Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated to combine the gel-forming properties of alginate with the healing properties of hyaluronate. Gels were prepared by diffusion of calcium into alginate-hyaluronate mixtures, with an alginate content of 20 mg/mL. The hyaluronate source was shown to have significant effect on the aspect and the properties of the gels. The gels have viscoelastic behaviour and the transient measurements carried out in creep mode could be interpreted through a Kelvin-Voigt generalised model: experimental data led to the steady state hardness and a characteristic viscosity of the gel. Gels prepared from Na rooster comb hyaluronate with weight ratio up to 0.50 have satisfactory mechanical properties, and fully stable gels are obtained after a few days; on the contrary, use of lower molecular weight hyaluronate led to loose gels for hyaluronate contents over 0.25. Gel formation was investigated by measurements of the exchange fluxes between the calcium chloride solution and the forming gel, which allowed thorough investigations of the occuring diffusion phenomena of water, calcium ion and hyaluronate. Strong interactions of water with hyaluronate reduce significantly the rate of weight loss from the gel beads and allows higher water content in steady-state gels. Calcium content in the gel samples could be correlated to the actual alginate concentration, whatever the nature and the weight ratio of hyaluronate.     

Synthesis and rheological properties of responsive thickeners based on polysaccharide architectures

New thermothickening copolymers were synthesized by grafting responsive poly(ethylene oxide-co-propylene oxide) [PEPO] onto three different polysaccharide backbones: carboxymethylcellulose [CMC], alginate [ALG], and carboxylated dextran [DEX]. The coupling reaction between carboxylic groups of biopolymers and the terminal amine of PEPO was activated at low temperature ( T < 10 degrees C) in water by using carbodiimide and N-hydroxysuccinimide. In these conditions it was shown that the formation of amide bonds strongly depends on the concentration of reactive groups, which is limited by the viscosity of the polymer sample. While a full conversion was obtained for the low molecular weight dextran, the efficiency of grafting remains low (between 30 to 40%) for CMC and alginate, which give a solution of high viscosity even at low concentration. When studied in the semidilute regime, all the copolymer solutions clearly exhibit thermothickening behavior with a large and reversible increase of viscosity upon heating. The association temperature and the gelation threshold were shown to depend on polymer concentration as it is expected from the phase diagram of PEPO precursor. Similarly, the influence of added salt on PEPO solubility in water has been used to control the self-assembling behavior of copolymer formulations. The relative comparison between the three copolymers reveals that the amplitude of the viscosity jump induced by heating mainly depends on the proportion of responsive material inside the macromolecular architecture rather than the dimensions of the main chain. The high increase of viscosity, which can reach several orders of magnitude between 20 degrees C and body temperature, clearly demonstrates the potentiality of these copolymers in biomedical applications like injectable gels for tissue engineering.     

A comparison of the reactivity of alginate and pectate esters with gelatin

Aqueous solutions of highly esterified propylene glycol alginate and gelatin interact rapidly in mildly alkaline conditions to form a gel with a very high melting point. The interaction involves the formation of amide bonds between the ester and uncharged amino groups on the protein.Neither high-methoxyl pectin nor highly esterified propylene glycol pectate formed thermostable gels with gelatin, and the lack of reactivity was not due to differences between pectate and alginate in viscosity, rate of depolymerisation or rate of saponification. Pectate esters will react, however, with low molecular weight diamines in anhydrous conditions.It is suggested that the different reactivity of the uronides in water reflects differences in the geometries of their glycosidic links between monomers, and that in alginate it is the mannuronic residues that are involved in these reactions.     

Some properties of alginate gels derived from algal sodium alginate

Alginic acid in soluble sodium alginate turns to insoluble gel after contact with divalent metal ions, such as calcium ions. The sodium alginate character has an effect on the alginate gel properties. In order to prepare a suitable calcium alginate gel for use in seawater, the effects of sodium alginate viscosity and M/G ratio (the ratio of D-mannuronate to L-guluronate) on the gel strength were investigated. The wet tensile strengths of gel fibers derived from high viscosity sodium alginate were higher than those from low viscosity sodium alginate. The tensile strength increased with diminishing sodium alginate M/G ratio. Among the gel fibers tested, the gel fiber obtained from a sodium alginate I-5G (1% aqueous solution viscosity = 520 mPa·s, M/G ratio = 0.6) had the highest wet tensile strength. After 13 days treatment in seawater, the wet tensile strength of the gel fiber retained 36% of the original untreated gel strength. For sodium alginates with similar viscosities, the seawater tolerance of low M/G ratio alginate was greater than that of the high M/G ratio one. This study enables us to determine a suitable calcium alginate gel for use in seawater.     

Bacterial Alginate Produced by a Mutant of Azotobacter vinelandii

The optimum conditions in shaken flasks for production of bacterial alginate by mutant C-14 of Azotobacter vinelandii NCIB 9068 and a comparison of the properties of bacterial and algal alginates were investigated. The largest amount of bacterial alginate was obtained in about 110 h by a culture grown on optimum medium at 34°C and 170-rpm shaking speed. The viscosity of the culture broth was 18,400 cps and the alginate concentration reached 6.22 g/liter. The viscosity of the purified bacterial alginate was as high as 11,200 cps at a low concentration (0.6%). A greater than fivefold concentration of algal alginate was required to reach the same viscosity at a low shear rate. A solution of bacterial alginate was more pseudoplastic than that of algal alginate was. No significant differences were observed in other properties of bacterial and algal alginates such as gel formation with calcium ion, thermostability, and effect of temperature, pH, and sodium chloride on viscosity.     

Amphiphilic derivatives of sodium alginate and hyaluronate: synthesis and physico-chemical properties of aqueous dilute solutions

This paper reports on the synthesis and the physico-chemical characterisation of various amphiphilic derivatives of two natural polysaccharides, sodium alginate and sodium hyaluronate, in which a rather small proportion of the carboxylic groups (≤10% mol) was esterified by long alkyl chains (C₁₂H₂₅ or C₁₈H₃₇).

The derivatives thus prepared were characterised by gas chromatography, ¹H and ¹³C n.m.r. spectroscopy and size exclusion chromatography coupled to a multi-angle laser light scattering detection. The tendency of these water-soluble compounds to hydrophobic association in aqueous solutions was evidenced firstly in dilute regime using capillary viscometry as well as fluorescence spectroscopy in the presence of a molecular probe, 1,1-dicyano-(4′-N,N-dimethylaminophenyl)-1,3-butadiene.     

Measurements of reducing end groups on bovine vitreous-humour hyaluronic acid by reaction with [14C]cyanide.

Bovine vitreous-humour sodium hyaluronate was purified by precipitation with cetylpyridinium chloride, CsCl-density-gradient sedimentation and gel-permeation chromatography. The number of reducing end groups in two similarly prepared hyaluronate samples was determined by reaction with K14CN, and measurements of intrinsic viscosity were performed to determine whether this reaction caused degradation of the hyaluronate. The intrinsic viscosity of one hyaluronate sample was 192ml/g, compared with a value of 187ml/g after reaction with [14C]cyanide, which indicates that the labelling reaction did not cause depolymerization of the hyaluronate. The Mr calculated from these viscosity values is approx. 60000. Fractionation of the [14C]cyanide-labelled hyaluronate by gel chromatography showed that it was composed of a polydisperse population of molecules with calculated chain lengths, based on the ratio of [14C]cyanide to uronic acid, ranging in molecular weight from 9000 to 264000, with an average Mr of 63200. On the basis of these measurements it is concluded that reaction with [14C]cyanide does not cause degradation of bovine vitreous-humour hyaluronate polysaccharide chains and that reaction with [14C]cyanide can be used to determine the molecular weight of this hyaluronate.     

Salt effects on the cross-linking mechanism of cupric-induced sol—gel transition in alginate solutions

The viscosity behaviour of alginate-Cu²⁺-NaCl systems has been experimentally examined at various concentrations of cupric and sodium salts. Dependence of the intrinsic viscosity of alginate as a function of NaCl concentration is discussed to supplement the previous study which shows a similar behaviour to that found for other polyelectrolytes in aqueous solution in the presence of an added salt. The effects of sodium ions on the cupric association in cupric-induced alginate solutions were investigated by means of viscosity measurements. The mechanisms of complex formation in the presence of the simple added salt were studied. It was found that, at a given NaCl concentration, the viscosity of the mixture will pass through a maximum with increasing cupric concentration. The amounts of cupric cations corresponding to the maximum depends on the concentration of NaCl in the solution. Comparison of salt effects on the viscosity behaviour of alginate solutions during sol—gel transition reveals that an optimum NaCl concentration of 10⁻² mol 1⁻¹ exists where the viscosity of the mixture gives a maximum value at a certain cupric amount. This result indicates that salt effects play an important role in the sol—gel transition of the polyelectrolyte solutions. The observed phenomenon was interpreted in terms of conformational change of polyelectrolyte chain due to the addition of salt resulting in a different cross-linking mode in the system.     

Influence of oligoguluronates on alginate gelation, kinetics, and polymer organization

Structural polysaccharides of the alginate family form gels in aqueous Ca2+-containing solutions by lateral association of chain segments. The effect of adding oligomers of alpha-l-guluronic acid (G blocks) to gelling solutions of alginate was investigated using rheology and atomic force microscopy (AFM). Ca-alginate gels were prepared by in situ release of Ca2+. The gel strength increased with increasing level of calcium saturation of the alginate and decreased with increasing amount of free G blocks. The presence of free G blocks also led to an increased gelation time. The gel point and fractal dimensionalities of the gels were determined based on the rheological characterization. Without added free G blocks the fractal dimension of the gels increased from df = 2.14 to df = 2.46 when increasing [Ca2+] from 10 to 20 mM. This increase was suggested to arise from an increased junction zone multiplicity induced by the increased concentration of calcium ions. In the presence of free G blocks (G block/alginate = 1/1) the fractal dimension increased from 2.14 to 2.29 at 10 mM Ca2+, whereas there was no significant change associated with addition of G blocks at 20 mM Ca2+. These observations indicate that free G blocks are involved in calcium-mediated bonds formed between guluronic acid sequences within the polymeric alginates. Thus, the added oligoguluronate competes with the alginate chains for the calcium ions. The gels and pregel situations close to the gel point were also studied using AFM. The AFM topographs indicated that in situations of low calcium saturation microgels a few hundred nanometers in diameter develop in solution. In situations of higher calcium saturation lateral association of a number of alginate chains are occurring, giving ordered fiber-like structures. These results show that G blocks can be used as modulators of gelation kinetics as well as local network structure formation and equilibrium properties in alginate gels.     

Extracellular Poly(α-L-guluronate)lyase from Corynebacterium sp.: Purification, Characteristics, and Conformational Properties

Extracellular alginate lyase was purified from the culture supernatant of Corynebacterium sp. isolated from the sewage of a sea tangle processing factory in order to elucidate the structure—function relationship of alginate lyase. The electrophoretically homogeneous enzyme was shown to have a molecular mass of 27 kDa by sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis (PAGE) and by gel filtration, with an isoelectric point of 7.3. The molecular mass from amino acid analysis was 28.644 kDa. The optimal pH and temperature for the enzyme reaction were around 7.0 and 55°C, respectively. Metal compounds such as MnCl₂ and NiCl₂ increased the enzyme activity. The enzyme was identified as the endolytic poly(α-L-guluronate)lyase, which was active on poly(α-L-1,4-guluronate) and caused a rapid decrease in the viscosity of alginate solution. Measurement of the far-UV circular dichroic spectrum of the enzyme molecule gave a spectrum with a deep trough at 215nm accompanied by a shallow one at around 237 nm, and with a high peak at 197 nm and a much lower one at 230 nm. This spectrum was most likely to be that of the β-form of the enzyme molecule and resembled poly(β-D-mannuronate)lyase from Turbo cornutus (wreath shell) and poly(α-L-guluronate)lyase from Vibrio sp. (marine bacterium). The near-UV circular dichroic spectrum was characteristic for aromatic amino acid residues. In the presence of 6 M urea, these spectra changed drastically in the near-UV and a little in the far-UV with the disappearance of the enzyme activity. Removal of the denaturant in the enzyme solution by dialysis restored both the activity and inherent circular dichroic spectra. The β-sheets observed in alginate lyases as the major ordered structure seem to be a common conformation for the lyases.     

Viscosity study of interactions between sodium alginate and CTAB in dilute solutions at different pH values

Interactions between anionic polyelectrolyte sodium alginate and the cationic surfactant cetytrimethylammonium bromide (CTAB) have been investigated by viscosity measurement techniques. The polymer–surfactant interactions are observed between alginate and CTAB at different pH in dilute solution. The results show that the rheological response of alginate dilute solutions is sensitive to a change of pH in the low pH range. The steady shear and intrinsic viscosity measurements reveal that the strong association between alginate and CTAB by electrostatic attraction above pH 5.0. However, as the pH value of solution decrease from 5.0 to 3.0, the strong association between alginate and CTAB is affected by not only electrostatic attraction but also hydrophobic interaction.     

Role of acetylation on metal induced precipitation of alginates

Acetylation dramatically effects both the solution properties and the metal induced precipitation of alginates. The presence of acetyl groups on both bacterial and seaweed alginate polymers marginally increased the weight average molecular weight (M_w) of each polymer by 7% and 11%, respectively. Acetylated bacterial alginate showed a significant increase in solution viscosity compared to its deacetylated counterpart. However microbial acetylation of seaweed alginate did not change its solution viscosity. Acetylation altered the calcium induced precipitation of both alginates. The presence of acetyl groups decreased the ability of each polymer to bind with calcium but increased their ability to bind with ferric Ion (Fe³⁺). By controlling the degree of acetylation on the alginate chains, it was possible to modify solution viscosity and cation induced precipitation of these polymers.     

Inter-Grade and Inter-Batch Variability of Sodium Alginate Used in Alginate-Based Matrix Tablets

The purpose of this study is to characterize the inter-grade and inter-batch variability of sodium alginate used in the formulation of matrix tablets. Four different grades and three batches of one grade of sodium alginate were used to prepare matrix tablets. Swelling, erosion, and drug release tests of sodium alginate matrix tablets were conducted in a USP dissolution apparatus. Substantial differences in swelling and erosion behavior of sodium alginate matrix tablets were evident among different viscosity grades. Even different batches of the same grade exhibit substantial differences in the swelling and erosion behavior of their matrix tablets. The erosion behavior of sodium alginate matrix tablets can be partly explained by their rheological properties (both apparent viscosity and viscoelasticity) in solution. Sodium alginate with higher apparent viscosity and viscoelasticity in solution show slower erosion rate and higher swelling rate. Compacts prepared from grades or batches with higher viscosity and higher viscoelasticity show slower drug release. For grades or batches with similar apparent viscosities, apparent viscosities of sodium alginate solution at low concentration alone are not sufficient to predict the functionality of sodium alginate in matrix tablets. Viscoelastic properties of sodium alginate solutions at one high concentration corresponding to the polymer gel state, may be suitable indicia of the extended release behavior of sodium alginate matrix tablets.     

Effect of dietary chitosans with different viscosity on plasma lipids and lipid peroxidation in rats fed on a diet enriched with cholesterol

To investigate the effect of dietary chitosan on lipid metabolism, male SD (Sprague-Dawley) rats were fed a cholesterol-enriched diet containing 5% cellulose (CE), 5% chitosan (CCS; high viscosity), or 5% chitosan (FCS; low viscosity) for 4 weeks. The two types of chitosan with a comparable degree of deacetylation had a different molecular weight and intrinsic viscosity. Significantly (p < 0.05) lower plasma total cholesterol, LDL-cholesterol and VLDL-cholesterol concentrations were observed in the rats fed on the chitosan diets. In addition, chitosan significantly increased the fecal cholesterol and triglyceride contents. Although no significant difference in body weight was found among the dietary groups, the rats fed on the chitosan diets had lower relative liver weight when compared with those fed on the cellulose diet. Both of the chitosan groups had significantly lower liver total lipid and total cholesterol contents compared to the cellulose group, although the FCS group was less effective. The plasma and liver thiobarbituric acid reactive substances (TBAR) values were similar in the CE and FCS groups, while the CCS group had increased liver TBAR values. Although a significant increase in liver glucose-6-phosphate dehydrogenase activity was observed in the CCS group, no significant change was found in the FCS group. The observed influence of chitosans with different viscosity on the plasma lipid level, liver lipids and lipid peroxidation suggests that, while the hypocholesterolemic action of chitosans with different viscosity was similar, changes in the liver lipids and liver peroxidation status depended on their molecular weight when the deacetylation degree was comparable.     

Interactions of cartilage proteoglycans with hyaluronate. The role of the hyaluronate acetamido groups.

Hyaluronate oligomers were treated with anhydrous hydrazine in the presence of hydrazine sulfate to remove the N-acetyl groups. Complete deacetylation could not be achieved without extensive degradation of the oligosaccharide chain. Partially deacetylated oligomers exhibited decreased inhibition of cartilage proteoglycan-hyaluronate interaction as compared to the unreacted starting material; re-N-acetylation by reaction with acetic anhydride restored the inhibitory activity to a great extent. When the hydrazine-treated oligosaccharides were reacted with other acyl anhydrides, the inhibitory potency was restored to an extent which was inversely related to the size of the acyl group. Thus, for maximal interaction between hyaluronate and proteoglycan, the glucosamine residue of hyaluronate must be N-acylated with a minimally sized acyl group.     

Alginate Gel; An Embedding Medium for Facilitating the Cutting and Handling of Frozen Sections

Small pieces of formalin-fixed tissue are infiltrated first with a 1% and then with a 2% solution of a low viscosity sodium alginate (a salt of a polymannuronic acid obtained from seaweed). This tissue is then transferred to a solution of a high molecular weight sodium alginate containing colloidally dispersed tricalcium phosphate. When a freshly prepared solution of gluconolactone is added, a calcium alginate gel is gradually formed—the lactone slowly hydrolyses to produce the free acid which liberates calcium ions from the colloidal phosphate. A block of gel containing the tissue is then cut out. If desired, it can be further hardened in a buffered calcium acetate solution and its cutting properties improved by soaking in 20% alcohol. At room temperature, enzymes such as the cholinesterases and phosphatases are not affected, but the procedure can be carried out at 0° C if desired. The gel does not crack and makes possible the cutting of coherent, serial frozen sections of many tissues. The alginate preparations used were supplied by Messrs. Alginate Industries Limited, Walter- House, Bedford Street, Strand, London, W.C.2.     

Human umbilical cord hyaluronate neutral sugar content and carbohydrate-protein linkage studies

Paper chromatography of neural sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neural sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced with NaBH₄-PdCl₂, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular weight to about one-half and an appreciable decrease in the specific rotation of hyaluronate, suggesting a separation of the two antiparallel chains of the double helical hyaluronate. The umbilical cord hyaluronate contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.     

Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 x 10(8) purified phage per ml for 24 h at 37 degrees C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10(10) and 10(12) phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

Dose-dependent suppression of hunger by a specific alginate in a low-viscosity drink formulation

Addition of specific types of alginates to drinks can enhance postmeal suppression of hunger, by forming strong gastric gels in the presence of calcium. However, some recent studies have not demonstrated an effect of alginate/calcium on appetite, perhaps because the selected alginates do not produce sufficiently strong gels or because the alginates were not sufficiently hydrated when consumed. Therefore, the objective of the study was to test effects on appetite of a strongly gelling and fully hydrated alginate in an acceptable, low-viscosity drink formulation. In a balanced order crossover design, 23 volunteers consumed a meal replacement drink containing protein and calcium and either 0 (control), 0.6, or 0.8% of a specific high-guluronate alginate. Appetite (six self-report scales) was measured for 5 h postconsumption. Relevant physicochemical properties of the drinks were measured, i.e., product viscosity and strength of gel formed under simulated gastric conditions. Hunger was robustly reduced (20-30% lower area under the curve) with 0.8% alginate (P < 0.001, analysis of covariance), an effect consistent across all appetite scales. Most effects were also significant with 0.6% alginate, and a clear dose-response observed. Gastric gel strength was 1.8 and 3.8 N for the 0.6 and 0.8% alginate drinks, respectively, while product viscosity was acceptable (<0.5 Pa.s at 10 s(-1)). We conclude that strongly gastric-gelling alginates at relatively low concentrations in a low-viscosity drink formulation produced a robust reduction in hunger responses. This and other related studies indicate that the specific alginate source and product matrix critically impacts upon apparent efficacy.     

Structure-activity relationship between carboxylic acids and T cell cycle blockade

This study was designed to examine the potential structure-activity relationship between carboxylic acids, histone acetylation and T cell cycle blockade. Toward this goal a series of structural homologues of the short-chain carboxylic acid n-butyrate were studied for their ability to block the IL-2-stimulated proliferation of cloned CD4+ T cells. The carboxylic acids were also tested for their ability to inhibit histone deacetylation. In addition, Western blotting was used to examine the relative capacity of the carboxlic acids to upregulate the cyclin kinase-dependent inhibitor p21cip1 in T cells. As shown earlier n-butyrate effectively inhibited histone deacetylation. The increased acetylation induced by n-butyrate was associated with the upregulation of the cyclin-dependent kinase inhibitor p21cip1 and the cell cycle blockade of CD4+ T cells. Of the other carboxylic acids studied, the short chain acids, C3-C5, without branching were the best inhibitors of histone deacetylase. This inhibition correlated with increased expression of the cell cycle blocker p21cip1, and the associated suppression of CD4+ T cell proliferation. The branched-chain carboxylic acids tested were ineffective in all the assays. These results underline the relationship between the ability of a carboxylic acid to inhibit histone deacetylation, and their ability to block T cell proliferation, and suggests that branching inhibits these effects.