Culture of chondrocytes in alginate gel: Variations in conditions of Gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes

Summary Sodium alginate, which gels in the presence of calcium ions, is commonly used for culture of anchorage-independent cells, such as chondrocytes. Normally, the gel appears microscopically homogeneous but, depending on the conditions of gelation, it may contain a varying number of small channels that extend inward from the surface. We have examined the influence of these channels on the morphology of cultured chondrocytes entrapped in alginate beads. Growth-plate or articular chondrocytes cultured in alginate normally proliferate and form rounded cell clusters but, in alginate beads containing numerous channels, many chondrocytes become aligned and form columns similar to those in the growth plate in vivo. As the pattern of cellular growth and morphology in alginate is profoundly influenced by the presence of channels in the gel, further studies were conducted to determine what specific conditions of gelation affect their formation. The channels are especially numerous when both the alginate and the gelling solutions lack sodium ions or other monovalent cations. The channels are cavities in the gel formed by particulate blocking of the rapid diffusion of calcium ions from the gelling solution into the boundary of the calcium alginate solution, and hence they extend inward from cells at the surface of the alginate gel. An understanding of the conditions under which these channels develop makes it possible either to avoid their formation or, alternatively, to enhance the number of channels in order to encourage proliferating cells to grow in radial columns, rather than in a less organized pattern characteristic of most culture systems.     

Phase separation in calcium alginate gels

Alginates are polysaccharides consisting of beta-D-mannuronate and alpha-L-guluronate units. In the presence of bivalent cations like calcium the guluronate blocks form physically cross-linked gels. The gelation properties of alginates play an important role in the stability of extracellular polymer substances and in the food industry. When stock solutions of Ca2+ ions and alginate are mixed, the gelation starts before the Ca2+ ions are evenly distributed, which leads to non-uniform gels. In this contribution, Ca alginate gels were prepared by in situ gelation using glucono-delta-lactone and CaCO3. In this way, uniform gels could be prepared directly in the measuring cell. Below a critical concentration, highly viscous solutions were obtained, which were below the critical point of gel formation. In these solutions at low rotational speeds a Schlieren peak arose, which became smaller and steeper with increasing time until a new meniscus could be detected. This behaviour is in contrast to the peak broadening due to diffusion after a synthetic boundary was formed. Evaluation of the data leads to negative diffusion coefficients. It has been shown by others that the mutual diffusion coefficient must be negative in the spinodal region. This phenomena is known as uphill diffusion and leads to phase separation of a binary system. The formation of the gel phase in this case is therefore discussed as uphill diffusion.     

Inhomogeneous alginate gel spheres: an assessment of the polymer gradients by synchrotron radiation-induced X-ray emission, magnetic resonance microimaging, and mathematical modeling

It has been previously demonstrated that calcium alginate gels prepared by dialysis often exhibit a concentration inhomogeneity being the polymer concentration considerably lower in the center of the gel than at the edges. Inhomogeneity may be a preferred structure in microcapsules due to low porosity and higher stability so that it is interesting to evaluate the polymer gradient in spherically symmetrical small alginate beads (1.0-0.7 mm diameter) obtained in different conditions. In this paper, two complementary techniques have been used to investigate this aspect. The concentration gradient of alginate has been analyzed by measuring both the spatial distribution of calcium ions in sections of alginate gel spheres, by means of x-ray fluorescence spectroscopy, and the T2 relaxation behavior on intact gel beads using magnetic resonance microimaging. The experimentally determined gradients from three-dimensional gels provide data to reevaluate the parameter estimates in the recently reported mathematical model for alginate gel formation (A. Mikkaelsen and A. Elgsaeter, Biopolymers, 1995, Vol. 36, pp. 17-41). The model may account for the gels being less inhomogeneous when nongelling sodium or magnesium ions are added during gelation.     

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.     

Diffusion characteristics of calcium alginate gels.

The diffusivity of a protein solute (bovine serum albumin) within calcium alginate gels made from sodium alginate of different guluronic acid content was determined. It was found that protein diffusion within alginate gels, prepared to be isotropic in structure, was greatest for gels prepared from sodium alginate of low guluronic acid content as opposed to those prepared from sodium alginate of high guluronic acid content. This finding was explained in terms of the difference in flexibility of the polymer backbone of the two alginates. The greater the polymer backbone flexibility, the greater the solute diffusivity within the gel.     

An Edible-to-insects Calcium Alginate Gel as a Carrier for Entomopathogenic Nematodes

A carrier for entomopathogenic nematodes based on an edible-to-insects calcium alginate gel was developed. The alginate system was produced by external setting through an interaction between an aqueous sodium alginate mixture and calcium ions under acidic conditions. Sodium hexa-metaphosphate was used to control gel formation. Yeast extract used in the gel as a phagostimulant for Spodoptera littoralis larvae improved the insect's relative consumption rate and digestibility. The nematodes in the gel effectively controlled the larvae in a 24-h leaf bioassay, although nematode survival in the gel was ~ 50%. Gels subjected to 31% relative humidity (RH) prior to larval feeding became desiccated and were inedible to insects. However, gels at 61% RH supported larval feeding, although the water loss from the gel due to evaporation from 200-400-mg gel cubes at this humidity exceeded 50%. The gel might be a useful delivery system for nematodes against insects infesting the plant canopy in greenhouses.     

Temperature dependence of the elastic properties of alginate gels

The moduli of elasticity of calcium and lead alginate gels increase with time after preparation, and the temperature dependence of the rate of syneresis suggests an activation energy of 8?12 x 10⁴ J.mol?¹ for the formation of new junctions. At zero time, a negative temperature-dependence was found for the elastic force measured at a low degree of deformation (4%). Deformation of the gels was associated with an increase in entropy and internal energy. When the calcium ions in a preformed calcium alginate gel were exchanged for lead ions, which have a higher affinity for alginate, the modulus increased due to an enhanced increase in internal energy with deformation. Reversal of the sequence of introducing the two types of ions gave the opposite effect. The data suggest that the junctions are “weak points” in the gels, and that even small deformations can cause partial rupture.     

Studies on the characteristics of alginate gels in relation to their use in separation and immobilized applications

Studies on diffusion of NAD and hemoglobin from calcium and barium gels are reported where alginate grade, concentration, and gel dimensions were varied. These show that NAD diffusion characteristics are unaffected by alginate and ion concentrations; however, hemoglobin diffusion is affected by alginate concentration. Both hemoglobin and NAD diffusion patterns were shown to be affected by alginate gel dimensions. Studies are reported that show that polymannuronic alginate gels posses good porosity characteristics while polyguluronic alginates from gels with lower porosity, specifically with respect to high-molecular-weight compounds. These findings are discussed with the view to the use of alginate gels for immobilization, solids separation, and diffusion chromatography techniques.     

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.     

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.     

Glucose oxidase release from calcium alginate gel capsules

Diffusion of glucose oxidase within calcium alginate gel capsules has been assayed and the experimental data fitted to a simple semi-empirical power equation, which is used to analyse the solute release from polymeric devices. It was found that an increase in the concentration of sodium alginate and calcium chloride gives rise to a reduction in the enzyme leakage. This was verified when glucose oxidase (GOD) diffusion percentages were compared in capsules with thicknesses of the same order of magnitude but obtained under different experimental conditions. So, the use of sodium alginate and calcium chloride solutions of concentrations 0.5% w/v and 2.6% w/v, respectively, lead to a diffusion percentage of 25 +/- 2. This percentage was reduced to 8 +/- 3 when sodium alginate and calcium chloride concentrations were fixed at 1% w/v and 4% w/v, respectively, even though the thicknesses of the capsules were of the same order of magnitude.     

Mechanical properties of hydrocolloid gels filled with internally produced CO2 gas bubbles.

Agar (2%), alginate (1% algin), and kappa-carrageenan (1.5%) gel specimens were prepared from mother solutions that contained 0-2.5% sodium bicarbonate (agar and carrageenan) or calcium carbonate (alginate). Upon immersion in a citric acid bath (0-2%), the carbonate reacted with the diffusing acid to produce numerous carbon dioxide bubbles. The compressive strength and deformability of the gas-filled gels so produced were determined using a Universal testing machine and compared with those of pure gels and gels containing the carbonate but not subjected to the process after various immersion times. While the agar and alginate gels retained considerable mechanical integrity even after several hours, the carrageenan gels disintegrated after about 2-5 h. Under similar conditions, the number of bubbles produced in the agar gels was about twice that in the alginate gels, an observation that cannot be explained solely by stoichiometric considerations.     

Properties of spherical calcium pectate and alginate gels and their use in diffusion chromatography,solids separations and immobilization of enzymes and cells

Water-soluble acidic polysaccharides—deesterified pectins and carboxy-derivatives of starch—precipitated with calcium ions were tested as precursors of spherical calcium gels. Pectates prepared from apple or citrus pectin, similarly to alginates, are compounds forming spherical calcium gels stable in aqueous medium which have a relatively highly reproducible mass, particle size, water content, shape, mechanical strength and shearing. Both the liquid-solid partition of low- and high-molar-mass solutes and its kinetics proved to be reasonable features. Distribution of pore size in the above materials was estimated. Detailed pictures of surface and of the interior of calcium beads in the scanning electron microscope are presented. The possible use of calcium beads as enzyme carriers, as affinity matrixes and entrapment materials for diffusion chromatography, solids separations and bioindication of a specific water pollution was evaluated. Calcium alginate beads were always used as reference material.     

Diffusion coefficients of glucose and ethanol in cell-free and cell-occupied calcium alginate membranes

The diffusivities of glucose and ethanol in cell-free and cell-occupied membranes of calcium alginate were measured in a diffusion cell. The lag time analysis was used. Diffusivities decreased with increasing alginate concentration and were comparable with those in water for a 2% alginate membrane. Glucose and ethanol concentrations had no effect on the respective diffusion coefficients. The ratio of ethanol diffusivity to glucose diffusivity in 2 and 4% alginate agreed closely with the inverse ratio of the hydrodynamic raii for the two molecules in water, indicating that the hydrodynamic theory of diffusion in liquids may be applicable to diffusion in dilute alginate gels. Also, the presence of 20% dead yeast cells had no effect on the diffusivities. The data reported can be used to study reaction and diffusion in immobilized cell reactors and cell physiology under immobilized conditions.     

Formation of effective channels in alginate gel for immobilization of anchorage-dependent animal cells

Summary The conditions for formation of effective channels in alginate gels for growth of anchorage-dependent animal cells were examined. Many channels were formed in the gels by adding a low concentration solution of a high molecular weight polymer of alginate to a high concentration solution of divalent cations. It is recommended that an alginate with a high molecular weight and a low mannuronic acid/guluronic acid ratio be gelled by contact with strontium ions for the cultivation of immobilized anchorage-dependent cells because the gels produced have many channels and are mechanically strong.     

Alginate as immobilization material: III. Diffusional properties

The rate of diffusion of serum albumin (MW 6.9 x 10(4) D) out of beads of calcium alginate gels depends upon the concentration and uronic acid composition of the alginate (ManA/GulA ratio), the conditions under which the beads are produced, the pH, and the temperature. The diffusion coefficient decreases with increasing alginate concentration, and (ManA/GulA) ratio and with decreasing pH. Diffusion out of the beads, in which the alginate is uniformly distributed (homogeneous gel), is faster than out of the beads in which the alginate is concentrated at the surface (inhomogeneous gel). The temperature dependence of the diffusion coefficient follows the Arrhenius law, with an activation energy of approximately 23 kJ x mol(-1).     

Ionic and acid gel formation of epimerised alginates; the effect of AlgE4

AlgE4 is a mannuronan C5 epimerase converting homopolymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 resulted in different amounts of alternating sequences. Both ionically cross-linked alginate gels as well as alginic acid gels were prepared from the epimerised alginates. Gelling kinetics and gel equilibrium properties were recorded and compared to results obtained with the original non-epimerised alginates. An observed reduced elasticity of the alginic acid gels following epimerisation by AlgE4 seems to be explained by the generally increased acid solubility of the alternating sequences. Ionically (Ca(2+)) cross-linked gels made from epimerised alginates expressed a higher degree of syneresis compared to the native samples. An increase in the modulus of elasticity was observed in calcium saturated (diffusion set) gels whereas calcium limited, internally set alginate gels showed no change in elasticity. An increase in the sol-gel transitional rate of gels made from epimerised alginates was also observed. These results suggest an increased possibility of creating new junction zones in the epimerised alginate gel due to the increased mobility in the alginate chain segments caused by the less extended alternating sequences.     

Utilization in alginate beads for Cu(II) and Ni(II) adsorption of an exopolysaccharide produced by <Emphasis Type="Italic">Chryseomonas luteola</Emphasis> TEM05

Copper and nickel adsorption onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and the immobilized C. luteola TEM05 from aqueous solutions were studied. After that, the multi metal ions containing these ions together were prepared and partial competitive adsorptions of these mixtures were also investigated. The metal adsorption of gel beads were carried out at pH 6.0, 25 °C. The maximum adsorption capacities in Langmuir isotherm for calcium alginate, calcium alginate + EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 1.505, 1.989, 1.976, 1.937 mmol/g dry weight for Cu(II) and 0.996, 1.224, 1.078, 1.219 mol/g dry weight for Ni(II), respectively.The competitive biosorption capacities of the carrier for all metal ions were lower than single conditions.     

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.     

Degradation of partially oxidized alginate and its potential application for tissue engineering

Alginate has been widely used in a variety of biomedical applications including drug delivery and cell transplantation. However, alginate itself has a very slow degradation rate, and its gels degrade in an uncontrollable manner, releasing high molecular weight strands that may have difficulty being cleared from the body. We hypothesized that the periodate oxidation of alginate, which cleaves the carbon-carbon bond of the cis-diol group in the uronate residue and alters the chain conformation, would result in promoting the hydrolysis of alginate in aqueous solutions. Alginate, oxidized to a low extent (approximately 5%), degraded with a rate depending on the pH and temperature of the solution. This polymer was still capable of being ionically cross-linked with calcium ions to form gels, which degraded within 9 days in PBS solution. Finally, the use of these degradable alginate-derived hydrogels greatly improved cartilage-like tissue formation in vivo, as compared to alginate hydrogels.