In the last decade, skeletal muscle has been recognized as an endocrine organ able to release molecules that may act as paracrine or endocrine factors, namely myokines. Among these, irisin is secreted upon muscle contraction after physical exercise (PE) and has been demonstrated to yield anabolic effects on different cell types. Recently, irisin has been shown to improve cortical bone mass, geometry and strength, hence resembling the effect of PE. It has also been reported that irisin levels in the serum and synovial fluid of patients with knee osteoarthritis (OA) were negatively correlated with OA severity. Therefore, we hypothesized that irisin may improve cartilage metabolism and blunt the osteoarthritic process. Human osteoarthritic chondrocytes (hOAC) were isolated from osteochondral specimens of patients undergoing total knee joint replacement. After hOAC treated with irisin showed a significant higher GAG content compared to the control group (p < 0.01). Moreover, irisin was able to reduce the expression of catabolic (MMP-1, -13, iNOS) and pro-inflammatory (IL-1, IL-6) markers, while incrementing the expression of TIMP-1 and -3 (p < 0.001). Our results showed that irisin was able to stimulate GAG synthesis and diminish extracellular matrix catabolism in hOAC, demonstrating the existence of a cross-talk between cartilage and muscle possibly supporting the beneficial role of PE on cartilage homeostasis.
Aims. Osteoarthritis (OA) is characterized by persistent destruction of articular cartilage. It has been found that microRNAs (miRNAs) are closely related to the occurrence and development of OA. The purpose of the present study was to investigate the mechanism of miR-486 in the development and progression of OA. Methods. The expression levels of miR-486 in cartilage were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN), matrix metalloproteinase (MMP)-13, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) in SW1353 cells at both messenger RNA (mRNA) and protein levels was determined by qRT-PCR, western blot, and enzyme-linked immunosorbent assay (ELISA). Double luciferase reporter gene assay, qRT-PCR, and western blot assay were used to determine whether silencing information regulator 6 (SIRT6) was involved in miR-486 induction of
Aims. Tert-butylhydroquinone (tBHQ) has been identified as an inhibitor of oxidative stress-induced injury and apoptosis in human neural stem cells. However, the role of tBHQ in osteoarthritis (OA) is unclear. This study was carried out to investigate the role of tBHQ in OA. Methods. OA animal model was induced by destabilization of the medial meniscus (DMM). Different concentrations of tBHQ (25 and 50 mg/kg) were intraperitoneally injected in ten-week-old female mice.
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.
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.
Summary Statement. IL-1β stimulation of human OA chondrocytes induces NFκB, ERK1/2, c-JUN, IκB and P38 signalling pathways. Pre-treatment with cannabinoid WIN-55 for 48 hours inhibits certain pathways, providing mechanisms for cannabinoids inhibitory actions on IL-1β induced cartilage degradation. Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) breakdown in osteoarthritis (OA) and their expression is regulated by nuclear factor kappa B (NFκB). In addition signalling pathways ERK1/2, c-JUN, IκB and P38 are activated in OA and are induced by inflammatory cytokine interleukin 1 (IL-1). Cannabinoids have been shown to reduce joint damage in animal models of arthritis. Synthetic cannabinoid WIN-55, 212-2 mesylate (WIN-55) significantly reduces IL-1β induced expression of MMP-3 and -13 in human OA chondrocytes, indicating a possible mechanism via which cannabinoids may act to prevent ECM breakdown. Here the effects of WIN-55 on IL-1β induced NFκB, ERK1/2, c-JUN, IκB and P38 phosphorylation in human OA chondrocytes has been investigated. Primary human chondrocytes were obtained from articular cartilage removed from patients with symptomatic OA during total knee replacement (Ethic approval:SMB002). Cartilage tissue was graded macroscopically 0–4 using the Outerbridge Classification method.
HACs from five OA patients (passage 1) were cultured in cytokine-free medium, under 280 or 380 mOsm respectively, under standard 2D Introduction
Methods
Introduction: Autologous chondrocyte implantation (ACI) has been developed in order to repair cartilage successfully. Experimental models are based on osteochondral defects with potentially triphasic chondrogenic system: periosteal flaps, bone marrow cells and transplanted chondrogenic cells. All these three have chondrogenic activity so it is difficult to determinate the role of the implanted cells unless appropriate control is set up. The purpose of this study is to determinate if the inoculation of chondrocytes under periosteal flaps does improve the chondrogenic potential of periosteal flaps. MATERIALS AND Methods: 10 New Zealand rabbits, 8 months old were used. Right knees served as study group (ACI Group; N5:
Purpose. Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold. Method.
Objective. To study the effect of hyaluronic acid (HA) on local anaesthetic
chondrotoxicity in vitro. Methods.
Osteoarthritis (OA) is a prevalent joint disorder with inflammatory response and cartilage deterioration as its main features. Dihydrocaffeic acid (DHCA), a bioactive component extracted from natural plant ( In vitro, interleukin-1 beta (IL-1β) was used to establish the mice OA chondrocytes. Cell counting kit-8 evaluated chondrocyte viability. Western blotting analyzed the expression levels of collagen II, aggrecan, SOX9, inducible nitric oxide synthase (iNOS), IL-6, matrix metalloproteinases (MMPs: MMP1, MMP3, and MMP13), and signalling molecules associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Immunofluorescence analysis assessed the expression of aggrecan, collagen II, MMP13, and p-P65. In vivo, a destabilized medial meniscus (DMM) surgery was used to induce mice OA knee joints. After injection of DHCA or a vehicle into the injured joints, histological staining gauged the severity of cartilage damage.Aims
Methods
The use of intra-articular corticosteroid injections for their anti-inflammatory effects is widespread amongst clinicians. Despite their use in both rheumatoid arthritis and osteoarthritis, the effect of these agents on articular chondrocytes is not fully established. Previous reports suggest a detrimental effect on cartilage explants resulting from inhibition of matrix synthesis. 1. However it has also been suggested that the beneficial effects in vivo may be due to prevention of inflamed synovium causing cartilage degradation. 2. Our aim was to assess the effect of a commercially available preparation of methylprednisolone (MP), at clinical doses, on articular chondrocytes cultured in vitro. Bovine articular chondrocytes were isolated by sequential digestion with pronase and collagenase and seeded in 2% alginate at 1x10. 7. cells/ml. The constructs were cultured for up to 15 days in standard culture medium (DMEM + 20% Fetal calf serum) containing varying concentrations of MP, including doses equivalent to those found in vivo. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA and glycosaminoglycans. Further constructs were fixed in 4% paraformaldehyde for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.
Aim: To determine the pattern of gene expression induced in cultured human chondrocytes in response to compressive mechanical loads. Methods:
The use of intra-articular hyaluronic acid injections for the treatment of early osteoarthritis is in widespread clinical use. Hyaluronate (HA) is a major component of connective tissue. 1. and is available commercially for the intra-articular injective treatment of osteoarthritis of the knee and periarthritis of the shoulder. Although it is known to improve intra-articular lubrication it is also thought to promote articular cartilage structure and prevent catabolism of matrix proteoglycans in osteoarthritis. Clinical studies have shown beneficial effects lasting for many months after cessation of therapy unlike anti-inflammatory drugs that have relatively short term relieving effects. 2,. 3. . Documentation of the true chondroprotective effects of hyaluronic acid (HA) at the cellular level is lacking and therefore this study aimed to identify the effects of HA on chondrocytes cultured in vitro. Bovine articular chondrocytes were isolated by sequential digestion with pronase and collagenase and seeded in 2% alginate at 1x10. 7. cells/ml. The constructs were cultured for up to 14 days in standard culture medium (DMEM + 20% Fetal calf serum) containing varying concentrations of HA (Sigma), including doses equivalent to those found in vivo. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA, glycosaminoglycans and Collagen. Further constructs were fixed in 4% paraformaldehyde for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.
Introduction: In the growth plate, chondrocyte swelling (hypertrophy) is a crucial event during endochondral ossification and bone lengthening, accounting for ~80% of the increase in bone length (. 1. ,. 3. ). The swelling is dramatic (~10x) and closely regulated. Failure of chondrocyte hypertrophy may underlie the chondrodysplasias of the vertebrate skeleton (. 1. ). However, the mechanisms which control cell swelling are poorly understood although there must be a key role for chondrocyte osmolyte transporters which are sensitive to an increase in cell volume. We have used confocal scanning laser microscopy (CLSM) to study volume regulation by living in situ growth plate chondrocytes at varying degrees of hypertrophy. Methods: Bovine growth plates were taken from the ends of young (~12d) bovine ribs. In situ growth plate chondrocytes at the proliferative through to hypertrophic stages were fluorescently-labelled (calcein-AM; 5μM), imaged (Zeiss CLSM510) and volumes determined quantitatively as described (. 2. ). An acute osmotic challenge (280-140mOsm) was delivered by perfusion to determine volume-regulatory capacity by cells in the various zones. Results: The resting volumes of proliferative and hypertrophic cells were 550±63μm. 3. and 5227±1974μm. 3. respectively. Reducing osmolarity resulted in a rapid (within ~1min) cell swelling, proliferative and hypertrophic chondrocytes increasing in volume by 126±2% and 146±5% (n=5) respectively.
Programmed cell death (PCD) contributes to the pathogenesis of many diseases including osteoarthritis. The principal method is apoptosis that has a well-defined and very characteristic morphology and biochemistry. The aim of the present study was examine whether the mechanism of cell death in OA chondrocytes was classical apoptosis. Rat thymocytes were used as controls since these cells are known to undergo classical apoptosis. Human OA cartilage was obtained from femoral head of patients (50 – 80 years) who were undergoing joint replacement surgery. Pieces of OA samples were processed into paraffin and sections incubated with the following antibodies: M3O, an antibody that recognizes the cleavage of cytokeratin 18 by caspases; annexin V, which recognizes phosphatidylserine “flip-flop” that occurs early in the apoptotic process; bcl-2, a protein whose presence protects apoptosis and c-myc, a transcription factor thought to be associated with apoptosis. To induce apoptosis, some samples were incubated with etoposide and staurosporine. In sections of thymus we noticed the presence of numerous apoptotic bodies. The number increased when the tissue was treated with etoposide and staurosporine. Some thymocytes were immunopositive for M3O and annexin V, and the number of positive cells increased when treated for 2h with etoposide.
It was aimed to investigate the isolated effect of hydrostatic pressure on chondrocyte metabolism.
Purpose and Background: There is increasing evidence that events within the diseased intervertebral disc (IVD) are mediated by locally synthesised cytokines. A prominent histological, imaging and surgical feature of IVD disease is degradation of the cartilaginous discal matrix. Whilst the mechanism by which this is mediated is unknown, in other situations where connective tissues are degraded degradation is the result of production of matrix-degrading enzymes by local connective tissue cells stimulated by cytokines, particularly the beta isoform of interleukin-1 (IL-1β). Included amongst these disorders is osteoarthritis (OA) of diarthrodial joints. OA has many similarities to the discal “degeneration” seen in mechanical back pain syndromes. In the current study, we have used a combination of in-situ techniques to establish if IL-1β is responsible for stimulating matrix degradation in the IVD. Methods: Using a combination of radioactive in-situ hybridisation (ISH) and competitive in situ zymography (ISZ) we have studied expression of IL-1β and IL-1R – its type 1 receptor (ISH) and matrix degradation (ISZ) in five diseased lumbar IVD taken at spinal fusion surgery and 10 cadaveric IVD (five normal and five diseased). The nucleus pulposus (NP) was separated from the annulus fibrosus and diced into 0.5cm cubes. Half the cubes (typically three) were fixed in formalin and processed into paraffin wax for ISH, and half were used for ISZ. For ISH, 5 μm sections of paraffin-embedded tissue were reacted with cDNA probes radiolabelled with 35S to 580 and 530 base segments of the IL-1β and IL-1R molecules. Hybridisation was disclosed using autoradiography. For ISZ, 50 μm vibratome sections were placed into wells on microscope slides precoated with gelatin. Sections were incubated for 10 days, half in culture medium and half in medium supplemented with human recombinant IL-1 receptor antagonist (IL-1Ra – an inhibitor of IL-1). Sections were photographed at daily intervals to detect evidence of gel degradation. Results:
Osteoarthritis (OA) is characterised by the progressive loss of the articular cartilage. This is accompanied by change in phenotype from cells expressing chondrocytic genes to cells, termed ‘degradative’ chondrocytes, that express aggrecanases and collagenases. To understand the cellular events involved, human articular cartilage was obtained from femoral heads after arthroplasty due to OA, fracture of the neck of femur (#NOF) due to osteoporosis, or from a 14 year old male (CDH). Samples were graded according to the new OARSI system (. Osteoarthritis and Cartilage. , . 2006. , . 14. :. 13. –29. ) and par-affin sections were immunostained for c-Myc (marker of cellular activation), S100 (typically expressed in chon-drocytes), Sox-9 (expressed in early-stage chondrocytes) and nucleostemin (a stem-cell marker). In addition, some specimen were incubated with fluorescent probes to identify metabolically activated cells (CellTracker green). All chondrocytes, irrespective of OA grade, were immunopositive for S100, but there were differences in the other parameters. Cartilage from the 14-year old (OARSI grade =0) was characterized by no loss of proteoglycans (safranin-O) in the superficial zone and absence of c-Myc, Sox-9 and nucleostemin in all articular chondrocytes. In #NOF cartilage, proteoglycan loss was evident in the very superficial zone. Many chondro-cytes in that zone showed bright green fluorescence with CellTracker-green and were c-Myc positive, consistent with cellular activation. Sox-9 and nucleostemin were absent. Mid-zone and deep zone chondrocytes showed no change. In low-grade OA samples (OARSI = 1-2), the zone of proteoglycan loss had increased, the Cell-Tracker-green/c-Myc positive chondrocytes in that zone had divided to form clusters of 4-8 cells. Occasional cells were positive for nucleostemin. Mid-zone and deep zone chondrocytes still showed no change. In high-grade fib-rillated OA cartilage (OARSI = 3-4) the superficial and mid zones had been eroded, leaving the deep zone at the surface.
The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a hypertrophic OA-like cell morphology. Our aim was to investigate cause and effects of PGE2 secretion in MSC-based cartilage neogenesis and hypertrophy and identify molecular mechanisms responsible for adverse effects in OA analgesia. Human bone marrow-derived MSCs were cultured in chondrogenic medium with TGFβ (10ng/mL) and treated with PGE2 (1µM), celecoxib (COX-2 inhibitor; 0.5µM), AH23848/AH6809 (PGE2 receptor antagonists; 10µM), or DMSO as a control (n=3–4). Assessment criteria were proteoglycan deposition (histology), chondrocyte/hypertrophy marker expression (qPCR), and ALP activity. PGE2 secretion was measured (ELISA) after TGFβ withdrawal (from day 21, n=2) or WNT inhibition (2µM IWP-2 from day 14; n=3). Strong decrease in PGE2 secretion upon TGFβ deprivation or WNT inhibition identified both pathways as PGE2 drivers. Homogeneous proteoglycan deposition and Although TGFβ and WNT are known pro-arthritic signaling pathways, they appear to induce a PGE2-mediated antihypertrophic effect that can counteract pathological cell changes in chondrocytes. Hampering this rescue mechanism via COX inhibition using NSAIDs thus risks acceleration of OA progression, indicating the need of OA analgesia adjustment.