Oxidized alginate-cross-linked alginate/gelatin hydrogel fibers for fabricating tubular constructs with layered smooth muscle cells and endothelial cells in collagen gels

Hydrogel fibers that possessed a cell-adhesive surface and were degradable via enzymatic reactions were developed for fabricating tubular constructs with smooth muscle cell (SMC) and endothelial cell (EC) layers, similar to native blood vessels, in collagen gels. The fibers were prepared by soaking hydrogel fibers prepared from a solution of sodium alginate and gelatin containing bovine ECs (BECs) in medium containing oxidized alginate (AO). BECs soaked in 8.0% (w/v) AO showed no reduction in viability within 3 h of soaking. Furthermore, mouse SMCs (MSMCs) adhered and proliferated on the AO-cross-linked hydrogels. Based on these results, we prepared AO-cross-linked hydrogel fibers containing BECs, covered their surface with MSMCs, and embedded them in collagen gels. We then degraded the fibers using alginate lyase to obtain channels in the collagen gels. Histological analysis of the released ECs using a specific fluorescent dye revealed the formation of tubular structures with layered BECs and MSMCs.     

Optical anisotropy of calcium-induced alginate gels

Alginate gels formed by diffusion of calcium ions into solutions of sodium alginate were found to exhibit optical anisotropy depending on preparation conditions. When observed under crossed nicols, the anisotropic alginate gels showed a birefringence pattern which is characteristic of radial orientation of polymer chains. Calcium alginate gels were prepared from different concentrations of sodium alginate and calcium ion, and the conditions for formation of the anisotropic gels were determined. The gel-formation process was measured by monitoring the development of the birefringent layer and was compared with the model in which the diffusion of calcium ions dominates gel formation.     

Conjugative plasmid transfer between Pseudomonas strains within alginate bead microcosms: effect of the internal gel structure.

Because microorganisms frequently live in an immobilized state in natural habitats, a cell-confined system was used to study bacterial conjugation. Two Pseudomonas putida strains were introduced together within calcium alginate gels. Different alginate beads were designed by varying the polysaccharide and the gelation solution concentrations. Microscopic examinations showed that 2% gels were quite homogeneous, but that 1.5% and 1% gels were rather heterogeneous. In these two last cases, shaft-shaped macrostructures were present. They were colonized during the culture by great densities of highly motile bacteria. Gene transfers due to conjugation were investigated in such alginate gel bead microcosms, in batch and continuous cultures. High-initial transfer frequencies were detected whatever the gel, but no conjugation events seemed to occur with further growth in the beads. Transfer frequency values were roughly similar in the different tested systems. Alginate gels used as artificial microcosms may be valuable to study the effect of cell microenvironment on genetic transfers in complex systems.     

Growth and viability of mycelial fragments of white-rot fungi on some hydrogels

The viability of mycelial fragments of Trametes versicolor and Irpex lacteus and their growth on selected hydrogels are described. The size of mycelial fragments of the fungi did not significantly influence their viability. Alginate hydrogel films supported fungal growth better than agarose, carrageenan, chitosan and gelatin films, and had the highest mechanical strength but were less hydrophilic than the other hydrogels. All commercial alginates that were tested supported aseptic growth of fungal fragments without prior sterilization of the hydrogel solution. The viability of mycelial fragments in the hydrogel solutions was higher for some commercial alginates than that in laboratory grade alginate. The mechanical strength and hydrophilicity of hydrogels from alginate type Sobalg FD 155 and Meer HV were comparable to that of laboratory grade alginate. Sterilization and pH of the alginate hydrogel did not significantly influence the growth of T. versicolor mycelial fragments but affected the growth of I. lacteus. Concentrations of alginate in the range of 1–2% in the hydrogel did not affect the growth of entrapped mycelial fragments of these fungi. Received 25 June 1997/ Accepted in revised form 07 March 1998     

Controlling rigidity and degradation of alginate hydrogels via molecular weight distribution

The mechanical rigidity and degradation rate of hydrogels utilized as cell transplantation vehicles have been regarded as critical factors in new tissue formation. However, conventional approaches to accelerate the degradation rate of gels deteriorate their function as a mechanical support in parallel. We hypothesized that adjusting the molecular weight distribution of polymers that are hydrolytically labile but capable of forming gels would allow one to alter the degradation rate of the gels over a broad range, while limiting the range of their elastic moduli (E). We investigated this hypothesis with binary alginate hydrogels formed from both ionically and covalently cross-linked partially oxidized (1% uronic acid residues), low [molecular weight (MW) approximately 60,000 g/mol] and high MW alginates (MW approximately 120,000 g/mol) in order to examine the utility of this approach with various cross-linking strategies. Increasing the fraction of low MW alginates to 0.50 maintained a value of E similar to that for the high MW alginate gels but led to faster degradation, irrespective of the cross-linking mode. This result was attributed to a faster separation between cross-linked domains upon chain breakages for the low MW alginates, coupled with their faster chain scission than the high MW alginates. The more rapidly degrading oxidized binary hydrogels facilitated the formation of new bone tissues from transplanted bone marrow stromal cells, as compared with the nonoxidized high MW hydrogels. The results of these studies will be useful for controlling the physical properties of a broad array of hydrogel-forming polymers.     

Rheology and nanostructure of hydrophobically modified alginate (HMA) gels and solutions

The effect of hydrophobic modification on the mechanical and structural characteristics of hydrophobically modified alginate (HMA) solutions and hydrogels were evaluated. The HMA systems consisted of alkyl chains, C₈, grafted onto alginate backbones. With an increase in degree of substitution of hydrophobic tails, the association became stronger in solution, but same was not true for gels. The contribution of ionic crosslinking was found to be the dominant factor in determining the mechanical strength of hydrogels. Rheological measurements of 2 wt% HMA gels reveal formation of a strongly crosslinked network with an elastic modulus close to 100 kPa. Small-angle X-ray scattering (SAXS) experiments indicate that HMA assembles into a disordered structure with regions rich in the hydrophobic domain surrounded by a crosslinked hydrophilic network.     

Formulation of organic and inorganic hydrogel matrices for immobilization of β‐glucosidase in microfluidic platform

The aim of this study was to formulate silica and alginate hydrogels for immobilization of β‐glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate–polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β‐glucosidase was loaded into glass–silicon–glass microreactors and catalysis of 4‐nitrophenyl β‐d ‐glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.     

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.     

Cellular cross-linking of peptide modified hydrogels

Peptide modification of hydrogel-forming materials is being widely explored as a means to regulate the phenotype of cells immobilized within the gels. Alternatively, we hypothesized that the adhesive interactions between cells and peptides coupled to the gel-forming materials would also enhance the overall mechanical properties of the gels. To test this hypothesis, alginate polymers were modified with RGDSP-containing peptides and the resultant polymer was used to encapsulate C2C12 myoblasts. The mechanical properties of these gels were then assessed as a function of both peptide and cell density using compression and tensile tests. Overall, it was found that above a critical peptide and cell density, encapsulated myoblasts were able to provide additional mechanical integrity to hydrogels composed of peptide-modified alginate. This occurred presumably by means of cell-peptide cross-linking of the alginate polymers, in addition to the usual Ca++ cross-linking. These results are potentially applicable to other polymer systems and important for a range of tissue engineering applications.     

An anomalous behavior of trypsin immobilized in alginate network

Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N _α-benzoyl-l-arginine-4-nitroanilide hydrochloride (l-BAPNA), and in the presence or absence of d-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 °C. It was found that at 37 and 42 °C a strong acceleration of the reaction was obtained, whereas at 10 °C and at room temperature, the presence of d-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with d-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus l-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.     

Alginate production by an <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> mutant unable to produce alginate lyase

Alginate is an industrially relevant linear copolymer composed of beta-1,4-linked D-mannuronic acid and its C-5 epimer L-guluronic acid. The rheological and gel-forming properties of alginates depend on the molecular weight and the relative content of the two monomers. Alginate produced by Azotobacter vinelandii was shown to be degraded towards the end of the culture, an undesirable situation in terms of potential alginate applications. A gene ( algL) encoding the alginate lyase activity AlgL is present within the alginate biosynthetic gene cluster of A. vinelandii. We constructed strain SML2, an A. vinelandii strain carrying a non-polar mutation within algL. No alginate lyase activity was detected in SML2. Under 3% dissolved oxygen tension, higher values of maximum mean molecular weight alginate were obtained (1240 kDa) with strain SML2, compared to those from the parental strain ATCC 9046 (680 kDa). These data indicate that AlgL activity causes the drop in the molecular weight of alginate produced by A. vinelandii.     

Adsorbents for Apheresis Prepared from Polysaccharides of Algae that Threaten Ecosystem Services

In this work, we investigated whether materials isolated from algae that threaten ecosystems can be used for human benefit. We converted acidic polysaccharides (ulvan) from the alga Ulva pertusa into soft hydrogel materials. In addition to ulvan, the hydrogels also contained alginate in a polyion complex with chitosan. Cross‐linking the hydrogel with glutaraldehyde reduced polysaccharide elution from the polyion complex gel. We also found that both ulvan? chitosan and alginate? chitosan gels were able to remove urea and heavy metals from aqueous solution. This is clinically significant, since during apheresis, toxic compounds such as urea have to be removed from the bloodstream of patients. Importantly, albumin was not removed by the hydrogels, implying that this vital protein can be returned to the bloodstream following dialysis.     

Using light scattering measurements to study the effects of monovalent and divalent cations on alginate aggregates

Light scattering measurements were used to assess the effectsof selected divalent and monovalent cations on alginate aggregationin vitro. Alginate, formed with either strontium, calcium orcobalt was partially dissolved with sodium. Calcium-alginatewas also partially dissolved with two other monovalent cations,lithium and potassium. Phosphate, when added to a solution containingcalcium-alginate, scrubbed algin-ate-bound calcium as well asfree calcium in solution. These findings provide an explanationfor an alternative approach for breaking down cell wall alginate. Key words: Alginate aggregates, monovalent cations, divalent cations, light scattering     

Alginate lyase: Structure,property, and application

Alginate is a linear polysaccharide in which β-D-mannuronate (M) and its epimer, α-L-guluronate (G), are covalently (1–4)-linked in different sequences. Alginate is mainly used as a food additive to modify food texture due to its high viscosity and gelling property. Alginate lyase can degrade alginate by cleaving the glycosidic bond through a β-elimination reaction, generating oligomer with 4-deoxy-L-erythro-hex-4-enepyranosyluronate at the nonreducing end. Alginate oligosaccharides have been shown to stimulate the growth of human endothelial cells and the secretion of cytotoxic cytokines from human macrophage. Alginate can be converted into unsaturated monosaccharide by saccharification process using endolytic and exolytic alginate lyases, thus alginate lyases have potential as key biocatalyst for application of alginate as a renewable source for biochemicals and biofuels in near future. In this paper, structures and functions of various alginate lyases are reviewed. Prospects on future applications of alginate lyases are also discussed.     

Photodegradable Iron(III) Cross-Linked Alginate Gels

Biocompatible photoresponsive materials are of interest for targeted drug delivery, tissue engineering, 2D and 3D protein patterning, and other biomedical applications. We prepared light degradable hydrogels using a natural alginate polysaccharide cross-linked with iron(III) cations. The "hard" iron(III) cations used to cross-link the alginate hydrogel were found to undergo facile photoreduction to "soft" iron(II) cations in the presence of millimolar concentrations of sodium lactate. The "soft" iron(II) cations have a decreased ability to cross-link the alginate which results in dissolution of the hydrogel and the formation of a homogeneous solution. The photodegradation is done using long wave UV or visible light at neutral pH. The very mild conditions required for the photodegradation and the high rate at which it occurs suggest applications for iron(III) cross-linked alginate hydrogels as light-controlled biocompatible scaffolds.     

Stability of hydrogels used in cell encapsulation: An in vitro comparison of alginate and agarose

The present studies were undertaken to evaluate the in vitro gel stability of the hydrogels alginate and agarose. Gel strength (of alginate and agarose) and protein diffusion (of alginate only) were shown to correlate with gel stability and to be useful techniques to monitor gel stability over time. The gel strengths of alginate and agarose were followed for a 90-day period using gel strength as a measure of gel stability. The gel strength of agarose diminished in the presence of cells because the cells likely interfered with the hydrogen bond formation required for agarose gelation. In the presence of cells, the gel strength of agarose decreased by an average of 25% from time 0 to 60 days, thereafter maintaining that value to 90 days. The gel strength of calcium- or barium-crosslinked alginate decreased over 90 days, with an equilibrium gel strength being achieved after 30 days. The presence of cells did not further decrease alginate gel strength. The gel strengths of calcium- and barium-crosslinked alginates were similar at 60 days-350 +/- 20 g and 300 +/- 60 g, respectively-indicating equivalence in their stability. The stability of calcium-crosslinked sodium alginate gels over a 60-day time period was monitored by diffusion of proteins ranging in molecular weight from 14.5 to 155 kD. From these diffusion measurements, the average pore size of the calcium-crosslinked alginate gels was estimated, using a semi-empirical model, to increase from approximately 176 to 289 A over a period of 60 days. (c) 1996 John Wiley & Sons, Inc.     

Influence of the composition and preparation method on the morphology and swelling behavior of alginate–chitosan hydrogels

In this study, a 2⁴ factorial experimental design was employed in order to evaluate the influence of the reaction conditions and preparation method on alginate–chitosan hydrogel properties. Alginate content, pH, chitosan molecular weight and the hydrogel preparation method were the independent variables and the reaction yield, particle size, swelling degree and point of zero surface charge were the dependent variables. The results showed that hydrogels were spherical with an average diameter of 5.0 ± 2.0 μm. Reaction yield varied according to the parameters, and chitosan molecular weight showed the greatest influence. Furthermore, the swelling degree and point of zero surface charge showed a linear dependence on the alginate content. In this regard, the study showed that hydrogels with a specific charge and swelling degree can be obtained by controlling the alginate content using the equation here provided to give an enhanced and site-specific controlled drug release.     

Studies on immobilization of the thermophilic bacterium Thermus aquaticus YT-1 by entrapment in various matrices

Summary Immobilization of the thermophilic bacterium Thermus aquaticus YT-1 has been studied using various entrapment techniques. Alginate, -carrageenan, agar, agarose and polyacrylamide were tested as supports during operation at 65°C at which the cells are known to produce protease when grown free in solution. Alginate showed toxic effects and no viability was observed after entrapment in Ca alginate or even after exposure of free living cells to sodium alginate. Polyacrylamide was observed to be the best support. Protease activity was closely related to the appearance of free cells in the medium.     

Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: application in lipase-catalysed reactions.

Alginate was evaluated as an immobilization matrix for enzyme-catalyzed reactions in organic solvents. In contrast to most hydrogels, calcium alginate was found to be stable in a range of organic solvents and to retain the enzyme inside the gel matrix. In hydrophobic solvents, the alginate gel (greater than 95% water) thus provided a stable, two-phase liquid system. The lipase from Candida cylindracea, after immobilization in alginate beads, catalysed esterification and transesterification in n-hexane under both batch and continuous-flow conditions. The operational stability of the lipase was markedly enhanced by alginate entrapment. In the esterification of butanoic acid with n-butanol, better results were obtained in the typical hydrophilic calcium alginate beads than in less hydrophilic matrices. The effects of substrate concentration, matrix area, and polarity of the substrate alcohols and of the organic solvent on the esterification activity were examined. The transesterification of octyl 2-bromopropanoate with ethanol was less efficient than that of ethyl 2-bromopropanoate with octanol. By using the hydrophilic alginate gel as an immobilization matrix in combination with a mobile hydrophobic phase, a two-phase liquid system was achieved with definite advantages for a continuous, enzyme-catalysed process.