Olav Smidsrød

Alginate as immobilization matrix for cells

In recent years, entrapment of cells within spheres of Ca2+ alginate has become the most widely used technique for immobilizing living cells. This versatile method includes applications ranging from immobilization of living or dead cells in bioreactors, immobilization of plant protoplasts for micropropagation and immobilization of hybridoma cells for production of monoclonal antibodies, to entrapment of animal cells for implantation of artificial organs. This review evaluates the potential of this method on the basis of the current knowledge of structural and functional relationships in alginate gels.

Alginate based new materials

Present and future applications of alginates are mainly linked to the most striking feature of the alginate molecule; i.e. a sol/gel transition in the presence of multivalent cations, e.g. Ca2+, almost independent on temperature. These very mild conditions, combined with the fact that alginates are highly characterised and understood both in the liquid and in the gel phase, makes this biopolymer unique compared to other gelling polysaccharides. Only pectins resemble alginate in the sol/gel transition behaviour, but this system can hardly be said to be as well characterised and understood as the alginates. The properties of alginate solutions and gels suggest biomedical and pharmaceutical uses. In this paper, the question of the specifications required by a polymer for applications in some biomedical areas will be discussed.

Alginate polycation microcapsules: I. Interaction between alginate and polycation

The interactions between alginate and polycations have been studied by using different labelling techniques. Binding of poly-L-lysine (PLL) to alginate in the gel state is mainly governed by the amount of dissociable negative charges on the bead surface. PLL was found to bind more rapidly to gel beads made from alginate with a high content of mannuronic acid. The binding was enhanced by increasing the alginate concentration on the surface by making inhomogeneous beads. When the capsules were stored in the presence of cations with high affinity for alginate (Ca2+, Sr2+), PLL was washed off. Less PLL is bound to strontium alginate than to calcium alginate beads. Two mechanisms appear to be responsible for the binding of sodium alginate to alginate PLL capsules (coating): (i) an electrostatic interaction between the soluble coating material and excess positive charges on PLL on the surface; (ii) the formation of a calcium alginate gel on the surface owing to leaching of calcium ions from the core. The stability and efficiency of the coating as a function of molecular size and sequential structure of the coating polymer have also been investigated.

A p.m.r. study of the composition and sequence of uronate residues in alginates

Abstract The sequence and composition of uronate residues in intact alginate samples have been obtained by high-resolution 1H-n.m.r. spectroscopy. The viscosity problem was overcome by a slight, controlled depolymerization of the alginate samples before the 1H-n.m.r. spectra were recorded at 90°. The mannuronate (M)/guluronate (G) molar ratio was obtained from the intensities of the signals for the anomeric protons. A sequence-dependent deshielding of H-5 of the guluronate residues made it possible to determine the fractions of the four possible doublets of nearest neighbours along the chain. The results were wholly consistent with 13C-n.m.r. data. Significant deviation from comparable results obtained by chemical analysis appeared only for samples containing a large fraction of the mixed doublets.

Uronic acid sequence in alginate from different sources

Abstract Alginate may be considered as a block co-polymer of D -mannuronic and L -guluronic acids, and consists of three types of blocks: homopolymeric blocks of mannuronic acid (MM) and of guluronic acid (GG), and blocks with an alternating sequence (MG). The block composition of alginates has been characterized by a simple chemical method involving partial hydrolysis with acid, followed by fractional precipitation of the acid-resistant part of the alginate. Alginates from eleven different species of brown algae have been examined and, for five species, alginates from different tissues have been compared. The results indicate that young tissue is rich in MM blocks, and that the difference between the alginates from different species is mainly due to the alginates from the older parts of the plants. Extracellular alginates from two types of bacteria have been examined.

Determination of the degree of N-acetylation and the distribution of N-acetyl groups in partially N-deacetylated chitins (chitosans) by high-field n.m.r. spectroscopy☆

The composition and sequence of 2-acetamido-2-deoxy-beta-D-glucose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucose (GlcN) residues in partially N-deacetylated chitosans, prepared under homogeneous and heterogeneous conditions, have been determined by 1H-n.m.r. spectroscopy. It was necessary to depolymerise the chitosan slightly by treatment with nitrous acid before spectroscopy. A sequence-dependent deshielding of H-1 of the GlcNAc residues made it possible to determine the proportions of the four possible diads. Chitosan prepared by N-deacetylation under homogeneous conditions gave values for the diad frequencies that were roughly consistent with a random distribution of the N-acetyl groups. Samples prepared under heterogeneous conditions have a frequency of the GlcNAc-GlcNAc diad slightly higher than for a random (Bernoullian) distribution. The chitosans, prepared under both homogeneous and heterogeneous conditions, with a degree of acetylation of 50% were soluble at neutral pH.

Microcapsules of alginate-chitosan – I: A quantitative study of the interaction between alginate and chitosan

The binding of chitosan to alginate beads was studied quantitatively by using radioactive labelled fractions of chitosan. The alginate-chitosan capsules were made either by dropping a solution of sodium alginate into a solution containing chitosan or by incubating calcium alginate beads in a solution of chitosan. The first procedure yielded a binding of 0.015 microg chitosan per mm2 of capsule surface, while the latter procedure yielded over 2 microg mm(-2). The maximum obtained weight ratio of chitosan to alginate in a microcapsule after 24 h was 0.40. The binding of chitosan was markedly increased by reducing the number average molecular weight of chitosan below 20000 Da and by increasing the porosity of the alginate gel. The porosity was increased by producing homogeneous gels, and by adding calcium chloride to the chitosan solution during the membrane forming stage. The effect of calcium ions on the porosity of the gel was studied by experiments involving release of blue dextran from calcium alginate beads. The binding of chitosan was also found to increase with decreasing fraction of N-acetylations, FA, on chitosan in the range of FA = 0.3 to FA = 0, and with increasing pH in the range from pH 4 to 6. Capsules with a diameter of 500 microm had a higher weight ratio of chitosan to alginate after 24 h of binding than the capsules with the larger diameter of 1500 microm.

Molecular basis for some physical properties of alginates in the gel state

Alginate is a binary linear heteropolymer containing 1,4-linked β-D-mannuronic and α-L-guluronic acid residues in varying proportions. The monomers are arranged in a blockwise fashion along the chain; it contains the two homopolymeric blocks (MM and GG) together with blocks of the alternating sequence (MG) in the same molecule. Data from light scattering and viscometric experiments show that their relative unperturbed dimensions increase in the order: MG-blocks < MM-blocks < GG-blocks. Ion exchange data show that GG-blocks are characterized by a selective binding of calcium ions in solution, a strong autocooperative binding of calcium between the chains in the gel state, and hystrisis due to a slow dissociation of the cooperatively-formed functions between the chains. The two other blocks have much lower selectivity for calcium ions, no autocooperative binding mechanism and no detectable hysteresis. The mechanical strength of calcium alginate gels is mainly due to junctions formed by the GG-blocks. the modulus of rigidity of gels formed by different cations is directly dependent on their ability to bind to the polyuronides by a cooperative inter-chain binding mechanism.

INDUCTION OF CYTOKINE PRODUCTION FROM HUMAN MONOCYTES STIMULATED WITH ALGINATE

Summary Alginates are polysaccharides with gel-forming properties composed of 1,4-linked β-D-mannuronic acid (M), α-L-guluronic acid (G), and alternating (MG) blocks. Alginate can be used as a matrix for implanted cells in vivo. In this study, we have examined the ability of alginates and their components to stimulate human monocytes to produce tumor necrosis factor-α, interleukin-6, and interleukin-1. Alginates stimulated the monocytes to produce high levels of all three cytokines. Low G alginates were approximately 10 times more potent in inducing cytokine production compared with high G alginates. The M-blocks and the MG-blocks, but not the G-blocks, stimulated the cytokine production. The results demonstrate that the mannuronic acid residues are the active cytokine inducers in alginates.

Inhomogeneous polysaccharide ionic gels

Abstract Polysaccharide ionotropic gels formed by diffusion of calcium ions into solutions of sodium alginate or pectate exhibited various degrees of inhomogeneity, in the sense that the polymer concentration was much higher at the surface than in the center of the gels. This was in contrast to the uniform gels obtained when κ-carrageenan was dialyzed against potassium ions and gellan gum against potassium and lead ions. The non-uniform distribution of polymer in alginate and pectate depended on parameters such as polymer concentration and molecular size, and the concentration of the gel inducing ion in the outer reservoir. For alginate a high fraction of l -guluronate residues slightly increased the inhomogeneity, whereas the presence of non-gelling cations strongly decreased it. A theory, limited to diffusion of reactants in infinite long tubes, compared with relevant experiments, suggested that the gelation was controlled by the relative rate of diffusion of polymer and gelling cations and that the gelling in the absence of non-gelling cations was irreversible and stoichiometric. A new simple technique for generating homogeneous calcium alginate gels by diffusion was also demonstrated.