Cytocompatibility and antibacterial activity of a PHBV membrane with surface-immobilized water-soluble chitosan and chondroitin-6-sulfate

A water-soluble chitosan (WSC)/chondroitin-6-sulfate (ChS) polyelectrolyte complex (PEC) is covalently immobilized onto the surface of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) membranes via ozone-induced oxidation and poly(acrylic acid) (PAA) graft polymerization. To characterize the modified membranes, X-ray photoelectron spectroscopy (XPS) and water contact angle measurements are performed. It is shown that by coupling WSC as a spacer, the amount of ChS immobilized can be significantly increased. The water contact angle decreases with the amount of PAA, WSC, and ChS immobilized, which indicates the improving hydrophilicity. After WSC- and PEC-immobilization modification, the PHBV membranes possess antibacterial activity against S. aureus, E. coli, P. aeruginosa, and Methicilin resistant Staphylococus aureus (MRSA). According to the L929 fibroblast cell growth inhibition index, the as-prepared PHBV membranes are non-cytotoxic. In addition, the in-vitro evaluation of L929 fibroblast attachment, proliferation, and viability of PEC-immobilized PHBV membranes are ascertained to be superior to those of immobilized WSC or ChS alone. The overall results demonstrate that WSC/ChS PEC immobilization can not only improve the hydrophilicity and cytocompatibility of the PHBV membrane, but also endows antibacterial activity. [GRAPH: SEE TEXT] The bacterial survival ratio of as-prepared PHBV membranes (n=3).     

Complex impedance spectroscopy to investigate degradable chondroitin–poly(amino-serinate) complexes

Sodium chondroitin-4-sulfate and poly(amino-serinate) bromide can interact to form a degradable polyelectrolyte complex. The structure of each polymer and of their complex before and after degradation is investigated by complex impedance spectroscopy. Poly(amino-serinate) bromide and sodium chondroitin-4-sulfate exhibit dc conductivity and dielectric relaxation phenomena in the 10²–10⁶ Hz range. On the contrary, no dc conductivity and dielectric relaxation are observed in the polyelectrolyte complex before degradation. After degradation, the released chondroitin-4-sulfate is re-complexed with additional poly(amino-serinate) bromide. Contrary to the original parent complex the restored complex exhibits both dc conductivity and dielectric relaxation phenomena. This difference is assigned to structural defects due to the presence of residual poly(amino-serinate) oligomers which compete with the newly added poly(amino-serinate) to complex the released chondroitin-4-sulfate. This outcome is interpreted assuming the displacement of the low molecular by higher molecular weight chains in contrast to the behavior usually reported for this type of polymeric systems.     

Surface immobilization of chondroitin 6-sulfate/heparin multilayer on stainless steel for developing drug-eluting coronary stents

A thin layer of gold was sputtered onto SUS316L stainless steel (SS) sheet. After thiolizing the Au layer with dimercaptosuccinic acid (DMSA), layers of chondroitin 6-sulfate (ChS) and heparin (HEP) were alternatively immobilized on the Au-treated SS. The resulting stent would be both anti-atherogenic and anti-thrombogenic. After repeating one to five cycles, one to five layers of polyelectrolyte complex (PEC) of ChS/HEP were successfully fabricated. A model drug, sirolimus, was loaded in the ChS/HEP layers. The SS-ChS-HEP surface was examined by X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) measurement. Biological tests including hemocompatibility, drug release pattern, and the inhibition of smooth muscle cell proliferation were also performed. The results show that the multilayer of ChS/HEP exhibits longer blood clotting time than pure SS substrates. Therefore, this biopolymer multilayer can avoid thrombosis on the stainless. The releasing rate of sirolimus can be controlled through the number of ChS/HEP PEC layers. With a five-layer coating, sirolimus can be released continuously for more than 20 days. Furthermore, the multilayer ChS/HEP loaded with sirolimus can suppress specifically to the growth of smooth muscle cells to avoid restenosis. This suggests that the PEC multilayer of ChS/HEP modified-SS could be applied in making drug-eluting stents.     

Profile analysis of chondroitin sulfates in human urine and serum

A highly sensitive high-performance liquid chromatographic method with fluorometric postcolumn labeling using 2-cyanoacetamide was developed for the profile analysis of chondroitin sulfates (ChS) in normal human urine and serum. Over-sulfated disaccharide units such as di- or trisulfated unsaturated disaccharides in urine were estimated and unsaturated 6-sulfated disaccharide (delta Di-6S) was found as a major component from ChS in urine, although only small amounts of delta Di-6S from ChS were present in serum.     

Interaction of hydroxyapatite with sodium chondroitin sulfate and calcium chondroitin sulfate in an aqueous phase

The suspension pH at a given concentration of hydroxyapatite (HAP) decreased with the concentration of calcium chondroitin-6-sulfate (CaChs), whereas it increased with the concentration of sodium chondroitin-6-sulfate (Na2Chs). The former effect is due to the increase in the concentration of H+ by ion exchange between H+ on the surface of HAP and Ca2+ of CaChs, and the latter is due to the protonation of phosphate ion (Pi) released from the surface of HAP. The absorbed amount of chondroitin-6-sulfate anion (Chs) by HAP was higher with CaChs than with Na2Chs over the concentration range examined. The equilibrium concentration of Pi decreased with increasing concentration of added CaChs because the concentration of free Ca2+, which dissociates from CaChs, regulates the free concentration of Pi through the restriction of the solubility product of HAP (Ksp). In contrast, that in the presence of Na2Chs increased with increasing concentration of added Na2Chs owing to the anion exchange between Chs and Pi of the HAP surface. The total concentration of Ca2+, which was released from HAP into the solution phase, increased after passing through a minimum with increasing concentration of added Na2Chs. That is, the concentration of Ca2+ free from Chs decreased with an increase in the concentration of released Pi owing to the restriction of the solubility product, whereas that of Ca2+ bound by Chs increased with increasing concentration of added Na2Chs through the ion exchange of Na+ with Ca2+. It was confirmed by the dialysis method that the value of Ksp was almost constant around 10, although HAP dissolves incongruently in the presence of Na2Chs.     

Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes

A method for the immunodetection of several natural complex polysaccharides (glycosaminoglycans) after their separation by conventional agarose gel electrophoresis, blotting and immobilizing on nitrocellulose membranes derivatized with the cationic detergent cetylpyridinium chloride (CPC), and direct and specific immunodetection by antibodies is described. This new approach is based on the principles that were used to develop the Western blot, and is applied to the separation of the glycosaminoglycans purified from normal human urine. After migration in agarose gel electrophoresis, chondroitin sulfate samples of different origin were blotted and transferred onto nitrocellulose membranes treated with CPC. Immunodetection was performed using the anti-chondroitin-6-sulfate antibody that specifically recognizes intact chondroitin-6-sulfate. By calculating the ratio between the antibody staining (epitope) and alcian blue staining (mass), the epitope density expressed as a percentage, i.e., the number of repetitive epitopes per mass, was obtained. These values were in agreement with the quantitation of 6-sulfated groups of chondroitin sulfate performed by the evaluation of unsatured disaccharide-6-sulfate (DeltaDi6S) produced after treatment with chondroitinase ABC and separated by high-performance liquid chromatography (HPLC). Furthermore, immunodetection of heparan sulfate was performed using the anti-heparan sulfate antibody.     

Fiber period of chondroitin sulfate and its mechanical history

Without elongational treatments, the fiber period of chondroitin-4-sulfate (0.87 nm) is shorter than in the elongated case (0.96 nm). The fiber period may change corresponding to its mechanical history. © 1996 John Wiley & Sons, Inc.     

Effect of water sorption on the dielectric behavior of calcium chondroitin-4-sulfate

The dielectric properties of calcium chondroitin-4-sulfate were studied as a function of frequency, temperature, and water content. The dielectric constant changes very little with water content below “monolayer” coverage, and after that the dielectric constant follows the shape of the adsorption isotherm. The dielectric behavior is discussed as affected by interfacial polarization and by the increase in the rotational freedom of the polymer side chains.     

Towards a model of the mineral-organic interface in bone: NMR of the structure of synthetic glycosaminoglycan- and polyaspartate-calcium phosphate composites

We have synthesised three materials-chondroitin sulphate (ChS, a commercial product derived from shark cartilage and predominantly chondroitin-6-sulphate (Ch-6-S)) bound to pre-formed hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)), HA formed in the presence of ChS and poly-Asp bound to pre-formed HA-to generate models for the mineral-organic interface in bone. The three materials have been investigated by (13)C cross polarisation magic-angle spinning (CPMAS) NMR, (13)C{(31)P} rotational echo double resonance (REDOR) and powder x-ray diffraction (XRD) in order to verify their composition and to determine the nature of their binding to HA. Our results show that for HA formed in the presence of Ch-6-S, all carbon atoms in the Ch-6-S having contact with mineral phosphate. We propose that HA in this case forms all around the Ch-6-S polymer rather than along one face of it as is more commonly supposed in cases of templating by organic molecules. However, Ch-6-S binding to pre-formed HA probably occurs via a surface layer of water on the mineral rather than to the mineral directly. In contrast, poly-Asp binds closely to the pre-formed HA surface and so is clearly able to displace at least some of the surface-bound water.     

Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography--electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases

Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases with a resolution of 1.5 on a diphenyl phase, and baseline separations were readily achieved on two carbonaceous phases with resolutions routinely exceeding three on graphitic carbon-coated zirconia (Zr-CARB) and resolutions as high as 19 on porous graphitic carbon (Hypercarb). An examination of a selected few conjugated estrogens in the complex drug substance by LC-MS on Hypercarb is presented.     

Chondroitin-4-sulfate: a bioactive macromolecule to foster vascular healing around stent-grafts after endovascular aneurysm repair

Deficient healing after endovascular aneurysm repair with a stent-graft is thought to be related to pro-apoptotic environment in abdominal aortic aneurysms and inertness of graft materials. We developed a bioactive coating containing chondroitin-4-sulfate and assessed its potential to improve cell adhesion, viability and resistance to apoptosis on PET surfaces. Coatings of collagen type I and CS were prepared and characterized by DMMB, FT-IR, DSC, SEM and contact angle goniometry. Preliminary cell culture experiments with vascular smooth muscle cells showed increased adhesion and viability in serum-free medium on CS-coated surfaces compared to control PET films.     

Conformation of the Unsaturated Uronic Acid Residues of Glycosamtnoglycan Disaccharides

Abstract

Molecular mechanics calculations (using the REFINE package) have been performed on a series of disaccharides obtained by cleavage of glycosaminoglycans with lyases, in order to examine the effect of chemical environment on the conformation of the 4,5-unsaturated uronic acid residue. The disaccharides were derived from heparin and heparan sulfate (1–5), hyaluronic acid (6), chondroitin (7), chondroitin-4-sulfate (8), and chondroitin-6-sulfate (9). The ¹H NMR spectra were analysed for the above compounds and for the unsaturated uronic acid residues of a low-molecular weight E. Coli K5 polysaccharide (10), as well as for the alditol derivative (11) of compound 8; the wide variation of the interproton vicinal coupling constants as a function of configuration and position of the glycosidic linkages and of the sulfation pattern is unequivocally interpreted in terms of equilibrium between two distinct ring forms, namely ²H₁ and ¹H₂. The equilibrium is semi-quantitatively explained by the results of the present energy calculations.     

Detection of non-typical porphyrin isomers in human urines by ion-pair reversed-phase high-performance liquid chromatography

An improved ion-pair reversed-phase high-performance liquid chromatographic system has been developed for the separation of uroporphyrin isomers I, II and III, whereas the isomers III and IV could not be resolved. Application of this method to the analysis of urines from porphyric patients indicated the presence of small amounts of the non-typical uroporphyrin isomer II. The questionable presence of the isomer IV was confirmed by acid-catalyzed decarboxylation to the corresponding coproporphyrin isomers, which were completely separated by a modified ion-pair method at elevated column temperatures. These procedures enabled the detection of small fractions of the atypical isomers II (1-3%) and IV (8-15%) besides the normal isomers I and III in urines of patients suffering from attacks of acute intermittent porphyria. Because such urines contain large amounts of porphobilinogen, the nonenzymatic self-condensation of porphobilinogen to uroporphyrinogens was studied under mild reaction conditions. In these experiments quite similar isomeric compositions were observed as compared to those in urines of patients with acute intermittent porphyria. Thus the non-typical uroporphyrin isomers II and IV present in human urines originate from a simple non-enzymatic condensation of porphobilinogen.     

Liquid chromatographic assay for constituent disaccharides of hyaluronic acid and chondroitin sulphate isomers

An improved high-performance liquid chromatographic (HPLC) method for unsaturated disaccharides prepared from hyaluronic acid and various chondroitin sulphate and dermatan sulphate isomers was developed, which involves an ion-exchange resin prepared from a sulphonated styrene-divinylbenzene copolymer. The retention times of the individual unsaturated disaccharides were unique and reproducible, the disaccharides appearing in the following order: unsaturated non-sulphated disaccharide derived from hyaluronic acid, then unsaturated 6-sulphated, non-sulphated and 4-sulphated disaccharides from chondroitin sulphate isomers. Unsaturated disulphated disaccharide G had a much shorter retention time than the unsaturated non-sulphated disaccharide derived from hyaluronic acid. The contents of these individual unsaturated disaccharides could be determined with similar sensitivities on the basis of their ultraviolet absorbance. Selective and unique retention times and good resolutions were found for various unsaturated disulphated and trisulphated disaccharides. The proposed method can be used to determine various chondroitin sulphate and dermatan sulphate isomers in addition to hyaluronic acid in amounts as small as 100 ng to 8 micrograms. The practicality of this method was verified by its application to the separation and determination of the different types of chondroitin sulphate and dermatan sulphate isomers derived from human arteries in the presence of appreciable amounts of hyaluronic acid.     

Spectroscopic study on the interaction between methylene blue and chondroitin 4-sulfate and its analytical application.

The interaction of Methylene Blue (MB) with chondroitin-4-sulfate (CHS) has been investigated using spectroscopic techniques, including UV-Vis absorption, Rayleigh resonance scattering (RRS), and circular dichroism (CD). The addition of CHS caused a decrease in the absorbance of MB at 664 nm with a new absorption band appearing at 570 nm, enhanced RRS at 314 nm and 560 nm, and also resulted in an intense CD signal at 568 nm. The Scatchard model has been applied to calculating the binding constant and the number of binding sites. The calculated parameters are consistent with the experimental results. The factors affecting the interaction were investigated. Quantitative spectroscopic methods were developed for the first time. They are based on the fact that a decrease in the absorption at 664 nm and an enhancement of the RRS intensity at 314 nm are proportional to the concentration of CHS added in a certain range. Satisfactory results were obtained on the determination of synthetic samples.     

Separation of chondroitin sulfate and hyaluronic acid fragments by centrifugal precipitation chromatography

Centrifugal precipitation chromatography (CPC) was applied for the first time to the separation of fragments of chondroitin sulfate (ChS) and hyaluronic acid (HA). The separation was performed using a gradient elution system between ethanol and water since solubility of these biopolymers highly depends on the concentration of ethanol in aqueous solution. ChS and HA were each eluted into several peaks through a flow-through UV detector at 275 nm, despite they have almost no absorbance at this wavelength in an aqueous solution. The separation was also confirmed by redissolving the dried fraction in water and measuring the absorbance at 210 nm. These results suggest that the CPC system can detect small precipitates of these biopolymers by light scattering at 275 nm. The separated fragments of biopolymers are not easily characterized because no suitable analytical method is available for identification of these compounds. However, the overall results demonstrate that CPC may be a useful separation of biopolymers such as glycosaminoglycans which quantitatively produce precipitates in an organic solvent mixture.     

Quantification of eicosapentaenoic and docosahexaenoic acid geometrical isomers formed during fish oil deodorization by gas-liquid chromatography

Long-chain polyunsaturated fatty acids (LC-PUFAs) of the n-3 series and especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively) have important biological properties. The main dietary sources of LC-PUFAs are fish and fish oil. Geometrical isomerization is one of the main reactions happening during the thermal treatment of polyunsaturated fatty acids. Refined fish oils are used to supplement food products in LC-PUFAs and the quality of these nutritional ingredients have to be controlled. In the present study, a suitable method for the quantification of EPA and DHA geometrical isomers in fish oils by gas-liquid chromatography (GC) is presented. A highly polar capillary column (CP-Sil 88, 100 m) operating under optimal conditions was used. Method selectivity was studied by GC-mass spectrometry. The performance characteristics of the quantification method were studied using samples of fish oil deodorized at 220 degrees C for 3 h. The linearity of the method was assessed by analyzing composite samples obtained by mixing fish oil deodorized at 220 degrees C with semi-refined fish oil (control). Precision was evaluated by analyzing the same samples in triplicate. Results showed that the validated method is suitable to quantify low amounts of geometrical (trans) isomers of EPA and DHA in refined fish oils. The limits of quantification of the EPA and DHA geometrical isomers are 0.16 and 0.56 g/100 g of fish oil, for EPA and DHA, respectively. Commercially available LC-PUFA oil samples were evaluated by using the validated method. The results show that the oils analyzed contain low amounts (<1% of total fatty acids) of geometrical isomers of EPA and DHA.     

In vitro biocompatibility of three‐dimensional chitosan scaffolds immobilized with chondroitin‐6‐sulfate

Cellular‐compatible scaffolds were prepared using a three‐dimensional micro‐porous chitosan (CS) non‐woven fabric immobilized by glutaraldehyde (GA), followed by the immobilization of chondroitin‐6‐sulfate (ChS). To characterize the immobilizing process, tensile analysis, and scanning electron microscopy (SEM) were performed. The cell seeding efficiency and proliferation test were evaluated using L929 fibroblasts. The chitosan scaffolds showed high water vapor transmission rate and antibacterial activity. In addition, ChS‐immobilized scaffolds exhibited higher cell seeding efficiency and fibroblasts proliferation. These results demonstrated that the CS non‐woven fabrics grafted with GA and immobilized with ChS could be an appropriate candidate for wound healing and artificial scaffolds in the clinical applications. Copyright © 2007 John Wiley & Sons, Ltd.     

Liquid and gas chromatographic separation of isomeric methylated dibenzothiophenes

Trace amounts of a dibenzothiophene (DBT) series in complex matrices were detected by gas chromatography with flame photometric detection (FPD) after purification and isolation by normal-phase high performance liquid chromatography. The gas chromatographic behavior of eleven methylated DBTs was studied on six common stationary phases. Complete resolution of the four monomethyl DBT isomers was only achieved on a capillary column coated with a methylphenylsilicone phase gummified in situ by heat-curing. The determination of the relative distribution of the four monomethylated isomers is proposed as a powerful method for monitoring oil pollution in marine environment.     

Evaluation of Chitosan-Based Films Containing Gelatin,Chondroitin 4-Sulfate and ZnO for Wound Healing

In this work, chitosan-based films containing gelatin and chondroitin-4-sulfate (C4S) with and without ZnO particles were produced and tested in vitro to investigate their potential wound healing properties. Chitosans were produced from shrimp-head processing waste by alkaline deacetylation of chitin to obtain chitosans differing in molecular weight and degree of deacetylation (80 ± 0.5%). The film-forming solutions (chitosan, C4S and gelatin) and ZnO suspension showed no toxicity towards fibroblasts or keratinocytes. Chitosan was able to agglutinate red blood cells, and film-forming solutions induced no hemolysis. Film components were released into solution when incubated in PBS as demonstrated by protein and sugar determination. These data suggest that a stable, chitosan-based film with low toxicity and an ability to release components would be able to establish a biocompatible microenvironment for cell growth. Chitosan-based films significantly increased the percentage of wound healing (wound contraction from 65 to 86%) in skin with full-thickness excision when compared with control (51%), after 6 days. Moreover, histological analysis showed increased granulation tissue in chitosan and chitosan/gelatin/C4S/ZnO films. Chitosan-based biopolymer composites could be used for improved biomedical applications such as wound dressings, giving them enhanced properties.