Clusterin secretion is attenuated by the proinflammatory cytokines interleukin‐1β and tumor necrosis factor‐α in models of cartilage degradation

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro-death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine-stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real-time polymerase chain reaction. MMP-3, MMP-13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down-regulated 7-days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker

Chondrogenic differentiation of human chondrocytes cultured in the absence of ascorbic acid

Bioreactor systems will likely play a key role in establishing regulatory compliant and cost‐effective production systems for manufacturing engineered tissue grafts for clinical applications. However, the automation of bioreactor systems could become considerably more complex and costly due to the requirements for additional storage and liquid handling technologies if unstable supplements are added to the culture medium. Ascorbic acid (AA) is a bioactive supplement that is commonly presumed to be essential for the generation of engineered cartilage tissues. However, AA can be rapidly oxidized and degraded. In this work, we addressed whether human nasal chondrocytes can redifferentiate, undergo chondrogenesis, and generate a cartilaginous extracellular matrix when cultured in the absence of AA. We found that when chondrocytes were cultured in 3D micromass pellets either with or without AA, there were no significant differences in their chondrogenic capacity in terms of gene expression or the amount of glycosaminoglycans. Moreover, 3D pellets cultured without AA contained abundant collagen Types II and I extracellular matrix. Although the amounts of Collagens II and I were significantly lower (34% and 50% lower) than in pellets cultured with AA, collagen fibers had similar thicknesses and distributions for both groups, as shown by scanning electron microscopy imaging. Despite the reduced amounts of collagen, if engineered cartilage grafts can be generated with sufficient properties that meet defined quality criteria without the use of unstable supplements such as AA, bioreactor automation requirements can be greatly simplified, thereby facilitating the development of more compact, user‐friendly, and cost‐effective bioreactor‐based manufacturing systems

Impaired Mineral Ion Metabolism in a Mouse Model of Targeted Calcium-Sensing Receptor (CaSR) Deletion from Vascular Smooth Muscle Cells

Background Impaired mineral ion metabolism is a hallmark of CKD–metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR. Methods To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells (SM22αCaSRΔflox/Δflox). Results Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D3), and parathyroid hormone levels. Renal tubular α-Klotho protein expression was increased in KO mice but vascular α-Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice. Conclusions These results suggest that, in addition to CaSR’s established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis

Bioreactor manufactured cartilage grafts repair acute and chronic osteochondral defects in large animal studies

Objectives Bioreactor‐based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost‐effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor‐based manufacturing system for the production of cartilage grafts. Materials & Methods All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor‐based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. Results The results of these studies demonstrate the safety and feasibility of the bioreactor‐based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell‐free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor‐manufactured grafts may result in a more robust repair in the longer term. Conclusion By demonstrating the safety and efficacy of bioreactor‐generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor‐based manufacturing system for the production of human cartilage grafts for clinical applications

The role of the cruciate ligaments in guinea pig spontaneous osteoarthritis

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N-cadherin and b-catenin signalling regulates vascular smooth muscle cell proliferation and cell cycle genes

Vascular smooth muscle cell (VSMC) proliferation is a key event in the pathogenesis of vascular diseases. Dismantling of cadherin junctions and translocation of b-catenin to the cytoplasm occurs in proliferating VSMCs. To determine whether N-cadherin and b-catenin signalling are essential in the regulation of VSMC proliferation we over-expressed, using adenoviral delivery, full length N-Cadherin, ICAT (an endogenous inhibitor of b-catenin signalling), or a dominant negative (dn) mutant of the b-catenin transcriptional co-factor, TCF-4. N-cadherin, ICAT and dnTCF-4 over-expression significantly reduced the proliferation of isolated human VSMCs, detected by BrdU incorporation, by 55 ± 6% (p < 0.003), 80 ± 5% (p < 0.001) and 46 ± 7% (p < 0.03), respectively. Similar effects were observed in medial saphenous vein segments infected with the ICAT and dnTCF-4 adenoviruses (51 ± 12% (p < 0.04) and 56 ± 9% (p < 0.02) of control, respectively). Co-transfection with a dnTCF4 construct in the ISS10 human SMC line significantly lowered TCF and cyclin D1 reporter activity by 73 ± 1% (p < 0.03) and 67 ± 4% (p < 0.001) respectively, and elevated p21 reporter activity by 108 ± 19% (p < 0.03). N-cadherin, ICAT and dnTCF-4 over-expression significantly lowered levels of cyclin D1 mRNA by 19 ± 2% (p < 0.01), 34 ± 6% (p < 0.02) and 36 ± 8% (p < 0.03), and lowered levels of cyclin D1 protein by 22 ± 4% (p < 0.04), 44 ± 6% (p < 0.03) and 48 ± 12% (p < 0.01), respectively. In contrast, over-expression of ICAT and dnTCF4 in isolated human VSMCs significantly elevated p21 mRNA levels by 49 ± 14% (p < 0.03) and 55 ± 15% (p < 0.04), and elevated p21 protein levels by 196 ± 45% (p < 0.03) and 454 ± 72% (p < 0.03) respectively. We have demonstrated that increasing N-cadherin mediated cell–cell contacts and inhibiting b-catenin signalling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We suggest that the N-cadherin and b-catenin signalling pathway is a key modulator of VSMC proliferation via the regulation of these two b-catenin responsive genes

Expression of lumican in the synovial fluid of patients with osteoarthritis (OA)

Introduction Fragmentation of small leucine rich proteogly-cans (SLRPs), including decorin, biglycan, lumican, keratocanand fibromodulin, has been shown to occur in osteoarthriticarticular cartilage (Melrose et al. 2008). We have previouslyshown an increased expression of lumican and keratocan, inosteoarthritic articular cartilage (Kerr et al. 2006). The long-term aim of this project is to develop ELISAs for the detectionof SLRP metabolites, and validate these potential biomarkerswith synovial fluid and serum samples from a large cohort ofnormal and osteoarthritic patients. Initially, we determinedwhether lumican could be detected in synovial fluid ofpatients with and without osteoarthritis. We also investigatedthe expression of lumican in an in vitro, cultured explantmodel of OA