the results of model establishment. After the model was successfully made, the rabbits were randomly divided into a model group, a CBN group and a regular acupuncture group, ten rabbits in each one.Rabbits in the CBN group and the regular acupuncture group were treated at "Neixiyan" (EX-LE 4), "Dubi" (ST 35), "Yinlingquan" (SP 9), "Zusanli" (ST 36) and "Liangqiu" (ST 34). The CBN group applied CBN, and the depth of needling was appropriate with needles reaching bone; the regular acupuncture group applied regular acupuncture. The electroacupuncture(EA) device was used in the two groups, 20 min per treatment, once a day.Five days of treatment were taken as one course, and totally 4 courses were given with an interval of 2 days between courses. The normal group received identical fixation as model group. After treatment, magnetic resonance imaging (MRI) was used to perform imaging observation on knee; transmission electron microscopy (TEM) was used to observe the cell structure of knee joint cartilage;HE staining was used to observe the pathological change of knee; TUNEL was used to observe the apoptotic index; the expressions of type-Ⅱ collagen proteins and mRNA were measured by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR); the serum PⅡCP and CTX-Ⅱ levels were measured using enzyme-linked immunosorbent assay (ELISA). After treatment, compared with the model group, in the CBN group and regular acupuncture group the articular cavity effusion was reduced without the subchondral bone edema; the cell structure of knee joint cartilage was regular with less apoptosis; the expressions of type-Ⅱ collagen proteins and mRNA were significantly increased (all P<0.05), the PⅡCP levels were significantly increased (both P<0.05), but the CTX-Ⅱ levels were significantly decreased (both P<0.05).The differences of the expressions of type-Ⅱ collagen proteins and mRNA, the levels of PⅡCP and CTX-Ⅱ between the CBN group and the regular acupuncture group were significant (all P<0.05); the differences between the CBN group and the normal group were non-significant (all P>0.05). CBN can significantly improve the pathological status of cartilage of KOA, reduce apoptosis, and is likely to regulate the expressions of PⅡCP and CTX-Ⅱ to promote the type-Ⅱ collagen, which is superior to regular acupuncture.

Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage.

ObjectiveTo identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA.MethodsOA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non‐OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase–polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex.ResultsRNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin‐1β (IL‐1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50‐associated cyclooxygenase 2–extragenic RNA (PACER) and 2 novel chondrocyte inflammation–associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non‐OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL‐1–stimulated secretion of proinflammatory cytokines.ConclusionThe inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation‐driven cartilage degeneration in OA.

Mechanical properties of normal and osteoarthritic human articular cartilage

Isotropic hyperelastic models have been used to determine the material properties of normal human cartilage, but there remains an incomplete understanding of how these properties may be altered by osteoarthritis. The aims of this study were to (1) measure the material constants of normal and osteoarthritic human knee cartilage using isotropic hyperelastic models; (2) determine whether the material constants correlate with histological measures of structure and/or cartilage tissue damage; and (3) quantify the abilities of two common isotropic hyperelastic material models, the neo-Hookean and Yeoh models, to describe articular cartilage contact force, area, and pressure. Small osteochondral specimens of normal and osteoarthritic condition were retrieved from human cadaveric knees and from the knees of patients undergoing total knee arthroplasty and tested in unconfined compression at loading rates and large strains representative of weight-bearing activity. Articular surface contact area and lateral deformation were measured concurrently and specimen-specific finite element models then were used to determine the hyperelastic material constants. Structural parameters were measured using histological techniques while the severity of cartilage damage was quantified using the OARSI grading scale. The hyperelastic material constants correlated significantly with OARSI grade, indicating that the mechanical properties of cartilage for large strains change with tissue damage. The measurements of contact area described anisotropy of the tissue constituting the superficial zone. The Yeoh model described contact force and pressure more accurately than the neo-Hookean model, whereas both models under-predicted contact area and poorly described the anisotropy of cartilage within the superficial zone. These results identify the limits by which isotropic hyperelastic material models may be used to describe cartilage contact variables. This study provides novel data for the mechanical properties of normal and osteoarthritic human articular cartilage and enhances our ability to model this tissue using simple isotropic hyperelastic materials.

Diet, nutrition and osteoarthritis

Osteoarthritis (OA) is the fastest growing cause of disability worldwide[1]. In the absence of effective therapies, patients may wish to take some control of their own condition by making dietary changes that have the potential to ameliorate symptoms or reduce disease progression. A number of dietary factors have been associated with OA symptoms or progression. Most notably, in those overweight, weight reduction of ≥10% has the potential to lead to important changes in pain and function[2]. Losing weight also reduces pain-associated inflammation[3]. Weight loss combined with physical activity has an even greater capacity to improve pain and function[4]. It has been suggested that OA is a metabolic disease in which lipids essentially contribute to the pathophysiology of cartilage degradation[5]. Dietary long-chain ω-3 PUFA may affect articular cartilage composition and appear to have beneficial effects in OA[5]. In a US cohort of individuals with, or at high risk of, knee OA, there was a significant inverse relationship between total n-3 PUFAs and patella-femoral cartilage loss[6]. A positive association has been shown between elevated serum cholesterol and OA; hypercholesterolemia (OR 1.61; 95% CI 1.06-2.47) and high serum cholesterol (3rd vs. 1st tertile: OR 1.73; 95% CI 1.02-2.92) were independently associated with generalized OA in the Ulm study [7]. Hence there may be a potential benefit in adopting dietary cholesterol-lowering strategies (such as consumption of sterol/stanol spreads/drinks). Vitamin D affects the state of multiple articular structures. The evidence for association between the vitamin D biomarker, serum 25(OH)D, and OA was assessed in a systematic review. For knee radiographic OA progression and cartilage loss, there was strong evidence for an association with low 25(OH)D[8]. Vitamin K is important in cartilage metabolism as an inhibitor of extracellular matrix calcification and a promoter of cell survival/proliferation. In the US MOST study, vitamin K deficiency was associated with incident radiographic knee OA and MRI-based cartilage lesions (RR 2.39; 95% CI, 1.05-5.40) compared with no deficiency[9]. Hence, dietary recommendations are the following: ▪ lose weight, if overweight, preferably combined with exercise; ▪ reduce plasma cholesterol by dietary means; ▪ at least for a trial period, increase intake of long-chain n-3 fatty acids preferably by eating oily fish twice a week; ▪ aim for a safe level of sun exposure, eat rich vitamin-D dietary sources or take vitamin D supplements, ≤ 25 μg/d; ▪ increase vitamin K intake by eating green leafy vegetables.

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

IntroductionRecent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy.Methods In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.Results In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10.ConclusionsIn articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.

Genome-wide DNA methylation study of hip and knee cartilage reveals embryonic organ and skeletal system morphogenesis as major pathways involved in osteoarthritis.

BackgroundEvidence suggests that epigenetics plays a role in osteoarthrits (OA). The aim of the study was to describethe genome wide DNA methylation changes in hip and knee OA and identify novel genes and pathwaysinvolved in OA by comparing the DNA methylome of the hip and knee osteoarthritic cartilage tissues withthose of OA-free individuals.MethodsCartilage samples were collected from hip or knee joint replacement patients either due to primary OA or hip fractures as controls. DNA was extracted from the collected cartilage and assayed by Illumina Infinium HumanMethylation450 BeadChip array, which allows for the analysis of >480,000 CpG sites. Student T-test was conducted for each CpG site and those sites with at least 10 % methylation difference and a p value <0.0005 were defined as differentially methylated regions (DMRs) for OA. A sub-analysis was also done for hip and knee OA separately. DAVID v6.7 was used for the functional annotation clustering of the DMR genes. Clustering analysis was done using multiple dimensional scaling and hierarchical clustering methods.ResultsThe study included 5 patients with hip OA, 6 patients with knee OA and 7 hip cartilage samples from OA-free individuals. The comparisons of hip, knee and combined hip/knee OA patients with controls resulted in 26, 72, and 103 DMRs, respectively. The comparison between hip and knee OA revealed 67 DMRs. The overall number of the sites after considering the overlaps was 239, among which 151 sites were annotated to 145 genes. One-fifth of these genes were reported in previous studies. The functional annotation clustering of the identified genes revealed clusters significantly enriched in skeletal system morphogenesis and development. The analysis revealed significant difference among OA and OA-free cartilage, but less different between hip OA and knee OA.ConclusionsWe found that a number of CpG sites and genes across the genome were differentially methylated in OA patients, a remarkable portion of which seem to be involved in potential etiologic mechanisms of OA. Genes involved in skeletal developmental pathways and embryonic organ morphogenesis may be a potential area for further OA studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-015-0745-5) contains supplementary material, which is available to authorized users.

Aspartic acid racemization reveals a high turnover state in knee compared with hip osteoarthritic cartilage

We investigated tissue turnover in healthy and osteoarthritic cartilage. We challenge long held views that osteoarthritis (OA) is dominated by a similar turnover process in all joints and present evidence that hip and knee cartilage respond very differently to OA. d- and l-Aspartate (Asp) were quantified for whole cartilage, collagen and non-collagenous components of cartilage obtained at the time of joint replacement. We computed the Asp racemization ratio (Asp-RR = d/d + l Asp), reflecting the proportion of old to total protein, for each component. Compared with hip OA, knee OA collagen fibrils (P < 0.0001), collagen (P = 0.007), and non-collagenous proteins (P = 0.0003) had significantly lower age-adjusted mean Asp-RRs consistent with elevated protein synthesis in knee OA. Knee OA collagen had a mean hydroxyproline/proline (H/P) ratio of 1.2 consistent with the presence of type III collagen whereas hip OA collagen had a mean H/P ratio of 0.99 consistent with type II collagen. Based on Asp-RR, the relative age was significantly different in knee and hip OA (P < 0.0005); on average OA knees were estimated to be 30 yrs 'younger', and OA hips 10 yrs 'older' than non-OA. The metabolic response to OA was strikingly different by joint site. Knee OA cartilage evinced an anabolic response that appeared to be absent in hip OA cartilage. These results challenge the long held view that OA cartilage is capable of only minimal repair and that collagen loss is irreversible.

Sam68 Promotes NF-κB Activation and Apoptosis Signaling in Articular Chondrocytes during Osteoarthritis.

To investigate the expression of Sam68 in articular cartilage of knee osteoarthritis (OA) and the relationship between Sam68 and NF-κB activation and apoptosis signaling in OA articular chondrocytes. Sam68 expression in normal and osteoarthritic cartilage was assessed by immunohistochemistry and RT-PCR on both meniscal/ligamentous injury (MLI)-induced OA rat model and the clinical human OA cartilage tissues. Sam68 expression in chondrocytes under tumor necrosis factor-alpha (TNF-α) stimuli was also assessed by immunoblot. Inhibiting Sam68 in chondrocytes under TNF-α stimuli was conducted using small interfering RNA (siRNA) and its influence on the expression of apoptotic marker and catabolic genes was examined by immunoblot. The mechanism of how Sam68 stimulates NF-κB activity was determined by co-immunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of TNF-α-treated chondrocytes for p65 and Sam68. Sam68 expression was increased in OA cartilage tissues and chondrocytes under TNF-α stimuli. Inhibition of Sam68 by siRNA significantly decreased the expression of apoptotic markers (cleaved caspase-3 and cleaved PARP) in chondrocytes following TNF-α-stimulation. Sam68 knockdown suppressed Iκ-B degradation and p65 nuclear transportation in TNF-α-treated chondrocytes, indicating a suppressed NF-κB activation. Upon TNF-α exposure, the nuclear transportation of Sam68 and its interaction with p65 was detected in chondrocytes. Furthermore, Sam68 knockdown also alleviated the TNF-α-induced catabolic marker (MMP13, ADAMTS5, iNOS and IL-6) expression. The highly expressed Sam68 promotes NF-κB signaling activation, catabolic gene expression and cellular apoptosis in TNF-α-treated chondrocytes, which may provide better insights into the pathophysiology of OA and a potential target for its treatment.

Mesenchymal stromal cells and rheumatic disorders

Mesenchymal stromal cells (MSC), often incorrectly called stem cells, have been the intense focus of in vitro studies and animal models of rheumatic and other diseases over more than a decade. Despite multiple plausible mechanisms of action and a plethora of positive in vivo animal studies, few randomised controlled clinical trials have demonstrated meaningful clinical benefit in any condition so far. This could be due to confusion in cell product terminology, complexity of clinical study design and execution or agreement on meaningful outcome measures. It could also indicate that MSC as currently employed are ineffective.Within the rheumatic diseases, SLE, rheumatoid arthritis (RA) and osteoarthritis (OA) have received most attention. Uncontrolled multiple trial data from over 300 SLE patients have been published from one centre suggesting a positive outcome; one single centre comparative study in 172 RA was positive and no human studies have compared intra-articular MSC therapy to non-MSC techniques for osteoarthritis in the absence of surgery. Two randomised studies suggested benefit from the addition of bone marrow derived MSC and peripheral blood mononuclear cells added to surgical “standard of care” in knee OA and knee focal cartilage defects, respectively.The possible reasons for this apparent mismatch between expectation and clinical reality will be discussed.

Spatial variations in magnetic resonance-based diffusion of articular cartilage in knee osteoarthritis

PurposeTo evaluate a pulse sequence combining stimulated echo diffusion preparation with a 3D segmented spoiled gradient echo (SPGR) acquisition for diffusion tensor imaging (DTI) of knee cartilage in healthy and osteoarthritis (OA) populations for early diagnosis and characterization of OA. MethodsDiffusion-weighted images of 40 subjects (20 healthy, 20 OA) at baseline and 20 subjects (10 healthy, 10 OA) at one year were obtained. The subjects were classified according to Kellgren Lawrence (KL) and whole organ magnetic resonance imaging scoring (WORMS) method acquired at 3T. Cartilage full thickness and laminar mean diffusivity (MD) and fractional anisotropy (FA) values were quantified. The reproducibility of MD and FA values was assessed in five healthy human subjects based on test–retest scans. ResultsIn general, the full thickness MD values were higher in subjects with knee OA compared to healthy controls in both the baseline and follow up cohort. Laminar analysis MD and FA results were significantly different (p<0.05) between the bone-articular and articular layer with the articular layer having higher MD and lower FA value compared to the bone layer. The global reproducibility error was 6.5% for MD and 11.6% for FA. ConclusionThe diffusion-weighted stimulated echo-based sequence may be used as a valuable tool for early diagnosis and characterization of knee OA at 3T in the future.

Elevated expression of periostin in human osteoarthritic cartilage and its potential role in matrix degradation via matrix metalloproteinase-13.

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and up-regulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 µg/ml (EC50 0.5-1 μg/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/β-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by up-regulating MMP-13 through canonical Wnt signaling.

Effects of acupuncture and moxibustion on the levels of matrix metalloproteinase-1 and interleukin-1β in articular cartilage during the development of knee osteoarthritis caused by immobilization in rabbits

Objective To investigate the effects of warming-needle acupuncture on the levels of matrix metalloproteinase-1 (MMP-1) and interleukin-1β (IL-1β) in the articular cartilage in knee osteoarthritis induced by immobilization in rabbits. Methods A total of 30 adult male Japanese white rabbits were randomly assigned into three groups by random number table method: a normal group, a model group and a treatment group, with 10 rabbits in each group. Knee osteoarthritis was induced by 6 weeks of immobilization. The rabbits in the treatment group received warming-needle acupuncture after modeling at points of “Neixiyan” (EX-LE4), “Dubi” (ST35), “Zusanli” (ST36) and “Yanglingquan” (GB34) for 4 weeks. The level of MMP-1 and IL-1β in the articular cartilage of the knee were determined with radioimmunological assay. Results There were significant difference in the levels of MMP-1(0.16 ± 0.02 ng/mg, 0.37 ± 0.02 ng/mg, and 0.28 ± 0.03 ng/mg; F=258.251) and IL-1β (0.21 ± 0.01 pg/mg, 0.34 ± 0.02 pg/mg, and 0.31 ± 0.04 pg/mg; F=127.112) among the normal group, model group and treatment group. The levels of MMP-1 and IL-1β both in the model group and treatment group were significant higher than those in the normal group (all P<0.01), while the levels of MMP-1 and IL-1β in the treatment group were significant lower than those in the model group (all P<0.01). Conclusion Warming-needle acupuncture can effectively reduced the levels of MMP-1 and IL-1β in the articular cartilage in knee osteoarthritis induced by immobilization in rabbits. Key words: Osteoarthritis, knee; Matrix metalloproteinase 1; Interleukin-1β; Acupuncture-moxibustion; Cartilage; Rabbits

Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis.

Amyloid deposits are prevalent in osteoarthritic (OA) joints. We undertook this study to define the dominant precursor and to determine whether the deposits affect chondrocyte functions. Amyloid deposition in human normal and OA knee cartilage was determined by Congo red staining. Transthyretin (TTR) in cartilage and synovial fluid was analyzed by immunohistochemistry and Western blotting. The effects of recombinant amyloidogenic and nonamyloidogenic TTR variants were tested in human chondrocyte cultures. Normal cartilage from young donors did not contain detectable amyloid deposits, but 7 of 12 aged normal cartilage samples (58%) and 12 of 12 OA cartilage samples (100%) had Congo red staining with green birefringence under polarized light. TTR, which is located predominantly at the cartilage surfaces, was detected in all OA cartilage samples and in a majority of aged normal cartilage samples, but not in normal cartilage samples from young donors. Chondrocytes and synoviocytes did not contain significant amounts of TTR messenger RNA. Synovial fluid TTR levels were similar in normal and OA knees. In cultured chondrocytes, only an amyloidogenic TTR variant induced cell death as well as the expression of proinflammatory cytokines and extracellular matrix-degrading enzymes. The effects of amyloidogenic TTR on gene expression were mediated in part by Toll-like receptor 4, receptor for advanced glycation end products, and p38 MAPK. TTR-induced cytotoxicity was inhibited by resveratrol, a plant polyphenol that stabilizes the native tetrameric structure of TTR. These findings are the first to suggest that TTR amyloid deposition contributes to cell and extracellular matrix damage in articular cartilage in human OA and that therapies designed to reduce TTR amyloid formation might be useful.

Mitochondrial biogenesis is impaired in osteoarthritis chondrocytes but reversible via peroxisome proliferator-activated receptor γ coactivator 1α.

The etiology of chondrocyte mitochondrial dysfunction in osteoarthritis (OA) is not completely understood. OA chondrocytes are deficient in the metabolic biosensors active AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT-1), which modulate the mitochondrial biogenesis "master regulator" peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Moreover, PGC-1α critically mediates AMPK anticatabolic activity in chondrocytes. The aim of this study was to test the hypothesis that mitochondrial biogenesis is deficient in human OA chondrocytes and that this deficiency functionally increases chondrocyte procatabolic responses, which are reversed by activation of the AMPK/SIRT-1/PGC-1α pathway. We assessed the expression and activity (phosphorylation) of AMPKα, SIRT-1, and PGC-1α in human knee chondrocytes and human and mouse knee cartilage, and we defined and compared the content and function of mitochondria, including oxidative phosphorylation and expression of mitochondrial biogenesis factors (mitochondrial transcriptional factor A [TFAM] and nuclear respiratory factors [NRFs]). Human knee OA chondrocytes had a decreased mitochondrial biogenesis capacity, which was linked to reduced AMPKα activity and decreased expression of SIRT-1, PGC-1α, TFAM, NRF-1, and NRF-2. Human knee OA and aging mouse knee cartilage had decreased expression of TFAM and ubiquinol-cytochrome c reductase core protein, a subunit of mitochondrial complex III, in situ. Chondrocyte TFAM knockdown inhibited mitochondrial biogenesis and enhanced procatabolic responses to interleukin-1β. Finally, activation of AMPK by A-769662 increased PGC-1α expression via SIRT-1 and reversed impairments in mitochondrial biogenesis, oxidative phosphorylation, and intracellular ATP in human knee OA chondrocytes. Mitochondrial biogenesis is deficient in human OA chondrocytes, and this deficiency promotes chondrocyte procatabolic responses. TFAM-mediated activation of the AMPK/SIRT-1/PGC-1α pathway reverses these effects, suggesting translational potential of pharmacologic AMPK activators to limit OA progression.