Glycosaminoglycan Depletion Research Articles

Non-invasive MRI Assessments of Tissue Microstructures and Macromolecules in the Eye upon Biomechanical or Biochemical Modulation.

The microstructural organization and composition of the corneoscleral shell (CSS) determine the biomechanical behavior of the eye, and are important in diseases such as glaucoma and myopia. However, limited techniques can assess these properties globally, non-invasively and quantitatively. In this study, we hypothesized that multi-modal magnetic resonance imaging (MRI) can reveal the effects of biomechanical or biochemical modulation on CSS. Upon intraocular pressure (IOP) elevation, CSS appeared hyperintense in both freshly prepared ovine eyes and living rat eyes using T2-weighted MRI. Quantitatively, transverse relaxation time (T2) of CSS increased non-linearly with IOP at 0–40 mmHg and remained longer than unloaded tissues after being unpressurized. IOP loading also increased fractional anisotropy of CSS in diffusion tensor MRI without apparent change in magnetization transfer MRI, suggestive of straightening of microstructural fibers without modification of macromolecular contents. Lastly, treatments with increasing glyceraldehyde (mimicking crosslinking conditions) and chondroitinase-ABC concentrations (mimicking glycosaminoglycan depletion) decreased diffusivities and increased magnetization transfer in cornea, whereas glyceraldehyde also increased magnetization transfer in sclera. In summary, we demonstrated the changing profiles of MRI contrast mechanisms resulting from biomechanical or biochemical modulation of the eye non-invasively. Multi-modal MRI may help evaluate the pathophysiological mechanisms in CSS and the efficacy of corneoscleral treatments.

Effects of Chondroitinase ABC-Mediated Proteoglycan Digestion on Decellularization and Recellularization of Articular Cartilage.

Articular cartilage has a limited capacity to heal itself and thus focal defects often result in the development of osteoarthritis. Current cartilage tissue engineering strategies seek to regenerate injured tissue by creating scaffolds that aim to mimic the unique structure and composition of native articular cartilage. Decellularization is a novel strategy that aims to preserve the bioactive factors and 3D biophysical environment of the native extracellular matrix while removing potentially immunogenic factors. The purpose of this study was to develop a procedure that can enable decellularization and recellularization of intact articular cartilage matrix. Full-thickness porcine articular cartilage plugs were decellularized with a series of freeze-thaw cycles and 0.1% (w/v) sodium dodecyl sulfate detergent cycles. Chondroitinase ABC (ChABC) was applied before the detergent cycles to digest glycosaminoglycans in order to enhance donor chondrocyte removal and seeded cell migration. Porcine synovium-derived mesenchymal stem cells were seeded onto the decellularized cartilage scaffolds and cultured for up to 28 days. The optimized decellularization protocol removed 94% of native DNA per sample wet weight, while collagen content and alignment were preserved. Glycosaminoglycan depletion prior to the detergent cycles increased removal of nuclear material. Seeded cells infiltrated up to 100 μm into the cartilage deep zone after 28 days in culture. ChABC treatment enhances decellularization of the relatively dense, impermeable articular cartilage by reducing glycosaminoglycan content. ChABC treatment did not appear to affect cell migration during recellularization under static, in vitro culture, highlighting the need for more dynamic seeding methods.

Heparan sulfate proteoglycans mediate Aβ-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells.

BackgroundSubstantial evidence suggests that amyloid-β (Aβ) species induce oxidative stress and cerebrovascular (CV) dysfunction in Alzheimer’s disease (AD), potentially contributing to the progressive dementia of this disease. The upstream molecular pathways governing this process, however, are poorly understood. In this report, we examine the role of heparan sulfate proteoglycans (HSPG) in Aβ-induced vascular smooth muscle cell (VSMC) dysfunction in vitro.ResultsOur results demonstrate that pharmacological depletion of HSPG (by enzymatic degradation with active, but not heat-inactivated, heparinase) in primary human cerebral and transformed rat VSMC mitigates Aβ1-40- and Aβ1-42-induced oxidative stress. This inhibitory effect is specific for HSPG depletion and does not occur with pharmacological depletion of other glycosaminoglycan (GAG) family members. We also found that Aβ1-40 (but not Aβ1-42) causes a hypercontractile phenotype in transformed rat cerebral VSMC that likely results from a HSPG-mediated augmentation in intracellular Ca2+ activity, as both Aβ1-40-induced VSMC hypercontractility and increased Ca2+ influx are inhibited by pharmacological HSPG depletion. Moreover, chelation of extracellular Ca2+ with ethylene glycol tetraacetic acid (EGTA) does not prevent the production of Aβ1-40- or Aβ1-42-mediated reactive oxygen species (ROS), suggesting that Aβ-induced ROS and VSMC hypercontractility occur through different molecular pathways.ConclusionsTaken together, our data indicate that HSPG are critical mediators of Aβ-induced oxidative stress and Aβ1-40-induced VSMC dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0073-8) contains supplementary material, which is available to authorized users.

Glycosaminoglycan chemical exchange saturation transfer at 3T MRI in asymptomatic knee joints.

Biochemical alterations such as glycosaminoglycan (GAG) depletion occur early in the course of osteoarthritis, but cannot be detected with standard magnetic resonance techniques. With glycosaminoglycan chemical exchange saturation transfer (gagCEST), a biochemical imaging technique, it is feasible to detect biochemical components in knee joint cartilage. To establish baseline values for gagCEST magnetic resonance imaging (MRI) in knee joint cartilage at 3 Tesla (T). Twenty volunteers (8 women, 12 men; mean age, 24.55 ± 2.35 years;age range, 21-29 years) with no history or clinical findings indicative of knee joint pathologies underwent MRI at 3T. The imaging protocol included three-dimensional (3D) double-echo steady-state sequence for morphological cartilage assessment and a prototype 3D CEST pulse sequence to evaluate the CEST effects in six cartilage regions of the knee joint: (i) lateral femoral condyle; (ii) medial femoral condyle; (iii) lateral tibial plateau; (iv) medial tibial plateau; (v) patella; and (vi) trochlea. We used the asymmetry of the magnetization transfer ratio (MTRasym) parameter to quantify the gagCEST effects in these regions. Regional differences revealed high MTRasym values in the patellar (1.62% ± 1.19%) and the trochlear (1.17% ± 1.29%) cartilages, and low MTRasym values in the medial femoral condyle (0.41% ± 0.58%) and the lateral tibial plateau (0.52% ± 0.53%) cartilages. Regional differences in the gagCEST values must be considered when conducting gagCEST imaging of knee joint cartilage. In the future gagCEST imaging may be an additional feature in the evaluation of the biochemical composition of knee joint cartilage.

Glycosaminoglycan chemical exchange saturation transfer of lumbar intervertebral discs in patients with spondyloarthritis.

To assess glycosaminoglycan (GAG) content of lumbar intervertebral discs (IVD) in patients with spondyloarthritis (SpA) using glycosaminoglycan chemical exchange saturation transfer (gagCEST). Ninety lumbar intervertebral discs of nine patients with SpA and nine age-matched healthy controls (eight patients with ankylosing spondylitis; one patient with spondylitis related to inflammatory bowel disease; mean age: 44.1 ± 14.0 years; range: 27-72 years) were examined with a 3T magnetic resonance imaging (MRI) scanner in this prospective study. The MRI protocol included standard morphological, sagittal T2 -weighted (T2 w) images to assess Pfirrmann score of the five lumbar IVDs (L1 to S1) and biochemical imaging with gagCEST to calculate a region of interest analysis of nucleus pulposus (NP) and annulus fibrosus (AF). Prior to statistical testing of gagCEST effects (MTRasym values in percent) in patients and controls, IVDs were classified according to the Pfirrmann score. Significantly lower gagCEST values of NP and AF were found in SpA patients compared with healthy volunteers (NP: 1.41% ± 0.41%, P = 0.001; 95% confidence interval, CI [0.600%-2.226%]; AF: 1.19% ± 0.32%, P < 0.001; CI [0.560%-1.822%]) by comparing the differences of the means. Pooled nondegenerative IVDs (Pfirrmann 1 and 2) had significantly lower gagCEST effects in patients suffering from SpA compared with healthy controls in NP (P < 0.001; CI [1.176%-2.337%]) and AF (P < 0.001; CI [0.858%-1.779%]). No significant difference of MTRasym values was found in degenerative IVDs between patients and controls in NP (P = 0.204; CI [-0.504%-2.170%]). GagCEST analysis of morphologically nondegenerative IVDs (Pfirrmann score 1 and 2) in T2 w images demonstrated significantly lower GAG values in patients with spondyloarthritis in NP and AF, possibly representing a depletion of GAG in spondyloarthritis in the absence of morphologic degeneration.

Ultrashort Echo Time T1ρ Is Sensitive to Enzymatic Degeneration of Human Menisci.

The aim of the study was to determine whether quantitative ultrashort echo time (UTE) -T1ρ magnetic resonance (MR) measurements are sensitive to proteoglycan degradation in human menisci by trypsin digestion. Conventional and quantitative UTE-T1ρ MR sequences were performed on 4 meniscal samples using a 3T scanner. Magnetic resonance imaging was performed before and after 4, 8, and 12 hours of trypsin solution immersion, inducing proteoglycan loss. One sample was used as a control. Digest solutions were analyzed for glycosaminoglycan (GAG) content. The UTE-T1ρ studies were analyzed for quantitative changes. Images showed progressive tissue swelling, fiber disorganization, and increase in signal intensity after GAG depletion. The UTE-T1ρ values tended to increase with time after trypsin treatment (P = 0.06). Cumulative GAG loss into the bath showed a trend of increased values for trypsin-treated samples (P = 0.1). Ultrashort echo time T1ρ measurements can noninvasively detect and quantify severity of meniscal degeneration, which has been correlated with progression of osteoarthritis.

Stimulation of Superficial Zone Protein/Lubricin/PRG4 by Transforming Growth Factor-β in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans.

Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-β. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-β1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-β1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-β1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-β1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-β1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-β1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-β1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-β1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-β1 and exogenous HS.

High-bandwidth AFM-based rheology is a sensitive indicator of early cartilage aggrecan degradation relevant to mouse models of osteoarthritis

Murine models of osteoarthritis (OA) and post-traumatic OA have been widely used to study the development and progression of these diseases using genetically engineered mouse strains along with surgical or biochemical interventions. However, due to the small size and thickness of murine cartilage, the relationship between mechanical properties, molecular structure and cartilage composition has not been well studied. We adapted a recently developed AFM-based nano-rheology system to probe the dynamic nanomechanical properties of murine cartilage over a wide frequency range of 1Hz to 10kHz, and studied the role of glycosaminoglycan (GAG) on the dynamic modulus and poroelastic properties of murine femoral cartilage. We showed that poroelastic properties, highlighting fluid–solid interactions, are more sensitive indicators of loss of mechanical function compared to equilibrium properties in which fluid flow is negligible. These fluid-flow-dependent properties include the hydraulic permeability (an indicator of the resistance of matrix to fluid flow) and the high frequency modulus, obtained at high rates of loading relevant to jumping and impact injury in vivo. Utilizing a fibril-reinforced finite element model, we estimated the poroelastic properties of mouse cartilage over a wide range of loading rates for the first time, and show that the hydraulic permeability increased by a factor ~16 from knormal=7.80×10−16±1.3×10−16m4/Ns to kGAG-depleted=1.26×10−14±6.73×10−15m4/Ns after GAG depletion. The high-frequency modulus, which is related to fluid pressurization and the fibrillar network, decreased significantly after GAG depletion. In contrast, the equilibrium modulus, which is fluid-flow independent, did not show a statistically significant alteration following GAG depletion.

Glycosaminoglycans, Hyperglycemia, and Disease

Diabetes is a widespread disease with many clinical pathologies. Despite numerous pharmaceutical strategies for treatment, the incidence of diabetes continues to increase. Hyperglycemia, observed in diabetes, causes endothelial injury resulting in microvascular and macrovascular complications such as nephropathy, retinopathy, neuropathy, and increased atherosclerosis. Proteoglycans are chemically diverse macromolecules consisting of a protein core with glycosaminoglycans (GAGs) attached. Heparan sulfate proteoglycans are important compounds found on the endothelial cell membrane and in the extracellular matrix, which play an important role in growth regulation and serve as a reservoir for cytokines and other bioactive molecules. Endothelial cells are altered in hyperglycemia by a reduction in heparan sulfate and upregulation and secretion of heparanase, an enzyme that degrades heparan sulfate GAGs on proteoglycans. Reactive oxygen species, increased in diabetes, also destroy GAGs. Preservation of heparan sulfate proteoglycans on endothelial cells may be a strategy to prevent angiopathy associated with diabetes. The use of GAGs and GAG-like compounds may increase endothelial heparan sulfate and prevent an increase in the heparanase enzyme. Elucidating the mechanisms of GAG depletion and its significance in endothelial health may help to further understand, prevent, and treat cardiovascular complications associated with diabetes. Further studies examining the role of GAGs and GAG-like compounds in maintaining endothelial health, including their effect on heparanase, will determine the feasibility of these compounds in diabetes treatment. Preservation of heparan sulfate by decreasing heparanase may have important implications not only in diabetes, but also in cardiovascular disease and tumor biology.

High-Bandwidth AFM-Based Rheology Reveals that Cartilage is Most Sensitive to High Loading Rates at Early Stages of Impairment

Utilizing a newly developed atomic-force-microscopy-based wide-frequency rheology system, we measured the dynamic nanomechanical behavior of normal and glycosaminoglycan (GAG)-depleted cartilage, the latter representing matrix degradation that occurs at the earliest stages of osteoarthritis. We observed unique variations in the frequency-dependent stiffness and hydraulic permeability of cartilage in the 1 Hz-to-10 kHz range, a frequency range that is relevant to joint motions from normal ambulation to high-frequency impact loading. Measurement in this frequency range is well beyond the capabilities of typical commercial atomic force microscopes. We showed that the dynamic modulus of cartilage undergoes a dramatic alteration after GAG loss, even with the collagen network still intact: whereas the magnitude of the dynamic modulus decreased two- to threefold at higher frequencies, the peak frequency of the phase angle of the modulus (representing fluid-solid frictional dissipation) increased 15-fold from 55 Hz in normal cartilage to 800 Hz after GAG depletion. These results, based on a fibril-reinforced poroelastic finite-element model, demonstrated that GAG loss caused a dramatic increase in cartilage hydraulic permeability (up to 25-fold), suggesting that early osteoarthritic cartilage is more vulnerable to higher loading rates than to the conventionally studied “loading magnitude”. Thus, over the wide frequency range of joint motion during daily activities, hydraulic permeability appears the most sensitive marker of early tissue degradation.

Quantitative parametric MRI of articular cartilage: a review of progress and open challenges

With increasing life expectancies and the desire to maintain active lifestyles well into old age, the impact of the debilitating disease osteoarthritis (OA) and its burden on healthcare services is mounting. Emerging regenerative therapies could deliver significant advances in the effective treatment of OA but rely upon the ability to identify the initial signs of tissue damage and will also benefit from quantitative assessment of tissue repair in vivo. Continued development in the field of quantitative MRI in recent years has seen the emergence of techniques able to probe the earliest biochemical changes linked with the onset of OA. Quantitative MRI measurements including T(1), T(2) and T(1ρ) relaxometry, diffusion weighted imaging and magnetisation transfer have been studied and linked to the macromolecular structure of cartilage. Delayed gadolinium-enhanced MRI of cartilage, sodium MRI and glycosaminoglycan chemical exchange saturation transfer techniques are sensitive to depletion of cartilage glycosaminoglycans and may allow detection of the earliest stages of OA. We review these current and emerging techniques for the diagnosis of early OA, evaluate the progress that has been made towards their implementation in the clinic and identify future challenges in the field.

GAG depletion increases the stress-relaxation response of tendon fascicles, but does not influence recovery

Cyclic and static loading regimes are commonly used to study tenocyte metabolism in vitro and to improve our understanding of exercise-associated tendon pathologies. The aims of our study were to investigate if cyclic and static stress relaxation affected the mechanical properties of tendon fascicles differently, if this effect was reversible after a recovery period, and if the removal of glycosaminoglycans (GAGs) affected sample recovery. Tendon fascicles were dissected frombovine-foot extensors and subjected to 14% cyclic (1Hz) or static tensile strain for 30min. Additional fascicles were incubated overnight in buffer with 0.5U chondroitinase ABC or in buffer alone prior to the static stress-relaxation regime. To assess the effect of different stress-relaxation regimes, a quasi-static test to failure was carried out, either directly post loading or after a 2h recovery period, and compared with unloaded control fascicles. Both stress-relaxation regimes led to a significant reduction in fascicle failure stress and strain, but this was more pronounced in the cyclically loaded specimens. Removal of GAGs led to more stress relaxation and greater reductions in failure stress after static loading compared to controls. The reduction in mechanical properties was partially reversible in all samples, given a recovery period of 2h. This has implications for mechanical testing protocols, as a time delay between fatiguing specimens and characterization of mechanical properties will affect the results. GAGs appear to protect tendon fascicles from fatigue effects, possibly by enabling sample hydration.

Time-dependent mechanical properties of aortic valve cusps: Effect of glycosaminoglycan depletion

Aortic valve (AV) performance is closely linked to its structural components. Glycosaminoglycans (GAGs) are thought to influence the time-dependent properties of living tissues. This study investigates the effect of GAGs on the viscoelastic behaviour of the AV. Fresh porcine AV cusps were either treated enzymatically to remove GAGs or left untreated (control). The specimens were tested for stress relaxation and tensile properties under equibiaxial load conditions. The stress relaxation curves were fitted using a double exponential decay equation and the early relaxation constant (τ1) and late relaxation constant (τ2) calculated for each specimen. Immunohistochemistry confirmed the successful depletion of both sulphated and non-sulphated GAGs from the AV cusps. A statistical increase in τ1 was found for both the radial and circumferential directions between the control and –GAGs group (radial, control 17.37s vs. –GAGs 25.65s; circumferential, control 21.47s vs. –GAGs 27.37s). It was also found that τ1 differed between the two directions for the control group but not after GAG depletion (control, radial 17.37s vs. circumferential 21.47s; –GAGs, radial 25.65s vs. circumferential 27.37s). No effect on stiffness was found. The results showed that the presence of GAGs influences the mechanical viscoelastic properties of the AV but has no effect on the stiffness. The natural anisotropy, which reflects the relaxation kinematics, is lost after GAG depletion.

Hylan G-F 20 maintains cartilage integrity and decreases osteophyte formation in osteoarthritis through both anabolic and anti-catabolic mechanisms

To perform a molecular mechanism-based investigation of the chondroprotective potential of hylan G-F 20. The effects of hylan G-F 20 on IL-1β-induced glycosaminoglycan (GAG) depletion and matrix metalloproteinase (MMP) expression in bovine and human cartilage explants were evaluated. Three weekly intra-articular hylan G-F 20 or control injections were administered 4weeks post-operatively to rabbits with surgically induced osteoarthritis (OA). Cartilage histopathologic scores and osteophyte size were evaluated at 1, 4, and 8weeks post-injections. Histomorphometry and immunostaining were used to quantify cartilage area and type II collagen (Col II) intensity, and real-time polymerase chain reaction (PCR) was used to examine the mRNA levels of Col2A1, MMP-13, -16 and IL-1β at 1week. Hylan G-F 20 retained GAG in IL-1β-exposed bovine and human cartilage explants and abrogated IL-1β-mediated increases in MMP-1, -3, and -13 in human explant culture. Hylan G-F 20‒treated OA joints had significantly better cartilage integrity at 1 and 4weeks post-treatment and significantly smaller osteophytes at 4weeks compared with control. Col2A1 mRNA increased with hylan G-F 20 treatment, which correlated with a trend toward increased Col II immunostaining. MMP-13 and -16 mRNAs increased in OA cartilage, but were not significantly altered by hylan G-F 20. IL-1β mRNA was undetectable in cartilage and unaltered in the synovium. Hylan G-F 20 improved cartilage integrity and decreased osteophyte formation in the rabbit model of OA. Our results suggest that hylan G-F 20 may stimulate cartilage repair by increasing Col II, and inhibit IL-1β-mediated matrix degradation by decreasing MMPs.

The Regional Contribution of Glycosaminoglycans to Temporomandibular Joint Disc Compressive Properties

Understanding structure-function relationships in the temporomandibular joint (TMJ) disc is a critical first step toward creating functional tissue replacements for the large population of patients suffering from TMJ disc disorders. While many of these relationships have been identified for the collagenous fraction of the disc, this same understanding is lacking for the next most abundant extracellular matrix component, sulfated glycosaminoglycans (GAGs). Though GAGs are known to play a major role in maintaining compressive integrity in GAG-rich tissues such as articular cartilage, their role in fibrocartilaginous tissues in which GAGs are much less abundant is not clearly defined. Therefore, this study investigates the contribution of GAGs to the regional viscoelastic compressive properties of the temporomandibular joint (TMJ) disc. Chondroitinase ABC (C-ABC) was used to deplete GAGs in five different disc regions, and the time course for >95% GAG removal was defined. The compressive properties of GAG depleted regional specimens were then compared to non-treated controls using an unconfined compression stress-relaxation test. Additionally, treated and non-treated specimens were assayed biochemically and histologically to confirm GAG removal. Compared to untreated controls, the only regions affected by GAG removal in terms of biomechanical properties were in the intermediate zone, the most GAG-rich portion of the disc. Without GAGs, all intermediate zone regions showed decreased tissue viscosity, and the intermediate zone lateral region also showed a 12.5% decrease in modulus of relaxation. However, in the anterior and posterior band regions, no change in compressive properties was observed following GAG depletion, though these regions showed the highest compressive properties overall. Although GAGs are not the major extracellular matrix molecule of the TMJ disc, they are responsible for some of the viscoelastic compressive properties of the tissue. Furthermore, the mechanical role of sulfated GAGs in the disc varies regionally in the tissue, and GAG abundance does not always correlate with higher compressive properties. Overall, this study found that sulfated GAGs are important to TMJ disc mechanics in the intermediate zone, an important finding for establishing design characteristics for future tissue engineering efforts.

Articular Cartilage Glycosaminoglycans Inhibit the Adhesion of Endothelial Cells

Articular cartilage undergoes severe loss of proteoglycan and its constituent glycosaminoglycans (GAGs) in osteoarthritis. We hypothesize that the low GAG content of osteoarthritic cartilage renders the tissue susceptible to pathological vascularization. This was investigated using an in vitro angiogenesis model assessing endothelial cell adhesion to GAG-depleted cartilage explants. Bovine cartilage explants were treated with hyaluronidase to deplete GAG content and then seeded with fluorescently tagged human endothelial cells (HMEC-1). HMEC-1 adherence was assessed after 4 hr and 7 days. The effect of hyaluronidase treatment on GAG content, chondrocyte viability, and biochemical composition of the extracellular matrix was also determined. Hyaluronidase treatment reduced the GAG content of cartilage explants by 78 ± 3% compared with that of controls (p < 0.0001). GAG depletion was associated with significantly more HMEC-1 adherence on both the surface (superficial zone) and the underside (deep zone) of the explants (both p < 0.0001). The latter provided a more favorable environment for extended culture of HMEC-1 compared with the articulating surface. Hyaluronidase treatment altered the immunostaining for chondroitin sulfate epitopes, but not for lubricin. Our results support the hypothesis that articular cartilage GAGs are antiadhesive to endothelial cells and suggest that chondroitin sulfate and/or hyaluronan are responsible. The loss of these GAGs in osteoarthritis may allow osteochondral angiogenesis resulting in disease progression.

The Immunosuppressant FTY720 (Fingolimod) enhances Glycosaminoglycan depletion in articular cartilage

BackgroundFTY720 (Fingolimod) is a novel immunosuppressive drug investigated in clinical trials for organ transplantation and multiple sclerosis. It acts as a functional sphingosine-1-phosphate (S1P) receptor antagonist, thereby inhibiting the egress of lymphocytes from secondary lymphoid organs. As S1P is able to prevent IL-1beta induced cartilage degradation, we examined the direct impact of FTY720 on cytokine induced cartilage destruction.MethodsBovine chondrocytes were treated with the bioactive phosphorylated form of FTY720 (FTY720-P) in combination with IL-1beta or TNF-alpha. Expression of MMP-1,-3.-13, iNOS and ADAMTS-4,-5 and COX-2 was evaluated using quantitative real-time PCR and western blot. Glycosaminoglycan depletion from cartilage explants was determined using a 1,9-dimethylene blue assay and safranin O staining.ResultsFTY720-P significantly reduced IL-1beta and TNF-alpha induced expression of iNOS. In contrast FTY720-P increased MMP-3 and ADAMTS-5 mRNA expression. Furthermore depletion of glycosaminoglycan from cartilage explants by IL-1beta and TNF-alpha was significantly enhanced by FTY720-P in an MMP-3 dependent manner.ConclusionsOur results suggest that FTY720 may enhance cartilage degradation in pro-inflammatory environment.

Regional variation in the mechanical role of knee meniscus glycosaminoglycans

High compressive properties of cartilaginous tissues are commonly attributed to the sulfated glycosaminoglycan (GAG) fraction of the extracellular matrix (ECM), but this relationship has not been directly measured in the knee meniscus, which shows regional variation in GAG content. In this study, biopsies from each meniscus region (outer, middle, and inner) were either subjected to chondroitinase ABC (CABC) to remove all sulfated GAGs or not. Compressive testing revealed that GAG depletion in the inner and middle meniscus regions caused a significant decrease in modulus of relaxation (58% and 41% decreases, respectively, at 20% strain), and all regions exhibited a significant decrease in viscosity (outer: 29%; middle: 58%; inner: 62% decrease). Tensile properties following CABC treatment were unaffected for outer and middle meniscus specimens, but the inner meniscus displayed significant increases in Young's modulus (41% increase) and ultimate tensile stress (40% increase) following GAG depletion. These findings suggest that, in the outer meniscus, GAGs contribute to increasing tissue viscosity, whereas in the middle and inner meniscus, where GAGs are most abundant, these molecules also enhance the tissue's ability to withstand compressive loads. GAGs in the inner meniscus also contribute to reducing the circumferential tensile properties of the tissue, perhaps due to the pre-stress on the collagen network from increased hydration of the ECM. Understanding the mechanical role of GAGs in each region of the knee meniscus is important for understanding meniscus structure-function relationships and creating design criteria for functional meniscus tissue engineering efforts.

Assessment of glycosaminoglycan concentration changes in the intervertebral disc via chemical exchange saturation transfer.

In this study, it is shown that the chemical exchange saturation transfer (CEST) method for hydroxyl protons can be used to detect changes in glycosaminoglycan (GAG) concentration in the intervertebral disc. The method, termed gagCEST, was demonstrated ex vivo by correlating the CEST effect with the fixed charge density (FCD) of the nucleus pulposus (NP), as well as by correlating tissue CEST images with their corresponding (23)Na images. Incubation of five NP samples with trypsin produced samples with varying GAG content (n = 19). A good correlation was found between the -OH CEST effect and FCD, as well as with the N-acetyl signal amplitude. gagCEST images in vitro further illustrated the amount of detail obtainable from this contrast mechanism when compared with conventional imaging. The large concentration of GAG and the relatively long T(1) of water in NP make the method sensitive, in particular, for the assessment of GAG depletion in this tissue. It is the loss of GAG in NP that indicates the early stage of disc degeneration.

Determination of the prevalence and severity of metacarpophalangeal joint osteoarthritis in Thoroughbred racehorses via quantitative macroscopic evaluation

To determine the prevalence and severity of osteoarthritis in the metacarpophalangeal joints of Thoroughbred racehorses via development and validation of a quantitative macroscopic evaluation system. Metacarpophalangeal joints from 50 Thoroughbred racehorses. Joints were collected from horses that died or were euthanized within 60 days of racing. Metacarpophalangeal joints were assessed for osteoarthritic degeneration by use of macroscopic and histologic scoring systems, polarized light microscopy, and cartilage biochemical analysis. The global macroscopic score for the entire metacarpophalangeal joint was based on factors that reflected the size and severity of lesions as well as the involvement of weight-bearing surfaces. One-third of all 2- and 3-year-old horses had partial-or full-thickness cartilage lesions and osteoarthritis. Osteoarthritis severity increased until age 6 in this population. Significant correlations were found between macroscopic grade and age, cause of death, glycosaminoglycan depletion, and loss of superficial cartilage zone polarized light intensity. The macroscopic system devised for this study had good correlations with quantitative methods. Two-and 3-year-old horses had full-thickness cartilage lesions that may have been career limiting. Year-to-year attrition and a small population of older horses may have led to underestimation of the prevalence of osteoarthritis in older horses. The macroscopic scoring system was reliable when used by nonexpert and expert users.