Studies of chitosan: II. Preparation and characterization of chitosan/poly(vinyl alcohol)/gelatin ternary blend films

Chitosan/poly(vinyl alcohol)/gelatin (CS/PVA/GA) ternary blend films were prepared by solution blending method in this study. The thermal properties of the CS/PVA/GA ternary blend films were examined by differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The melting point of the CS/PVA/GA ternary blend film was increased when the amount of GA in the blend film was increased based upon the DSC thermal analysis. Results of X-ray diffraction (XRD) analyses indicated that the intensity of diffraction peak at 19 degrees of PVA became lower and broader with increasing the amount of GA in the CS/PVA/GA ternary blend film. Although CS, PVA, and GA are hydrophilic biodegradable polymers, the results of water contact angle measurements are still as high as 83 degrees, 68 degrees, and 66 degrees, respectively. A minimum water contact angle (56 degrees) was observed when the ternary blend film contains 50 wt.% GA (i.e. GA5). This behavior is primarily due to the reorientation of polar functional groups toward to the top surface of CS/PVA/GA ternary blend films.     

Modification of collagen with a natural cross-linker, procyanidin

We have investigated the modification of collagen with a natural plant polyphenol, procyanidin under acidic conditions. Fourier transform infrared spectroscopy (FTIR) and Atomic force microscopy (AFM) studies demonstrate that the hydrogen bond interactions between collagen and procyanidin does not destroy the triple helix conformation of collagen, and the fibril aggregation occurs because of the cross-linking with procyanidin. The water contact angle (WCA) tests indicate that the hydrophobicity of the procyanidin modified collagen films can be improved. Whereas, the water vapor permeability (WVP) of the films decrease with the increasing procyanidin content due to the formation of denser structure. Moreover, differential scanning calorimetry (DSC) and thermogravimetric (TG) measurements reveal that the collagen/procyanidin films have improved thermal stability in comparison with pure collagen. The present study reveals that procyanidin stabilizes collagen as a cross-linker and preserves its triple helical structure.     

Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate) films: effects of epoxidation and curing

Medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers derived from linseed oil (PHA-L) have a relatively small molar mass and contain a high concentration of unsaturated side-chains. As such, these polymers are amorphous and take on the consistency of a viscous liquid at room temperature. In order to increase the application potential of this material, the side-chain olefinic groups of PHA-L were converted to epoxy derivatives (PHA-LE) using m-chloroperoxybenzoic acid (m-CPBA). Epoxidation resulted in a 37% conversion of olefinic to epoxy groups. The epoxy groups enhanced the PHA-LE film susceptibility to crosslinking upon exposure to air. PHA-LE films began to crosslink and stiffen in less than 25 days, whereas PHA-L films began to crosslink between days 50 and 75. The PHA-LE films showed an increase in tensile strength (TS, from 4.8 to 20.7 MPa) and Young's modulus (YM, from 12.9 to 510.6 MPa) between 25 and 100 days. In contrast, PHA-L had a TS of 25.0 MPa and YM of 767.8 MPa after 100 days. Epoxidation helped induce crosslink formation; however, aging for 100 days ultimately resulted in crosslinked films from both PHA-L and PHA-LE with higher strength and durability than the original materials.     

A new approach for estimating the crosslink density of covalently crosslinked ionic polysaccharide gels

For an ideal polysaccharide gel with a known total polymer chain contour length, crosslinks all of the same functionality and elastic chains all with the same contour length and stiffness, the gel crosslink density can readily be determined from measurements of the maximum volume of the swollen gel (Moe et al., (1991) Food Hydrocolloids, 5, (1/2), 119–123. In the case of randomly crosslinked polysaccharide gels, where the chain contour length between two adjacent crosslinks may vary greatly, it is often much more difficult to determine the crosslink density. This paper reports on an attempt to extend the use of maximum gel volume measurements to estimate crosslink density for the latter type of gel. This is done by calculating the maximum swelling volume for polymer networks with four-functional crosslinks, known elastic chain mean contour length and standard deviation. The numerical analysis involves the calculation of the equilibrium force at each crosslink as the network expands. This allows a detailed study of how the distribution of individual polymer chain contour lengths affects the maximum swelling volume. The computer simulation results are compared with the results from experimental measurements of the maximum volume of swollen covalently crosslinked sodium alginate gels.     

Participation of the alpha 2(I) chain of bovine skin collagen in the formation of mature crosslinks

It is shown that regions of unreduced, insoluble cow hide collagen, represented by the peptides alpha 1(I)-CB6, alpha 2(I)-CB4 and the alpha 2(I)-CB3,5, are involved in the formation of unreducible acid-stable and mature-type crosslinks. The characteristic ratio of the CNBr peptides in soluble type I collagen was found to be changed in the insoluble collagen of cow hides. The intensity of the bands of alpha 1(I)-CB6, alpha 2(I)-CB4 and alpha 2(I)-CB3,5, shown by dodecyl sulfate polyacrylamide gel electrophoresis, is significantly reduced in such samples, which indicates an involvement of these peptides in crosslink formation. The purified highly polymeric CNBr peptide fraction was also investigated to confirm the participation of the alpha 2 chain of type I collagen in mature crosslink formation. Chymotryptic digests of such material contain peptides which originate from alpha 2(I)-CB4, alpha 2(I)-CB3,5, and alpha 1(I)-CB6. Finally, acid hydrolysates of crosslinked material were screened carefully for crosslinks down to concentrations of 1 in 1000 amino acids. Only two compounds were detected, one identified as "hydroxyaldol-histidine" and the other an as yet unknown compound. These results indicate that both the alpha 1(I) and the alpha 2(I) chains are involved in mature crosslink formation and that the polymeric CNBr peptide fraction contains components crosslinked by so far uncharacterized, nonreducible crosslinks.     

In vitro deposition of hydroxyapatite on cortical bone collagen stimulated by deformation-induced piezoelectricity

In the present work, we have studied the effect of the piezoelectricity of elastically deformed cortical bone collagen on surface using a biomimetic approach. The mineralization process induced as a consequence of the piezoelectricity effect was evaluated using scanning electron microscopy (SEM), thermally stimulated depolarization current (TSDC), and differential scanning calorimetry (DSC). SEM micrographs showed that mineralization occurred predominantly over the compressed side of bone collagen, due to the effect of piezoelectricity, when the sample was immersed in the simulated body fluid (SBF) in a cell-free system. The TSDC method was used to examine the complex collagen dielectric response. The dielectric spectra of deformed and undeformed collagen samples with different hydration levels were compared and correlated with the mineralization process followed by SEM. The dielectric measurements showed that the mineralization induced significant changes in the dielectric spectra of the deformed sample. DSC and TSDC results demonstrated a reduction of the collagen glass transition as the mineralization process advanced. The combined use of SEM, TSDC, and DSC showed that, even without osteoblasts present, the piezoelectric dipoles produced by deformed collagen can produce the precipitation of hydroxyapatite by electrochemical means, without a catalytic converter as occurs in classical biomimetic deposition.     

Structure-activity relationship study of WSS25 derivatives with anti-angiogenesis effects

WGEW, an α(1-4) linked glucan with an α(1-4) linked branch attached to C-6, was isolated from the rhizoma of Gastrodia elata Bl. WSS25, a sulfated derivative of WGEW, was reported to inhibit angiogenesis by disrupting BMP2/Smad/Id1 signaling pathway. However, the structure-activity relationship (SAR) for WSS25 is not known. To study the SAR, seven sulfated saccharides derived from WGEW degradation products, six sulfated polysaccharides with varying degrees of substitution, and four aminopropylated, carboxymethylated, phosphorylated, and acetylated derivatives of WGEW were prepared. A sulfated, unbranched product of polysaccharide was also obtained. The structural features of these derivatives were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy. An HMEC-1 cell tube formation assay was employed to measure the antiangiogenic effect of the derivatives. The results indicated that only sulfated polysaccharides with molecular weights of more than 41,000?Da could inhibit HMEC-1 cell tube formation. The inhibition effect was dependent on the presence of a sulfate group, since the tube formation was not blocked by aminopropylated, carboxymethylated, phosphorylated, or acetylated WGEW. A higher degree of sulfate substitution on the polysaccharide led to a stronger inhibitory effect, and the degree of sulfate substitution between 0.173 and 0.194 was found to be optimal. Interestingly, the WGEW side chain was not required for anti-tube formation activity. All these preliminary results may provide a clue for further modification of the core structure of WSS25 to discover polysaccharide derivatives as novel anti-angiogenic inhibitors.     

Yeast β(1-3),(1-6)-d-glucan films: Preparation and characterization of some structural and physical properties

Cell wall polysaccharide suspensions (mainly β-glucan) was isolated from baker's yeasts (Saccharomyces cerevisiae) and used for the preparation of films. Glycerol was added as a plasticizer. Purity and composition of the films were tested by elemental analysis, enzymatic assay of α- and β-glucans, monosaccharide composition analysis (total hydrolysis, HPAEC) and vibration spectroscopy (FTIR, FT Raman). Surface properties and the degree and type of crystallinity, together with ageing effects, were estimated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray diffraction (XRD). Mechanical and thermal properties were characterized by tensile tests and difference scanning calorimetry (DSC), respectively. The prepared films were water insoluble, compact, non-porous, exhibit no pronounced crystallinity and consist of granular-like and fibre microstructures, which could be assigned as cell wall residues and released polysaccharide macromolecules. Certain structural changes in the film surface during one-year shelf storage can be related to reorientation and decomposition of surface macromolecules due to reaction with the ambient atmosphere, rather than to crystallization phenomena.     

Effects of Ca crosslinking on structure and properties of waterborne polyurethane-carboxymethylated guar gum films

Films from waterborne polyurethane (WPU) and carboxymethylated guar gum (CMGG) with different contents (20–80 wt%) were prepared through solution casting method, and then were crosslinked with calcium chloride. The effect of CMGG content on the miscibility, morphology and physical properties of the blend films is investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, density measurements, differential scanning calorimetry, dynamic mechanical thermal analysis, thermogravimetric analysis, water sensitivity measurements, solvent-swelling and tensile tests. The results reveal that the uncrosslinked films exhibit good miscibility when CMGG content is lower than 60 wt%, whereas typical “sea-island” structure occurs when the CMGG content further increases. After crosslinking with calcium ion, the blend films form a relatively dense architecture, which leads to better miscibility, higher storage modulus and thermal stability. The crosslinked films also exhibit better tensile strength (11.6–56.5 MPa) and solvent-resistance than that of the uncrosslinked films over the entire composition range. A model describing the configuration of Ca²⁺-chelating structure was proposed to illustrate the different structures of the two series of the blend films.     

Studies of ferroelectric properties of collagen

Dielectric hysteresis loop and differential thermal analysis (DTA) studies of collagen were performed for temperatures ranging from a room temperature to 393 K. The material studied was collagen from Achilles tendon of a bullock, which contained 10% of water. The DTA curve for collagen with 10% of water showed a clear minimum at 353 K. The character of the curve proved that around 353 K an endothermic transformation of collagen took place which could be connected with the transition spiral-ball. A hypothetical dielectric hysteresis loop shows a maximum shift along the direction of the electric field applied in the vicinity of 375 K. Low frequencies of the applied electric field are connected with a great dielectric loss influencing the interpretation of collagen ferroelectricity.     

The effect of crosslinking by bis(3,5-dibromosalicyl) fumarate on the autoxidation of hemoglobin

Bis(3,5-dibromosalicyl) fumarate was used to crosslink hemoglobin both in the oxy and deoxy states. This double headed diaspirin was known to crosslink oxy Hb A selectively between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxy Hb A between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R. Y., et al. (1986) J. Biol. Chem. 261, 9929). The autoxidation at 37 degrees C of oxy alpha 99 crosslinked hemoglobin was found to be 1.8 times as fast as that of Hb A while that of the oxy beta 82 crosslinked hemoglobin was only 1.2 times as fast. After 5 hours the formation of methemoglobin in the alpha crosslinked Hb A is 21.3% compared to 10.8% in beta crosslinked Hb A and 6.4% in Hb A. These results may effect the proposed use of alpha 99 crosslinked hemoglobin as a blood substitute by demonstrating the need for protection from autoxidation during storage.     

Quantitative Evaluation of Collagen Crosslinks and Corresponding Tensile Mechanical Properties in Mouse Cervical Tissue during Normal Pregnancy

The changes in the mechanical integrity of the cervix during pregnancy have implications for a successful delivery. Cervical collagens are known to remodel extensively in mice with progressing gestation leading to a soft cervix at term. During this process, mature crosslinked collagens are hypothesized to be replaced with immature less crosslinked collagens to facilitate cervical softening and ripening. To determine the mechanical role of collagen crosslinks during normal mouse cervical remodeling, tensile load-to-break tests were conducted for the following time points: nonpregnant (NP), gestation day (d) 6, 12, 15, 18 and 24 hr postpartum (PP) of the 19-day gestation period. Immature crosslinks (HLNL and DHLNL) and mature crosslinks (DPD and PYD) were measured using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). There were no significant changes in the total immature crosslink density (HLNL+DHLNL mol per collagen mol) throughout normal mouse gestation (range: 0.31–0.49). Total mature crosslink density (PYD+DPD mol per collagen mol) decreased significantly in early softening from d6 to d15 (d6: 0.17, d12: 0.097, d15: 0.026) and did not decrease with further gestation. The maturity ratio (total mature to total immature crosslinks) significantly decreased in early softening from d6 to d15 (d6: 0.2, d15: 0.074). All of the measured crosslinks correlated significantly with a measure of tissue stiffness and strength, with the exception of the immature crosslink HLNL. This data provides quantitative evidence to support the hypothesis that as mature crosslinked collagens decline, they are replaced by immature collagens to facilitate increased tissue compliance in the early softening period from d6 to d15.     

Molecular mobility of elastin: effect of molecular architecture

The thermal and dielectric properties of elastin and two soluble derivatives (kappa-elastin and derived elastin peptides from enzymatic elastolysis) were investigated in the freeze-dried state in a wide temperature range (from -180 to +220 degrees C). The glass transition of these amorphous proteins was studied by differential scanning calorimetry (DSC). The dielectric relaxations of both proteins were followed by thermally stimulated currents (TSC), an isochronal dielectric spectrometry running at variable temperature, analogous to a low-frequency spectroscopy (10(-3)-10(-2) Hz) and by dynamic dielectric spectroscopy (DDS), performed isothermally with the frequency varying from 10(-2) to 3 x 10(6) Hz. The combination of TSC and DDS experiments and the determination of the activation parameters of the relaxation times inform about the molecular mobility of the proteins, both in the glassy state and in the liquid state. Major differences between the relaxation behavior of elastin and its soluble derivatives have been discussed and correlated with the molecular architecture of the proteins.     

Comparative study of carrageenans from reproductive and sterile forms of <Emphasis Type="Italic">Tichocarpus crinitus</Emphasis> (Gmel.) Rupr (Rhodophyta,Tichocarpaceae)

A comparative study of the structure and properties of the sulfated polysaccharides (carrageenans) isolated from the vegetative and reproductive forms of the red alga Tichocarpus crinitus was performed. The polysaccharides were separated into the gelling (KCl-insoluble) and non-gelling (KCl-soluble) fractions by precipitation with 4% KCl. The total content of polysaccharides extracted from the reproductive form of the alga was 1.8-fold more than that extracted from the vegetative form, and in the first case, the gelling polysaccharides mostly accumulated. The gelling polysaccharides from the vegetative form have the highest molecular weight (354 kD). According to the results of FT-IR and 13C-NMR spectroscopy, the gelling polysaccharide fractions from both forms are kappa/beta carrageenans. The differences concern the content of the kappa- and beta-disaccharide units and the presence of a small content of the sulfated disaccharide segments (precursors of the kappa-carrageenans) in the polysaccharide from the reproductive form of the alga. The non-gelling polysaccharide fractions from both forms of the plant are mixtures of sulfated galactans with a low content of 3,6-anhydrogalactose.     

Structure and anticoagulant activity of a sulfated galactan from the red alga, Gelidium crinale. Is there a specific structural requirement for the anticoagulant action?

Marine red algae are an abundant source of sulfated galactans with potent anticoagulant activity. However, the specific structural motifs that confer biological activity remain to be elucidated. We have now isolated and purified a sulfated galactan from the marine red alga, Gellidium crinale. The structure of this polysaccharide was determined using NMR spectroscopy. It is composed of the repeating structure -4-alpha-Galp-(1-->3)-beta-Galp1--> but with a variable sulfation pattern. Clearly 15% of the total alpha-units are 2,3-di-sulfated and another 55% are 2-sulfated. No evidence for the occurrence of 3,6-anhydro alpha-galactose units was observed in the NMR spectra. We also compared the anticoagulant activity of this sulfated galactan with a polysaccharide from the species, Botryocladia occidentalis, with a similar saccharide chain but with higher amounts of 2,3-di-sulfated alpha-units. The sulfated galactan from G. crinale has a lower anticoagulant activity on a clotting assay when compared with the polysaccharide from B. occidentalis. When tested in assays using specific proteases and coagulation inhibitors, these two galactans showed significant differences in their activity. They do not differ in thrombin inhibition mediated by antithrombin, but in assays where heparin cofactor II replaces antithrombin, the sulfated galactan from G. crinale requires a significantly higher concentration to achieve the same inhibitory effect as the polysaccharide from B. occidentalis. In contrast, when factor Xa instead of thrombin is used as the target protease, the sulfated galactan from G. crinale is a more potent anticoagulant. These observations suggest that the proportion and/or the distribution of 2,3-di-sulfated alpha-units along the galactan chain may be a critical structural motif to promote the interaction of the protease with specific protease and coagulation inhibitors.     

Crosslinking of phycobiliproteins from the cyanobacterium Mastigocladus laminosus with bis-imidates: localization of an intrasubunit and an intersubunit crosslink in C-phycocyanin

The light-harvesting pigment-protein complexes allophycocyanin (AP), C-phycocyanin (PC) and phycoerythrocyanin (PEC) of the cyanobacterium Mastigocladus laminosus consist of alpha- and beta-subunits containing about 170 amino-acid residues each. These two subunits form an alpha,beta-monomer, three of which build up a disc-shaped trimer. In this study these phycobiliproteins were crosslinked with bis-imidates. Various spacer lengths of the reagent and various aggregation states of the phycobiliprotein were tested. An intersubunit crosslink could be verified in all three phycobiliproteins. PC-trimers were crosslinked with the homobifunctional reagent dimethyl pimelimidate having a maximal crosslinking distance of 10 A. Two crosslinks could be identified: an intramonomer intersubunit crosslink with a yield of 48% and an intrasubunit crosslink within alpha PC (57%). These products were chemically and enzymatically fragmented and the small crosslinked peptides were isolated and then identified by amino-acid analysis. The following amino acids were crosslinked: alpha-Val 1 with beta-Ala 1 and alpha-Lys 62 with alpha-Lys 134. Both crosslinks could be localized within the known three-dimensional structure of PC.     

In Vitro Anti-influenza Virus Activities of Sulfated Polysaccharide Fractions from Gracilaria lemaneiformis

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent?

The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked beta-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked alpha-fucose and one or two nonsulfated, 4-linked beta-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.     

Collagen crosslinking: isolation of hydroxyaldol-histidine, a naturally-occurring crosslink.

A new trifunctional crosslink, termed hydroxyaldol-histidine, was isolated from cow skin collagen. This compound was not reducible by sodium borohydride; it was characterized by PMR spectroscopy and by low and high resolution mass spectroscopy of volatile derivatives. This crosslink is identical to an unknown amino acid previously detected in pure collagen-derived peptides. We postulate that it arises by condensation of peptidyl allysine, hydroxyallysine and histidine. This is the first example of a non-borohydride reducible crosslink found in collagen.     

The effect of different methods of cross-linking of collagen on its dielectric properties

This paper reports on the effect of different methods of collagen cross-linking on its dielectric properties. In order to obtain collagen-hyaluronic acid (HA) scaffolds, collagen was first dehydrated by a combination of thermal and vacuum drying (DHT) and then treated with the chemical reagent carbodiimide (EDC/NHS) for final cross-linking. The measurements of the relative permittivity varepsilon' and the dielectric loss varepsilon' for all materials were carried over the frequency range of 10 Hz-100 kHz and at temperatures from 22 to 260 degrees C. The results for these samples reveal distinct relaxation processes at low temperatures, below 140 degrees C and at higher temperatures as broad peak around 230 degrees C. The first and second relaxation are associated with changes in the secondary structure of collagen accompanied by the release of water and with the denaturation of dry collagen, respectively. The influence of cross-linking on the permittivity of collagen is significant over the entire temperature range.