Aims: As inflammation plays a key role in the etiology of intervertebral disc degeneration, we suggest a possible contribution of pro-inflammatory gene polymorphisms in the pathogenesis of adolescent idiopathic scoliosis (AIS). The nucleus pulposus of scoliotic discs responds to exogenous stimuli by secreting interleukin-6 (IL-6) and other inflammatory cytokines. The association between matrix metalloproteinases (MMPs) and disc degeneration has been reported by several investigators. A human MMP-3 promoter 5A/6A gene polymorphism regulates MMP-3 genes expression, while the G/C polymorphism of the promoter region of IL-6 gene influences levels and functional activity of the IL-6 protein. Methods: We conducted a case-control study to investigate whether the 5A/6A polymorphism of the MMP-3 gene and the G/C polymorphism of the promoter region of IL-6 gene were associated with susceptibility to AIS. Results: The frequency of the 5A/5A genotype of MMP-3 gene polymorphism in patients with scoliosis was almost 3 times higher than in controls (30.2 % vs 11.2 %, p 0.001) and the frequency of the G/G genotype of IL-6 gene polymorphism in patients with scoliosis was almost 2 times higher than in controls (52.8 % vs 26.2 %, p < 0.001). 5A/5A genotype of MMP-3 gene polymorphism and G/G genotype of IL-6 gene polymorphism are independently associated with an higher risk of scoliosis (odds ratio respectively 3.34 and 10.54). Conclusions: This is the first study that has evaluated the possibility that gene variants of IL-6 and MMPs might be associated with scoliosis and suggests that MMP-3 and IL-6 promoter polymorphisms constitute important factors for the genetic predisposition to scoliosis

Aims. Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. Methods. Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology. Results. Berberine inhibited proliferation and adhesion of RA-FLS cells, and significantly reduced the expression of MMP-1, MMP-3, RANKL, and TNF-α. Transcriptional results suggested that berberine intervention mainly regulated forkhead box O (FOXO) signal pathway, prolactin signal pathway, neurotrophic factor signal pathway, and hypoxia-inducible factor 1 (HIF-1) signal pathway. Conclusion. The effect of berberine on RA was related to the regulation of RAS/mitogen-activated protein kinase/FOXO/HIF-1 signal pathway in RA-FLS cells. Cite this article: Bone Joint Res 2023;12(2):91–102

A variety of scaffolds, including collagen-based membranes, fleeces and gels are seeded with osteoblasts and applied for the regeneration of bone defects. However, different materials yield different outcomes, despite the fact that they are generated from the same matrix protein, i.e. type I collagen. Recently we showed that in fibroblasts MMP-3 is induced upon attachment to matrix proteins in the presence of TGFbeta. Aim: To investigate the regulation of matrix metalloproteinases (MMPs) and interleukins (IL) in osteoblasts upon attachment to type I collagen (col-1) in comparison to laminin -1 (LM-111) in the presence or absence of costimulatory signals provided by transforming growth factor beta (TGFbeta). Methods: Osteoblasts were seeded in col-1–and LM-111-coated flasks and activated by the addition of TGFbeta. Mock-treated cells served as controls. The expression of genes was investigated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunocytochemistry and ELISA. Results: Attachment of osteoblasts to col-1 or LM-111 failed to activate the expression of MMPs or ILs. In contrast, TGFbeta induced the expression of MMP-3, MMP-9, and MMP-13, IL-6 and IL-16 mRNAs. MMP-3 was found to be elevated in supernatants of activated cells. No difference was found in the expression of MMP-1, IL-8 and IL–18. Interestingly, the expression of IL-1beta mRNA was not activated by TGFbeta alone, but it was activated by attachment of osteoblasts to LM-111 in the presence of TGFbeta. Conclusion: In contrast to fibroblasts, attachment of osteoblasts to col-1 or LM-111 had no effect on the induction of MMPs and ILs. TGFbeta induced the expression of MMPs and ILs in these cells but only MMP-3 was released. The results show significant differences between osteoblasts and fibroblasts in the effects of attachment to scaffold materials. This may have important consequences for tissue engineering of bone and for wound healing after surgery

Aims. Rheumatoid arthritis (RA), which mainly results from fibroblast-like synoviocyte (FLS) dysfunction, is related to oxidative stress. Advanced oxidation protein products (AOPPs), which are proinflammatory mediators and a novel biomarker of oxidative stress, have been observed to accumulate significantly in the serum of RA patients. Here, we present the first investigation of the effects of AOPPs on RA-FLSs and the signalling pathway involved in AOPP-induced inflammatory responses and invasive behaviour. Methods. We used different concentrations of AOPPs (50 to 200 µg/ml) to treat RA-FLSs. Cell migration and invasion and the expression levels of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), and MMP-13 were investigated. Western blot and immunofluorescence were used to analyze nuclear factor-κB (NF-κB) activation. Results. AOPPs promoted RA-FLS migration and invasion in vitro and significantly induced the messenger RNA (mRNA) and protein expression of TNF-α, IL-6, MMP-3, and MMP-13 in dose- and time-dependent manners. Moreover, AOPPs markedly activated the phosphorylation of nuclear factor-κB (NF-κB) p65 protein, which triggered inhibitory kappa B-alpha (IκBα) degradation, NF-κB p65 protein phosphorylation, and NF-κB p65 translocation into the nucleus. Furthermore, treatment with a neutralizing antibody specific to receptor for advanced glycation end products (RAGE) significantly suppressed aggressive behaviour and inflammation, decreased TNF-α, IL-6, MMP-3, and MMP-13 expression, and blocked AOPP-induced NF-κB pathway activation. Conclusion. The results indicate that AOPPs can enhance aggressive behaviour and the inflammatory response in RA-FLSs via the RAGE–NF-κB pathway. These results present AOPPs as a new class of potentially important mediators of progressive disease in RA patients. Cite this article: Bone Joint Res 2021;10(4):259–268

Aims. This study aimed to define the histopathology of degenerated humeral head cartilage and synovial inflammation of the glenohumeral joint in patients with omarthrosis (OmA) and cuff tear arthropathy (CTA). Additionally, the potential of immunohistochemical tissue biomarkers in reflecting the degeneration status of humeral head cartilage was evaluated. Methods. Specimens of the humeral head and synovial tissue from 12 patients with OmA, seven patients with CTA, and four body donors were processed histologically for examination using different histopathological scores. Osteochondral sections were immunohistochemically stained for collagen type I, collagen type II, collagen neoepitope C1,2C, collagen type X, and osteocalcin, prior to semiquantitative analysis. Matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 levels were analyzed in synovial fluid using enzyme-linked immunosorbent assay (ELISA). Results. Cartilage degeneration of the humeral head was associated with the histological presentation of: 1) pannus overgrowing the cartilage surface; 2) pores in the subchondral bone plate; and 3) chondrocyte clusters in OmA patients. In contrast, hyperplasia of the synovial lining layer was revealed as a significant indicator of inflammatory processes predominantly in CTA. The abundancy of collagen I, collagen II, and the C1,2C neoepitope correlated significantly with the histopathological degeneration of humeral head cartilage. No evidence for differences in MMP levels between OmA and CTA patients was found. Conclusion. This study provides a comprehensive histological characterization of humeral cartilage and synovial tissue within the glenohumeral joint, both in normal and diseased states. It highlights synovitis and pannus formation as histopathological hallmarks of OmA and CTA, indicating their roles as drivers of joint inflammation and cartilage degradation, and as targets for therapeutic strategies such as rotator cuff reconstruction and synovectomy. Cite this article: Bone Joint Res 2024;13(10):596–610

The interleukin-6/gp130-associated Janus Kinases/STAT3 axis is known to play an important role in mediating inflammatory signals, resulting in production of matrix metalloproteinase-3 (MMP-3). The hip joints with rapidly destructive coxopathy (RDC) demonstrate rapid chondrolysis, probably by increased production of MMP-3 observed in the early stage of RDC. In the recent study, no apparent activation of STAT3 has been shown in the synovial tissues obtained from the osteoarthritic joint at operation. However, no data are currently available on STAT3 activation in the synovial tissues in the early stage of RDC. This study aimed to elucidate STAT3 activation in the synovial tissues in the early stage of RDC. Synovial tissues within 7 months from the disease onset were obtained from four RDC patients with femoral head destruction and high serum levels of MMP-3. RDC synovial tissues showed the synovial lining hyperplasia with an increase of CD68-positive macrophages and CD3-positive T lymphocytes. STAT3 phosphorylation was found in the synovial tissues by immunohistochemistry using anti-phospho-STAT3 antibody. The majority of phospho-STAT3-positive cells were the synovial lining cells and exhibited negative expression of macrophage or T cell marker. Treatment with tofacitinib, a Janus Kinase inhibitor, resulted in a decrease in phospho-STAT3-positive cells, especially with high intensity, indicating effective suppression of STAT3 activation in RDC synovial tissues. Inhibitory effect of tofacitinib could act through the Janus Kinase/STAT3 axis in the synovial tissues in the early stage of RDC. Therefore, STAT3 may be a potential therapeutic target for prevention of joint structural damage in RDC. Acknowledgements: This study was supported by Katakami Foundation for Clinical Research

Rapidly progressive osteoarthritis of the hip (RPOH) is an unusual subset of osteoarthritis. It is characterized by rapid joint space loss, chondroly­sis, and sometimes marked femoral head and acetabular destruction as a late finding. The exact pathogenetic mechanism is unknown. Potential causes of RPOH include subchondral insufficiency fracture resulting from osteoporosis, increasing posterior pelvic tilt as a mechanical factor, and high serum levels of matrix metalloproteinase (MMP)-3 as biological factors. This study was aimed to identify some markers that associate with the destructive process of RPOH by analyzing the proposed pathological factors of the disease, MMP-3, pelvic tilt, and osteoporosis. Of female patients who visited our hospital with hip pain from 2012 through 2018, this study enrolled female patients with sufficient clinical records including the onset of hip pain, age and body mass index (BMI) at the onset, a series of radiographs during the period of >12 months from the onset of hip pain, and hematological data of MMP-3 and C-reactive protein (CRP). We found the hip joints of 31 patients meet the diagnostic criteria of RPOH, chondrolysis >two mm in one year, or 50% joint space narrowing in one year. Those patients were classified into two groups, 17 and 14 patients with and without subsequent femoral head destruction in one year shown by computed tomography, respectively. Serum MMP-3 and CRP were measured with blood samples within one year after the hip pain onset. The cortical thickness index (CTI) as an indicator of osteoporosis and pelvic tilt parameters were evaluated on the initial anteroposterior radiograph of the hip. These factors were statistically compared between the two groups. This study excluded male patients because RPOH occurs mainly in elderly females and the reference intervals of MMP-3 are different between males and females. There was no difference in age at onset or bone mass index between the RPOH patients with and without subsequent femoral head destruction. Serum levels of MMP-3 were significantly higher in the RPOH patients with the destruction (152.1 ± 108.9 ng/ml) than those without the destruction (66.8 ± 27.9 ng/ml) (P = 0.005 by Mann-Whitney test). We also found increased CRP in the patients with femoral head destruction (0.725 ± 1.44 mg/dl) compared with those without the destruction (0.178 ± 0.187 mg/dl) (P = 0.032 by Mann-Whitney test). No difference in the duration between the hip pain onset and the blood examination was found between the two groups. There was no significant difference in CTI or pelvic tilt between the two groups. The pathological condition that may increase serum MMP-3 and CRP could be involved in femoral head destruction after chondrolysis of the hip in patients with RPOH

The patellofemoral joint is an important source of symptoms in osteoarthritis of the knee. We have used a newly designed surgical model of patellar strengthening to induce osteoarthritis in BALB/c mice and to establish markers by investigating the relationship between osteoarthritis and synovial levels of matrix metalloproteinases (MMPs). Osteoarthritis was induced by using this microsurgical technique under direct vision without involving the cavity of the knee. Degeneration of cartilage was assessed by the Mankin score and synovial tissue was used to determine the mRNA expression levels of MMPs. Irrigation fluid from the knee was used to measure the concentrations of MMP-3 and MMP-9. Analysis of cartilage degeneration was correlated with the levels of expression of MMP. After operation the patellofemoral joint showed evidence of mild osteoarthritis at eight weeks and further degenerative changes by 12 weeks. The level of synovial MMP-9 mRNA correlated with the Mankin score at eight weeks, but not at 12 weeks. The levels of MMP-2, MMP-3 and MMP-14 mRNA correlated with the Mankin score at 12 weeks. An increase in MMP-3 was observed from four weeks up to 16 weeks. MMP-9 was notably increased at eight weeks, but the concentration at 16 weeks had decreased to the level observed at four weeks. Our observations suggest that MMP-2, MMP-3 and MMP-14 could be used as markers of the progression of osteoarthritic change

Aims. Interleukin (IL)-1β is one of the major pathogenic regulators during the pathological development of intervertebral disc degeneration (IDD). However, effective treatment options for IDD are limited. Suramin is used to treat African sleeping sickness. This study aimed to investigate the pharmacological effects of suramin on mitigating IDD and to characterize the underlying mechanism. Methods. Porcine nucleus pulposus (NP) cells were treated with vehicle, 10 ng/ml IL-1β, 10 μM suramin, or 10 μM suramin plus IL-1β. The expression levels of catabolic and anabolic proteins, proinflammatory cytokines, mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB-related signalling molecules were assessed by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence analysis. Flow cytometry was applied to detect apoptotic cells. The ex vivo effects of suramin were examined using IDD organ culture and differentiation was analyzed by Safranin O-Fast green and Alcian blue staining. Results. Suramin inhibited IL-1β-induced apoptosis, downregulated matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5, and upregulated collagen 2A (Col2a1) and aggrecan in IL-1β-treated NP cells. IL-1β-induced inflammation, assessed by IL-1β, IL-8, and tumour necrosis factor α (TNF-α) upregulation, was alleviated by suramin treatment. Suramin suppressed IL-1β-mediated proteoglycan depletion and the induction of MMP-3, ADAMTS-4, and pro-inflammatory gene expression in ex vivo experiments. Conclusion. Suramin administration represents a novel and effectively therapeutic approach, which could potentially alleviate IDD by reducing extracellular matrix (ECM) deposition and inhibiting apoptosis and inflammatory responses in the NP cells. Cite this article: Bone Joint Res 2021;10(8):498–513

Mechanical loading regulates the metabolism of chondrocytes in cartilage1. Nowadays, studies exploring the in vitro response of cartilage towards loading often rely on bioreactor experiments applying only compressive loading. This is likely not sufficiently representative for the complex multi-directional loading profile in vivo (i.e. where typical compressive and shear loading are both present). The impact of multi-axial loading is specifically relevant in the context of the onset of osteoarthritis (OA) due to joint destabilization. Here, alterations in the 3D loading profile, and in particular increased shear forces, are suggested to initiate catabolic molecular responses leading to cartilage degeneration3. However, in vitro/ex vivo data confirming this hypothesis are currently lacking. Therefore, we aim to investigate how increased shear loading affects the metabolism and ECM deposition of a healthy chondrogenic cell line and if this response is different in osteoarthritic primary chondrocytes. A murine chondrogenic precursor cell line (ATDC5) and primary human osteoarthritic articular chondrocytes (hOACs) were encapsulated in 2.2% alginate disks and cultured in DMEM medium for three days. Hydrogels seeded with the different cell groups were loaded in the TA ElectroForce BioDynamic Bioreactor and subjected to following loading conditions: (a) 10% compression at 1Hz for 1h, (b) 10% compression and 10° shear loading at 1Hz for 1h. Unloaded constructs were used as control. After loading, hydrogel constructs were stabilized in culture medium for 2 hours, to facilitate adequate gene expression responses, before being dissolved and snap frozen. RNA was isolated and gene expression levels specific for anabolic pathways, characterized by extracellular matrix (ECM) genes (Col2a1, Aggrecan and Perlecan), catabolic processes (MMP-3 and MMP-13) and chondrogenic transcription factor (Sox9) were evaluated using RT-qPCR. The TA ElectroForce BioDynamic Bioreactor was successfully set-up to mimic cartilage loading. In ATDC5 cells, compression elicits an increase in all measured ECM genes (Col2a1, Aggrecan and Perlecan) compared to unloaded controls, suggesting an anabolic response. This upregulation is decreased when adding additional shear strain. In contrast to ATDC5 cells, the anabolic response of proteoglycans Aggrecan and Perlecan to compressive loading was lower in osteoarthritic chondrocytes, and Col2a1 expression appeared decreased. Adding shear strain reversed this effect on Col2a1 expression. Multi-directional loading increased transcription factor Sox9 expression compared to compression in both ATDC5 and OA chondrocytes. In OA chondrocytes, both loading regimens increased MMP-3 and MMP-13 expression. Shear loading reduces the anabolic effect of compressive loading in both cell types. OA cells presented more catabolic response to mechanical loading compared to precursors, given the increase in catabolic enzymes MMP-3 and MMP-13

Introduction. Chondrocytes are enveloped within the pericellular matrix (PCM), a structurally intricate network primarily demarcated by the presence of collagen type VI microfibrils and perlecan, resembling a protective cocoon. The PCM serves pivotal functions in facilitating cell mechanoprotection and mechanotransduction. The progression of osteoarthritis (OA) is associated with alterations in the spatial arrangement of chondrocytes, transitioning from single strings to double strings, small clusters, and eventually coalescing into large clusters in advanced OA stages. Changes in cellular patters coincide with structural degradation of the PCM and loss of biomechanical properties. Here, we systematically studied matrix metalloproteinases (MMPs), their distribution, activity, and involvement in PCM destruction, utilizing chondrocyte arrangement as an OA biomarker. Methods. Cartilage specimens were obtained from 149 osteoarthritis (OA) patients, and selected based on the predominant spatial pattern of chondrocytes. Immunoassays were employed to screen for the presence of various MMPs (-1, -2, -3, -7, -8, -9, -10, -12, -13). Subsequently, the presence and activity of elevated MMPs were further investigated through immunolabeling, western blots and zymograms. Enzymatic assays were utilized to demonstrate the direct involvement of the targeted MMPs in the PCM destruction. Results. Screening revealed increased levels of MMP-1, -2, -3, -7, and -13, with their expression profile demonstrating a distinct dependency on the stage of degeneration. We found that MMP-2 and -3 can directly compromise the integrity of collagen type VI, whereas MMP-3 and MMP-7 disrupt perlecan. Conclusions. Presence of both pro- and active forms of MMP-2, -3, and -7 in OA-induced patterns, along with their direct involvement in collagen type VI and perlecan degradation, underscores their crucial role in early PCM destruction. Given the early stages of the disease already exhibit heightened MMP expression, this understanding could inform early targeted therapies aimed at arresting abnormal PCM remodelling. Acknowledgments. Faculty of Medicine of the University of Tübingen (grant: 2650-0-0)

Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration

Osteoarthritis (OA) is the most prevalent degenerative joint disease that is a leading cause of disability worldwide. Existing therapies of OA only address the symptoms. Liraglutide is a well-known anti-diabetic medication that is used to treat type 2 diabetes and obesity. In inflammatory and post-traumatic OA animal models, liraglutide has demonstrated anti-inflammatory, pain-relieving, and cartilage-regenerating effects1 . The objective of this study is to investigate liraglutide's ability to reduce inflammation and promote anabolism in human OA chondrocytes in vitro. Pellets formed with human OA chondrocytes were cultured with a chondrogenic medium for one week to form cartilage tissue. Afterward, pellets were cultured for another 2 weeks with a chondropermissive medium. The OA group was treated with IL-1β to mimic an inflammatory OA condition. The drug group was treated with 0.5 or 10 µM liraglutide. On days 0, 1, and 14, pellets were collected. Conditioned medium was collected over the 2 weeks culture period. The gene and protein expression levels of regenerative and inflammatory biomarkers were evaluated and histological analyzes were performed. Results showed that the nitric oxide release of the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups were lower than the OA group. The DNA content of the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups were higher than the OA group on day 14. The RT-qPCR results showed that the anabolism (ACAN, COMP, and COL2) markers were higher expressed in the OA + 0.5 µM liraglutide and OA + 10 µM liraglutide groups when compared with the OA group. The inflammation (CCL-2 and IL-8) markers and catabolism markers (MMP-1, MMP-3, ADAMTS4, and ADAMTS5) had lower expression levels in the OA + liraglutide groups compared to the OA group. The histomorphometric analysis (Figure 1) supported the RT-qPCR results. The results indicate that liraglutide has anabolic and anti-inflammatory effects on human OA chondrocyte pellets. Acknowledgments: This project has received funding from the Eurostars-2 joint program with co-funding from the European Union Horizon 2020 research and innovation program. The funding agencies supporting this work are (in alphabetical order of participating countries): France: BPI France; Germany: Project Management Agency (DLR), which acts on behalf of the Federal Ministry of Education and Research (BMBF); The Netherlands: Netherlands Enterprise Agency (RVO); Switzerland: Innosuisse (the Swiss Innovation Agency). For any figures and tables, please contact the authors directly

Osteoarthritis (OA) is a multifactorial debilitating disease that affects over four million Canadians. Although the mechanism(s) of OA onset is unclear, the biological outcome is cartilage degradation. Cartilage degradation is typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II) – partly due to the up-regulation of catabolic enzymes - aggrecanases a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). There is currently no treatment that will prevent or repair joint damage, and current medications are aimed mostly at pain management. When pain becomes unmanageable arthroplastic surgery is often performed. Interest has developed over the presence of calcium crystals in the synovial fluid of OA patients, as they have been shown to activate synovial fibroblasts inducing the expression of catabolic agents. We recently discovered elevated levels of free calcium in the synovial fluid of OA patients and raised the question on its role in cartilage degeneration. Articular cartilage was isolated from 5 donors undergoing total hip replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase and expanded in DMEM supplemented with 10% heat-inactivated FBS. OA and normal human articular chondrocytes (PromoCell, Heidelberg, Germany) were transferred to 6-well plates in culture medium containing various concentrations of calcium (0.5, 1, 2.5, and 5 mM CaCl2), and IL-1β. Cartilage explants were prepared from the same donors and included cartilage with the cortical bone approximately 1 cm2 in dimension. Bovine articular cartilage explants (10 months) were used as a control. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. Immunohistochemistry was performed on cartilage explants to measure expression of Col X, MMP-13, and alkaline phosphatase. The sulfated glycosaminoglycan (GAG, predominantly aggrecan) content of cartilage was analyzed using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay, and aggregan fragmentation was determined by Western blotting using antibody targeted to its G1 domain. Western blotting was also performed on cell lysate from both OA and normal chondrocytes to measure aggrecan, Col II, MMP-3 and −13, ADAMTS-4 and −5. Ca2+ significantly decreased the proteoglycan content of the cartilage explants as determined by the DMMB assay. The presence of aggrecan and Col II also decreased as a function of calcium, in both the human OA and bovine cartilage explants. When normal and OA chondrocytes were cultured in medium supplemented with increasing concentrations of calcium (0.5–5 mM Ca2+), aggrecan and Col II expression decreased dose-dependently. Surprisingly, increasing Ca2+ did not induce the release of MMP-3, and −13, or ADAMTS-4 and-5 in conditioned media from OA and normal chondrocytes. Interestingly, inhibition of the extracellular calcium-sensing receptor CaSR) reversed the effects of calcium on matrix protein synthesis. We provide evidence that Ca2+ may play a direct role in cartilage degradation by regulating the expression of aggrecan and Col II through activation of CaSR

The purpose of this study was to investigate the possible relationship between matrix metalloproteinase-3 (MMP-3) promoter 5A/6A polymorphism and intervertebral disc (IVD) degeneration in the older generation. One of the important steps in IVD degeneration is disc matrix degradation by matrix degrading enzymes such as MMPs. MMP-3 is one of the potent proteoglycan degrading enzymes and has been suggested to play an important role in IVD degradation. A common 5A/6A polymorphism in the promoter region of the human MMP-3 gene has been identified. This polymorphism was reported to be involved in the regulation of MMP-3 gene expression (the 5A allele has 2-fold higher promoter activity than 6A). We now hypothesize that IVD degeneration is associated with MMP-3 promoter 5A/6A polymorphism. Forty-nine elderly Japanese volunteers (mean age 74.3 years, range 64–94 years) were studied. Each lumbar disc was graded according to the radiographic classification system of IVD degeneration described by Kellgren and Lawrence. The 5A/6A polymorphism was determined with both single strand conformation polymorphism (SSCP) and polymerase chain reaction with allele-specific primers (AS-PCR). Two subjects (4%) with 5A5A genotype, 16 (33%) with 5A6A, and 31 (63%) with 6A6A were observed. Genotype was totally independent of age and sex. There was a significantly larger number of IVDs graded 2 and higher in the 5A/5A+5A/6A than in the 6A/6A (p< 0.05). The degenerative scores of lumber discs were also distributed more highly in the 5A/5A+5A/6A than in the 6A/6A (p=0.0029). Many environmental factors have been reported to accelerate IVD degeneration. Recently, genetic factors have also been highlighted as possible risk factors. The 5A allele of the human MMP-3 promoter is a possible risk factor for acceleration of IVD degeneration in people aged over 64 years old. We conclude that MMP-3 plays a key role in the degeneration of IVD in the older generation

Osteoarthritis (OA) is characterised by progressive erosion of articular cartilage, which, once started, cannot be halted. The breakdown of cartilage is mediated by proteases, including MMP-3 and -13. These may initially be derived from the synovium but are also produced by OA chondrocytes, particularly in later stages of the disease. In normal articular chondrocytes, the proteases are not expressed and it has previously been shown that this is due, in part, to silencing by epigenetic mechanisms, in particular DNA methylation at so-called CpG sites (Arthritis & Rheumatism 52:3110–24). In OA, chondrocytes increasingly produce the enzymes and stably transmit the abnormal gene expression to daughter cells. This aberrant expression has been shown to be associated with an epigenetic “un-silencing” via demethylation of specific CpG sites within the promoter regions. Why and how this demethylation takes place is not known. The pro-inflammatory cytokine IL-1beta is of potential importance in OA, where temporary synovitis could provide the cytokine. Moreover, it is well established that IL-1beta upregulates MMPs in chondrocyte monolayer cultures. We investigated whether the IL-1 mediated induction of MMPs was associated with DNA demethylation. Control chondrocytes were isolated from non-OA articular cartilage, obtained with ethical permission from patients with a femoral neck fracture, and expanded in monolayer culture. The cells from each patient were divided into pre-culture control, no-treatment control culture and IL-1 treated culture. When confluent, simultaneous RNA and DNA extraction was carried out. mRNA expression was analysed by RT-PCR and the methylation status of specific CpG sites within the promoters of MMP-3, -13, and IL-1â was determined in the same samples, using methylation-sensitive restriction enzymes and PCR. The pre-culture controls expressed type II collagen and low levels of MMP-3, but not MMP-13 nor IL-1beta. All IL-1 treated samples expressed high levels of MMP-3, -13, and, surprisingly, IL-1beta itself. As predicted, the large increases in MMP-3 and IL-1beta were associated with some loss of methylation at specific CpG sites in the promoter of these mediators with the strongest correlation between IL-1beta expression and promoter demethylation. IL-1beta thus induced its own expression, which was associated with loss of DNA methylation at one specific CpG site in the IL-1 promoter. If these in vitro results have relevance for the in vivo situation, then these findings suggest the following mechanisms for OA progression: An initial inflammatory episode in the synovium could induce IL-1beta in surface chondrocytes. Since this induction is associated with loss of DNA methylation, IL-1beta is now part of the expression repertoire of these chondrocytes and this abnormal expression is stably transmitted to daughter cells. IL-1 then could diffuse deeper into the cartilage to induce its own expression in adjacent chondrocytes, thus providing a continuous supply of IL-1beta even after synovial inflammation had abated. This may explain the unremitting progression of OA

Introduction: Modic changes are vertebral endplate changes visible in magnetic resonance imaging (MRI), which associate with degenerative intervertebral disc disease. Twin studies suggest that intervertebral disc degeneration and low back pain may be primarily explained by genetic factors. There are, however, no studies on genetic factors in Modic changes. Materials and methods: Eleven variations in eight genes (COL9A2, COL9A3, COL11A2, IL1A, IL1B, IL6, MMP-3 and VDR) were genotyped in an occupational cohort of 159 male train engineers and 69 male paper mill workers. All the study subjects were MRI scanned and evaluated for Modic changes. Results: Out of 228 subjects studied, 128 (56%) were found to have Modic change at one or more disc levels. 15% of them had exclusively Modic type I while 32% had exclusively Modic II changes. 10% of the subjects had both type I and type II changes. When single nucleotide polymorphisms (SNPs) were analyzed independently, none of them significantly associated with Modic changes. However, when the gene-gene interactions were evaluated IL1A and MMP-3 polymorphisms together associated with type II Modic changes (OR 3.2, 95% CI 1.2–8.5; p = 0.038). Furthermore, IL-1 gene cluster together with MMP-3 polymorphism associated significantly with type II Modic changes (OR = 8.14, 95% CI 1.72–38.44; p = 0.008). Discussion: This is the first study evaluating the role of genetic factors in relation to Modic changes. Genetic variations in IL-1 cluster and MMP-3 gene were found together to associate significantly with type II Modic changes

Introduction: After a meniscus trauma, preservation of the meniscus is the most important surgical goal. The use of scaffolds colonized with meniscus cells (fibrochondrocytes) to reconstruct meniscal defects seem to be a promising way for the treatment of a meniscus trauma. The goal of our investigations was the analysis of expression of different anabolic and catabolic factors in human fibrochondrocytes after seeding these cells onto a collagen I scaffold to investigate the regenerative potential of such a construct for the treatment of meniscus tears. Material and Methods: Human meniscus tissue was digested in collagenase and dispase and cells were characterized by immunohistochemistry. To test scaffolds, we used a commercially available bovine collagen I matrix approved for surgical purposes. The scaffold was colonized with human fibrochondrocytes in a density of 106 cells per cm2. Cells expanded at the same ínoculation density w/o scaffold served as mock-controls. After 14 and 28 days in culture, the cells were extracted from the scaffold by aid of collagenase (Sigma, Deisenhofen, FGR) and analyzed for the expression of different factors, including IL-1β, IL-6, TGF-β, TIMP-1, TIMP-3, MMP-1, and MMP-3 using a quantitative RT-PCR-technology. Results: Bovine collagen I matrices could be colonized with human fibrochondrocytes. After 14 and 28 days of incubation on the scaffolds, the cells show the same mRNA expression levels of IL-1β, TIMP-1, TIMP-3, and TGF-β when compared to controls. In contrast, after 14 days IL-6 (12.7-fold ± 4.4, p< 0.001), MMP-1 (11.3-fold ± 2.4, p< 0.001), and MMP-3 (13.7-fold ± 6.8, p< 0.031) were upregulated on transcription levels in the scaffold when compared to controls after the same period of culture. After 28 days of culture in scaffold the expression of MMP-3 was upregulated 78.2-fold (± 7.4, p< 0.0001), MMP-1 (71.3-fold ± 5.9, p< 0.0001) and IL-6 was elevated 98.9-fold (± 9.1, p< 0.0001) compared to controls. Discussion/Conclusion: We were able to cultivate and characterize human fibrochondrocytes from menisci of the knee joint colonized onto a bovine collagen I matrix. We could show that meniscus cells revealed a significantly increased expression of MMP-1 and MMP-3, and also a significant elevation of IL-6 mRNA after 14 and 28 days of culture. No changes were found in the expression levels of IL-1β, TGF-β, and the TIMPs. This suggests that the meniscus cells colonized onto a bovine collagen I scaffold produce a considerable amount of catabolic or inflammatory factors. This may lead to a destruction of the scaffold-matrix itself and the extracellular matrix of the meniscus. Secondly, IL-6 could induce a global inflammation around the scaffold by activating the IL-6 inflammation cascade

Idiopathic osteoarthritis (OA) is a complex, late-onset disease whose causes are still unknown. In spite of tremendous efforts, the search for the genes pre-disposing towards osteoarthritis has so far met with little success. We hypothesize that epigenetic changes play a major role in the pathology of OA. Epigenetics refers to stable, heritable, but potentially reversible modifications of gene expression that do not involve mutations in the DNA sequence, for example DNA methylation or histone modification. Epigenetic changes are gene and cell-type specific, may arise sporadically with increasing age or be provoked by environmental factors. To investigate whether epigenetic changes are significant factors in OA, we examined the DNA methylation status of the promoter regions of three genes that are expressed by OA, but not by normal, articular chondrocytes, namely MMP-3 (stromelysin-1), MMP-9 (gelatinase B) and MMP-13 (collagenase3). We hypothesized that these genes are silenced in normal chondrocytes by methylation of the cytosines of CpG dinucleotides in the respective promoter regions, but that abnormal expression is associated with a de-methylation, leading to eunsilencing f of gene expression. Cartilage was obtained from the femoral heads of 16 OA and 10 femoral neck fracture (#NOF) patients, which served as controls due to the inverse relationship between osteoporosis and OA. The cartilage was milled in a freezer mill with liquid nitrogen, DNA was extracted with a Qiagen kit, digested with methylation sensitive restriction enzymes, followed by PCR amplification. These enzymes will cut at their specific cleavage sites only if the CpGs is not methylated and thus allow us to determine methylation status of specific CpG sites. Results. Less than 5% of the chondrocytes in superficial layer from #NOF cartilage expressed degradative enzymes, whereas all cloned chondrocytes from advanced-stage OA cartilage were immunopositive. The overall % of CpG demethylation in the promoters of control patients (whose chondrocytes did not express the enzymes) was 20.1%, whereas 48.6% of CpG sites were demethylated in degradative chondrocytes of OA patients (p< 0.001). For MMP-13, the increase in demethylation between control and OA was from 4 . . 20%; for MMP-9 from 47 . . 81% and for MMP-3 from 30 . . 57%. However, not all available CpG sites were equally demethylated. Some sites were uniformly methylated in both OA and controls, others were demethylated even in controls. However, there was at least one crucial site for each degradative enzyme, where the differences in the degree of methylation were greatest and statistically different. These sites were at −110 for MMP-13; −36 for MMP-9; −635 for MMP-3. There was no relation between the % demethylation and the patient fs age and no apparent difference between males and females. Conclusions: We have demonstrated an association between abnormal gene expression of MMP-3, MMP-9 and MMP-13 and promoter DNA demethylation. This epigenetic dysregulation of genes appeared to be clonally inherited by daughter cells and may be typical for osteoarthritis and other complex, late-onset diseases

Introduction. Osteoarthritis (OA) has historically been thought of as a degenerative joint disease, but inflammation and angiogenesis are increasingly being recognised as contributing to the pathogenesis, symptoms and progression of OA. b-dystroglycan (b-DG) is a pivotal element of the transmembrane adhesion molecule involved in cell-extracellular matrix adhesion and angiogenesis. Matrix metalloproteinases (MMPs) are the main enzymes responsible for cartilage extracellular matrix breakdown and are also implicated in both angiogenesis and b-DG degradation in a number of malignancies. We aimed to investigate the expression and localisation of b-DG and MMP-3, -9, and -13 within cartilage, synovium and synovial fluid and establish their roles in the pathogenesis of OA. Methods. Following ethical committee approval, cartilage, synovium and synovial fluid were obtained from the hip joints of 5 osteoarthritic (patients undergoing total hip replacement) and 5 control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for b-DG expression using Western Blotting and for the distribution of b-DG, MMP-3, -9, and -13 using immunohistochemistry on paraffin embedded tissue. Results. Whilst no significant expression of b-DG was found in cartilage or synovial fluid, b-DG was expressed in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Moreover, b-DG was expressed in endothelium of blood vessels of OA synovium, but not in the normal endothelium. In the endothelium of osteoarthritic synovial blood vessels, b-DG co-localised with MMP -3 and -9. Discussion. Our results demonstrate that b-DG does not act as a cell adhesion molecule binding chondrocytes to the ECM. However, specific immunolocalisation of b-DG within endothelium of inflamed OA blood vessels suggests that b-DG may play a role in angiogenesis associated with OA. Its co-localisation with MMP-3 and -9, previously reported to also have pro-angiogenic roles, may be linked. Further research is required to understand these roles more fully