Zone Of Articular Cartilage Research Articles

Oxidant damage in Kashin‐Beck disease and a rat Kashin‐Beck disease model by employing T‐2 toxin treatment under selenium deficient conditions

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy, but the mechanisms underlying its pathogenesis remains unclear. This study compares antioxidant capacity and lipid peroxidation using a novel model, in which rats were administered a selenium-deficient diet for 4 weeks prior to their exposure to T-2 toxin for 4 weeks. Changes in cell morphology and empty chondrocyte lacunae indicative of cell death, as well as cartilage proteoglycan loss in the deep zone of articular cartilage of knee joints were observed in rats with selenium-deficient diet plus T-2 toxin treatment. These changes were similar to those observed previously in KBD. The levels of thiobarbituric acid reactive substances (TBARS), indicative of lipid peroxidation in serum and cartilage, were significantly increased in all experimental groups compared to the normal diet group, while the levels of antioxidants, measured as total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidases (GPX), in serum and cartilage were significantly lower than that in the normal diet group. The mRNA expression of those antioxidants in cartilage tissue was significantly reduced by T-2 toxin alone or by selenium-deficient diet plus T-2 toxin treatment. These results indicate that increasing TBARS and decreasing antioxidants in serum and cartilage by T-2 toxin treatment with a selenium-deficient nutritional status may alter oxidative stress in joint tissues and contribute to the pathological process of cartilage damage in KBD.

Histopathology of chondronecrosis development in knee articular cartilage in a rat model of Kashin–Beck disease using T-2 toxin and selenium deficiency conditions

The objective of this study is to observe pathogenic lesions of joint cartilages in rats fed with T-2 toxin under a selenium deficiency nutrition status in order to determine possible etiological factors causing Kashin-Beck disease (KBD). Sprague-Dawley rats were fed selenium-deficient or control diets for 4 weeks prior to their being exposed to T-2 toxin. Six dietary groups were formed and studied 4 weeks later, i.e., controls, selenium-deficient, low T-2 toxin, high T-2 toxin, selenium-deficient diet plus low T-2 toxin, and selenium-deficient diet plus high T-2 toxin. Selenium deficiencies were confirmed by the determination of glutathione peroxidase activity and selenium levels in serum. The morphology and pathology (chondronecrosis) of knee joint cartilage of experimental rats were observed using light microscopy and the expression of proteoglycans was determined by histochemical staining. Chondronecrosis in deep zone of articular cartilage of knee joints was seen in both the low and high T-2 toxin plus selenium-deficient diet groups, these chondronecrotic lesions being very similar to chondronecrosis observed in human KBD. However, the chondronecrosis observed in the rat epiphyseal growth plates of animals treated with T-2 toxin alone or T-2 toxin plus selenium-deficient diets were not similar to that found in human KBD. Our results indicate that the rat can be used as a suitable animal model for studying etiological factors contributing to the pathogenesis (chondronecrosis) observed in human KBD. However, those changes seen in epiphyseal growth plate differ from those seen in human KBD probably because of the absence of growth plate closure in the rat.

Analysis of Collagen Type II and X in Ageing and Osteoarthritic Articular Cartilage

Objective: Collagens identified in articular cartilage include the cartilage specific collagen types II, IX, X and XI as well as the more universally distributed types III, V, VI, XII and XIV. Type II collagen is the predominant and characteristic collagen of the articular cartilage, composed of a triple helix of three identical α chains. The hypertrophic chondrocytes of calcified zone of articular cartilage synthesize type X collagen in addition to the type II collagen. The present study was conducted to observe the changes in the collagenase activity in osteoarthritis by detecting TCA fragment of the α chain of collagen type II. The expression of native collagen type X was also observed in the osteoarthritic articular cartilage by SDS-PAGE and western blotting. Methods: 22 osteoarthritic cartilage specimens were obtained from patients with primary osteoarthritis (46-81years) undergoing total knee replacement. 12 age-matched (41-86years) and 14 young (16-40years) non-osteoarthritic control cartilage specimens were obtained from the cadavers in the department of Anatomy and from patients undergoing lower limb amputation in Trauma center of PGIMER, Chandigarh. The cartilage specimens were processed for SDS-PAGE and western blotting. Results: Analysis of the α-chymotrypsin extracts of the articular cartilage for TCA fragment (75kDa) of collagen type II and native collagen type X (60kDa) revealed a much higher content in the osteoarthritic group. Conclusions: The features of the articular cartilage from the osteoarthritic group were markedly different from the non-osteoarthritic age-matched articular cartilage suggesting that OA is not an inevitable feature of aging.

Expression of cartilage oligomeric matrix protein in articular cartilage in response to bone morphogenetic protein-7 and tumour necrosis factor : peer reviewed original article

Introduction: The extracellular matrix of articular cartilage consists of collagens, proteoglycans and noncollageneous proteins. Cartilage oligomeric matrix protein (COMP) is a non-collagenous protein isolated from articular cartilage. It is localised in articular cartilage and the proliferative and hypertrophic zones of the epiphyseal growth plate. Aim: The aim of the study was to investigate the role of bone morphogenetic proteins (BMP-7) and tumour necrosis factor (TNF-α) on expression of COMP in articular cartilage. Methods: Stifle joints (n=12) from three month-old calves were obtained from a local abattoir. Articular chondrocytes were isolated from three distinct zones of cartilage and cultured as monolayers in a serum-free chemically defined medium. Results: BMP-7 increased COMP accumulation in dose and time dependent manner. TNF-α was however found to significantly reduce COMP accumulation Conclusion: BMP-7 was shown to be important regulators of the COMP gene in different zones of articular cartilage

The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse

A chondrocyte progenitor population isolated from the surface zone of articular cartilage presents a promising cell source for cell-based cartilage repair. In this study, equine articular cartilage progenitor cells (ACPCs) and equine bone marrow-derived stromal cells (BMSCs) were compared as potential cell sources for repair. Clonally derived BMSCs and ACPCs demonstrated expression of the cell fate selector gene, Notch-1, and the putative stem cell markers STRO-1, CD90 and CD166. Chondrogenic induction revealed positive labelling for collagen type II and aggrecan. Collagen type X was not detected in ACPC pellets but was observed in all BMSC pellets. In addition, it was observed that BMSCs labelled for Runx2 and matrilin-1 antibodies, whereas ACPC labelling was significantly less or absent. For both cell types, osteogenic induction revealed positive von Kossa staining in addition to positive labelling for osteocalcin. Adipogenic induction revealed a positive result via oil red O staining in both cell types. ACPCs and BMSCs have demonstrated functional equivalence in their multipotent differentiation capacity. Chondrogenic induction of BMSCs resulted in a hypertrophic cartilage (endochondral) phenotype, which can limit cartilage repair as the tissue can undergo mineralisation. ACPCs may therefore be considered superior to BMSCs in producing cartilage capable of functional repair.

Cartilage Intermediate Layer Protein 2 (CILP-2) Is Expressed in Articular and Meniscal Cartilage and Down-regulated in Experimental Osteoarthritis

Using transcriptome profiling to determine differential gene expression between the permanent mouse articular cartilage and the transient growth plate cartilage, we identified a highly expressed gene, Cilp2, which is expressed differentially by articular chondrocytes. CILP-2 is highly homologous to CILP-1 (cartilage intermediate layer protein 1), which is expressed in the intermediate zone of articular cartilage and has been linked to cartilage degenerative diseases. We demonstrated that Cilp2 has a restricted mRNA distribution at the surface of the mouse articular cartilage during development, becoming localized to the intermediate zone of articular cartilage and meniscal cartilage with maturity. Although the extracellular CILP-2 protein localization is broadly similar to CILP-1, CILP-2 appears to be more localized in the deeper intermediate zone of the articular cartilage extracellular matrix at maturity. CILP-2 was shown to be proteolytically processed, N-glycosylated, and present in human articular cartilage. In surgically induced osteoarthritis in mice, Cilp1 and Cilp2 gene expression was dysregulated. However, whereas Cilp1 expression was increased, Cilp2 gene expression was down-regulated demonstrating a differential response to mechanically induced joint destabilization. CILP-2 protein was reduced in the mouse osteoarthritic cartilage. Ultrastructural analysis also suggested that CILP-2 may be associated with collagen VI microfibrils and thus may mediate interactions between matrix components in the territorial and inter-territorial articular cartilage matrix. mRNA expression analysis indicated that whereas Cilp1 and Cilp2 are expressed most abundantly in cartilaginous tissues, expression can be detected in muscle and heart.

Phenotyping of chondrocytes from human osteoarthritic cartilage: chondrocyte expression of beta integrins and correlation with anatomic injury

Chondrocyte-ECM (extracellular matrix) interactions are believed to play a pivotal role in the development and metabolic homeostasis of articular cartilage. Cell surface adhesion molecules have been reported to modulate chondrocyte binding to ECM (collagen, fibronectin, laminin) and they also act as transducers of critical signals in many biological processes such as growth, differentiation, migration and matrix synthesis. Recently, it has been shown that normal human articular chondrocytes strongly express beta1 integrins, which are constituted by a common chain (beta1) and a variable alphachain, but the behaviour of these molecules in human osteoarthritic cartilage has not been extensively investigated. We studied the expression of beta integrins (beta1-5, alpha1-6, av chains), LFA-1, ICAM-1 and CD44, on freshly isolated chondrocytes obtained from 10 osteoarthritic patients undergoing surgical knee replacement. Chondrocytes were isolated by enzymatic digestion from three zones of each articular cartilage with a differing degree of macroscopic and microscopic damage. Integrin expression and cell cycle analysis were carried out by flowcytometry. Chondrocytes from costal cartilages of 5 human foetuses were also studied. Chondrocytes from osteoarthritic cartilage expressed high levels of beta1 integrin and, at different percentages, all the alphachains. The alphachain most frequently expressed was alpha1, foilowed by alpha3, alpha5, alpha2, alphav. Integrin expression decreased from the least to the most damaged zone of articular cartilage and cell cycle analysis showed that proliferating chondrocytes (S phase) were prevalent on the latter zone. beta2, beta3, beta2, beta5, CD44, LFA-1/ICAM-1 complex were very low expressed. Fetal chondrocytes strongly expressed beta1 and beta5 chains. These data provide evidence to show that integrin expression on human chondrocytes changes in osteoarthritis and suggest that perturbations of chondrocyte-ECM signalling occur in the development of the disease. The different pattern of expression of beta1 and beta5 chains on adult and fetal chondrocytes leads to speculate that integrins play a key role in control of cartilage morphogenesis and differentiation.

Engineering articular cartilage with spatially-varying matrix composition and mechanical properties from a single stem cell population using a multi-layered hydrogel

Despite significant advances in stem cell differentiation and tissue engineering, directing progenitor cells into three-dimensionally (3D) organized, native-like complex structures with spatially-varying mechanical properties and extra-cellular matrix (ECM) composition has not yet been achieved. The key innovations needed to achieve this would involve methods for directing a single stem cell population into multiple, spatially distinct phenotypes or lineages within a 3D scaffold structure. We have previously shown that specific combinations of natural and synthetic biomaterials can direct marrow-derived stem cells (MSC) into varying phenotypes of chondrocytes that resemble cells from the superficial, transitional, and deep zones of articular cartilage. In this current study, we demonstrate that layer-by-layer organization of these specific biomaterial compositions creates 3D niches that allow a single MSC population to differentiate into zone-specific chondrocytes and organize into a complex tissue structure. Our results indicate that a three-layer polyethylene glycol (PEG)-based hydrogel with chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) incorporated into the top layer (superficial zone, PEG:CS:MMP-pep), CS incorporated into the middle layer (transitional zone, PEG:CS) and hyaluronic acid incorporated in the bottom layer (deep zone, PEG:HA), creates native-like articular cartilage with spatially-varying mechanical and biochemical properties. Specifically, collagen II levels decreased gradually from the superficial to the deep zone, while collagen X and proteoglycan levels increased, leading to an increasing gradient of compressive modulus from the superficial to the deep zone. We conclude that spatially-varying biomaterial compositions within single 3D scaffolds can stimulate efficient regeneration of multi-layered complex tissues from a single stem cell population.

An Atypical Degenerative Osteoarthropathy in Hyp Mice is Characterized by a Loss in the Mineralized Zone of Articular Cartilage

Patients with X-linked hypophosphatemia (XLH) develop enthesophytes and osteophytes secondary to articular cartilage degeneration and together are the primary cause of morbidity in adult patients so afflicted. We have previously characterized the enthesopathy in Hyp mice, a murine model of XLH. We now extend these studies to the synovial joint in order to characterize potential cellular changes in articular cartilage that may predispose patients to the osteoarthropathy of XLH. We report that, despite highly elevated levels of alkaline phosphatase activity throughout articular cartilage, there is a complete loss in the mineralized zone of articular cartilage as assessed by von Kossa staining of mineral and as quantified by EPIC-microCT analysis and evidence of vascular invasion. We also identify the downregulation of extracellular matrix (ECM) factors identified as regulators of terminally differentiated mineralizing articular chondrocytes. There is also a striking increase in the histochemical staining of sulfated proteoglycans, a change that may reflect the loss of a transitional tissue that reduces mechanical stress at the interface between cartilage and subchondral bone. The failure of mineralizing articular chondrocytes to develop in the hypophosphatemic state suggests that phosphate may be a key regulator of chondrocyte mineralization. Accordingly, we find that the appropriate zonal arrangement and phenotypic markers of articular cartilage are significantly reestablished by phosphate-replacement therapy. Given the turnover and maintenance of articular cartilage ECM, the identification of early and abnormal cellular changes unique to XLH will undoubtedly aid in a more effective management of this disease to minimize the onset of degenerative osteoarthropathy.

Differential response of cartilage oligomeric matrix protein (COMP) to morphogens of bone morphogenetic protein/transforming growth factor-β family in the surface, middle and deep zones of articular cartilage

Cartilage oligomeric matrix protein (COMP) is a prominent non-collagenous component of the extracellular matrix (ECM) in articular cartilage. The regulation of COMP synthesis and secretion is critical for the understanding of cartilage homeostasis in health and disease. We therefore investigated the role of bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) superfamily members on COMP. Articular chondrocytes were isolated from three distinct zones (surface, middle and deep) and cultured as monolayers in serum-free chemically defined medium. Protein levels of COMP were determined in the medium by enzyme-linked immunosorbent assay. The mRNA expression was determined by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR). TGFβ1 significantly stimulated the expression of COMP at the mRNA and protein levels in the superficial zone in a time-dependent manner. An unexpected discovery was that surface chondrocytes were more responsive to TGFβ isoforms than those in the deep layer. However, BMP-7 and growth differentiation factor-5 (GDF-5) also upregulate COMP expression; the effects were not as potent as those of TGFβ1. Activins A, B and AB demonstrated no effects on COMP in any of the zones. In conclusion, COMP synthesis is differentially regulated by TGFβ1 in the surface and middle zones of bovine articular cartilage.

Identification of an extraordinary bone phenotype in a patient with alkaptonuria

Event Abstract Back to Event Identification of an extraordinary bone phenotype in a patient with alkaptonuria AM Taylor1*, JS Davidson2, JC Jarvis1, LR Ranganath1, JA Gallagher1 and A Boyde3 1 University of Liverpool, Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, United Kingdom 2 Royal Liverpool and Broadgreen University Hospitals Trust, Department of Trauma & Orthopaedics, United Kingdom 3 Queen Mary University of London, Institute of Dentistry, United Kingdom A 59 year old man with known alkaptonuria (AKU) presented for surgery due to ochronotic osteoarthropathy and underwent a total hip replacement. The resected femoral head showed uniform ochronosis of the articular cartilage with the presence of a large osteophyte on the neck of femur, also demonstrating ochronosis. Surrounding capsular tissues displayed patchy ochronotic pigmentation. An ultrastructural and histological analysis of patient tissues was undertaken. Samples were processed for histology and for topographical 3D scanning electron microscopy (SEM) and quantitative back scattered electron SEM (qBSE-SEM). These analyses revealed an extraordinary phenotype including the presence of some novel microanatomical structures. Histological examination of the cartilage demonstrated the presence of ochronotic pigment throughout all zones of articular cartilage, both intra- and extracellularly. No similar pigmentation was observed in the calcified cartilage or bone matrices, but these latter tissues underwent significant structural modelling. Ochronosis was observed in osteoblasts, osteoclasts and osteocytes, with osteoclasts seen engulfing pigmented osteocytes from the bone matrix. The most striking feature was the resorption of subchondral bone and calcified cartilage such that in advanced stages there was complete loss of the subchondral bone plus calcified cartilage plate. The underlying trabecular bone also contained idiosyncratic architecture. Trabecular surfaces had numerous outgrowths that we have termed 'excrescences' of which three distinct types were recognized. The first type arose from the incomplete resorption of branching trabeculae and they were characterized by scalloped surfaces and rugged edges. The second type arose in a similar way but had been smoothed over by new bone deposition. The third type which resembled coarse stucco probably arose from resting surfaces that had been focally reactivated. These were poorly cemented to the prior trabecular wall. They had a disorganized arrangement of collagen fibres and contained poorly mineralised and hypermineralised regions. We propose these distinctive microanatomical structures do not arise as a result of pigmentation per se but because of abnormal osteoclast/osteoblast modelling secondary to altered mechanical loading or other aberrant signalling. The distinctive subchondral environment underneath pigmented cartilage in AKU provides a unique model to investigate the response of bone to altered mechanical loading. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Poster Topic: Abstracts Citation: Taylor A, Davidson J, Jarvis J, Ranganath L, Gallagher J and Boyde A (2011). Identification of an extraordinary bone phenotype in a patient with alkaptonuria. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00067 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Dr. AM Taylor, University of Liverpool, Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, Liverpool, United Kingdom, amtaylor@liv.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers AM Taylor JS Davidson JC Jarvis LR Ranganath JA Gallagher A Boyde Google AM Taylor JS Davidson JC Jarvis LR Ranganath JA Gallagher A Boyde Google Scholar AM Taylor JS Davidson JC Jarvis LR Ranganath JA Gallagher A Boyde PubMed AM Taylor JS Davidson JC Jarvis LR Ranganath JA Gallagher A Boyde Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Unique biomaterial compositions direct bone marrow stem cells into specific chondrocytic phenotypes corresponding to the various zones of articular cartilage

Numerous studies have reported generation of cartilage-like tissue from chondrocytes and stem cells, using pellet cultures, bioreactors and various biomaterials, especially hydrogels. However, one of the primary unsolved challenges in the field has been the inability to produce tissue that mimics the highly organized zonal architecture of articular cartilage; specifically its spatially varying mechanical properties and extra-cellular matrix (ECM) composition. Here we show that different combinations of synthetic and natural biopolymers create unique niches that can “direct” a single marrow stem cell (MSC) population to differentiate into the superficial, transitional, or deep zones of articular cartilage. Specifically, incorporating chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) into PEG hydrogels (PEG:CS:MMP-pep) induced high levels of collagen II and low levels of proteoglycan expression resulting in a low compressive modulus, similar to the superficial zone. PEG:CS hydrogels produced intermediate-levels of both collagen II and proteoglycans, like the transitional zone, while PEG:hyaluronic acid (HA) hydrogels induced high proteoglycan and low collagen II levels leading to high compressive modulus, similar to the deep zone. Additionally, the compressive moduli of these zone-specific matrices following cartilage generation showed similar trend as the corresponding zones of articular cartilage, with PEG:CS:MMP-pep having the lowest compressive modulus, followed by PEG:CS while PEG:HA had the highest modulus. These results underscore the potential for composite scaffold structures incorporating these biomaterial compositions such that a single stem-progenitor cell population can give rise to zonally-organized, functional articular cartilage-like tissue.

Hox11 genes establish synovial joint organization and phylogenetic characteristics in developing mouse zeugopod skeletal elements

Hox11 genes are essential for zeugopod skeletal element development but their roles in synovial joint formation remain largely unknown. Here, we show that the elbow and knee joints of mouse embryos lacking all Hox11 paralogous genes are specifically remodeled and reorganized. The proximal ends of developing mutant ulna and radius elements became morphologically similar and formed an anatomically distinct elbow joint. The mutant ulna lacked the olecranon that normally attaches to the triceps brachii muscle tendon and connects the humerus to the ulna. In its place, an ulnar patella-like element developed that expressed lubricin on its ventral side facing the joint and was connected to the triceps muscle tendon. In mutant knees, both tibia and fibula fully articulated with an enlarged femoral epiphyseal end that accommodated both elements, and the neo-tripartite knee joint was enclosed in a single synovial cavity and displayed an additional anterior ligament. The mutant joints also exhibited a different organization of the superficial zone of articular cartilage that normally exerts an anti-friction function. In conclusion, Hox11 genes co-regulate and coordinate the development of zeugopod skeletal elements and adjacent elbow and knee joints, and dictate joint identity, morphogenesis and anatomical and functional organization. Notably, the ulnar patella and tripartite knee joints in the mouse mutants actually characterize several lower vertebrates, including certain reptiles and amphibians. The re-emergence of such anatomical structures suggests that their genetic blueprint is still present in the mouse genome but is normally modified to the needs of the mammalian joint-formation program by distinct Hox11 function.

Expression of lysyl oxidase family in different zones of articular cartilage

Objective To observe the expression of lysyl oxidase (LOX) family in different zones of articular cartilage and preliminarily explore the effect of LOX family on the collage cross-linking of the articular cartilage. Methods Full-thickness cartilage explants were harvested from the medially patello-femoral groove of adult bovine animals. The cartilage explants consisted with the superficial zone,the transitional zone and the deep zone. RT-PCR was used to assess expression of LOX family in every zone of articular cartilage. Results The levels of LOX, LOXL2 and LOXL4 were descended from superficial zone to deep zone. No expressions of LOXL1 and LOXL3 were found in the cartilage. Conclusion Different expression of LOX family in different zones of articular cartilage may affect the configuration and the physiological and biochemical features of the collage in different zones. Key words: Lysyl oxidase; Cartilage, articular; Pattelofemoral joint

Integrity of articular cartilage on T2 mapping associated with meniscal signal change

Objective The purpose of this study was to investigate the relationship between T2 relaxation values (T2 RVs) within the superficial zone of articular cartilage and different types of meniscal degeneration/tear. Materials and methods A review of 310 consecutive knee MRIs which included an 8 echo T2 relaxation sequence, in patients referred for standard clinical indications, was performed independently and in blinded fashion by 2 observers. The posterior horns of the medial and lateral menisci were each evaluated and divided into 4 subgroups: Normal (control), Grade I/II meniscal signal, Grade III meniscal signal-simple tear (Grade III-S), and Grade III meniscal signal-complex tear (Grade III-C). After exclusion criteria were applied, the medial meniscal group consisted of 65 controls and 133 patients, while the lateral meniscal group consisted of 143 controls and 55 patients. T2 RVs were measured by an observer blinded to the clinical history and MRI grading. Measurements were obtained over the superficial zone of femoral and tibial articular cartilage adjacent to the center of the posterior horn of each meniscus to ensure consistency between measurements. Analysis of covariance adjusting for age and gender was used to compare T2 RVs between patients and controls. Results T2 RVs were significantly increased in patients with Grade III-C meniscal tears compared to controls over the medial tibial plateau (MTP; p = 0.0001) and lateral tibial plateau (LTP; p = 0.0008). T2 RVs were not increased in patients with Grade III-C meniscal tears over the medial femoral condyle (MFC; p = 0.11) or lateral femoral condyle (LFC; p = 0.99). Grade I/II meniscal signal was not associated with elevated T2 RVs over the MFC ( p = 0.15), LFC ( p = 0.69), MTP ( p = 0.42), or LTP ( p = 0.50). Grade III-S meniscal signal was not associated with elevated T2 RVs over the MFC ( p = 0.54), LFC ( p = 0.43), MTP ( p = 0.30), or LTP ( p = 0.38). Conclusion Grade III-C meniscal tears are associated with elevated T2 RVs in adjacent tibial articular cartilage. The results may have an impact on prognostication and treatment in order to delay or prevent the onset of osteoarthritis.

Specificity of Fourier Transform Infrared (FTIR) Microspectroscopy to Estimate Depth-Wise Proteoglycan Content in Normal and Osteoarthritic Human Articular Cartilage.

Background:Fourier transform infrared (FTIR) microspectroscopy is a promising method for estimating the depth-wise composition of articular cartilage. The aim was to compare the specificity of two earlier introduced, presumably proteoglycan (PG)–specific FTIR parameters (i.e., absorption in the carbohydrate region with and without normalization with Amide I absorption) to estimate the reference PG content of normal and osteoarthritic human articular cartilage. This study is a direct continuation of our earlier studies, from which the presented data are reanalyzed.Design:Earlier, FTIR microspectroscopy, digital densitometry, histological analyses, and polarized light microscopy were conducted in vitro for articular cartilage samples of human patellae (n = 72). In the present study, earlier data were combined and statistically reanalyzed in a depth-wise manner to clarify the specificity of FTIR parameters introduced for the estimation of PG content of articular cartilage. Digital densitometry for Safranin O-stained samples was used to indicate reference PG content.Results:Direct absorption of the carbohydrate region estimated well the PG content in the middle and deep zones of articular cartilage and appeared to be superior compared to carbohydrate values normalized with Amide I absorption. However, in the superficial zone, the specificity of both FTIR-derived PG parameters was limited.Conclusions:Limitations of current FTIR-based PG parameters in the superficial zone of articular cartilage should be recognized and carefully taken into account in future studies using FTIR microspectroscopy for PG content estimation. Further research is needed to improve the specificity of FTIR parameters for the estimation of PG content of articular cartilage.

Curcumin mediated suppression of nuclear factor-κB promotes chondrogenic differentiation of mesenchymal stem cells in a high-density co-culture microenvironment

IntroductionOsteoarthritis (OA) and rheumatoid arthritis (RA) are characterised by joint inflammation and cartilage degradation. Although mesenchymal stem cell (MSC)-like progenitors are resident in the superficial zone of articular cartilage, damaged tissue does not possess the capacity for regeneration. The high levels of pro-inflammatory cytokines present in OA/RA joints may impede the chondrogenic differentiation of these progenitors. Interleukin (IL)-1β activates the transcription factor nuclear factor-κB (NF-κB), which in turn activates proteins involved in matrix degradation, inflammation and apoptosis. Curcumin is a phytochemical capable of inhibiting IL-1β-induced activation of NF-κB and expression of apoptotic and pro-inflammatory genes in chondrocytes. Therefore, the aim of the present study was to evaluate the influence of curcumin on IL-1β-induced NF-κB signalling pathway in MSCs during chondrogenic differentiation.MethodsMSCs were either cultured in a ratio of 1:1 with primary chondrocytes in high-density culture or cultured alone in monolayer with/without curcumin and/or IL-1β.ResultsWe demonstrate that although curcumin alone does not have chondrogenic effects on MSCs, it inhibits IL-1β-induced activation of NF-κB, activation of caspase-3 and cyclooxygenase-2 in MSCs time and concentration dependently, as it does in chondrocytes. In IL-1β stimulated co-cultures, four-hour pre-treatment with curcumin significantly enhanced the production of collagen type II, cartilage specific proteoglycans (CSPGs), β1-integrin, as well as activating MAPKinase signaling and suppressing caspase-3 and cyclooxygenase-2.ConclusionsCurcumin treatment may help establish a microenvironment in which the effects of pro-inflammatory cytokines are antagonized, thus facilitating chondrogenesis of MSC-like progenitor cells in vivo. This strategy may support the regeneration of articular cartilage.

Zonal Chondrocyte Subpopulations Reacquire Zone-Specific Characteristics during in Vitro Redifferentiation

Background If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. Hypothesis Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture). Study Design Controlled laboratory study. Methods Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes). Results Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone. Conclusion Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion. Clinical Relevance Appreciation of the zonal differences can lead to important advances in cartilage tissue engineering. Findings support the use of hydrogels such as alginate for engineering zonal cartilage constructs.

070 AGE-RELATED BEHAVIOR OF COLLAGEN AND PROTEOGLYCAN IN SUPERFICIAL, MIDDLE AND DEEP ZONES OF PATELLAR CARTILAGE: FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR) ANALYSIS OF NORMAL RABBITS

Purpose: To detect the collagen content and proteoglycan content in the superficial, middle and deep zones of articular cartilage is a trend but still technical challenge. Fourier transform infrared spectroscopy (FTIR) is powerful tool to investigate the collagen and proteoglycan in cartilage matrix. The FTIR quantifies the collagen content and proteoglycan content on histologic sections of cartilage. Because the FTIR maps the distribution of collagen and proteoglycan on the sections, the collagen and proteoglycan can be analyzed in the superficial, middle and deep zones of cartilage. Age-related behavior of the collagen and proteoglycan in each zone of patellar cartilage has not been reported yet. Therefore, the objective of this pilot study is to investigate the behavior of the collagen and proteoglycan in each zone using the FTIR. Methods: Non-treated five rabbits of various ages (3-week, 8week, 6-month, 1-year, 2.5-year) were examined. Specimens of patella were removed from each rabbit and all specimens were observed macroscopically and confirmed that the cartilage surface was smooth and glossy. Specimens were then prepared and sagittal sections were cut for histology and for the FTIR. The sections stained with safranin-O/fast green were observed using a light microscopy and confirmed that no fibrillation or no degenerative change had occurred. The sections for the FTIR were not stained. The integrated area of collagen Amide I (wave, 1590-1720 cm-1) was defined as collagen content. The integrated area of proteogly-

Mechanical properties and gene expression of chondrocytes on micropatterned substrates following dedifferentiation in monolayer.

Chondrocytes in articular cartilage normally exhibit high expression of collagen II and aggrecan but rapidly dedifferentiate to a fibroblastic phenotype if passaged in culture. Previous studies have suggested that the loss of chondrocyte phenotype is associated with changes in the structure of the F-actin cytoskeleton, which also controls cell mechanical properties. In this study, we examined how dedifferentiation in monolayer influences the mechanical properties of chondrocytes isolated from different zones of articular cartilage. Atomic force microscopy was used to measure the mechanical properties of superficial and middle/deep zone chondrocytes as they underwent serial passaging and subsequent growth on fibronectin-coated, micropatterned self-assembled monolayers (MSAMs) that restored a rounded cell shape in 2D culture. Chondrocytes exhibited significant increases in elastic and viscoelastic moduli with dedifferentiation in culture. These changes were only partially ameliorated by the restoration of a rounded shape on micropatterned surfaces. Furthermore, intrinsic zonal differences in cell mechanical properties were rapidly lost with passage. These findings indicate that cell mechanical properties may provide additional measures of phenotypic expression of chondrocytes as they undergo dedifferentiation and possibly redifferentiation in culture.