Chondroitin Sulfate Content Research Articles

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.

Effects of different doses of soy isoflavones on bone tissue of ovariectomized rats

Aim Studies report that hormone replacement prevents osteoporosis, but there are doubts whether isoflavones are really efficient in this process. The aim of this study was to evaluate the effects of different doses of soy isoflavones on bone tissue of ovariectomized rats.Methods Forty female rats at the age of 6 months were ovariectomized and, after 3 months, the animals were divided into four groups: GI – Control (treated with drug vehicle); GII – treated with isoflavones (80 mg/kg per day); GIII – treated with isoflavones (200 mg/kg per day) and GIV – treated with isoflavones (350 mg/kg per day). Soy isoflavones were administered by gavage for 90 consecutive days. After treatment, the rats were euthanized and their distal femurs were removed for histological routine, histochemistry and biochemical study. Histological sections were stained with hematoxylin–eosin or subjected to picrosirius red and alcian blue methods. Shafts of femurs were submitted to biochemical assay and tibias were subjected to biophysical and biomechanical tests.Results In distal femurs, the trabecular bone volume was higher in the groups treated with isoflavones, being higher in GIV, while the cortical bone width and the presence of mature type I collagen fibers were higher in GII. At the trabecular bone region, the percentage of total glycosaminoglycans (GAGs) was higher in GII and the percentage of only sulfated GAGs was higher in GIII, while the higher content of chondroitin sulfate in shafts of femurs was seen in GIV. Biophysical and biomechanical tests in tibias did not differ among the groups.Conclusion Our data indicate that soy isoflavones improve bone quality in femurs of rats by increasing histomorphometric parameters, the content of GAGs and mature type I collagen fibers. These positive effects are dose-dependent and it was different in cortical and trabecular bone.

Plasmatic dermatan sulfate and chondroitin sulfate determination in mucopolysaccharidoses

The evaluation of plasmatic galactosaminoglycans, dermatan sulfate (DS) and chondroitin sulfate (CS) can be helpful in the early identification of MPS patients, also considering that primary storage of one type of GAG can lead to secondary accumulation of other lysosomal substrates. We explore the possibility to determine plasmatic DS and CS in numerous healthy pediatric (and sometimes adult) subjects depending on age and in patients affected by various forms of MPS. A highly sensitive HPLC separation and fluorescence detection was applied for plasma/serum DS and CS determination after a specific enzymatic treatment able to release their constituent disaccharides. DS and CS content decrease significantly with age in controls having high values in the first year (∼8μg/mL). A highly significant decrease was observed for 1–5-year-old (∼−33%) and 5–10-year-old (∼−65%) healthy subgroups. No further decrease was determined showing a stabilization after 5 years of age. MPS I Scheie and Hurler patients showed rather similar DS and CS content significantly higher than controls matched for age. Similarly, MPS II, III and IV subjects all presented significantly higher plasmatic DS and CS content compared to healthy subjects matched for age. The same trend was determined for the only patient affected by MPS VI. Plasmatic DS and CS analyzed by the present procedure may be a useful diagnostic and screening marker for various forms of MPS.

Determination of galactosamine impurities in heparin sodium using fluorescent labeling and conventional high-performance liquid chromatography

Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia.

Bonding and Fusion of Meniscus Fibrocartilage Using a Novel Chondroitin Sulfate Bone Marrow Tissue Adhesive

The weak intrinsic meniscus healing response and technical challenges associated with meniscus repair contribute to a high rate of repair failures and meniscectomies. Given this limited healing response, the development of biologically active adjuncts to meniscal repair may hold the key to improving meniscal repair success rates. This study demonstrates the development of a bone marrow (BM) adhesive that binds, stabilizes, and stimulates fusion at the interface of meniscus tissues. Hydrogels containing several chondroitin sulfate (CS) adhesive levels (30, 50, and 70 mg/mL) and BM levels (30%, 50%, and 70%) were formed to investigate the effects of these components on hydrogel mechanics, bovine meniscal fibrochondrocyte viability, proliferation, matrix production, and migration ability in vitro. The BM content positively and significantly affected fibrochondrocyte viability, proliferation, and migration, while the CS content positively and significantly affected adhesive strength (ranged from 60±17 kPa to 335±88 kPa) and matrix production. Selected material formulations were translated to a subcutaneous model of meniscal fusion using adhered bovine meniscus explants implanted in athymic rats and evaluated over a 3-month time course. Fusion of adhered meniscus occurred in only the material containing the highest BM content. The technology can serve to mechanically stabilize the tissue repair interface and stimulate tissue regeneration across the injury site.

RELATIONSHIP BETWEEN BIOCHEMICAL PARAMETERS OF THE SPINAL MOTION SEGMENT AND OUTCOME OF SURGERY FOR HERNIATED LUMBAR INTERVERTEBRAL DISC

Objective. To analyze natural biochemical changes in proteoglycans and glycosaminoglycans of patients with recurrent and nonrecurrent herniations of lumbar intervertebral discs. Material and Methods. Biochemical parameters of primary intervertebral disc hernias were prospectively investigated in 50 patients who underwent repeated surgery for recurrent hernia at the same level, and in 50 patients without recurrence. The quantity and quality of glycosaminoglycans in tissues of the hernia and surrounding annulus fibrosus were determined by analytical biochemistry methods. The properties of proteoglycans in the hernia tissue were studied by composite gel electrophoresis. Results. Type 1 recurrent hernia tissues are characterized by high content of water and glycosaminoglycans, but relatively few glycosaminoglycans tightly bound within the extracellular matrix (ECM) are retained in the tissue after its treatment with 4 M guanidinium chloride. Type 2 hernia tissues showed reduced content of water and glycosaminoglycans, and increased content of neutral hexoses. More than a half of them were tightly bound to ECM structures. On MRI evidence, Type 1 hernias correspond to Grade III disc degeneration, and Type 2 – to Grade IV. Biochemical profile of glycosaminoglycans in intervertebral disc tissues of the recurrence group reliably differed from that of the nonrecurrence group by the higher content of chondroitin sulfates and proteoglycans loosely bound to ECM structures. Conclusion. The tissue structure of nucleus pulposus and annulus fibrosus in recurrent and nonrecurrent intervertebral disc differs in qualitative and quantitative characteristics of glycosaminoglycans.

Corneal Stromal Cell Growth on Gelatin/Chondroitin Sulfate Scaffolds Modified at Different NHS/EDC Molar Ratios

A nanoscale modification strategy that can incorporate chondroitin sulfate (CS) into the cross-linked porous gelatin materials has previously been proposed to give superior performance for designed corneal keratocyte scaffolds. The purpose of this work was to further investigate the influence of carbodiimide chemistry on the characteristics and biofunctionalities of gelatin/CS scaffolds treated with varying N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) molar ratios (0–1) at a constant EDC concentration of 10 mM. Results of Fourier transform infrared spectroscopy and dimethylmethylene blue assays consistently indicated that when the NHS to EDC molar ratio exceeds a critical level (i.e., 0.5), the efficiency of carbodiimide-mediated biomaterial modification is significantly reduced. With the optimum NHS/EDC molar ratio of 0.5, chemical treatment could achieve relatively high CS content in the gelatin scaffolds, thereby enhancing the water content, glucose permeation, and fibronectin adsorption. Live/Dead assays and interleukin-6 mRNA expression analyses demonstrated that all the test samples have good cytocompatibility without causing toxicity and inflammation. In the molar ratio range of NHS to EDC from 0 to 0.5, the cell adhesion ratio and proliferation activity on the chemically modified samples significantly increased, which is attributed to the increasing CS content. Additionally, the materials with highest CS content (0.143 ± 0.007 nmol/10 mg scaffold) showed the greatest stimulatory effect on the biosynthetic activity of cultivated keratocytes. These findings suggest that a positive correlation is noticed between the NHS to EDC molar ratio and the CS content in the biopolymer matrices, thereby greatly affecting the corneal stromal cell growth.

Osmotic Pressure of Biomimetic Aggrecan from “Grafting “To Synthesis

IntroductionThe proteoglycan content of the nucleus pulposus (NP) decreases linearly with degeneration.1 This loss of proteoglycan is attributed to both a reduction of the synthesis of aggrecan by the NP cells and an increase degradation by MMP's and proteases, which attack the aggrecan protein core, resulting in aggrecan fragmentation.2-5 We envision a treatment strategy for restoration of the NP, by the addition of an enzymatically-resistant, biomimetic aggrecan (BioAGG) to the NP. This BioAGG, which “grafts” chondroitin sulfate (CS) bristles “to” a synthetic polymer core resistant to enzymatic degradation, will have osmotic pressure as a key functional property for application in the NP. We hypothesize that osmotic pressure can be impacted by both the number and density of the CS bristles incorporated, which will influence the fixed charge density and configurational entropy of this biomimetic macromolecule. Materials and MethodsTo synthesize BioAGG, linear CS molecules (bristles) with primary amine end groups were “grafted to” functional groups along poly-acrylic acid (PAA) chains. Reactions were performed with two PAA molecules at different lengths (250 kDa and 10 kDa) and varying ratios (mol/mol) of CS molecules to AA functional groups (CS; AA). Reaction kinetics was monitored using fluorescamine assay. Resulting macromolecules were purified via membrane dialysis. Polymer samples were dialyzed for 96 hours against DI water (50K MWCO) and lyophilized. Their chemical structure was confirmed with 1H NMR (300 MHz UnityInova NMR Spectrometer, 30 mg/mL samples in D2O).Using a custom membrane osmometer, osmotic pressures of the synthesized molecules were measured and compared to CS alone and an unreacted mixture of PAA and CS. All solutions were tested at the same fixed charge density based on calculations of fixed charge density. ResultsThree different molecules were fabricated using two molecular weights of the PAA core (250 kDa and 10 kDa). For PAA 250-CS samples, we varied the bristle density by controlling the molar ratio of CS to acrylic acid monomer units (CS: AA). PAA250-CS-high with “high” bristle density was synthesized with CS: AA at 1:15, and PAA250-CS-low with “low” bristle density was synthesized with CS:AA at 1:30. The molar ratio of CS: AA for PAA10-CS was kept constant at 1:10. Our theoretical estimation of the molecular weight of BioAGG samples has been done from conjugation data for CS, as determined using a fluorescamine assay, which is sensitive to the reduction in primary amines (PA) on CS. Results of these calculations are seen in Table 1.Table 1 Table 1MoleculeMolecular Wt (kDa)Number of BristlesBristle Spacing (nm)PAA250-CS-high≈2000≈854 to 8PAA250-CS-low≈1100≈4010 to 15PAA250-CS-high≈250≈93 to 5Chemical structures of the synthesized molecules were confirmed via 1H-NMR showing characteristic peaks for CS and corresponding polymer backbones for all purified compounds. No degradation of CS bristles after the synthesis and purification was observed. At the highest concentrations examined (25 mg/mL PAA250: CS) no gelation was observed. Osmotic pressure result of the PAA250-CS-high was 560 kPA about 8× higher than 70 kPA for CS alone (Fig. 1). Interestingly, when the same concentrations of PAA250 and CS are just mixed in solution (PAA250 + CS mixture), the osmotic pressure is close to that of CS alone (∼70 kPA). The same PAA250 with a lower CS bristle density, (PAA250-CS-low) is about 20% lower than the high-density version (410 kPA). A lower molecular weight with high bristle density (PAA10-CS) has an intermediate osmotic pressure between the PAA250-CS and CS alone (140 kPA). ConclusionBioAGG molecules were synthesized using a “grafting to” strategy incorporating CS bristles on PAA polymer backbones. The resultant molecules showed osmotic pressures in excess of those for the fixed charge alone. Moreover, the number and density of CS side chains influences the osmotic pressure supporting a hypothesis that the electrostatic repulsion and conformation entropy derived from the 3D macromolecular bottle-brush structure contributes to the osmotic pressure. BioAGG shows promise for mimicking physical and osmotic properties of natural aggrecan.Acknowledgment Coulter Foundation GrantI confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredOlczyk K. Zeitschrift für Rheumatologie 1994;53:19Roberts S, et.al. Spine 2000;25:3005Goupille P, et.al. Spine 1998;23:1612Le Maitre C, et.al. Biochemical Society Transactions 2007;35:652Kiani C, et al. Cell Research 2002;12:19

Osmotic Pressure of Biomimetic Aggrecan from “Grafting “To Synthesis

Introduction The proteoglycan content of the nucleus pulposus (NP) decreases linearly with degeneration.1 This loss of proteoglycan is attributed to both a reduction of the synthesis of aggrecan by the NP cells and an increase degradation by MMP's and proteases, which attack the aggrecan protein core, resulting in aggrecan fragmentation.2-5 We envision a treatment strategy for restoration of the NP, by the addition of an enzymatically-resistant, biomimetic aggrecan (BioAGG) to the NP. This BioAGG, which “grafts” chondroitin sulfate (CS) bristles “to” a synthetic polymer core resistant to enzymatic degradation, will have osmotic pressure as a key functional property for application in the NP. We hypothesize that osmotic pressure can be impacted by both the number and density of the CS bristles incorporated, which will influence the fixed charge density and configurational entropy of this biomimetic macromolecule. Materials and Methods To synthesize BioAGG, linear CS molecules (bristles) with primary amine end groups were “grafted to” functional groups along poly-acrylic acid (PAA) chains. Reactions were performed with two PAA molecules at different lengths (250 kDa and 10 kDa) and varying ratios (mol/mol) of CS molecules to AA functional groups (CS; AA). Reaction kinetics was monitored using fluorescamine assay. Resulting macromolecules were purified via membrane dialysis. Polymer samples were dialyzed for 96 hours against DI water (50K MWCO) and lyophilized. Their chemical structure was confirmed with 1H NMR (300 MHz UnityInova NMR Spectrometer, 30 mg/mL samples in D2O). Using a custom membrane osmometer, osmotic pressures of the synthesized molecules were measured and compared to CS alone and an unreacted mixture of PAA and CS. All solutions were tested at the same fixed charge density based on calculations of fixed charge density. Results Three different molecules were fabricated using two molecular weights of the PAA core (250 kDa and 10 kDa). For PAA 250-CS samples, we varied the bristle density by controlling the molar ratio of CS to acrylic acid monomer units (CS: AA). PAA250-CS-high with “high” bristle density was synthesized with CS: AA at 1:15, and PAA250-CS-low with “low” bristle density was synthesized with CS:AA at 1:30. The molar ratio of CS: AA for PAA10-CS was kept constant at 1:10. Our theoretical estimation of the molecular weight of BioAGG samples has been done from conjugation data for CS, as determined using a fluorescamine assay, which is sensitive to the reduction in primary amines (PA) on CS. Results of these calculations are seen in Table 1 . [Table: see text] Chemical structures of the synthesized molecules were confirmed via 1H-NMR showing characteristic peaks for CS and corresponding polymer backbones for all purified compounds. No degradation of CS bristles after the synthesis and purification was observed. At the highest concentrations examined (25 mg/mL PAA250: CS) no gelation was observed. Osmotic pressure result of the PAA250-CS-high was 560 kPA about 8× higher than 70 kPA for CS alone (Fig. 1). Interestingly, when the same concentrations of PAA250 and CS are just mixed in solution (PAA250 + CS mixture), the osmotic pressure is close to that of CS alone (∼70 kPA). The same PAA250 with a lower CS bristle density, (PAA250-CS-low) is about 20% lower than the high-density version (410 kPA). A lower molecular weight with high bristle density (PAA10-CS) has an intermediate osmotic pressure between the PAA250-CS and CS alone (140 kPA). Conclusion BioAGG molecules were synthesized using a “grafting to” strategy incorporating CS bristles on PAA polymer backbones. The resultant molecules showed osmotic pressures in excess of those for the fixed charge alone. Moreover, the number and density of CS side chains influences the osmotic pressure supporting a hypothesis that the electrostatic repulsion and conformation entropy derived from the 3D macromolecular bottle-brush structure contributes to the osmotic pressure. BioAGG shows promise for mimicking physical and osmotic properties of natural aggrecan. Acknowledgment Coulter Foundation Grant I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Olczyk K. Zeitschrift für Rheumatologie 1994;53:19 Roberts S, et.al. Spine 2000;25:3005 Goupille P, et.al. Spine 1998;23:1612 Le Maitre C, et.al. Biochemical Society Transactions 2007;35:652 Kiani C, et al. Cell Research 2002;12:19

Initial application of EPIC-μCT to assess mouse articular cartilage morphology and composition: effects of aging and treadmill running

The current study was undertaken to adapt Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-μCT) to mouse articular cartilage (AC), which presents a particular challenge because it is thin (30 μm) and has a small volume (0.2-0.4 mm(3)), meaning there is only approximately 2-4 μg of chondroitin sulfate (CS) glycosaminoglycan per joint surface cartilage. Using 6 μm isotropic voxels and the negatively charged contrast agent ioxaglate (Hexabrix), we optimized contrast agent concentration and incubation time, assessed two methods of tissue preservation (formalin fixation and freezing), examined the effect of ex vivo chondroitinase ABC digestion on X-ray attenuation, assessed accuracy and precision, compared young and skeletally mature cartilage, and determined patterns of degradation in a murine cartilage damage model induced by treadmill running. The optimal concentration of the contrast agent was 15%, formalin fixation was preferred to freezing, and 2 h of incubation was needed to reach contrast agent equilibrium with formalin-fixed specimens. There was good agreement with histologic measurements of cartilage thickness, although μCT over-estimated thickness by 13% (5 μm) in 6-week-old mice. Enzymatic release of 0.8 μg of chondrotin sulfate (about 40% of the total) increased X-ray attenuation by 17%. There was a 15% increase in X-ray attenuation in 14-week-old mice compared to 6-week-old mice (P < 0.001) and this corresponded to 65% decrease in CS content at 14 weeks. The older mice also had reductions of 33% in cartilage thickness and 44% in cartilage volume (P < 0.001). Treadmill running induced a 16% decrease in cartilage thickness (P = 0.012) and a 12% increase in X-ray attenuation (P = 0.006) in 14-week-old mice. This technique enables non-destructive visualization and quantification of murine femoral AC in three dimensions with anatomic specificity and should prove to be a useful new tool in studying degeneration of cartilage in mouse models.

Evaluation of intramuscularly administered sodium pentosan polysulfate for treatment of experimentally induced osteoarthritis in horses

To assess clinical, radiographic, histologic, and biochemical effects of sodium pentosan polysulfate (NaPPS) administered IM for treatment of experimentally induced osteoarthritis in horses. 18 horses. Osteoarthritis was induced arthroscopically in 1 middle carpal joint of all horses. Nine horses received NaPPS (3 mg/kg, IM) on study days 15, 22, 29, and 36. Nine control horses received the same volume of saline (0.9% NaCl) solution IM on study days 15, 22, 29, and 36. Clinical, radiographic, gross, histologic, histochemical, and biochemical findings as well as findings of synovial fluid analysis were evaluated. No adverse treatment-related events were detected. Induced osteoarthritis caused a substantial increase in lameness, response to flexion, joint effusion, radiographic findings, synovial membrane inflammation, and articular cartilage fibrillation. Articular cartilage fibrillation was substantially reduced by NaPPS treatment, and concentrations of chondroitin sulfate 846 epitope were significantly increased in the synovial fluid of osteoarthritic and non-osteoarthritic joints of treated horses. Results indicated that NaPPS has some beneficial disease-modifying effects and may be a therapeutic option for osteoarthritis in horses.

Chondroitin Sulfate in Solution: Effects of Mono- and Divalent Salts.

Chondroitin sulphate (CS) is a linear sulfated polysaccharide found in cartilage and other tissues in the body. Small angle neutron scattering (SANS) and dynamic light scattering (DLS) measurements are made on semi-dilute CS solutions to determine ion induced changes in the local order of the CS chains and in their dynamic properties. In salt-free CS solutions SANS detects the correlation peak due to local ordering between adjacent chains in which the characteristic interchain distance is d ≈ 57 Å. In both monovalent and divalent salts (NaCl and CaCl2) aligned linear regions are distinguishable corresponding to distance scales ranging from the length of the monomer unit (8 Å) to about 1000 Å. With increasing calcium ion concentration, the scattering intensity increases. Even in the presence of 200 mM CaCl2, however, neither phase separation nor cross-linking occurs. DLS in the CS solutions reveals two characteristic relaxation modes, the fast mode corresponding to the thermal concentration fluctuations. The collective diffusion coefficient D decreases with increasing calcium ion concentration and exhibits a power law function of the single variable c/J, where c is the CS concentration and J is the ionic strength of the salt in the solution. This result implies that the effect of the sodium and calcium ions on the dynamic properties of CS solutions is fully accounted for by the ionic strength.

Formulation and evaluation of chondroitin sulphate tablets of aceclofenac for colon targeted drug delivery.

The aim of the present study was to develop a single unit, site-specific matrix tablets of aceclofenac allowing targeted drug release in the colon with a microbially degradable polymeric carrier, chondroitin suphate (CS) and to coat the optimized batches with a pH dependent polymeric. The tablets were prepared by wet granulation method using starch mucilage as a binding agent and HPMC K-100 as a swellable polymer. Chondroitin Sulphate and drug and physical mixture were characterized by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The tablets were tested for their in-vitro dissolution characteristics in various simulated gastric fluids for their suitability as a colon-specific drug delivery system and also the tablets were evaluated for physicochemical properties, drug content, water percentage swelling and erosion characteristics. The dissolution data demonstrates that the 10% w/w increase in coating level of the pH dependent polymer (Eudragit L-100 and Eudragit S-100 in a ratio of 1 : 4 prevented the drug release in the simulated gastric fluid (pH 1.2-SGF) and the simulated intestinal fluid (pH 7.4-SIF). The dissolution rate of the tablet is dependent upon the concentration of Chondroitin sulphate in the simulated colonic fluid (SCF). The rapid increase in release of aceclofenac in SCF was revealed as due to the degradation of the Chondroitin sulphate membrane by bacterial enzymes. The studies confirmed that, the designed system could be used potentially as a carrier for colon delivery of aceclofenac by regulating drug release in stomach and the small intestine.

Nanoscale modification of porous gelatin scaffolds with chondroitin sulfate for corneal stromal tissue engineering

Recent studies reflect the importance of using naturally occurring biopolymers as three-dimensional corneal keratocyte scaffolds and suggest that the porous structure of gelatin materials may play an important role in controlling nutrient uptake. In the current study, the authors further consider the application of carbodiimide cross-linked porous gelatin as an alternative to collagen for corneal stromal tissue engineering. The authors developed corneal keratocyte scaffolds by nanoscale modification of porous gelatin materials with chondroitin sulfate (CS) using carbodiimide chemistry. Scanning electron microscopy/energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy showed that the amount of covalently incorporated polysaccharide was significantly increased when the CS concentration was increased from 0% to 1.25% (w/v). In addition, as demonstrated by dimethylmethylene blue assays, the CS content in these samples was in the range of 0.078–0.149 nmol per 10 mg scaffold. When compared with their counterparts without CS treatment, various CS-modified porous gelatin membranes exhibited higher levels of water content, light transmittance, and amount of permeated nutrients but possessed lower Young’s modulus and resistance against protease digestion. The hydrophilic and mechanical properties of scaffolds modified with 0.25% CS were comparable with those of native corneas. The samples from this group were biocompatible with the rabbit corneal keratocytes and showed enhanced proliferative and biosynthetic capacity of cultured cells. In summary, the authors found that the nanoscale-level modification has influence on the characteristics and cell-material interactions of CS-containing gelatin hydrogels. Porous membranes with a CS content of 0.112 ± 0.003 nmol per 10 mg scaffold may hold potential for use in corneal stromal tissue engineering.

Association between Human Plasma Chondroitin Sulfate Isomers and Carotid Atherosclerotic Plaques.

Several studies have evidenced variations in plasma glycosaminoglycans content in physiological and pathological conditions. In normal human plasma GAGs are present mainly as undersulfated chondroitin sulfate (CS). The aim of the present study was to evaluate possible correlations between plasma CS level/structure and the presence/typology of carotid atherosclerotic lesion. Plasma CS was purified from 46 control subjects and 47 patients undergoing carotid endarterectomy showing either a soft or a hard plaque. The concentration and structural characteristics of plasma CS were assessed by capillary electrophoresis of constituent unsaturated fluorophore-labeled disaccharides. Results showed that the concentration of total CS isomers was increased by 21.4% (P < 0.01) in plasma of patients, due to a significant increase of undersulfated CS. Consequently, in patients the plasma CS charge density was significantly reduced with respect to that of controls. After sorting for plaque typology, we found that patients with soft plaques and those with hard ones differently contribute to the observed changes. In plasma from patients with soft plaques, the increase in CS content was not associated with modifications of its sulfation pattern. On the contrary, the presence of hard plaques was associated with CS sulfation pattern modifications in presence of quite normal total CS isomers levels. These results suggest that the plasma CS content and structure could be related to the presence and the typology of atherosclerotic plaque and could provide a useful diagnostic tool, as well as information on the molecular mechanisms responsible for plaque instability.

Biological functionalization of dental implants with collagen and glycosaminoglycans—A comparative study

Biological implant surface coatings are an emerging technology to increase bone formation. Such an approach is of special interest in anatomical regions like the maxilla. In the present study, we hypothesized that the coating of titanium implants with components of the organic extracellular matrix increases bone formation and implant stability compared to an uncoated reference. The implants were coated using collagen-I with either two different concentrations of chondroitin sulfate (CS) or two differentially sulfated hyaluronans. Implant coatings were characterized biochemically and with atomic force microscopy. Histomorphometry was used to assess bone-implant contact (BIC) and bone-volume density (BVD) after 4 and 8 weeks of submerged healing in the maxilla of 20 minipigs. Further, implant stability was measured by resonance frequency analysis (RFA). Implants containing the lower CS concentration had significantly more BIC, compared to the uncoated reference at both times of interest. No significant increase was measured from week 4 to 8. Differences in BVD and RFA were statistically not significant. A higher concentration of CS and the application of sulfated hyaluronans showed no comparable increase in BIC. This study demonstrates a positive effect of a specific collagen-glycosaminoglycan combination on early bone formation in vivo.

Chondroitin sulphate enhances the antitumor activity of gemcitabine and mitomycin-C in bladder cancer cells with different mechanisms

Non-muscle invasive bladder cancer is the most common type of bladder cancer in Western countries. The glycosaminoglycan (GAG) layer at the bladder surface non-specifically blocks the adherence of bacteria, ions and molecules to the bladder epithelium and bladder cancer cells express CD44 that binds GAG. Currently, there are few options other than cystectomy for the management of non-muscle invasive bladder cancer with intravesical chemotherapy using several drugs such as gemcitabine (GEM) and mitomycin-C (MMC) with poor prophylactic activity. In this study, we investigated the effects of the GAG chondroitin sulphate (CS) on the growth inhibition of human bladder cancer cell lines HT-1376 and effects of the combination between GEM or MMC with CS. We have found that CS, MMC and GEM induced 50% growth inhibition at 72h. Therefore, we have evaluated the growth inhibition induced by different concentration of CS in combination with MMC or GEM, respectively, at 72h. We have observed, at Calcusyn analysis, a synergism when HT-1376 cells were treated with CS in combination with MMC or GEM, respectively, if used at an equimolar ratio. We have also found that CS/GEM combination induced a strong potentiation of apoptosis with the consequent activation of caspases9 and 3. On the other hand, HT-1376 cells were necrotic if exposed to the CS/MMC combination and no signs of caspase activation was observed. In conclusion, in the human bladder cancer cell line HT-1376 pharmacological combination of CS with GEM or MMC resulted in a strong synergism on cell growth inhibition.

Plasma Concentration of Glucosamine and Chondroitin Sulfate in Horses after an Oral Dose

This study was conducted to study absorption of glucosamine (GlucN) and chondroitin sulfate (CS) in horses immediately after feeding. Six mature mares were used in a replicated 3 × 3 Latin-square-designed experiment. The experiment consisted of three 15-day periods, which included 10 days of diet adaptation followed by a 5-day sampling period. Blood was drawn after feeding on one day during each sampling period. Horses were fed a control diet (40% hay, 60% concentrate) balanced to meet NRC requirements for maintenance of mature horses (NRC, Nutrient requirements of horses, 1989). In one experimental diet, 2.0 g CS and 5.5 g GlucN were added to the basal ration at each feeding. In the other experimental diet, 3.5 g CS and 8.5 g GlucN were added to the basal ration at each feeding. After collections, blood was centrifuged and plasma was harvested and stored until analyzed for the presence of each compound. Analyses for plasma GlucN were performed using high-performance liquid chromatography. CS in the plasma was analyzed using a color reagent, dimethylmethylene blue, followed by ultraviolet spectrophotometry. There were no significant differences ( P < .05) in the concentration of either CS or GlucN in plasma, when comparing the three different diets. This leads to a conclusion that these compounds were not absorbed intact through the intestinal wall into the bloodstream. This poses a question as to whether oral forms of these compounds are absorbed and are able to migrate to joints through the blood to improve joint function.

Analysis of the Deep Digital Flexor Tendon in Rats Submitted to Stretching after Immobilization

Few studies have analyzed the effect of stretching after immobilization on the structural and biochemical properties of tendons. Here, the effect of stretching and immobilization on the proximal (p), intermediate (i), and distal (d) regions of the deep digital flexor tendon in rats was analyzed. The d region was subjected to compression and tension forces, the i region was subjected to compressive forces and the p region received tension forces. Rats were separated into five groups: GI—control for GII; GII—immobilized rats; GIII—control for GIV and GV groups; GIV—immobilized and stretched rats; and GV—immobilized rats which were allowed free cage activity. GII showed a higher molecular organization in the d and p regions as detected by measuring optical retardation, a lower concentration of hydroxyproline in the i region and a significant decrease in noncollagenous proteins found in the three regions of the tendon. Regarding the glycosaminoglycans, diminishing dermatan sulfate and the absence of chondroitin sulfate in the i region were observed in GII when compared to GI. However, in the same region of GIV, higher concentrations of chondroitin and dermatan sulfate were observed along with a strong metachromasy. An increase in hydroxyproline content in the i region and a higher molecular organization in the d and p regions were observed in GIV. Apparently, the active isoforms of metalloproteinase-2 also increased after stretching in all regions. These results suggest that stretching after immobilization contributed to the increase in molecular organization and to the synthesis of extracellular matrix components.

Synthesis and characterization of some cellulose/chondroitin sulphate hydrogels and their evaluation as carriers for drug delivery.

Mixed hydrogels based on natural, biodegradable and biocompatible polysaccharides, such as cellulose (C) and chondroitin sulphate (CS) in various mixing ratios were prepared by a crosslinking technique and characterized by swelling behaviour, FTIR spectroscopy, scanning electron microscopy, toxicity and biocompatibility tests. The mixed cellulose/chondroitin sulphate hydrogels have been loaded with 7-[2-nitroxiacetyl-oxy-3-(4-acetyl-amino-phenoxy)-propyl]-8-morpholino-1,3-dimethyl-xanthine, a novel nitric oxide donor compound with a lower toxicity and a higher anti-inflammatory activity than its parent molecules, paracetamol and theophylline. Swelling and release kinetics have been also studied. It has been established that an increase of CS content in hydrogels composition leads to a higher swelling ratio for all formulations and to a decreased released amount of nitric oxide donor compound. It has been found that the swelling occurs by an anomalous swelling mechanism, while the release of nitric oxide donor compound follows a diffusion controlled mechanism.