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.

Aims. Femoroacetabular impingement (FAI) is a potential cause of hip osteoarthritis (OA). The purpose of this study was to investigate the expression profile of matrix metalloproteinases (MMPs) in the labral tissue with FAI pathology. Methods. In this study, labral tissues were collected from four FAI patients arthroscopically and from three normal hips of deceased donors. Proteins extracted from the FAI and normal labrums were separately applied for MMP array to screen the expression of seven MMPs and three tissue inhibitors of metalloproteinases (TIMPs). The expression of individual MMPs and TIMPs was quantified by densitometry and compared between the FAI and normal labral groups. The expression of selected MMPs and TIMPs was validated and localized in the labrum with immunohistochemistry. Results. On MMP arrays, most of the targeted MMPs and TIMPs were detected in the FAI and normal labral proteins. After data normalization, in comparison with the normal labral proteins, expression of MMP-1 and MMP-2 in the FAI group was increased and expression of TIMP-1 reduced. The histology of the FAI labrum showed disorderly cell distribution and altered composition of thick and thin collagen fibres. The labral cells expressing MMP-1 and MMP-2 were localized and their percentages were increased in the FAI labrum. Immunohistochemistry confirmed that the percentage of TIMP-1 positive cells was reduced in the FAI labrum. Conclusion. This study established an expression profile of MMPs and TIMPs in the FAI labrum. The increased expression of MMP-1 and MMP-2 and reduced expression of TIMP-1 in the FAI labrum are indicative of a pathogenic role of FAI in hip OA development. Cite this article:Bone Joint Res. 2020;9(4):173–181

Objectives. Re-rupture is common after primary flexor tendon repair. Characterization of the biological changes in the ruptured tendon stumps would be helpful, not only to understand the biological responses to the failed tendon repair, but also to investigate if the tendon stumps could be used as a recycling biomaterial for tendon regeneration in the secondary grafting surgery. Methods. A canine flexor tendon repair and failure model was used. Following six weeks of repair failure, the tendon stumps were analyzed and characterized as isolated tendon-derived stem cells (TDSCs). Results. Failed-repair stump tissue showed cellular accumulation of crumpled and disoriented collagen fibres. Compared with normal tendon, stump tissue had significantly higher gene expression of collagens I and III, matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF). The stump TDSCs presented both mesenchymal stem and haematopoietic cell markers with significantly increased expression of CD34, CD44, and CD90 markers. Stump TDSCs exhibited similar migration but a lower proliferation rate, as well as similar osteogenic differentiation but a lower chondrogenic/adipogenic differentiation capability, compared with normal TDSCs. Stump TDSCs also showed increasing levels of SRY-box 2 (Sox2), octamer-binding transcription factor 4 (Oct4), tenomodulin (TNMD), and scleraxis (Scx) protein and gene expression. Conclusion. We found that a failed repair stump had increased cellularity that preserved both mesenchymal and haematopoietic stem cell characteristics, with higher collagen synthesis, MMP, and growth factor gene expression. This study provides evidence that tendon stump tissue has regenerative potential. Cite this article: C-C. Lu, T. Zhang, R. L. Reisdorf, P. C. Amadio, K-N. An, S. L. Moran, A. Gingery, C. Zhao. Biological analysis of flexor tendon repair-failure stump tissue: A potential recycling of tissue for tendon regeneration. Bone Joint Res 2019;8:232–245. DOI: 10.1302/2046-3758.86.BJR-2018-0239.R1

Previous studies showed that telo-peptides degraded from type II collagen, a type of collagen fragments, could induce cartilage damage in bovine stifle joints. We aim to investigate the role of integrins (ITGs) and matrix metalloproteinases (MMPs) in collagen fragment-induced human cartilage damage that is usually observed in osteoarthritis (OA). We hypothesized that N-telopeptide (NT) derived from type II collagen could up-regulate the expression of β1 integrin (ITGB1) and then MMPs that may lead to osteoarthritic cartilage damage. Human chondrocytes were isolated from femoral head or tibial plateau of patients receiving arthroplasty (N = 24). Primary chondrocyte cultures were either treated with 30 µM NT, or 30 µM scrambled NT (SN), or PBS, or left untreated for 24 hrs. Total proteins and RNAs were extracted for examination of expression of ITGB1 and MMPs-3&13 with Western blotting and quantitative real-time PCR. Compared to untreated or PBS treated chondrocytes, NT-treated chondrocytes expressed significantly higher levels of ITGB1 and MMPs-3&-13. However, SN also up-regulated expression of ITGB1 and MMP-13. ITGB1 and MMPs-3&-13 might mediate the catalytic effect of NT, a type of collagen fragments, on human cartilage damage that is a hallmark of OA

Introduction. Within articular cartilage, chondrocytes reside within the pericellular matrix (PCM), collectively constituting the microanatomical entity known as a chondron. The PCM functions as a pivotal protective shield and mediator of biomechanical and biochemical cues. In the context of Osteoarthritis (OA), enzymatic degradation of the PCM is facilitated by matrix metalloproteinases (MMPs). This study delves into the functional implications of PCM structural integrity decline on the biomechanical properties of chondrons and impact on Ca. 2+. signaling dynamics. Method. Chondrons isolated from human cartilage explants were incubated with activated MMP-2, -3, or -7. Structural degradation of the pericellular matrix (PCM) was assessed by immunolabelling (collagen type VI and perlecan, n=5). Biomechanical properties of chondrons (i.e. elastic modulus (EM)) were analyzed using atomic force microscopy (AFM). A fluorescent calcium indicator (Fluo-4-AM) was used to record and quantify the intracellular Ca. 2+. influx of chondrons subjected to single cell mechanical loading (500nN) with AFM (n=7). Result. Each of the three MMPs disrupted the structural integrity of the PCM, leading to attenuated fluorescence intensity for both perlecan and collagen VI. A significant decrease of EM was observed for all MMP groups (p<0.005) with the most notable decrease observed for MMP-2 and MMP-7 (p<0.001). In alignment with the AFM results, there was a significant alteration in Ca. 2+. influx observed for all MMP groups (p<0.05), in particular for MMP-2 and MMP-7 (p<0.001). Conclusion. Proteolysis of the PCM by MMP-2, -3, and -7 not only significantly alters the biomechanical properties of articular chondrons but also affects their mechanotransduction profile and response to mechanical loading, indicating a close interconnection between these processes. These findings underscore the influence of an intact pericellular matrix (PCM) in protecting cells from high stress profiles and carry implications for the transmission of mechanical signaling during OA onset and progression

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)

Purpose: Articular cartilage is a physiologically hypoxic tissue with a gradient of oxygen tension ranging from about 10% oxygen at the cartilage surface to less than 1% in the deepest layers. The overall goal of the study was to determine whether an injectable allogeneic/autologous fibrin scaffolds in combination with mesenchymal stem cells (MSCs) is suitable for articular cartilage tissue engineering, and to determine the effect of hypoxic culture conditions on the stability of cell-fibrin scaffolds. The secondary goal was to enhance the accumulation of extracellular matrix (ECM) inside the fibrin scaffold under these conditions. Method: Chondroprogenitor clonal cell line RCJ3.1C5.18 (C5.18) and human mesenchymal stem cells (hMSCs) were encapsulated in fibrin hydrogel and fibrin glue scaffolds. The stabilization of fibrin scaffolds and development of ECM components were evaluated using zymography, SDS-polyacrylamide electrophoresis (SDS-PAGE), immunochemistry, spectrophotometry, RT-PCR including real time and histology (. Ahmed TA., et al. . Tissue Engineering. 2007. ;. 13. (7): . 1469. –77. ). Results: After encapsulation of C5.18 and hMSCs, fibrin gels quickly degraded under normoxic conditions (21 % oxygen) due to upregulation of plasminogen and matrix metalloproteinases (MMPs) genes especially MMP-2, -3, and -9. Protease inhibitors such as aprotinin and galardin (GM6001), in combination or separately, prevented the fibrin-C5.18 hydrogels breakdown for up to 5 weeks. Only a combination of aprotinin and galardin resulted in accumulation of ECM components such as collagen II and aggrecan. In contrast, fibrin-hMSCs hydrogels were found to be stable under hypoxic conditions (5% O2) for up to 4 weeks in the absence of inhibitors, suggesting that hypoxic conditions may downregulate the expression of the enzymes responsible for fibrin-hydrogel breakdown. Conclusion: These results suggest that in C5.18 and MSCs cell lines, expression of matrix metalloproteinases (MMPs) and plasmin is upregulated under normoxic conditions and is responsible for fibrin-hydrogel breakdown. Moreover, inhibition of both proteases is required to enhance the accumulation of ECM. However, fibrin hydrogel scaffolds were stabilized under low oxygen tension, which is more physiological than normoxia and therefore these constructs may be stable after implantation in the absence of protease inhibitors

The in situ increased production of matrix metalloproteinases (MMPs) has been associated with the development of periprosthetic osteolysis. The aim of the study was to compare the effect of Co. 2+. and Cr. 3+. ions on macrophages matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP (TIMP-1) expression. Using reverse transcription-polymerase chain reaction (RT-PCR), we showed that both Co. 2+. and Cr. 3+. ions induce the expression of MMP-1 and TIMP-1 in a dose-dependent manner. Since MMP-1 and TIMP-1 participate in the extracellular matrix degradation and tissue remodeling, our results suggest that the modulation of MMP-1 and TIMP-1 may contribute to the development of periprosthetic osteolysis. The in situ increased production of matrix metalloproteinases (MMPs) has been associated with the development of periprosthetic osteolysis. Aseptic loosening due to periprosthetic osteolysis is the major cause of total hip arthroplasty failure. Because of their potential for improved wear performance, there has been a revived interest in metal-metal bearings, made of cobalt-chromium-molybdenum alloys. However, metal particle and ion toxicity remains a major cause for concern. The aim of the study was to determine the effects of Co. 2+. and Cr. 3+. ions on the expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1), two proteins participating in the extracellular matrix degradation and tissue remodeling. Human U937 macrophages were incubated with Co. 2+. and Cr. 3+. ions. The expression of MMP-1 and TIMP-1 mRNAs was measured by reverse transcription-polymerase chain reaction (RT-PCR) and calculated as the ratio of the house keeping gene GAPDH expression. Results show that both Co. 2+. and Cr. 3+. ions induced in a dose-dependent manner the expression of PCR products (mRNAs) of MMP-1 (135 bp) and TIMP-1 (328 bp). Co. 2+. ions were more effective in inducing MMP-1 and TIMP-1 expression than Cr. 3+. ions. The induction of MMP-1 and TIMP-1 paralleled the induction of TNF-α mRNA expression. Our results demonstrate that the expression of MMP-1 and TIMP-1 were up regulated by incubating macrophages with Co. 2+. and Cr. 3+. ions, suggesting that metal ions contribute to tissue damage in the periprosthetic environment and that variations in MMP-1 and TIMP-1 expression may contribute to periprosthetic osteolysis

Intervertebral disc degeneration (IDD) is a major cause of low back pain, which affects 80% of the adult population at least once in their life. The pathophysiological conditions underlying IDD are still poorly understood. Genetic makeup, aging, smoking, physical inactivity and mechanical overloading, especially due to obesity, are among the strongest risk factors involved. Moreover, IDD is often associated with chronic inflammation within disc tissues, which increases matrix breakdown, glycosaminoglycan (GAG) loss and cell death. This micro-inflammatory environment is typical of several metabolic disorders, including diabetes mellitus (DM). As the etiopathogenesis of IDD in diabetic subjects remains scarcely understood, we hypothesised that this may be driven by a DM-induced inflammation leading to a combination of reduced GAG levels, decreased proteoglycan synthesis and increased matrix breakdown within the disc. The objective of the study was to investigate the pathogenesis of IDD in a murine model of type 1 DM (T1DM), namely non-obese diabetic (NOD) mouse. Total disc glycosaminoglycan (GAG) content, proteoglycan synthesis, aggrecan fragmentation mediated by matrix metalloproteinases (MMPs) and a Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS), glucose transporter (mGLUT1) gene expression and apoptosis (TUNEL assay) were assessed in NOD mice and wild-type euglycemic control mice. Spinal structural and molecular changes were analysed by micro-computed tomography (mCT), histological staining (Safranin-O and fast green) and quantitative immunofluorescence (anti-ADAMTS-4 and 5 antibodies). Statistical analysis was conducted considering the average of 35 samples ± standard error for each measurement, with 95% confidence intervals calculated to determine statistical significance (p-value < 0.05). IVDs of NOD mice showed increased disc apoptosis (p < 0.05) and higher aggrecan fragmentation mediated by ADAMTS (p < 0.05). However, ADAMTS-4 and −5 did not appear to be involved in this process. The total GAG content normalized to DNA and PG synthesis showed no statistically significant alterations, as well as Safranin O staining. Although not significantly, NOD mice showed reduced glucose uptake. In addition, the vertebral structure of NOD mice at mCT seemed not to be altered. These data demonstrate that DM may contribute to IDD by increasing aggrecan degradation and promoting cell apoptosis, which may represent early indicators of the involvement of DM in the pathogenesis of IDD

After anterior cruciate ligament (ACL) rupture, reconstructive surgery with a hamstring tendon autograft is often performed. Despite overall good results, ACL re-rupture occurs in up to 10% of the patient population, increasing to 30% of the cases for patients aged under 20 years. This can be related to tissue remodelling in the first months to years after surgery, which compromises the graft's mechanical strength. Resident graft fibroblasts secrete matrix metalloproteinases (MMPs), which break down the collagen I extracellular matrix. After necrosis of these fibroblasts, myofibroblasts repopulate the graft, and deposit more collagen III rather than collagen I. Eventually, the cellular and matrix properties converge towards those of the native ACL, but full restoration of the ACL properties is not achieved. It is unknown how inter-patient differences in tissue remodelling capacity contribute to ACL graft rupture risk. This research measured patient-specific tissue remodelling-related properties of human hamstring tendon-derived cells in an in vitro micro-tissue platform, in order to identify potential biological predictors for graft rupture. Human hamstring tendon-derived cells were obtained from remnant autograft tissue after ACL reconstructions. These cells were seeded in collagen I gels on a micro-tissue platform to assess inter-patient cellular differences in tissue remodelling capacity. Remodelling was induced by removing the outermost micro-posts, and micro-tissue compaction over time was assessed using transmitted light microscopy. Protein expression of tendon marker tenomodulin and myofibroblast marker α-smooth muscle actin (αSMA) were measured using Western blot. Expression and activity of remodelling marker MMP2 were determined using gelatin zymography. Cells were obtained from 12 patients (aged 12–51 years). Patient-specific variations in micro-tissue compaction speed or magnitude were observed. Up to 50-fold differences in αSMA expression were found between patients, although these did not correlate with faster or stronger compaction. Surprisingly, tenomodulin was only detected in samples obtained from two patients. Total MMP2 expression varied between patients, but no large differences in active fractions were found. No correlation of patient age with any of the remodelling-related factors was detected. Remodelling-related biological differences between patient tendon-derived cells could be assessed with the presented micro-tissue platform, and did not correlate with age. This demonstrates the need to compare this biological variation in vitro - especially cells with extreme properties - to clinical outcome. Sample size is currently increased, and patient outcome will be determined. Combined with results obtained from the in vitro platform, this could lead to a predictive tool to identify patients at risk for graft rupture

Human synovium harbours macrophages and T-cells that secrete inflammatory cytokines, stimulating chondrocytes to release proteinases like aggrecanases and matrix metalloproteinases (MMPs) during the development of Osteoarthritis (OA). Inflammation of the synovium is a key feature of OA, linked to several clinical symptoms and the disease progression. As a prelude to testing in an OA mouse model, we have used the tetracycline system (Tet) to modify mouse mesenchymal stem cells (mMSCs) to over-express viral interleukin 10 (vIL10), an anti-inflammatory cytokine, to modulate the osteoarthritic environment and prevent disease development. MSCs isolated from the marrow of C57BL/6J mice expressed CD90.2, SCA-1, CD105, CD140a, and were negative for CD34, CD45 and CD11b by flow cytometry. Adenoviral transduction of MSCs carrying CMVIL10 and TetON as test, and untransduced, AdNull and TetOFF as negative controls was successful and tightly controlled vIL10 production was demonstrated by CMVIL10 and TetON MSCs using a vIL10 ELISA kit. Co-incubation of vIL10MSC CM with lipopolysaccharide activated bone-marrow derived murine macrophages (BMDMs) resulted in reduction of TNF-α, IL-6 levels and elevated production of IL-10 by ELISA and high iNOS release by Griess assay. Co-culture of active macrophages with TetON MSCs, resulted in polarisation of macrophage cell population from M1 to M2 phase, with decrease in pro-inflammatory MHC-II (M1 marker) and increase in regulatory CD206 (M2 marker) expression over time. The PCR profiler array on MSC CM treated BMDMs, also showed changes in gene expression of critical pro-inflammatory cytokines and receptors involved in the TLR4 pathway. The biscistronic TetON transduced MSCs proved to be most immuno-suppressive and therefore feasible as efficient anti-inflammatory therapy that can utilised in vivo

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

Osteoarthritis (OA) is a debilitating disease and the most common joint disorder worldwide. Although the development of OA is considered multifactorial, the mechanisms underlying its initiation and progression remain unclear. A prominent feature in OA is cartilage degradation typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II). Cartilage homeostasis is maintained by the anabolic and catabolic activities of chondrocytes. Prolonged exposure to stressors such as mechanical loading and inflammatory cytokines can alter the phonotype of chondrocytes favoring cartilage catabolism, and occurs through decreased matrix protein synthesis and upregulation of catabolic enzymes such as aggrecanases (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). More recently, the endoplasmic reticulum (ER) stress response has been implicated in OA. The ER-stress response protects the cell from misfolded proteins however, excessive activation of this system can lead to chondrocyte apoptosis. Acute exposure of chondrocytes to IL-1β has been demonstrated to upregulate ER-stress markers (GADD153 and GRP78), however, it is unclear whether the ER-stress response plays a role on chronic IL-1β exposure. The purpose of this study was to determine whether modulating the ER stress response with tauroursodeoxycholic acid (TUDCA) in human OA chondrocytes during prolonged IL-1β exposure can alter its catabolic effects. Articular cartilage was isolated from donors undergoing total hip or knee 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-low glucose supplemented with 10% FBS. Chondrocytes were expanded in flasks for one passage before being prepared for micropellet culture. Chondrocyte pellets were cultured in regular growth medium (Control), medium supplemented with IL-1β [10 ng/mL], TUDCA [100 uM] or IL-1β + TUDCA for 12 days. Medium was replaced every three days. Cartilage explants were prepared from the donors undergoing knee replacement, and included cartilage with the cortical bone approximately 1 cm2 in dimension. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. RNA was extracted using Geneaid RNA Mini Kit for Tissue followed by cDNA synthesis. QPCR was performed using Cyber Green mastermix and primers for the following genes: ACAN (aggreacan), COL1A1, COL2A1, COL10A1, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13, on an ABI 7500 fast qPCR system. Although IL-1β did not significantly decrease the expression of matrix proteins, it did increase the expression of ADAMTS-4, −5, and MMP3 and −13 when compared to controls (Kruskal-Wallis, p < 0 .05, n=3). TUDCA treatment alone did not significantly increase the expression of catabolic enzymes but it did increase the expression of collagen type II. When IL-1β was coincubated with TUDCA, the expression of ADAMTS-4, ADAMTS-5, and MMP-13 significantly decreased by ∼40-fold, ∼10-fold, and ∼3-fold, respectfully. We provide evidence that the catabolic activities of IL-1β on human cartilage can be abrogated through modulation of the ER stress response

Objectives. Osteoarthritis (OA) is the most common form of arthritis, affecting approximately 15% of the human population. Recently, increased concentration of nitric oxide in serum and synovial fluid in patients with OA has been observed. However, the exact role of nitric oxide in the initiation of OA has not been elucidated. The aim of the present study was to investigate the role of nitric oxide in innate immune regulation during OA initiation in rats. Methods. Rat OA was induced by performing meniscectomy surgery while cartilage samples were collected 0, 7, and 14 days after surgery. Cartilage cytokine levels were determined by using enzyme-linked immunosorbent assay, while other proteins were assessed by using Western blot. Results. In the time course of the study, nitric oxide was increased seven and 14 days after OA induction. Pro-inflammatory cytokines including tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were decreased. L-NG-Nitroarginine methyl ester (L-NAME, a non-specific nitric oxide synthase inhibitor) significantly decreased cartilage nitric oxide and blocked immune suppression. Further, L-NAME decreased Matrix metalloproteinase (MMPs) and increased tissue inhibitor of metalloproteinase (TIMP) expression in meniscectomised rats. Conclusion. Nitric oxide-dependent innate immune suppression protects cartilage from damage in the early stages of OA initiation in rats. Cite this article: C-C. Hsu, C-L. Lin, I-M. Jou, P-H. Wang, J-S. Lee. The protective role of nitric oxide-dependent innate immunosuppression in the early stage of cartilage damage in rats: Role of nitric oxide in ca rtilage da mage. Bone Joint Res 2017;6:253–258. DOI: 10.1302/2046-3758.64.BJJ-2016-0161.R1

Cartilage calcification induces the synthesis of degrading enzymes, such as matrix metalloproteinases (MMPs) and prostaglandin E2 leading to tissue degeneration. The aim of the study was to investigate the effect of vitamin D on the calcification process in osteoarthritic cartilage. We evaluated the effect of vitamin D on klotho (KL), Fibroblast Growth Factor 23 (FGF23) and Fibroblast Growth Factor Receptor 1c (FGFR1c) mRNA and protein expression levels by real-time PCR and western blot analysis, respectively. Possible interactions between klotho and FGF23 on the receptor FGFR1c in normal chondrocytes were investigated using immunoprecipitation assay. The direct effect of 1,25 dihydroxyvitamin D3 (1,25D) on KL, FGF23 and FGFR1c promoter was also evaluated. We found that FGF23 and FGFR1c mRNA expression levels were significantly increased in osteoarthritic chondrocytes compared to normal, while KL mRNA levels were decreased (p=0.001 for all genes). We showed that klotho-FGF23-FGFR1c form complexes in normal chondrocytes and confirmed the participation of klotho in the initiation of FGF23-FGFR1c signalling. Treatment of normal chondrocytes with 1,25D resulted in a significant dose and time dependent increase of FGF23 and FGFR1c mRNA levels and in an increase of KL mRNA levels in osteoarthritic chondrocytes compared to untreated (p=0.001). We revealed, for the fist time, the presence of conserved, canonical VDREs in the proximal promoters of KL, FGF23 and FGFR1c. We propose a common regulatory scheme of mineral homeostasis and aging in osteoarthritic chondrocytes evidenced by the positive/negative feedback actions by KL, FGF23, FGFR1c and 1,25D, through binding of vitamin D receptor (VDR) on the promoters of the above mentioned genes

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

Matrix metalloproteinase enzymes (MMPs) play a crucial role in the remodeling of articular cartilage, contributing also to osteoarthritis (OA) progression. The pericellular matrix (PCM) is a specialized space surrounding each chondrocyte, containing collagen type VI and perlecan. It acts as a transducer of biomechanical and biochemical signals for the chondrocyte. This study investigates the impact of MMP-2, -3, and -7 on the integrity and biomechanical characteristics of the PCM. Human articular cartilage explants (n=10 patients, ethical-nr.:674/2016BO2) were incubated with activated MMP-2, -3, or -7 as well as combinations of these enzymes. The structural degradative effect on the PCM was assessed by immunolabelling of the PCM's main components: collagen type VI and perlecan. Biomechanical properties of the PCM in form of the elastic moduli (EM) were determined by means of atomic force microscopy (AFM), using a spherical cantilever tip (2.5µm). MMPs disrupted the PCM-integrity, resulting in altered collagen type VI and perlecan structure and dispersed pericellular arrangement. A total of 3600 AFM-measurements revealed that incubation with single MMPs resulted in decreased PCM stiffness (p<0.001) when compared to the untreated group. The overall EM were reduced by ∼36% for all the 3 individual enzymes. The enzyme combinations altered the biomechanical properties at a comparable level (∼36%, p<0.001), except for MMP-2/-7 (p=0.202). MMP-induced changes in the PCM composition have a significant impact on the biomechanical properties of the PCM, similar to those observed in early OA. Each individual MMP was shown to be highly capable of selectively degrading the PCM microenvironment. The combination of MMP-2 and -7 showed a lower potency in reducing the PCM stiffness, suggesting a possible interplay between the two enzymes. Our study showed that MMP-2, -3, and -7 play a direct role in the functional and structural remodeling of the PCM. Acknowledgements: This work was supported by the Faculty of Medicine of the University of Tübingen (grant number.: 2650-0-0)

Summary Statement. Intra-articular injection of humanised monoclonal anti-VEGF antibody (Bevacizumab, Avastin®) in a osteoarthritis rabbit model is related to positive restorative effects in terms of histopathologic evaluation. Introduction. Vascular endothelial growth factor (VEGF) is generally undetectable in adult human articular cartilage under physiological conditions. Upon exposure to pathological stimulation such as inflammation, hypoxia or accumulating mechanical stress, VEGF would be up regulated in hypertrophic chondrocytes of arthritic cartilage leading to osteophyte formation, disregulation of chondrocyte apoptosis and induction of catabolic factors, including matrix metalloproteinases (MMPs). This in vivo study aims to investigate the potential role of VEGF inhibition to treat Osteoarthritis (OA), through intra-articular injection of Bevacizumab, a humanised monoclonal anti-VEGF antibody, in a OA rabbit model. Methods. OA was induced in twelve adult male New Zealand rabbits surgically by monolateral Anterior Cruciate Ligament Transection (ACLT). The rabbits were randomly divided into two equal groups (experimental and control). Intra-articular injections of Bevacizumab or saline (control) were given 4 weeks after ACLT and were administered once a week for 4 time. Animal were sacrificed at 2 and 3 month time point an knee analyzed histologically and grossly. Histopathological variables such as the number of fibroblasts and inflammatory cells, collagenous matrix deposition, synovial hyperplasia, granulation tissue formation, vascular proliferation were evaluated. Results:The macroscopic evaluation of the knee in the experimental group revealed smooth joint surfaces of articular cartilage and no osteophyte formation compared to the control group that showed marked arthritis including synovial hypertrophy and osteophyte formation. Histologic assessment demonstrated, in the experimental group, significantly higher scores concerning number of microvessels, synovial hyperplasia, macrophage infiltration, collagenous matrix deposition, chondrocytes proliferation and apoptosis compared to the control group. Conclusion. In conclusion, VEGF modulation via intra-articular injection of Bevacizumab in a rabbit model of knee OA, resulted in reduction of articular cartilage degeneration through setting up an appropriate environment that prevent chondrocyte hypertrophy, apoptosis and osteophytes formation by blocking the intrinsic VEGF catabolic pathway, endochondral ossification, and the extrinsic VEGF-induced vascular invasion. VEGF-signaling inhibtion through Bevacizumab represent a potential way to treat OA

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

The purpose of this study was to profile the mRNA expression for the 23 known matrix metalloproteinases (MMPs), 4 tissue inhibitor of metalloproteinases (TIMPs) and 19 ADAMTSs (a disintegrin and metalloproteinase with thrombospontin motif) in Dupuytren's Disease and normal palmar fascia. Dupuytren's Disease (DD) is a fibroproliferative disorder affecting the palmar fascia, leading to contractures. The MMPs and ADAMTSs are related enzymes collectively responsible for turnover of the extracellular matrix. The balance between the proteolytic action of the MMPs and ADAMTSs and their inhibition by the TIMPs underpins many pathological processes. Deviation in favour of proteolysis is seen in e.g. invasive carcinomata, whereas an imbalance towards inhibition causes e.g. fibrosis. A group of patients with end-stage gastric carcinoma was treated with a broad spectrum MMP inhibitor in an attempt to reduce the rate of carcinoma advancement; a proportion developed a ‘musculoskeletal syndrome’ resembling DD. Tissue samples were obtained from patients undergoing surgery to correct contractures caused by DD and from healthy controls undergoing carpal tunnel decompression. The DD tissue was separated macroscopically into cord and nodule. Total RNA was extracted and mRNA expression analysed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), normalised to 18S rRNA. Comparing across all genes, the DD nodule, DD cord and normal palmar fascia samples each had a distinct mRNA expression profile. Statistically significant (p<0.05) differences in mRNA expression included: higher MMP-2, -7 and ADAMTS-3 levels in both cord and nodule; higher MMP-1, -14, TIMP-1 and ADAMTS-4 and -5 in nodule alone, lower MMP-3 in nodule and cord and lower TIMP-2, -3 and -4 and ADAMTS-1 and -8 levels in nodule alone. The distinct mRNA profile of each group suggests differences in extracellular proteolytic activity which may underlie the process of fascial remodelling in DD