Early Stages Of Cartilage Degeneration Research Articles

ABNORMAL CHONDROCYTE MORPHOLOGY, CLUSTERING, AND CELL SWELLING IN A SCRAPED HUMAN CARTILAGE MODEL OF OSTEOARTHRITIS

AbstractObjectivesOsteoarthritis (OA) is a complex joint disorder characterised by the loss of extracellular matrix (ECM) leading to cartilage degeneration. Changes to cartilage cell (chondrocyte) behaviour occur including cell swelling, the development of fine cytoplasmic processes and cell clustering leading to changes in cell phenotype and development of focal areas of mechanically-weak fibrocartilaginous matrix[1]. To study the sequence of events in more detail, we have investigated the changes to in situ chondrocytes within human cartilage which has been lightly scraped and then cultured with serum.MethodsHuman femoral heads were obtained with Ethical permission and consent from four female patients (mean age 74 yrs) undergoing hip arthroplasty following femoral neck fracture. Osteochondral explants of macroscopically-normal cartilage were cultured as a non-scraped control, or scraped gently six times with a scalpel blade and both maintained in culture for up to 2wks in Dulbecco's Modified Eagle's Medium (DMEM) with 25% human serum (HS). Explants were then labelled with CMFDA (5-chloromethylfluorescein-diacetate) and PI (propidium iodide) (10μM each) to identify the morphology of living or dead chondrocytes respectively. Explants were imaged using confocal microscopy and in situ chondrocyte morphology, volume and clustering assessed quantitatively within standardised regions of interest (ROI) using Imaris® imaging software.ResultsWithin 2wks of culture with HS, chondrocyte volume increased significantly from 412±9.3µm3 (unscraped) at day 0 to 724±16.6 µm3 (scraped) [N(n) = 4(380)] (P=0.0002). Chondrocyte clustering was a prominent feature of HS culture as the percentage of clusters in the cell population increased with scraping from 4.8±1.4% to 14.9±3.9% [N(n) = 4(999)] at week 2 (P=0.0116). In addition, the % of the chondrocyte population within clusters increased from approximately 38% to 60%, and the number of cells per cluster increased significantly from 3.2±0.08 to 4±0.22 (P=0.031). The development of abnormal ‘fibroblastic-like’ chondrocyte morphology demonstrating long (>5µm) cytoplasmic processes also occurred, however the time course of this was more variable. For some samples, clustering occurred before abnormal morphology, but for others the opposite occurred. Typically, by the second week, 17±2.64% of the cell population had processes and this increased to 22±4.02% [N(n) = 4(759)] with scraping.ConclusionsScraping the cartilage will remove surface constituents including lubricants (e.g. lubricin, hyaluronic acid, phospholipids), extracellular matrix constituents (collagen, proteoglycans – potentially the ‘lamina splendens’) and cells (chondrocytes and mesenchymal stromal cells (MSCs)). Although we do not know which of these component(s) is important, the effect is to dramatically increase the permeation of serum factors into the cartilage matrix and signal the development of cytoplasmic processes, cell clustering and swelling. It is notable that these cellular changes are similar to those occurring in early OA[1]. This raises the interesting possibility that scraped cartilage cultured with human serum recapitulates some of the changes to in situ chondrocytes during early stages of cartilage degeneration and as such, could be a useful model for following the deleterious changes to matrix metabolism.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

CHANGES TO THE F-ACTIN DISTRIBUTION OF THE CHONDROCYTE CYTOSKELETON IN EARLY OSTEOARTHRITIS

Cartilage degeneration and loss are key events in the initiation and progression of osteoarthritis (OA). Changes to chondrocyte volume and morphology (in the form of cytoplasmic processes) and thus cell phenotype are implicated, as they lead to the production of a mechanically-weakened extracellular matrix. The chondrocyte cytoskeleton is intimately linked to cell volume and morphology and hence we have investigated alterations to levels and distribution of chondrocyte F-actin that occur during early OA.The femoral heads (FH) from hip joints (N=16) were obtained with ethical permission and patient consent following femoral neck fracture. Cartilage was assessed as grade 0 (non-degenerate) and grade 1 (superficial fibrillation) using OARSI criteria. In situ chondrocyte volume and F-actin distribution were assessed using the fluorescent indicators (5-chloromethyl fluorescein diacetate (CMFDA)) and phalloidin, and imaged and quantified by confocal microscopy, ImarisTM and ImageJ software.There were no differences between the volume or total F-actin levels of in situ chondrocytes within the superficial zone of grade 0 (n=164 cells) compared to grade 1 (n=145) cartilage (P>0.05). However, a more detailed analysis of phalloidin labelling was performed, which demonstrated significant increases in both intense punctuate (IP) or intense areas (IA) (P<0.0001; P=0.0175 respectively). A preliminary analysis of IP and IA F-actin labelling suggested that while the former did not appear to be associated with changes to chondrocyte morphology, most of the cytoplasmic processes were associated with the presence of IA at the starting point of the protrusion.These results demonstrate marked changes to F-actin distribution in chondrocytes in the very early stages of cartilage degeneration as occurs in OA. These subtle changes are probably an early indication of a change to the chondrocyte phenotype and thus worthy of further study as they may lead to deleterious alterations to matrix metabolism and ultimately cartilage weakening.

Radiology Alchemy: GAN We Do It?

Radiology Alchemy: GAN We Do It?

Iron triggers the early stages of cartilage degeneration in vitro: The role of articular chondrocytes

ObjectiveArthropathy is a major clinical problem in patients with hemochromatosis, the most common genetic disorder of iron overload. The pathological features of hemochromatosis arthropathy (HA) are heterogeneous and its specific nature remains unknown. One important drawback is the lack of proper in vitro models. The aim of the present study was to set up a model to investigate the biological response of cartilage to iron exposure. DesignBovine articular cartilage explants were incubated with ferric citrate for up to 9 days. We evaluated chondrocyte viability, iron deposition, and biomarkers of cartilage degradation in the conditioned medium. ResultsIron accumulated within chondrocytes, which was associated with programmed cell death through chondroptosis. Iron treatment increased the release of sulfated glycosaminoglycans (sGAG), a component of the extracellular matrix, into the medium (p=0.0189). This was dependent on the presence of viable chondrocytes and was associated with increased activity of matrix-degrading metalloproteinases (MMP) (pro/active MMP-9, p=0.0317; pro MMP-2, p=0.0092; active MMP-2, p=0.0288). Co-treatment with the broad MMP/aggrecanase inhibitor prinomastat reduced iron-mediated sGAG release (0.02 ​μM, p=0.0425; 2 ​μM, p=0.0014), confirming that iron induces sGAG release via the activation of catabolic enzymes. Notably, iron-treated cartilage continued to release an increased amount of sGAG into the medium for 6 days after termination of the ferric citrate treatment (p=0.0259). ConclusionsIron triggers the early stages of cartilage degeneration. Removal of iron exposure does not prevent further damage to the cartilage, thus providing a possible explanation why HA is not prevented after iron depletion by phlebotomy treatment.

Articulation of an alumina-zirconia composite ceramic against living cartilage – An in vitro wear test

BackgroundThere is interest in minimally invasive solutions that reduce osteoarthritic symptoms and restore joint mobility in the early stages of cartilage degeneration. The aim of the present study was to evaluate an alumina-zirconia composite (AZC) as a counterface for articulation against live cartilage explants in comparison to the clinically relevant cobalt-chromium (CoCrMo) alloy. MethodsA four-station bioreactor designed to articulate against living tissue in an incubator was used for testing. Twelve 32 mm AZC and twelve 32 mm CoCrMo femoral heads with equal surface roughness made up both test groups. Each head articulated against a cartilage disk harvested from stifle joints of 24-week old steers. Test samples and free-swelling control cartilage samples were cultured in Mini ITS medium. Testing was conducted 3 h daily over 10 days applying a contact load of approximately 2 MPa. PG/GAG and hydroxyproline were analyzed using biochemical assays. Additionally, chondrocyte survival and Mankin score analyses were performed on histological slides. ResultsCells stayed alive during the course of the experiment, with cell survival values close to 80% at test completion in the superficial zone. There was no significant difference between AZC and free-swelling control tissue. However, cell count values were inferior for CoCrMo (p = 0.003). Tested tissue suffered mostly structural abnormalities. The PG/GAG content in medium was not different between CoCrMo and AZC (p = 0.315); however, the hydroxyproline release into medium was nearly 30% higher for CoCrMo (p = 0.024). ConclusionBased on average values, AZC induces less cell and tissue damage than CoCrMo. However, only the hydroxyproline measurements reached statistical significance, partially due to substantial scatter within both groups.

Quantifying birefringence in the bovine model of early osteoarthritis using polarisation-sensitive optical coherence tomography and mechanical indentation

Recent studies have shown potential for using polarisation sensitive optical coherence tomography (PS-OCT) to study cartilage morphology, and to be potentially used as an in vivo, non-invasive tool for detecting osteoarthritic changes. However, there has been relatively limited ability of this method to quantify the subtle changes that occur in the early stages of cartilage degeneration. An established mechanical indenting technique that has previously been used to examine the microstructural response of articular cartilage was employed to fix the bovine samples in an indented state. The samples were subject to creep loading with a constant compressive stress of 4.5 MPa and, when imaged using PS-OCT, enabled birefringent banding patterns to be observed. The magnitude of the birefringence was quantified using the birefringence coefficient (BRC) and statistical analysis revealed that PS-OCT is able to detect and quantify significant changes between healthy and early osteoarthritic cartilage (p < 0.001). This presents a novel utilization of PS-OCT for future development as an in vivo assessment tool.

Calcium-phosphate complex increased during subchondral bone remodeling affects earlystage osteoarthritis

An activation of osteoclasts and subchondral bone remodeling is a major histologic feature of early-stage osteoarthritis (OA), which can be accompanied by an increase of calcium (Ca) and phosphate (Pi) level in the subchondral milieu. Considering articular cartilage gets most of nutrition from subchondral bone by diffusion, these micro-environmental changes in subchondral bone can affect the physiology of articular chondrocytes. Here, we have shown that Ca is increased and co-localized with Pi in articular cartilage of early-stage OA. The Ca-Pi complex increased the production of MMP-3 and MMP-13 in the hypertrophic chondrocytes, which was dependent on nuclear factor-kappa B (NF-kB), p38 and extracellular signal-regulated kinase (Erk) 1/2 mitogen-activated protein (MAP) kinase and Signal transducer and activator of transcription 3 (STAT3) signaling. The Ca-Pi complexes increased the expression of endocytosis markers, and the inhibition of the formation of the Ca-Pi complex ameliorated the Ca-Pi complex-mediated increases of MMPs expression in hypertrophic chondrocytes. Our data provide insight regarding the Ca-Pi complex as a potential catabolic mediator in the subchondral milieu and support the pathogenic role of subchondral bone in the early stages of cartilage degeneration.

T2 MR imaging vs. computational modeling of human articular cartilage tissue functionality

The detection of early stages of cartilage degeneration remains diagnostically challenging. One promising non-invasive approach is to functionally assess the tissue response to loading by serial magnetic resonance (MR) imaging in terms of T2 mapping under simultaneous mechanical loading. As yet, however, it is not clear which cartilage component contributes to the tissue functionality as assessed by quantitative T2 mapping. To this end, quantitative T2 maps of histologically intact cartilage samples (n=8) were generated using a clinical 3.0-T MR imaging system. Using displacement-controlled quasi-static indentation loading, serial T2 mapping was performed at three defined strain levels and loading-induced relative changes were determined in distinct regions-of-interest. Samples underwent conventional biomechanical testing (by unconfined compression) as well as histological assessment (by Mankin scoring) for reference purposes. Moreover, an anisotropic hyperelastic constitutive model of cartilage was implemented into a finite element (FE) code for cross-referencing. In efforts to simulate the evolution of compositional and structural intra-tissue changes under quasi-static loading, the indentation-induced changes in quantitative T2 maps were referenced to underlying changes in cartilage composition and structure. These changes were parameterized as cartilage fluid, proteoglycan and collagen content as well as collagen orientation. On a pixel-wise basis, each individual component correlation with T2 relaxation times was determined by Spearman's ρs and significant correlations were found between T2 relaxation times and all four tissue parameters for all indentation strain levels. Thus, the biological changes in functional MR Imaging parameters such as T2 can further be characterized to strengthen the scientific basis of functional MRI techniques with regards to their perspective clinical applications.

Subchondral bone histology and grading in osteoarthritis.

ObjectiveOsteoarthritis (OA) has often regarded as a disease of articular cartilage only. New evidence has shifted the paradigm towards a system biology approach, where also the surrounding tissue, especially bone is studied more vigorously. However, the histological features of subchondral bone are only poorly characterized in current histological grading scales of OA. The aim of this study is to specifically characterize histological changes occurring in subchondral bone at different stages of OA and propose a simple grading system for them.Design20 patients undergoing total knee replacement surgery were randomly selected for the study and series of osteochondral samples were harvested from the tibial plateaus for histological analysis. Cartilage degeneration was assessed using the standardized OARSI grading system, while a novel four-stage grading system was developed to illustrate the changes in subchondral bone. Subchondral bone histology was further quantitatively analyzed by measuring the thickness of uncalcified and calcified cartilage as well as subchondral bone plate. Furthermore, internal structure of calcified cartilage-bone interface was characterized utilizing local binary patterns (LBP) based method.ResultsThe histological appearance of subchondral bone changed drastically in correlation with the OARSI grading of cartilage degeneration. As the cartilage layer thickness decreases the subchondral plate thickness and disorientation, as measured with LBP, increases. Calcified cartilage thickness was highest in samples with moderate OA.ConclusionThe proposed grading system for subchondral bone has significant relationship with the corresponding OARSI grading for cartilage. Our results suggest that subchondral bone remodeling is a fundamental factor already in early stages of cartilage degeneration.

Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse

Aggrecan loss in human and animal cartilage precedes clinical symptoms of osteoarthritis, suggesting that aggrecan loss is an initiating step in cartilage pathology. Characterizing early stages of cartilage degeneration caused by aging and overuse is important in the search for therapeutics. In this study, atomic force microscopy (AFM)-based force–displacement micromechanics, AFM-based wide bandwidth nanomechanics (nanodynamic), and histologic assessments were used to study changes in distal femur cartilage of wildtype mice and mice in which the aggrecan interglobular domain was mutated to make the cartilage aggrecanase-resistant. Half the animals were subjected to voluntary running-wheel exercise of varying durations. Wildtype mice at three selected age groups were compared. While histological assessment was not sensitive enough to capture any statistically significant changes in these relatively young populations of mice, micromechanical assessment captured changes in the quasi-equilibrium structural-elastic behavior of the cartilage matrix. Additionally, nanodynamic assessment captured changes in the fluid–solid poroelastic behavior and the high frequency stiffness of the tissue, which proved to be the most sensitive assessment of changes in cartilage associated with aging and joint-overuse. In wildtype mice, aging caused softening of the cartilage tissue at the microscale and at the nanoscale. Softening with increased animal age was found at high loading rates (frequencies), suggesting an increase in hydraulic permeability, with implications for loss of function pertinent to running and impact-injury. Running caused substantial changes in fluid–solid interactions in aggrecanase-resistant mice, suggestive of tissue degradation. However, higher nanodynamic stiffness magnitude and lower hydraulic permeability was observed in running aggrecanase-resistant mice compared to running wildtype controls at the same age, thereby suggesting protection from joint-overuse.

Reproducibility and regional variations of an improved gagCEST protocol for the in vivo evaluation of knee cartilage at 7T.

The objective was to establish a gagCEST protocol that would enable robust and reproducible assessment of the glycosaminoglycan (GAG) content in knee cartilage at 7T within a clinically feasible measurement time. Ten young healthy volunteers (mean age 26years, range 24-28, five males, five females) were examined on a 7T MR system. Informed consent was obtained from all individual participants prior to enrollment into the study. Each volunteer was measured twice for reproducibility assessment. The examined knee was immobilized using a custom-made fixation device. For the gagCEST measurement, a prototype segmented 3-D RF-spoiled gradient-echo sequence with an improved saturation scheme employing adiabatic pulses was used in a scan time of 19min. The asymmetry of the Z-spectra (MTRasym) in selected regions of interest in knee cartilage was calculated. Differences in MTRasym between different regions were evaluated using ANOVA and the Bonferroni corrected post hoc test. The improvement of the saturation scheme reduced the influence of field inhomogeneities, resulted in more uniform saturation, and allowed for good reproducibility in a reasonable measurement time (19min), as demonstrated by an intraclass correlation coefficient of 0.77. Improved fixation helped to reduce motion artifacts. Whereas similar MTRasym values were found for weight-bearing and non-weight-bearing femoral cartilage, lower values were observed in the trochlear groove (p=0.028), patellar (p=0.015) and tibial cartilage (p<0.001) when compared to non-weight-bearing femoral cartilage. Reasonable reproducibility and sensitivity to regional differences in GAG content suggests that the improved gagCEST protocol might be useful for assessing the biochemical changes in articular cartilage that are associated with early stages of cartilage degeneration.

T1ρ magnetic resonance: basic physics principles and applications in knee and intervertebral disc imaging.

T1ρ relaxation time provides a new contrast mechanism that differs from T1- and T2-weighted contrast, and is useful to study low-frequency motional processes and chemical exchange in biological tissues. T1ρ imaging can be performed in the forms of T1ρ-weighted image, T1ρ mapping and T1ρ dispersion. T1ρ imaging, particularly at low spin-lock frequency, is sensitive to B0 and B1 inhomogeneity. Various composite spin-lock pulses have been proposed to alleviate the influence of field inhomogeneity so as to reduce the banding-like spin-lock artifacts. T1ρ imaging could be specific absorption rate (SAR) intensive and time consuming. Efforts to address these issues and speed-up data acquisition are being explored to facilitate wider clinical applications. This paper reviews the T1ρ imaging's basic physic principles, as well as its application for cartilage imaging and intervertebral disc imaging. Compared to more established T2 relaxation time, it has been shown that T1ρ provides more sensitive detection of proteoglycan (PG) loss at early stages of cartilage degeneration. T1ρ has also been shown to provide more sensitive evaluation of annulus fibrosis (AF) degeneration of the discs.

The Circadian Clock in Murine Chondrocytes Regulates Genes Controlling Key Aspects of Cartilage Homeostasis

ObjectiveTo characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA).MethodsCartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription–polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis.ResultsMouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA.ConclusionThese results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently, circadian disruption (e.g., during aging) may compromise tissue homeostasis and increase susceptibility to joint damage or disease.

An eight‐channel transmit/receive multipurpose coil for musculoskeletal MR imaging at 7 T

MRI plays a leading diagnostic role in assessing the musculoskeletal (MSK) system and is well established for most questions at clinically used field strengths (up to 3 T). However, there are still limitations in imaging early stages of cartilage degeneration, very fine tendons and ligaments, or in locating nerve lesions, for example. 7 T MRI of the knee has already received increasing attention in the current published literature, but there is a strong need to develop new radiofrequency (RF) coils to assess more regions of the MSK system. In this work, an eight-channel transmit/receive RF array was built as a multipurpose coil for imaging some of the thus far neglected regions. An extensive coil characterization protocol and first in vivo results of the human wrist, shoulder, elbow, knee, and ankle imaged at 7 T will be presented. Eight surface loop coils with a dimension of 6 x 7 cm2 were machined from FR4 circuit board material. To facilitate easy positioning, two coil clusters, each with four loop elements, were combined to one RF transmit/receive array. An overlapped and shifted arrangement of the coil elements was chosen to reduce the mutual inductance between neighboring coils. A phantom made of body-simulating liquid was used for tuning and matching on the bench. Afterward, the S-parameters were verified on a human wrist, elbow, and shoulder. For safety validation, a detailed compliance test was performed including full wave simulations of the RF field distribution and the corresponding specific absorption rate (SAR) for all joints. In vivo images of four volunteers were assessed with gradient echo and spin echo sequences modified to obtain optimal image contrast, full anatomic coverage, and the highest spatial resolution within a reasonable acquisition time. The performance of the RF coil was additionally evaluated by in vivo B1 mapping. A comparison of B1 per unit power, flip angle distribution, and anatomic images showed a fairly homogeneous excitation for the smaller joints (elbow, wrist, and ankle), while for the larger joints, the shoulder and especially the knee, B1 inhomogeneities and limited penetration depth were more pronounced. However, the greater part of the shoulder joint could be imaged. In vivo images rendered very fine anatomic details such as fascicles of the median nerve and the branching of the nerve bundles. High-resolution images of cartilage, labrum, and tendons could be acquired. Additionally, turbo spin echo (TSE) and inversion recovery sequences performed very well. This study demonstrates that the concept of two four-channel transmit/receive RF arrays can be used as a multipurpose coil for high-resolution in vivo MR imaging of the musculoskeletal system at 7 T. Not only gradient echo but also typical clinical and SAR-intensive sequences such as STIR and TSE performed well. Imaging of small structures and peripheral nerves could in particular benefit from this technique.

MR diffusion weighted imaging experimental study on early stages of articular cartilage degeneration of knee

Objective To study the appearance of MR diffusion weighted imaging in early stages of cartilage degeneration and to detect its values. Methods In 20 goat left knees, intra- articular injection of 5 units of papain was performed causing a loss of cartilage proteoglycan. Twenty right knees were used as control group. MR diffusion weighted imaging was performed at 24 hours after intra-articular injection of papain. ADC of each part of articular cartilage was measured and compared with each other. The proteoglycan content was measured biochemically and histochemicaUy. Routine MRI and DWI were performed in 100 patients with osteoarthritis and 20 healthy people. The ADC of each interested part of articular cartilage was measured and compared with each other. Results In experimental control group, the ADCav of articular cartilage was (14.2±2.3)×10-4 mm2/s. In early stages of cartilage degeneration group, the ADCav of articular cartilage was (17.5±4.2) × 10-4 mm2/s. The ADCav of the control group was lower than that of the early stages of cartilage degeneration group (t = 2.709 ; P = 0.016) . The proteloglyean content of articular cartilage was 4.22×10 μg/kg in control group, and 0.82×10 μg/kg in experimental group at 24 hours after injection of papain. The difference between control group and experimental group was significant (t = 2.705, P = 0.018). In healthy people, the ADCav of articular cartilage was (7.6±2. 2) × 10-4 mm2/s. In osteoarthritis group, the ADCav of articular cartilage was (10.3±4. 2) × 10-4 mm2/s. The ADCav in the healthy group was significantly lower than that in the osteoarthritis group (t = 2.609, P = 0.014). Conclusion DWI is an useful method in detecting early stages of cartilage degeneration which can not be showed on routine sequences. Key words: Cartilage diseases; Knee joint; Diffusion magnetic resonance imaging

ADAM15 modulates outside‐in signalling in chondrocyte–matrix interactions

ADAM15 belongs to a family of transmembrane multi-domain proteins implicated in proteolysis, cell-cell and cell-matrix interactions in various disease conditions. In osteoarthritis (OA), ADAM15 is up-regulated in the chondrocytes already at early stages of cartilage degeneration where it seems to exert homeostatic effects likely associated with its ability to enhance integrin-mediated chondrocyte adhesion to the surrounding collagen matrix. The aim of our present study was, therefore, to characterize functional domains of ADAM15 involved in collagen II (CII) interaction and to analyse associated outside-in signalling events. Accordingly, ADAM15 and respective deletion mutants were stably transfected into the chondrocyte cell line T/C28a4. Transfected cells were adhered to CII and phosphoproteins analysed by Western blotting. Co-immunoprecipitation served to identify protein binding to ADAM15. Our results elucidate the prodomain as critical for the capacity of ADAM15 to enhance CII adhesion, thereby identifying for the first time a cell-adhesive role of a metalloproteinase prodomain. Moreover, the cytoplasmic tail of ADAM15 confers a modulatory effect on the autophosphorylation site Y397 of the focal adhesion kinase (FAK) during chondrocyte-collagen interaction. In conclusion, the newly uncovered impact of ADAM15 on signalling events that arise from chondrocyte interactions with its collagen matrix might contribute to the elucidation of the mechanism underlying its proposed chondroprotective role in degenerative cartilage disease.

High-resolution/high-contrast MRI of human articular cartilage lesions

Background Magnetic resonance microscopy (MRM) is an important experimental tool in the identification of early cartilage lesions.Methods Normal and degenerated cartilage samples were imaged at 11.74 T using a standard spin echo sequence. Quantitative MR measurements for T1, T2, and ADC were obtained and mapping for T2 and ADC was performed. The bi-exponential model for T2 relaxation was also explored. Histology was carried out for comparison with MR images.Results MR images of cartilage samples displaying early stages of degeneration were positively correlated to their histological appearance in 23-μm high-resolu-tion images and also with much shorter imaging times at 47-μm resolution. T2 maps enable delineation of the actual cartilage zones, distinguishing the super?cial zone in particular. The bi-exponential model can reflect cartilage components at different stages of degeneration.Interpretation At 11.74 T, with 23-μm resolution or with 47-μm resolution and shorter imaging times, MRM provides images that allow visualization of early stages of cartilage degeneration, including super?cial ?brillation. This has not been shown previously. The images also allow quantitative measurements (T1, T2, and ADC) in each cartilage region, which can be indicative of different stages of cartilage degeneration.

Glycosaminoglycan Content of Knee Cartilage Following Posterior Cruciate Ligament Rupture Demonstrated by Delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC)

Following joint trauma and during the early stages of cartilage degeneration, typical changes become apparent in tissue structure and composition, including the loss of glycosaminoglycan1,2. These changes often are not apparent on conventional magnetic resonance imaging or visible at arthroscopy, thereby precluding diagnosis. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage, or dGEMRIC, is an emerging in vivo method for the assessment of the biochemical composition of articular cartilage and provides a surrogate measure of glycosaminoglycan content3,4. When injected intravenously, the anionic contrast agent gadolinium diethylene triamine penta-acetic acid (Gd-DTPA2–) penetrates cartilage both from the synovial surface and from the subchondral bone3,5. Given sufficient time, the anionic contrast agent distributes inversely to the fixed negative charge associated with the cartilage glycosaminoglycan content in accordance with the Donnan principle of equilibrium. Gd-DTPA2-therefore distributes in relatively higher concentrations in regions of low glycosaminoglycan, and vice versa. Gd-DTPA2-has a concentration-dependent effect on the magnetic resonance imaging parameter T1; therefore, T1 imaging in the presence of Gd-DTPA2– (T1Gd) reflects the cartilage Gd-DTPA2–concentration and, hence, glycosaminoglycan concentration. We present the case of a patient (one of the authors [A.A.Y.]) who sustained an injury to the posterior cruciate ligament of the knee while enrolled as a “normal” control subject in a dGEMRIC study of the knee. This incidental occurrence provided us with a unique opportunity to visualize the temporal and spatial changes that occur in cartilage biochemistry following an acute posterior cruciate ligament rupture. Athirty-year-old man (one of the authors [A.A.Y.]) was involved in a motor-vehicle accident in which he sustained a direct blow to the anterior aspect of the proximal portion of the right tibia at 90° of knee flexion (a typical “dashboard” type of …

Immunohistochemical analysis of 3-B-(-) and 7-D-4 epitope expression in canine osteoarthritis.

To examine the distribution of the 3-B-3(-) and 7-D-4 epitopes in proteoglycans from morphologically normal and osteoarthritic (OA) canine articular cartilage. Cartilage samples from the femurs of stable and destabilized stifle joints of 9 dogs that had undergone transection of the cranial cruciate ligament were examined by immunohistochemistry. The 3-B-3(-) and 7-D-4 epitopes were expressed in the superficial zone of cartilage from the destabilized femorotibial joints in the early stages of developing OA. The staining patterns with these two antibodies differed, with 3-B-3(-) reactivity confined to the superficial and upper middle zones of the articular cartilage, and 7-D-4 reactivity more prominent in the matrix, extending into the deeper zones and increasing with progression of the lesion. Both epitopes were also expressed in the superficial and upper middle zones of areas peripheral to the lesions and were detectable before the loss of matrix and proteoglycans could be identified by histochemical staining with toluidine blue. In this study, the expression of atypical chondroitin sulfate proteoglycans was demonstrated in osteoarthritic canine cartilage, and the pattern of expression changed as the lesions progressed. The occurrence of 3-B-3(-) and 7-D-4 epitopes appears to be associated with changes in chondrocyte metabolism in the early stages of cartilage degeneration in experimental osteoarthritis.