Cartilage Chondroitin Sulfate Research Articles

Extraction, Physicochemical Properties, and In Vitro Antioxidant Activities of Chondroitin Sulfate from Bovine Nose Cartilage.

Beef is an important high-nutrition livestock product, and several byproducts, such as bovine cartilage, are produced during slaughter. To effectively utilize these agricultural and pastoral byproducts, combined (trypsin-papain) enzymolysis and cetylpyridine chloride purification methods were used to obtain chondroitin sulfate (CS) from the nasal cartilage of Shaanxi Yellow cattle. The effects of pH, temperature, and time on the CS yield during enzymatic hydrolysis were investigated, and the CS extraction process was optimized using response surface methodology. The best yield of CS was 21.62% under the optimum conditions of pH 6.51, temperature of 64.53°C, and enzymolysis time of 19.86 h. The molecular weight of CS from Shaanxi cattle nasal cartilage was 89.21 kDa, glucuronic acid content was 31.76 ± 0.72%, protein content was 1.12 ± 0.03%, and sulfate group content was 23.34 ± 0.08%. The nasal cartilage CS of the Yellow cattle showed strong DPPH•, •OH, and ABTS+• radical scavenging abilities and ferrous reduction ability in the experimental concentration range. This study could contribute to "turn waste into treasure" and improve the comprehensive utilization of regional characteristic biological resources.

A chondroitin sulfate purified from shark cartilage and bovine serum albumin interaction activity

Chondroitin sulfate (CS) was extracted and purified from shark cartilage, and its interaction with bovine serum albumin (BSA) were studied. The content of chondroitin sulfate in shark cartilage was 29.97 % using the 1,9-dimethyl-methylene blue method. The molecular weight of CS was determined to be 62.464 kDa by high-performance gel permeation chromatography. UV and FT-IR spectroscopy identified the characteristics of CS and its functional group information. NMR spectroscopy and disaccharide derivatization revealed that CS was predominantly composed of disulfated disaccharides, specifically ΔDi4,6S. Fluorescence quenching experiments indicated that the interaction between CS and BSA exhibited static quenching, with a binding site number of 1. The binding process was primarily mediated by van der Waals forces and hydrogen bonds. Furthermore, synchronous and 3D fluorescence spectroscopy demonstrated that CS had minimal impact on the polarity and hydrophobicity of the microenvironment surrounding Tyr and Trp residues. UV–vis absorption and circular dichroism (CD) spectroscopy demonstrated the altered structure of BSA. The molecular docking analysis revealed that CS formed hydrogen bonds and salt bridges with BSA, predominantly binding to the IIA substructure domain of BSA. Investigating the interaction between CS and BSA holds the potential for enhancing its applications in drug delivery and tissue engineering endeavors.

Fabrication of N‐methyl morpholine‐N‐oxide modified bacterial exopolysaccharide as natural scaffold toward effective cartilage regeneration in monosodium iodoacetate induced experimental osteoarthritis

AbstractA non‐pathogenic strain of Ochrobactrum pseudintermedium C1 produces potent surface‐active exopolysaccharides which exhibits a porous scaffold‐like architecture. Such a property is quite prospective in facilitating housing of cells for in vivo tissue regeneration. This surfactant exopolysaccharide has been fabricated for augmenting such regenerative properties. Different vital parameters of the fabricated EPS like surface morphology, swelling behavior, in vitro degradation study, cytotoxicity and biocompatibility have been evaluated which helped postulate the applicative profile of this natural scaffold. Further, Ochrobactrum‐borne fabricated EPS, when applied as a scaffold in monosodium iodoacetate induced experimental osteoarthritis in Wistar rat model, helped significant chondrogenesis without leaving any residual amount of scaffold. The functional restoration of knee joints, assessment of chondroitin sulfate in damaged cartilage, diminution of stress induced indicators like reactive oxygen metabolite and histopathological findings quite appreciably favored the objectives. Moreover, a single high dose exhibited considerable efficacy compared to repetitive targeted administration of the scaffold at regular intervals. Thus, this novel biological macromolecule offers a green and cost‐effective alternative to various commercially available synthetic scaffolds that promote tissue regeneration.

Comparation of the structural characteristics and biological activities of chondroitin sulfates extracted from notochord and backbone of Chinese sturgeon (Acipenser sinensis)

To compare the structural properties and biological activities of chondroitin sulfate (CS) in two different tissues of Chinese sturgeon (Acipenser sinensis) and Russian sturgeon (Acipenser gueldenstaedti), we extracted their backbone cartilage CS (Cart-CS) and notochord CS (Noto-CS), and analyzed the CS structural properties using chromatographic and spectroscopic methods. The molecular weights of Chinese sturgeon Cart-CS and Noto-CS were 54.7 and 25.4 kDa, respectively, and the molecular weights of Russian sturgeon were 50.0 and 38.4 kDa, respectively. The disaccharide composition results showed that Cart-CS was mainly composed of CS-C, while Noto-CS was almost composed of pure CS-A. The antioxidant activity of sturgeon CS and its effect on collagen fibril formation were discussed. Sturgeon CS exhibited higher antioxidant activity than shark and bovine CSs. Sturgeon CS inhibited the self-assemble of type I collagen into fibrils. The inhibition effect of Cart-CS was higher than that of Noto-CS. The high value-added utilization of Cart-CS and Noto-CS will increase the value of sturgeon by-products. Furthermore, the disaccharide composition of CS in sturgeon depends on tissues of origin, but not on species. It means that the CS of Chinese sturgeon can be substituted by the CS of other commercial sturgeon. That will contribute to the protection of endangered species of Chinese sturgeon from illegal fishing and increase the value of commercial sturgeon by-products.

Comprehensive analysis of chondroitin sulfate and aggrecan in the head cartilage of bony fishes: Identification of proteoglycans in the head cartilage of sturgeon

Cartilage in the head of sturgeon or salmon has been gaining attention as a rich source of functional chondroitin sulfate (CS) or proteoglycans. Although the cartilage was found in the heads of other bony fishes, the structure of CS and its core protein, especially aggrecan, was not fully investigated. In this study, comprehensive analysis of CS and aggrecan in the head cartilage of 10 bony fishes including sturgeon and salmon was performed. The 4-O-sulfation to 6-O-sulfation ratio (4S/6S ratio; S: sulfate residue) of CS in Perciformes was ≧1.0, while the 4S/6S ratios of CS from sturgeons and salmon were less than 0.5. Dot blotting and proteomic analysis revealed that aggrecan was a major core protein in head cartilage of all bony fishes. These results suggest that the head cartilage of bony fishes is a promising source for the preparation of CS or proteoglycans as a health food ingredient.

Anti-inflammatory and Antioxidant Activity of Peptides From Ethanol-Soluble Hydrolysates of Sturgeon (Acipenser schrenckii) Cartilage.

Research has shown that cartilage containing chondroitin sulfate and protein presents versatile bioactivities. Chondroitin sulfate in cartilage is beneficial to activate the immune system while the protein/peptide has not been fully understood. The current study investigated the antioxidant and anti-inflammatory properties of ethanol-soluble hydrolysates of sturgeon cartilage (ESCH) prepared through hot-pressure, enzymatic hydrolysis and ethanol extraction. UV spectrum, IR and agarose gel electrophoresis results suggested the successful exclusion of chondroitin sulfate from peptides. Nitric oxide (NO) floods in cells activated by inflammation. It was inhibited when administrated with ESCH. To further explain the observed anti-inflammatory activity, ESCH was separated with Sephadex G-15 into 3 components, among which F3 showed a higher NO inhibition rate and significantly reduced the production of the proinflammatory cytokine IL-6. In addition, the yield of IL-10 increased. Western blotting suggested that F3 downregulated the NO content and IL-6 level by suppressing Mitogen-activated protein kinases (MAPK) channels. Moreover, both ESCH and F3 showed DPPH and ABTS free radical scavenging abilities which was possibly related to the anti-inflammatory property. These results indicated that ESCH behaved anti-inflammatory and antioxidant activities. Cartilage may be a good source to produce anti-inflammatory peptides.

Combined detection of COMP and CS846 biomarkers in experimental rat osteoarthritis: a potential approach for assessment and diagnosis of osteoarthritis

BackgroundTo comprehensively evaluate the diagnostic value of serum cartilage oligomeric matrix protein (COMP) and chondroitin sulfate 846 epitope (CS846) biomarkers in osteoarthritis (OA), longitudinal and combined measurement of serum COMP and CS846 were performed at different stages in the pathological process of OA in a rat model of anterior cruciate ligament transection (ACLT).MethodsSixty male Sprague-Dawley rats were randomly divided into two groups, including a model group (n = 30) and a control group (n = 30). Rat models were established by ACLT surgery, and sham operations were performed on rats in the control group. Prior to surgery and at 2, 4, 6, 8, and 10 weeks after ACLT surgery, serum levels of COMP and CS846 biomarkers were determined using an enzyme-linked immunosorbent assay approach. Five rats per group were euthanized at 2, 4, 6, 8, and 10 weeks after surgery, after which tibial plateau specimens were collected. Macroscopic observation and histological examination were employed for rat tibial plateau. Histological changes in articular cartilage were evaluated according to Osteoarthritis Research Society International (OARSI) scoring criteria. The area under the curve (AUC) of COMP, CS846, and combined biomarkers was compared using receiver operating characteristic (ROC) curve.ResultsWithin 10 weeks after surgery, serum levels of COMP and CS846 in the model group were significantly higher when compared to those in the control group. Moreover, a significant correlation was observed between changes in COMP and CS846 levels. At each time point, macroscopic observations and OARSI scores were significantly increased in the development of OA. The AUC of combined biomarkers was higher compared to that of COMP and CS846 alone. Finally, a positive relationship was found between levels of COMP and CS846 and the OARSI score.ConclusionsIn this study, we found that combined detection of serum CS846 and COMP levels can be used for diagnosis and monitoring of OA progression.

Synthesis and Biocompatibility Characterizations of in Situ Chondroitin Sulfate-Gelatin Hydrogel for Tissue Engineering.

Novel hydrogel composed of both chondroitin sulfate (CS) and gelatin was developed for better cellular interaction through two step double crosslinking of N-(3-diethylpropyl)-N-ethylcarbodiimide hydrochloride (EDC) chemistries and then click chemistry. EDC chemistry was proceeded during grafting of amino acid dihydrazide (ADH) to carboxylic groups in CS and gelatin network in separate reactions, thus obtaining CS-ADH and gelatin-ADH, respectively. CS-acrylate and gelatin-TCEP was obtained through a second EDC chemistry of the unreacted free amines of CS-ADH and gelatin-ADH with acrylic acid and tri(carboxyethyl)phosphine (TCEP), respectively. In situ CS-gelatin hydrogel was obtained via click chemistry by simple mixing of aqueous solutions of both CS-acrylate and gelatin-TCEP. ATR-FTIR spectroscopy showed formation of the new chemical bonds between CS and gelatin in CS-gelatin hydrogel network. SEM demonstrated microporous structure of the hydrogel. Within serial precursor concentrations of the CS-gelatin hydrogels studied, they showed trends of the reaction rates of gelation, where the higher concentration, the quicker the gelation occurred. In vitro studies, including assessment of cell viability (live and dead assay), cytotoxicity, biocompatibility via direct contacts of the hydrogels with cells, as well as measurement of inflammatory responses, showed their excellent biocompatibility. Eventually, the test results verified a promising potency for further application of CS-gelatin hydrogel in many biomedical fields, including drug delivery and tissue engineering by mimicking extracellular matrix components of tissues such as collagen and CS in cartilage.

Preclinical evaluation of 99mTc labeled chondroitin sulfate for monitoring of cartilage degeneration in osteoarthritis

PurposeIn previous in-vitro and ex-vivo studies we proved the specific uptake of 99mTc radiolabeled chondroitin sulfate (CS) in human articular cartilage. As a logical next step for the clinical use for imaging osteoarthritis we investigated in-vivo uptake of 99mTcCS in dogs. ProceduresThe radiolabeling of CS Condrosulf (IBSA, Lugano, Switzerland) was performed using 25mg of CS and 20–40MBq/kg body weight of 99mTc by means of the tin method. In-vivo uptake of 99mTcCS was evaluated in dogs (n=12, castrated males, 4–9years, with 15–51kg body weight). 6 healthy dogs served as controls and 6 with clinical and radiological signs of osteoarthritis in the carpal, elbow, and tarsal joint were examined. The tracer was i.v. injected into the external cephalic vein. The uptake was monitored after 2, 4, 6 and 24h in healthy and osteoarthritic dogs using a planar gamma camera by regional planar or whole body ventral and dorsal acquisition. For whole body scintigraphy animals were under general anesthesia, for planar under sedation only. ResultsIn healthy control dogs we did not detect any specific uptake of 99mTcCS in the cartilage. In contrast, in the diseased dogs suffering from osteoarthritis a significant, specific, persistent uptake between 4 and 6h in tarsal, carpal and cubital joints was documented.Median target (joint) to background (mid antebrachium) ratio (T/B) in the OA joints after 4, 6, and 24h was significantly higher than in healthy controls. Target to background ratio using soft tissue as a background (T/S) a similar significantly higher than in healthy controls.In all osteoarthritic joints we found a significant positive correlation (r=0.8, n=20) between grade of disease (I–III) and T/B. When matching radiographic (X ray) changes in osteoarthritic joints (grade II and III) we found also a maximal uptake of 99mTcCS at the specific anatomical site of highest cartilage degeneration. None of the dogs experienced any side effects. ConclusionThese results suggest that 99mTcCS might become a promising diagnostic tool for imaging osteoarthritis. More extensive and detailed examinations are required, however, before extending this methodology for application in humans.

Structural analysis of isomeric chondroitin sulfate oligosaccharides using regioselective 6-O-desulfation method and tandem mass spectrometry

A strategy based on a regioselective 6-O-desulfation reaction and negative ion electrospray ionization tandem mass spectrometry (ESI-MS(n)) was developed for the structural delineation of isomeric chondroitin sulfate oligosaccharides. Product ions resulting from the glycosidic cleavage provided information about the number of sulfate groups in each sugar residue. After the regioselective 6-O-desulfation reaction, the number of sulfate groups on each residue was obtained using a tandem mass spectrometry analysis of the reaction product. The sulfation pattern could be obtained based on the product ions of analytes before and after the desulfation reaction. The strategy was demonstrated using a series of tetrasaccharides prepared from shark cartilage chondroitin sulfate D. Among the 12 identified tetrasaccharides, six structures had not been reported before.

Changes in the metabolism of chondroitin sulfate glycosaminoglycans in articular cartilage from patients with Kashin–Beck disease

To identify changes in the expression patterns of enzymes involved in chondroitin sulfate (CS) glycosaminoglycan (GAG) metabolism in articular cartilage proteoglycan (PG) isolated from adolescent patients with Kashin-Beck disease (KBD). Samples of articular cartilage were divided into two groups: Control samples (from five normal children), and KBD samples (from five KBD children) aged 3-12 years old. The morphology and pathology of hand joint cartilage were examined by histochemical staining. The localization and expression patterns of enzymes involved in CS GAG metabolism (i.e., PAPS synthetase 2 (PAPSS2), PAPS transporter 1 (PAPST1), Carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferases 15 (CHST15), Arylsulfatase B (ARSB) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS)) were performed using immuno-histochemical analyses. Positive immunostaining in articular cartilage was semi-quantified. Reduced aggrecan staining was observed in KBD samples compared with the control samples. The percentages of positive staining for the anabolic enzymes PAPSS2, PAPST1 and CHST15 in the upper and middle zones of KBD samples were significantly lower than that found in the Controls. In contrast, the percentages of positive staining in KBD samples for the catabolic enzymes ARSB and GALNS were significantly higher than the control samples. However, the staining for all of these GAG metabolism enzymes were hardly observed in the deep zones of KBD cartilage, suggesting that significant cell death and necrosis had occurred in this region. Our results indicate that alterations of enzymes involved in articular cartilage CS GAG metabolism on PGs in the articular cartilage play an important role in the onset and pathogenesis of KBD in adolescent children.

Treatment of experimentally induced osteoarthritis in horses using an intravenous combination of sodium pentosan polysulfate, N-acetyl glucosamine, and sodium hyaluronan.

To assess the effects of sodium pentosan polysulfate (PPS), N-acetyl glucosamine (NAG), and sodium hyaluronan (HA) in horses with induced osteoarthritis (OA). Experimental. Adult Standard bred horses (n = 16). OA was induced arthroscopically in 1 intercarpal joint; 8 horses were administered 3 mg/kg PPS, 4.8 mg/kg NAG, and 0.12 mg/kg HA (PGH), intravenously (IV), weekly and 8 horses were administered an equivalent volume of saline IV until study completion (day 70). Horses underwent a standardized treadmill exercise program. Clinical and radiographic findings and synovial fluid analysis were evaluated throughout the study. Macroscopic, histologic, histochemical, and biochemical findings were evaluated after necropsy. Comparisons of interest included OA and non-OA joints of saline treated horses and OA joints of PGH treated horses and OA joints of saline treated horses. Results were statistically analyzed with significance set at P < .05. OA caused increases in clinical assessment scores, synovial fluid variables, radiographic, macroscopic, and histologic cartilage scores, synovial fluid and cartilage chondroitin sulfate 846-epitope and glycosaminoglycan concentration. Total radiographic scores, total macroscopic joint pathology and macroscopic cartilage pathology scores were significantly reduced in horses treated with PGH compared with saline treated horses. Synovial fluid total protein concentration and white blood cell count were higher in OA joints of PGH treated horses compared with saline treated horses. There were no other significant differences between treatment groups. Improvements in macroscopic variables were not supported by other outcomes. Further evidence is needed before PGH can be recommended as a therapeutic option for osteoarthritis in horses.

Alterations in the chondroitin sulfate chain in human osteoarthritic cartilage of the knee

To determine whether the structure of chondroitin sulfate (CS) in cartilage is reflected by the degree of cartilage degeneration in patients with osteoarthritis (OA) of the knee and to determine how CS biosynthesis affects cartilage degeneration. Two osteoarthritic cartilage samples were obtained from medial femoral condyle (MFC) and lateral femoral condyle (LFC) of 24 knees with end-stage OA. The samples were assigned to two groups as follows: lesion and remote cartilage were adjacent to and remote from the osteoarthritic cartilage, respectively. Histological grade was determined according to the Mankin score. The CS concentration and chain length were determined using high-performance liquid chromatography (HPLC) and gel filtration chromatography, respectively. Expression of the gene encoding CS glycosyltransferase was evaluated using a real-time quantitative polymerase chain reaction (qPCR) assay. These results were compared between lesion and remote cartilage. The Mankin score indicated that lesion cartilage was more degraded compared with remote cartilage. Although the CS levels varied among individuals, the mean CS concentration and chain length were significantly lower and shorter in lesion cartilage than in remote cartilage, respectively (concentration: 12.04 vs 14.84 μg/mg wet weight, P = 0.021; chain length: 5.36 vs 6.19 kDa, P = 0.026). Three genes encoding CS glycosyltransferases (CHPF, CSGALNACT1, CSGALNACT2) were expressed at lower levels in lesion cartilage. In the osteoarthritic knee, the CS concentration and chain length were reduced closer to the more degraded cartilage with decreasing CS glycosyltransferase gene expression. Inhibition of CS glycosyltransferase gene expression may reduce CS chain length, which may contribute to OA progression.

Extraction, purification and characterisation of chondroitin sulfate in Chinese sturgeon cartilage

Chinese sturgeon (Acipenser sinensis), a rare species, is an important fishery resource in China. To save this species from overfishing, damming and pollution, artificial propagation has been developed rapidly in recent years. However, the cartilage from Chinese sturgeon, which contains an abundance of chondroitin sulfate (CS), is currently discarded as solid waste after consumption of the fish. The aim of this study was to investigate the extraction, purification and characterisation of CS from Chinese sturgeon cartilage. The optimal extraction parameters were a ratio of aqueous NaOH to cartilage powder of 9.2, a concentration of aqueous NaOH of 4.4% and an extraction time of 3.9 h. Under these optimal conditions the yield of crude CS from Chinese sturgeon cartilage was 26.51%, in agreement with the model prediction (26.54%). Purification by DEAE-52 cellulose and Sephadex G-100 column chromatography yielded a single fraction, CS-11. Its characterisation indicated that CS-11 was consistent with the polysaccharide backbone structure -4GlcAβ-3GalNβ- and was present in the form of chondroitin-4-sulfate and chondroitin-6-sulfate. The results of this study provide a basis for promoting the utilisation of Chinese sturgeon resources and are significant for the development and utilisation of CS from Chinese sturgeon cartilage in the food industry.

Highly sulfated hexasaccharide sequences isolated from chondroitin sulfate of shark fin cartilage: Insights into the sugar sequences with bioactivities

Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates and are linear polysaccharides consisting of variously sulfated repeating disaccharides, [-4GlcUAβ1-3GalNAcβ1-](n), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. CS chains containing D-disaccharide units [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)] are involved in the development of cerebellar Purkinje cells and neurite outgrowth-promoting activity through interaction with a neurotrophic factor, pleiotrophin, resulting in the regulation of signaling. In this study, to obtain further structural information on the CS chains containing d-disaccharide units involved in brain development, oligosaccharides containing D-units were isolated from a shark fin cartilage. Seven novel hexasaccharide sequences, ΔO-D-D, ΔA-D-D, ΔC-D-D, ΔE-A-D, ΔD-D-C, ΔE-D-D and ΔA-B-D, in addition to three previously reported sequences, ΔC-A-D, ΔC-D-C and ΔA-D-A, were isolated from a CS preparation of shark fin cartilage after exhaustive digestion with chondroitinase AC-I, which cannot act on the galactosaminidic linkages bound to D-units. The symbol Δ stands for a 4,5-unsaturated bond of uronic acids, whereas A, B, C, D, E and O represent [GlcUA-GalNAc(4-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(4-O-sulfate)], [GlcUA-GalNAc(6-O-sulfate)], [GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)], [GlcUA-GalNAc(4-O-, 6-O-sulfate)] and [GlcUA-GalNAc], respectively. In binding studies using an anti-CS monoclonal antibody, MO-225, the epitopes of which are involved in cerebellar development in mammals, novel epitope structures, ΔA-D-A, ΔA-D-D and ΔA-B-D, were revealed. Hexasaccharides containing two consecutive D-units or a B-unit will be useful for the structural and functional analyses of CS chains particularly in the neuroglycobiological fields.

Artra is a model of combination symptom-modifying therapy for osteoarthrosis and intervertebral osteochondrosis

The paper gives an update on therapy for osteoarthrosis (OA). Symptomatic sustained-release drugs, particularly structural analogues of the cartilage (glucosamine and chondroitin sulfate), that have the weightiest body of evidence is noted to occupy a special place in the treatment of OA. Therapy involving these agents contributes not only to the suppression of pain and the recovery of affected joint function, but also to the slow progression, normalization, or stabilization of structural changes in the hyaline cartilage, to the prevention of changes in the intact joint, and to improved quality of life in the patients.

Cell Surface Glycosylation and Glycosaminoglycan Composition Profiles in Immature and Mature Intervertebral Disks

IntroductionBack pain is a predominant cause of disability, and the major cause of this disability is intervertebral disk (IVD) degeneration.1 Proteoglycan (PG) content of the IVD is decreased during degeneration, and glycosaminoglycan (GAG) composition is altered.2 In this study, the cell surface glycosylation and GAG composition profiles of immature and mature ovine IVD tissue were profiled in an effort to understand potential signals involved in cell-extracellular matrix cross-talk during maturation and degeneration.Materials and MethodsIVD tissue from L3-L4 and L4-L5 lumbar segments were harvested from 3- and 11-month-old animals. One portion of the tissue was fixed for histochemical profiling with a panel of fluorescently-labeled lectins (Maackia amurensis agglutinin, Sambucus nigra agglutinin I, Ulex europaeus agglutinin I, Concanavalin A (jack bean lectin), Wisteria floribunda agglutinin). The remaining portion was digested with proteinase K and the sulfated GAG (s-GAG) content was quantified by dimethylmethylene blue (DMMB) assay. The digested tissue was treated with chondroitinase ABC (ChABC) and analyzed by HPLC.ResultsThe glycosylation expression level differed between annulus fibrosus (AF) and nucleus pulposus (NP) tissue as indicated by quantitative analysis using lectin histochemistry. All lectins stained cells and extracellular matrix (ECM) of the IVD independently of the tissue type. The cells demonstrated greater lectin binding compared to the ECM and the intensity of staining differed according to the tissue-type. With maturity, the expression profiles of cell surface glycosylation changed, notably for the n a-(2?6)-linked sialic acid (SNA-I) and terminal GalNAc (WFA staining) expression with a 10-fold and 2-fold increase of expression at 11 months old, respectively.DMMB assay showed that NP tissue contained threefold more s-GAGs than AF and cartilage in 3-month-old samples. In the 11-month-old IVD samples, no difference was observed between AF and NP, but overall content of s-GAG was higher than cartilage. HPLC analysis of the ChABC-digested IVD tissue revealed that the quantity and ratio of s-GAG components differed from NP to AF tissue. Moreover, a change in sulfation pattern was observed with the maturity of the disk (Fig. 1). Indeed, an inversion of the occurrence of Ddi-4S and Ddi-6S disaccharides was observed. A significant increase of Ddi-4S disaccharides with a significant decrease of Ddi-6S disaccharides was noted in the 11-month-old IVD AF tissue compared to 3 month old. A similar trend, albeit not significant, was observed for the NP tissue. Cartilage tissue did not present a significant difference in percentage of sulfated disaccharides with maturity.DownloadOpen in new tabDownload in PowerPointFigure 1 Percentage of nonsulfated and sulfated chondroitin sulfate in ovine nucleus pulposus (A), annulus fibrosus (B), and cartilage (C) at 3- and 11-month-old. Data were represented relative to the total sGAG and as mean ± standard error of the mean. *, ** represent significant differences at p < 0.05 and p < 0.01, respectively.ConclusionGAG sulfation pattern was shown to change with maturation. Sulfation is critical for maintaining water content and specific patterns of sulfation are influential in cellular signaling and differentiation events. Furthermore, high changes in glycosylation profiles at a cellular and extracellular level were observed. A better understanding of these phenomena (sulfation and glycosylation profiles changes) will allow us to design therapeutic strategies for IVD regeneration.Acknowledgements AO Foundation (S-09-7P), Science Foundation Ireland, Research Frontiers Programme (07/RFP/ENMF482), Science Foundation Ireland, 07/SRC/B1163.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredFritz JM, et al., Physical therapy for ccute low back pain: Associations with subsequent healthcare costs. Spine 2008;33(16):1800–1805O'Halloran D, et al. Tissue-engineering approach to regenerating the intervertebral disk. Tissue Engineering 2007;13(8):1927–1954

Knockout of ADAMTS5 does not eliminate cartilage aggrecanase activity but abrogates joint fibrosis and promotes cartilage aggrecan deposition in murine osteoarthritis models

To investigate the role of ADAMTS5 in murine osteoarthritis (OA), resulting from destabilization of the medial meniscus (DMM model) or from TGFb1 injection and enforced uphill treadmill running (TTR model). Wild-type (WT) and ADAMTS5-/- mice were subjected to either DMM or TTR and joints were evaluated for meniscal damage, cartilage changes, and fibrotic ingrowths from the joint margins. Cartilage lesions were quantified on an 8-point scoring system. Cartilage chondroitin sulfate (CS) content was evaluated by SafraninO staining and by quantitative electrophoresis (FACE). The abundance of aggrecan, versican, and specific aggrecanase-generated products was determined by Western analysis. Joint changes were similar for WT mice taken through either the DMM or the TTR model. ADAMTS5 ablation essentially eliminated cartilage erosion and fibrous overgrowth in both models. In the TTR model, ADAMTS5 ablation did not eliminate aggrecanase activity from the articular cartilage but blocked fibrosis and resulted in the accumulation of aggrecan in the articular cartilage. The cartilage protection provided by ADAMTS5 ablation in the mouse does not result from prevention of aggrecanase activity per se, but it appears to be due to a blockade of joint tissue fibrosis and a concomitant increase in cartilage aggrecan content.

Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(+/-6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study the linkage region are lacking.

The mobility of chondroitin sulfate in articular and artificial cartilage characterized by 13C magic‐angle spinning NMR spectroscopy

We have studied the molecular dynamics of one of the major macromolecules in articular cartilage, chondroitin sulfate. Applying (13)C high-resolution magic-angle spinning NMR techniques, the NMR signals of all rigid macromolecules in cartilage can be suppressed, allowing the exclusive detection of the highly mobile chondroitin sulfate. The technique is also used to detect the chondroitin sulfate in artificial tissue-engineered cartilage. The tissue-engineered material that is based on matrix producing chondrocytes cultured in a collagen gel should provide properties as close as possible to those of the natural cartilage. Nuclear relaxation times of the chondroitin sulfate were determined for both tissues. Although T(1) relaxation times are rather similar, the T(2) relaxation in tissue-engineered cartilage is significantly shorter. This suggests that the motions of chondroitin sulfate in natural and artificial cartilage are different. The nuclear relaxation times of chondroitin sulfate in natural and tissue-engineered cartilage were modeled using a broad distribution function for the motional correlation times. Although the description of the microscopic molecular dynamics of the chondroitin sulfate in natural and artificial cartilage required the identical broad distribution functions for the correlation times of motion, significant differences in the correlation times of motion that are extracted from the model indicate that the artificial tissue does not fully meet the standards of the natural ideal. This could also be confirmed by macroscopic biomechanical elasticity measurements. Nevertheless, these results suggest that NMR is a useful tool for the investigation of the quality of artificially engineered tissue.