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1.  Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism 
Annals of the rheumatic diseases  2008;67(0 3):iii75-iii82.
In osteoarthritis (OA), adult articular chondrocytes undergo phenotypic modulation in response to alterations in the environment owing to mechanical injury and inflammation. These processes not only stimulate the production of enzymes that degrade the cartilage matrix but also inhibit repair. With the use of in vitro and in vivo models, new genes, not known previously to act in cartilage, have been identified and their roles in chondrocyte differentiation during development and in dysregulated chondrocyte function in OA have been examined. These new genes include growth arrest and DNA damage (GADD)45β and the epithelial-specific ETS (ESE)-1 transcription factor, induced by bone morpho-genetic protein (BMP)-2 and inflammatory cytokines, respectively. Both genes are induced by NF-κB, suppress COL2A1 and upregulate matrix meatalloproteinase-13 (MMP-13) expression. These genes have also been examined in mouse models of OA, in which discoidin domain receptor 2 is associated with MMP-13-mediated remodelling, in order to understand their roles in physiological cartilage homoeostasis and joint disease.
doi:10.1136/ard.2008.098764
PMCID: PMC3939701  PMID: 19022820
2.  Analysis of mass spectrometry data from the secretome of an explant model of articular cartilage exposed to pro-inflammatory and anti-inflammatory stimuli using machine learning 
Background
Osteoarthritis (OA) is an inflammatory disease of synovial joints involving the loss and degeneration of articular cartilage. The gold standard for evaluating cartilage loss in OA is the measurement of joint space width on standard radiographs. However, in most cases the diagnosis is made well after the onset of the disease, when the symptoms are well established. Identification of early biomarkers of OA can facilitate earlier diagnosis, improve disease monitoring and predict responses to therapeutic interventions.
Methods
This study describes the bioinformatic analysis of data generated from high throughput proteomics for identification of potential biomarkers of OA. The mass spectrometry data was generated using a canine explant model of articular cartilage treated with the pro-inflammatory cytokine interleukin 1 β (IL-1β). The bioinformatics analysis involved the application of machine learning and network analysis to the proteomic mass spectrometry data. A rule based machine learning technique, BioHEL, was used to create a model that classified the samples into their relevant treatment groups by identifying those proteins that separated samples into their respective groups. The proteins identified were considered to be potential biomarkers. Protein networks were also generated; from these networks, proteins pivotal to the classification were identified.
Results
BioHEL correctly classified eighteen out of twenty-three samples, giving a classification accuracy of 78.3% for the dataset. The dataset included the four classes of control, IL-1β, carprofen, and IL-1β and carprofen together. This exceeded the other machine learners that were used for a comparison, on the same dataset, with the exception of another rule-based method, JRip, which performed equally well. The proteins that were most frequently used in rules generated by BioHEL were found to include a number of relevant proteins including matrix metalloproteinase 3, interleukin 8 and matrix gla protein.
Conclusions
Using this protocol, combining an in vitro model of OA with bioinformatics analysis, a number of relevant extracellular matrix proteins were identified, thereby supporting the application of these bioinformatics tools for analysis of proteomic data from in vitro models of cartilage degradation.
doi:10.1186/1471-2474-14-349
PMCID: PMC3878677  PMID: 24330474
Osteoarthritis; Cartilage; Biomarker; Interleukin 1 β; Carprofen; Bioinformatics; Machine learning
3.  Transport of anti-IL-6 antigen binding fragments into cartilage and the effects of injury 
The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules.
doi:10.1016/j.abb.2012.12.020
PMCID: PMC3596461  PMID: 23333631
cartilage; TNFα; Fab; transport; injury; osteoarthritis
4.  Cartilage homeostasis in health and rheumatic diseases 
As the cellular component of articular cartilage, chondrocytes are responsible for maintaining in a low-turnover state the unique composition and organization of the matrix that was determined during embryonic and postnatal development. In joint diseases, cartilage homeostasis is disrupted by mechanisms that are driven by combinations of biological mediators that vary according to the disease process, including contributions from other joint tissues. In osteoarthritis (OA), biomechanical stimuli predominate with up-regulation of both catabolic and anabolic cytokines and recapitulation of developmental phenotypes, whereas in rheumatoid arthritis (RA), inflammation and catabolism drive cartilage loss. In vitro studies in chondrocytes have elucidated signaling pathways and transcription factors that orchestrate specific functions that promote cartilage damage in both OA and RA. Thus, understanding how the adult articular chondrocyte functions within its unique environment will aid in the development of rational strategies to protect cartilage from damage resulting from joint disease. This review will cover current knowledge about the specific cellular and biochemical mechanisms that regulate cartilage homeostasis and pathology.
doi:10.1186/ar2592
PMCID: PMC2714092  PMID: 19519926
5.  Effects of short-term glucocorticoid treatment on changes in cartilage matrix degradation and chondrocyte gene expression induced by mechanical injury and inflammatory cytokines 
Arthritis Research & Therapy  2011;13(5):R142.
Introduction
Traumatic joint injury damages cartilage and causes adjacent joint tissues to release inflammatory cytokines, increasing the risk of developing osteoarthritis. The main objective of this study was to determine whether the combined catabolic effects of mechanical injury, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) on cartilage could be abolished by short-term treatment with glucocorticoids such as dexamethasone.
Methods
In an initial dexamethasone-dose-response study, bovine cartilage explants were treated with TNFα and increasing concentrations of dexamethasone. Bovine and human cartilage explants were then subjected to individual and combined treatments with TNFα, IL-6/sIL-6R and injury in the presence or absence of dexamethasone. Treatment effects were assessed by measuring glycosaminoglycans (GAG) release to the medium and synthesis of proteoglycans. Additional experiments tested whether pre-exposure of cartilage to dexamethasone could prevent GAG loss and inhibition of biosynthesis induced by cytokines, and whether post-treatment with dexamethasone could diminish the effects of pre-established cytokine insult. Messenger ribonucleic acid (mRNA) levels for genes involved in cartilage homeostasis (proteases, matrix molecules, cytokines, growth and transcription factors) were measured in explants subjected to combined treatments with injury, TNFα and dexamethasone. To investigate mechanisms associated with dexamethasone regulation of chondrocyte metabolic response, glucocorticoid receptor (GR) antagonist (RU486) and proprotein convertase inhibitor (RVKR-CMK) were used.
Results
Dexamethasone dose-dependently inhibited GAG loss and the reduction in biosynthesis caused by TNFα. The combination of mechanical injury, TNFα and IL-6/sIL-6R caused the most severe GAG loss; dexamethasone reduced this GAG loss to control levels in bovine and human cartilage. Additionally, dexamethasone pre-treatment or post-treatment of bovine explants lowered GAG loss and increased proteoglycan synthesis in cartilage explants exposed to TNFα. Dexamethasone did not down-regulate aggrecanase mRNA levels. Post-transcriptional regulation by dexamethasone of other genes associated with responses to injury and cytokines was noted. GR antagonist reversed the effect of dexamethasone on sulfate incorporation. RVKR-CMK significantly reduced GAG loss caused by TNFα + IL-6 + injury.
Conclusions
Short-term glucocorticoid treatment effectively abolished the catabolic effects exerted by the combination of pro-inflammatory cytokines and mechanical injury: dexamethasone prevented proteoglycan degradation and restored biosynthesis. Dexamethasone appears to regulate the catabolic response of chondrocytes post-transcriptionally, since the abundance of transcripts encoding aggrecanases was still elevated in the presence of dexamethasone.
doi:10.1186/ar3456
PMCID: PMC3308070  PMID: 21888631
6.  Polymer Mechanics as a Model for Short-Term and Flow-Independent Cartilage Viscoelasticity 
Articular cartilage is the load bearing soft tissue that covers the contacting surfaces of long bones in articulating joints. Healthy cartilage allows for smooth joint motion, while damaged cartilage prohibits normal function in debilitating joint diseases such as osteoarthritis. Knowledge of cartilage mechanical function through the progression of osteoarthritis, and in response to innovative regeneration treatments, requires a comprehensive understanding of the molecular nature of interacting extracellular matrix constituents and interstitial fluid. The objectives of this study were therefore to (1) examine the timescale of cartilage stress-relaxation using different mechanistic models and (2) develop and apply a novel (termed “sticky”) polymer mechanics model to cartilage stress-relaxation based on temporary binding of constituent macromolecules. Using data from calf cartilage samples, we found that different models captured distinct timescales of cartilage stress-relaxation: monodisperse polymer reptation best described the first second of relaxation, sticky polymer mechanics best described data from ∼1-100 seconds of relaxation, and a model of inviscid fluid flow through a porous elastic matrix best described data from 100 seconds to equilibrium. Further support for the sticky polymer model was observed using experimental data where cartilage stress-relaxation was measured in either low or high salt concentration. These data suggest that a complete understanding of cartilage mechanics, especially in the short time scales immediately following loading, requires appreciation of both fluid flow and the polymeric behavior of the extracellular matrix.
doi:10.1016/j.msec.2010.11.029
PMCID: PMC3087607  PMID: 21552375
Articular cartilage; osteoarthritis; polymer dynamics; reptation; viscoelasticity; biomechanics
7.  MECHANICAL LOAD INHIBITS IL-1 INDUCED MATRIX DEGRADATION IN ARTICULAR CARTILAGE 
Summary
Objective
Osteoarthritis is a disease process of cellular degradation of articular cartilage caused by mechanical loads and inflammatory cytokines. We studied the cellular response in native cartilage subjected to a mechanical load administered simultaneously with an inflammatory cytokine (IL-1), hypothesizing that the combination of load and cytokine would result in accelerated extracellular matrix (ECM) degradation.
Methods
Mature bovine articular cartilage was loaded for three days (stimulation) with 0.2 and 0.5 MPa stresses, with and without interleukin-1 (IL-1α, 10 ng/ml), followed by three days of no stimulation (recovery). Aggrecan and collagen loss were measured as well as aggrecan cleavage using monoclonal antibodies AF-28 and BC-3 for cleavage by aggrecanases (ADAMTS) and matrix metalloproteinases (MMPs), respectively.
Results
Incubation with IL-1 caused aggrecan cleavage by aggrecanases and MMPs during the three days of stimulation. A load of 0.5 MPa inhibited the IL-1-induced aggrecan loss while no inhibition was found for the 0.2 MPa stress. There was no collagen loss during the treatments but upon load and IL-1 removal proteoglycan and collagen loss increased. Load itself under these conditions was found to have no effect when compared to the unloaded controls.
Conclusions
A mechanical load of sufficient magnitude can inhibit ECM degradation by chondrocytes when stimulated by IL-1. The molecular mechanisms involved in this process are not clear but probably involve altered mechanochemical signal transduction between the ECM and chondrocyte.
doi:10.1016/j.joca.2009.07.012
PMCID: PMC2818235  PMID: 19747586
Cartilage; Mechanobiology; Load; IL-1; MMPs; ADAMTS
8.  Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: immunohistochemical studies with an active form specific antibody 
Annals of the Rheumatic Diseases  1999;58(3):175-181.
OBJECTIVE—The first complement component C1s was reported to have novel functions to degrade matrix components, besides its activities in the classic complement pathway. This study explores participation of C1s in articular cartilage degradation in rheumatoid arthritis (RA).
METHODS—Normal articular cartilage (n=6) and cartilage obtained from joints with RA (n=15) and osteoarthritis (OA, n=10) were immunostained using anti-C1s monoclonal antibodies PG11, which recognises both active and inactive C1s, and M241, which is specifically reactive to activated C1s. The effects of inflammatory cytokines on C1s production by human articular chondrocytes were also examined by sandwich ELISA.
RESULTS—In normal articular cartilage, C1s was negative in staining with both PG11 and M241. In contrast, degenerating cartilage of RA was stained with PG11 (14 of 15 cases), and in most of the cases (13 of 15 cases) C1s was activated as revealed by M241 staining. In OA, C1s staining was restricted in severely degrading part of cartilage (5 of 10 cases), and even in that part C1s was not activated. In addition, C1s production by chondrocytes in vitro was increased by an inflammatory cytokine, tumour necrosis factor α.
CONCLUSION—These results suggest that C1s activated in degenerative cartilage matrix of RA but not in that of OA. C1s is thought to participate in the pathogenesis of RA through its collagenolytic activity in addition to the role in the classic cascade.

 Keywords: complement C1s; articular cartilage; rheumatoid arthritis
PMCID: PMC1752845  PMID: 10364916
9.  Bony island within the articular cartilage of the knee in a child: a rare condition for early osteoarthritis 
Orthopedic Reviews  2011;3(1):e5.
Articular cartilage is a specific type of connective tissue composed of hydrated proteoglycans within a matrix of collagen fibrils. In the elderly population, it shows degenerative changes that may results in osteoarthritis. The more severe form of osteoarthritis occasionally demonstrates bone formation within the cartilage, which is designated as a bony protuberance, however, such lesions are rare in children. This report presents the case of a 10-year-old boy with a bony protuberance within the articular cartilage of the knee. The patient initially complained of knee pain and he subsequently developed flexion contracture. Radiological and arthroscopic examinations revealed a bony protuberance in the articular cartilage and degenerative changes of the cartilage above it. He was successfully treated by the removal of the bony protuberance and osteochondral grafting. The bony protuberance may have caused cartilage degradation since the thickness of the cartilage above it was thinner than that around the lesion. The bony protuberance within the articular cartilage formed in the younger population may be a possible cause of osteoarthritis. This case is a noteworthy with regard to the pathogenesis of osteoarthritis.
doi:10.4081/or.2011.e5
PMCID: PMC3143997  PMID: 21808717
bone protuberance; articular cartilage; osteoarthritis; child.
10.  Bony island within the articular cartilage of the knee in a child: a rare condition for early osteoarthritis 
Orthopedic Reviews  2011;3(1):e7.
Articular cartilage is a specific type of connective tissue composed of hydrated proteoglycans within a matrix of collagen fibrils. In the elderly population, it shows degenerative changes that may results in osteoarthritis. The more severe form of osteoarthritis occasionally demonstrates bone formation within the cartilage, which is designated as a bony protuberance, however, such lesions are rare in children. This report presents the case of a 10-year-old boy with a bony protuberance within the articular cartilage of the knee. The patient initially complained of knee pain and he subsequently developed flexion contracture. Radiological and arthroscopic examinations revealed a bony protuberance in the articular cartilage and degenerative changes of the cartilage above it. He was successfully treated by the removal of the bony protuberance and osteochondral grafting. The bony protuberance may have caused cartilage degradation since the thickness of the cartilage above it was thinner than that around the lesion. The bony protuberance within the articular cartilage formed in the younger population may be a possible cause of osteoarthritis. This case is a noteworthy with regard to the pathogenesis of osteoarthritis.
doi:10.4081/or.2011.e7
PMCID: PMC3144001  PMID: 21808719
bone protuberance; articular cartilage; osteoarthritis; child.
11.  Mechanically induced experimental knee osteoarthritis benefits from anti-inflammatory and immunomodulatory properties of simvastatin via inhibition of matrix metalloproteinase-3 
Background
We investigated the anti-inflammatory and immunomodulatory effect of simvastatin on articular cartilage via the inhibition of matrix metalloproteinase-3 (MMP-3), a matrix-degrading enzyme, in a mechanically induced experimental osteoarthritis (OA) animal model.
Materials and methods
Twenty-seven albino Wistar rats were divided in three groups of equal number. Unphysiologic loading of articular cartilage was simulated by transecting anterior cruciate ligaments of the right knees of 18 rats consisting of groups 1 and 2. Nine animals in group 2 received orally administered simvastatin 20 mg/kg per day by gavage for 8 weeks. Animals in group 3 were sham operated. All animals were sacrificed at postoperative 8 weeks. Effects of simvastatin on disease progression was evaluated by documenting OA changes in cartilage specimens using Osteoarthritis Research Society International (OARSI) OA cartilage histopathology assessment system scores combined with the percentage of MMP-3 expression in chondrocytes.
Results
Simvastatin treatment significantly down-regulated the percentage of MMP-3 expression in chondrocytes as assessed by immunohistochemistry methods. Suppression of this matrix-degrading enzyme by simvastatin also reduced OARSI scores, suggesting the potential for statins against OA progression.
Conclusions
Following knee trauma, OA initiates at the molecular level in a short period of time. Irreversible structural changes in cartilage that require demanding treatment strategies led us to focus on effective measures to prevent OA. Statins have immunomodulatory and anti-inflammatory properties independent from their serum-cholesterol-lowering effects. One of these widely used drugs, simvastatin, showed beneficial effects on OA progression and extent by reducing cartilage degradation in our experimental setting. If these results are confirmed by human trials, simvastatin might be considered by orthopedic surgeons as a disease-modifying drug during the early inflammatory phase of posttraumatic OA.
doi:10.1007/s10195-011-0154-y
PMCID: PMC3163794  PMID: 21863259
Simvastatin; Anterior cruciate ligament transection; Knee osteoarthritis; Matrix metalloproteinase-3
12.  Vulnerability of the superficial zone of immature articular cartilage to compressive injury 
Arthritis and rheumatism  2010;62(10):3016-3027.
Objective
The zonal composition and functioning of adult articular cartilage causes depth-dependent responses to compressive injury. In immature cartilage, shear and compressive modulus, collagen and glycosaminoglycan (GAG) content also vary with depth. However, there is little understanding of depth-dependent damage caused by injury. Since injury to immature knee joints most often causes articular cartilage lesions, our objectives were to characterize the zonal dependence of biomechanical, biochemical and matrix-associated changes cause by injury.
Methods
Superficial and deeper zones disks from bovine calves were biomechanically characterized, injured (50% compression, 100%/sec) and re-characterized. Tissue compaction upon injury, GAG-density, GAG loss and biosynthesis were measured. Collagen-fiber-orientation and matrix damage was assessed using histology, Diffraction-Enhanced-X-Ray-Imaging, and texture analysis.
Results
Injured superficial disks showed surface disruption, compaction by 20.3±4.3%, and immediate biomechanical impairment: dynamic stiffness decreased to 7.1±3.3% of its initial value and equilibrium modulus was below detection. Tissue areas apparently intact by histology showed clear textural alterations. Injured deeper zones disks showed collagen crimping but remained undamaged and biomechanically intact. Superficial zone disks did not lose GAG immediately after injury but lost 17.8±1.4% by 48h; deeper zones disks lost only 2.8±0.3% GAG. Biomechanical impairment was primarily associated with structural damage.
Conclusion
The soft superficial zone of immature cartilage is vulnerable to compressive injury causing superficial matrix disruption, extensive compaction, and textural alteration, and resulting in immediate loss of biomechanical function. In conjunction with delayed superficial GAG loss, these changes may predispose the articular surface to further softening, damage, and increased risk of developing secondary OA.
doi:10.1002/art.27610
PMCID: PMC2952071  PMID: 20556809
13.  OXIDANT CONDITIONING PROTECTS CARTILAGE FROM MECHANICALLY-INDUCED DAMAGE 
Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the anti-oxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that anti-oxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant pre-conditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 hours with tert-butyl hydrogen peroxide (tBHP) at non-lethal concentrations (25, 100, 250, 500 µM) for a varying number of times (1, 2 or 4) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post-compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 µM tBHP applied 4 times. RNA analysis revealed significant effects of 100 µM tBHP on gene expression. Catalase, hypoxia-inducible factor-1alpha (HIF-1α), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated 4 times with 100 µM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase-3 (MMP-3) expression. These findings demonstrate that repeated exposure of cartilage to sub-lethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide-induced changes in gene expression that could render chondrocytes more resistant to oxidative damage.
doi:10.1002/jor.21072
PMCID: PMC3708667  PMID: 20058262
Cartilage; mechanical stress; oxidant
14.  Mechanisms of disruption of the articular cartilage surface in inflammation. Neutrophil elastase increases availability of collagen type II epitopes for binding with antibody on the surface of articular cartilage. 
Journal of Clinical Investigation  1991;87(5):1531-1536.
We recently observed that specific antibodies to type II collagen do not bind in appreciable amounts to the intact surface of articular cartilage, whereas antibodies to the minor collagen types V, VI, and IX do. These results suggest that the outermost cartilage surface layer prevented interaction of the antibodies with the major collagen type in articular cartilage. The present studies were designed to investigate the pathogenic mechanisms involved in the disruption of the cartilage surface layer in inflammatory arthritis. Articular cartilage obtained from rabbits undergoing acute antigen-induced arthritis of 72 h duration showed a significant increase in binding of anti-type II antibody to cartilage surfaces compared with normal control cartilage (P less than 0.01). Augmentation of anti-type II binding was also observed upon in vitro incubation of bovine articular slices or intact rabbit patellar cartilage for 1 h with human polymorphonuclear neutrophils (PMN), PMN lysates, or purified human PMN elastase. This increase was not inhibited by sodium azide, nor was it enhanced by incubation of cartilage with the strong oxidant hypochlorous acid. Chondrocyte-mediated matrix proteoglycan degradation in cartilage explants cultured in the presence of cytokines failed to increase antibody binding appreciably. The augmentation in antibody binding seen with PMN lysates was inhibited by the nonspecific serine-esterase inhibitor PMSF, but not by the divalent metal chelator EDTA. The elastase-specific inhibitor AAPVCMK also inhibited most of the PMN-induced increase in antibody binding, whereas the cathepsin G-specific inhibitor GLPCMK was much less effective. Incubation of intact cartilage with purified human PMN elastase indicated that this serine esterase could account for the increase in anti-type II collagen antibody binding to intact cartilage surfaces. These studies suggest that in an inflammatory response, PMN-derived elastase degrades the outer layer of articular cartilage, exposing epitopes on type II collagen. They also help clarify the pathogenic mechanisms involved in early articular cartilage damage in inflammatory joint diseases.
PMCID: PMC295233  PMID: 1708782
15.  Cartilage healing: A review with emphasis on the equine model 
The Canadian Veterinary Journal  1990;31(8):565-572.
Articular cartilage is a remarkably resilient tissue capable of withstanding considerable stress and repeated loading. Since this tissue has no blood vessels, nerve elements, or lymphatics, it is not surprising that it has a limited capacity for repair when damaged. In the horse, cartilage damage occurs as an occupational hazard. Furthermore, developmental defects such as osteochondrosis can lead to osteochondritis dissecans. Resultant cartilage flaps, fissures, and poorly organized subchondral bone produce disruption of joint surfaces.
Veterinarians are often called upon to intervene when damaged cartilage has healed incompletely. Basic understanding of the physiology and repair mechanisms of cartilage is paramount to successfully managing such injuries. This literature review gives a brief overview of recently published clinical and experimental studies on the healing of cartilage. The discussion centers on the equine model.
PMCID: PMC1480835  PMID: 17423644
16.  New developments in osteoarthritis. Posttraumatic osteoarthritis: pathogenesis and pharmacological treatment options 
Joint trauma can lead to a spectrum of acute lesions, including osteochondral fractures, ligament or meniscus tears and damage to the articular cartilage. This is often associated with intraarticular bleeding and causes posttraumatic joint inflammation. Although the acute symptoms resolve and some of the lesions can be surgically repaired, joint injury triggers a chronic remodeling process in cartilage and other joint tissues that ultimately manifests as osteoarthritis in a majority of cases. The objective of the present review is to summarize information on pathogenetic mechanisms involved in the acute and chronic consequences of joint trauma and discuss potential pharmacological interventions. The focus of the review is on the early events that follow joint trauma since therapies for posttraumatic joint inflammation are not available and this represents a unique window of opportunity to limit chronic consequences.
doi:10.1186/ar3046
PMCID: PMC2911903  PMID: 20602810
17.  Mesenchymal chondroprogenitor cell origin and therapeutic potential 
Mesenchymal progenitor cells, a multipotent adult stem cell population, have the ability to differentiate into cells of connective tissue lineages, including fat, cartilage, bone and muscle, and therefore generate a great deal of interest for their potential use in regenerative medicine. During development, endochondral bone is formed from a template of cartilage that transforms into bone; however, mature articular cartilage remains in the articulating joints, where its principal role is reducing friction and dispersing mechanical load. Articular cartilage is prone to damage from sports injuries or ageing, which regularly progresses to more serious joint disorders, such as osteoarthritis. Osteoarthritis is a degenerative joint disease characterized by the thinning and eventual wearing of articular cartilage, and affects millions of people worldwide. Due to low chondrocyte motility and proliferative rates, and complicated by the absence of blood vessels, cartilage has a limited ability to self-repair. Current pharmaceutical and surgical interventions fail to generate repair tissue with the mechanical and cellular properties of native host cartilage. The long-term success of cartilage repair will therefore depend on regenerative methodologies resulting in the restoration of articular cartilage that closely duplicates the native tissue. For cell-based therapies, the optimal cell source must be readily accessible with easily isolated, abundant cells capable of collagen type II and sulfated proteoglycan production in appropriate proportions. Although a cell source with these therapeutic properties remains elusive, mesenchymal chondroprogenitors retain their expansion capacity with the promise of reproducing the structural or biomechanical properties of healthy articular cartilage. As current knowledge regarding chondroprogenitors is relatively limited, this review will focus on their origin and therapeutic application.
doi:10.1186/scrt49
PMCID: PMC3092148  PMID: 21371355
18.  Simulating the swelling and deformation behaviour in soft tissues using a convective thermal analogy 
Background
It is generally accepted that cartilage adaptation and degeneration are mechanically mediated. Investigating the swelling behaviour of cartilage is important because the stress and strain state of cartilage is associated with the swelling and deformation behaviour. It is well accepted that the swelling of soft tissues is associated with mechanical, chemical, and electrical events.
Method
The purpose of the present study was to implement the triphasic theory into a commercial finite element tool (ABAQUS) to solve practical problems in cartilage mechanics. Because of the mathematical identity between thermal and mass diffusion processes, the triphasic model was transferred into a convective thermal diffusion process in the commercial finite element software. The problem was solved using an iterative procedure.
Results
The proposed approach was validated using the one-dimensional numerical solutions and the experimental results of confined compression of articular cartilage described in the literature. The time-history of the force response of a cartilage specimen in confined compression, which was subjected to swelling caused by a sudden change of saline concentration, was predicted using the proposed approach and compared with the published experimental data.
Conclusion
The advantage of the proposed thermal analogy technique over previous studies is that it accounts for the convective diffusion of ion concentrations and the Donnan osmotic pressure in the interstitial fluid.
doi:10.1186/1475-925X-1-8
PMCID: PMC443818  PMID: 12685940
19.  ERK1/2 Activation Induced by Inflammatory Cytokines Compromises Effective Host Tissue Integration of Engineered Cartilage 
Tissue Engineering. Part A  2009;15(10):2825-2835.
Objective
Proinflammatory cytokines are known to provoke degradative signaling cascades that promote extracellular matrix disintegration in articular cartilage. Because integration of the repair tissue into the surrounding native cartilage to produce a mechanically stable interface has a profound impact on the viability and functionality of the restored joint surface, this study examined the effects of proinflammatory cytokines on the properties of tissue-engineered cartilage in the context of integration. Methods: Using an established in vitro cartilage defect model, we examined the integration of chondrocyte-laden agarose constructs into native articular cartilage and the biochemical and biomechanical alterations of these implants upon treatment with interleukin 1-β (IL1-β) and tumor necrosis factor-α (TNF-α). Additionally, we probed extracellular regulated kinase (ERK) signaling involvement in response to proinflammatory cytokines. Results: The time-dependent accumulation of extracellular matrix and concomitant increase in Young's modulus observed in the absence of cytokines was significantly decreased upon IL1-β and TNF-α treatment. Push-out test showed the highest interface strength in hybrid cultures maintained without cytokines, which was significantly lowered with IL1-β and TNF-α treatment. Histological characteristics of the interface region are consistent with the biochemical findings. Treatment with an inhibitor of ERK pathway antagonized the deleterious effects caused by both cytokines. Conclusion: This study is the first to show the functional catastrophic effects of IL1-β and TNF-α on the biochemical, structural, and integrative properties of tissue-engineered cartilage and their significant counteraction by the blockade of ERK signaling pathway. With the discovery of new potential chemical entities, ERK inhibitor may emerge as a new therapeutic approach for functional integration and mechanical integrity of an engineered cartilage to the host tissue and, therefore, enhance long-term viability and functionality of the restored joint surface.
doi:10.1089/ten.tea.2008.0663
PMCID: PMC2792059  PMID: 19243242
20.  Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β 
Arthritis Research & Therapy  2013;15(6):R223.
Introduction
Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen.
Methods
Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1β (10 ng/ml), carprofen (100 μg/ml), and carprofen (100 μg/ml) + IL-1β (10 ng/ml). Explants were incubated (37°C, 5% CO2) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs).
Results
Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1β stimulation increased the release of all three MMPs. IL-1β also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by β-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1β-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1β versus 52.91% ± 9.35% (SD) with carprofen + IL-1β.
Conclusions
Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.
doi:10.1186/ar4424
PMCID: PMC3978949  PMID: 24373218
21.  Articular cartilage studies and osteoarthrosis. 
Osteoarthrosis is characterized in the early stages by degradation of articular cartilage matrix. Clinical, radiological, and pathological studies have failed to reveal the factors which initiate the breakdown of cartilage and are not applicable to detailed sequential studies of the affected tissues at all stages in the disease. Therefore animal experiments have been employed to provide more information on degradation and repair process in cartilage. These studies have demonstrated: 1) Matrix protection and induced repair of mature articular cartilage by the use of oral aspirin after lacerative injury. 2) Establishment by the intra-articular injection of the plant enzyme papain of a model of osteoarthrosis in the rabbit hip which mimics human osteoarthrosis and is suitable for further experimental studies. 3) A proliferation of mature articular cartilage chondrocytes in response to loss of matrix, which indicates a latent repair capacity. 4) Repair of extensively damaged hip joints after femoral osteotomy by increased formation of subchondral new bone and formation of fibrocartilage on the articulating surfaces. These tissue repair processes are associated with an increase in vascularity of the femoral head and acetabulum produced by the osteotomy. 5) Succesful transplantation as allografts in both normal and arthrotic rabbit knees of aggregates of epiphysial chondrocytes isolated from their matrix. This method of joint surface replacement may have clinical applications.
Images
PMCID: PMC2388573  PMID: 1163951
22.  Reactive nitrogen and oxygen species in interleukin-1-mediated DNA damage associated with osteoarthritis 
Objective
Osteoarthritis (OA) is associated with increased levels of reactive nitrogen and oxygen species and pro-inflammatory cytokines, such as interleukin-1 (IL-1). Nitric oxide (NO) can mediate a number of the catabolic effects of IL-1 in articular cartilage. The aims of this study were to determine if OA cartilage shows evidence of DNA damage, and if IL-1 could induce DNA damage in non-OA cartilage by increasing NO or superoxide.
Methods
Articular chondrocytes were isolated from porcine femoral condyles and embedded in 1.2% alginate. The effects of 24 hrs incubation with IL-1, the nitric oxide synthase 2 (NOS2) selective inhibitor, the free radical scavenger SOD, the NO donor NOC18, or the combined NO and peroxynitrite donor SIN-1 on DNA damage were tested, using the “comet” assay. NO production was measured using the Griess assay. The type of oxidative damage present was assessed using a modified comet assay.
Results
OA cartilage had significantly more DNA damage than non-OA cartilage (p < 0.001). IL-1 caused an increase in DNA damage (p < 0.01), which was associated with increased NO production (p < 0.01). Both oxidative DNA strand breaks and base modifications of purines and pyrimidines were observed. IL-1-induced DNA damage was inhibited by a NOS2 inhibitor or by superoxide dismutase (p < 0.01). Furthermore, NOC-18 or SIN-1 caused DNA damage (p < 0.001).
Conclusion
Our work shows chondrocytes in osteoarthritic cartilage exhibit DNA damage, and that IL-1 induces DNA damage and reactive oxygen and nitrogen species in non-OA chondrocytes in alginate.
doi:10.1016/j.joca.2007.09.012
PMCID: PMC2430155  PMID: 17945515
23.  Post-translational aging of proteins in osteoarthritic cartilage and synovial fluid as measured by isomerized aspartate 
Introduction
Aging proteins undergo non-enzymatic post-translational modification, including isomerization and racemization. We hypothesized that cartilage with many long-lived components could accumulate non-enzymatically modified amino acids in the form of isomerized aspartate and that its liberation due to osteoarthritis (OA)-related cartilage degradation could reflect OA severity.
Methods
Articular cartilage and synovial fluid were obtained from 14 randomly selected total knee arthroplasty cases (56 to 79 years old) and non-arthritis cartilage from 8 trauma cases (51 to 83 years old). Paired lesional cartilage and non-lesioned OA cartilage were graded histologically using a modified Mankin system. Paired cartilage and synovial fluids were assayed for isomerized aspartate, phosphate-buffered saline/EDTA (ethylenediaminetetraacetic acid) extractable glycosaminoglycans, and total protein. Macroscopically normal non-lesioned OA cartilage was separated into superficial and deep regions when cartilage thickness was at least 3 mm (n = 6).
Results
Normalized to cartilage wet weight, normal cartilage and deep non-lesioned OA cartilage contained significantly (P < 0.05) more isomerized aspartate than superficial non-lesioned OA cartilage and lesioned cartilage. Synovial fluid isomerized aspartate correlated positively (R2 = 0.53, P = 0.02) and glycosaminoglycans correlated negatively (R2 = 0.42, P = 0.04) with histological OA lesion severity. Neither synovial fluid isomerized aspartate nor glycosaminoglycans nor total protein correlated with histological scores of non-lesioned areas.
Conclusions
We show for the first time that human cartilage and synovial fluid contain measurable quantities of an isomerized amino acid and that synovial fluid concentrations of isomerized aspartate reflected severity of histological OA. Further assessment is warranted to identify the cartilage proteins containing this modification and to assess the functional consequences and biomarker applications of this analyte in OA.
doi:10.1186/ar2675
PMCID: PMC2688206  PMID: 19371408
24.  Cells of the synovium in rheumatoid arthritis. Chondrocytes 
Rheumatoid arthritis (RA) is one of the inflammatory joint diseases in a heterogeneous group of disorders that share features of destruction of the extracellular matrices of articular cartilage and bone. The underlying disturbance in immune regulation that is responsible for the localized joint pathology results in the release of inflammatory mediators in the synovial fluid and synovium that directly and indirectly influence cartilage homeostasis. Analysis of the breakdown products of the matrix components of joint cartilage in body fluids and quantitative imaging techniques have been used to assess the effects of the inflammatory joint disease on the local remodeling of joint structures. The role of the chondrocyte itself in cartilage destruction in the human rheumatoid joint has been difficult to address but has been inferred from studies in vitro and in animal models. This review covers current knowledge about the specific cellular and biochemical mechanisms that account for the disruption of the integrity of the cartilage matrix in RA.
doi:10.1186/ar2292
PMCID: PMC2212563  PMID: 18001488
25.  Establishment of an Index with Increased Sensitivity for Assessing Murine Arthritis 
Purpose
Establish quantitative outcomes for assessing murine knee arthritis and develop an Arthritis Index that incorporates multiple outcomes into a single calculation that provides enhanced sensitivity.
Methods
Using an accepted model of meniscal/ligamentous injury (MLI)-induced osteoarthritis (OA), we assessed mouse knee arthritis using several approaches. Histology-based methods were performed to visualize joint tissues including articular cartilage and subchondral bone. Accepted histologic scoring methods and histomorphometry were performed to grade cartilage degeneration and determine articular cartilage area, respectively. MicroCT was used to visualize and quantify the bony structures of the joint including osteophytes and joint bone volume. A statistical algorithm was then developed that combined histologic scores and cartilage areas into a single Arthritis Index.
Results
MLI induced progressive, OA-like articular cartilage degeneration characterized by increasing (worsening) histologic score and decreasing cartilage area. MicroCT revealed osteophytes and increased joint bone volume between the femoral and tibial physes following MLI. Lastly, an Arthritis Index calculation was established, which incorporated histologic scoring and cartilage area. The Arthritis Index provided enhanced quantitative sensitivity in assessing the level of joint degeneration compared to either histologic scoring or cartilage area determination alone; when using the Index, between 29% and 43% fewer samples are needed to establish statistical significance in studies of murine arthritis.
Conclusions
Arthritis in the mouse knee can be quantitatively assessed by histologic scoring, measuring cartilage area and determining joint bone volume. Enhanced sensitivity can be achieved by performing the Arthritis Index calculation, a novel method for quantitatively assessing mouse knee arthritis.
doi:10.1002/jor.21368
PMCID: PMC3115505  PMID: 21374709
Osteoarthritis; Articular cartilage; Meniscal injury; Micro-computed tomography; Arthritis index

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