Relevance of rheological properties of gel beads for their mechanical stability in bioreactors

The mechanical stability of biocatalyst particles in bioreactors is of crucial importance for applications of immobilized-cell technology in bioconversions. The common methods for evaluation of the strength of polymer beads (mostly force-to-fracture or tensile tests) are, however, not yet proven to be relevant for the assessment of their mechanical stability in bioreactors. Therefore, we tested fracture properties of gel materials and investigated their relevance for abrasion in bioreactors. Abrasion of gel beads was assumed to be a continuous fracturing of the bead surface. At first, three rheological properties were considered: stress at fracture; strain at fracture; and the total fracture energy. If stress at fracture is the most important property, beads having a similar fracture energy, but a smaller stress at fracture, would abrade faster in a bioreactor than beads with a larger stress at fracture; if fracture energy the determining factor, beads that require less energy to fracture would abrade faster than those having a larger fracture energy for the same fracture stress. To determine this, beads of kappa-carrageenan and agar (at two different polymer concentrations) were tested for abrasion in four identical bubble columns under the same operating conditions. Agar beads were expected to abrade faster than those of carrageenan because agar had either a lower stress at fracture or a lower fracture energy. However, no correlation between fracture properties and abrasion rate was found in any of the combinations tested. Carrageenan beads abraded faster than those of agar in all combinations. Furthermore, both the stress and strain at fracture of agar and carrageenan beads decreased during the run and those of carrageenan decreased faster, suggesting that the gels are liable to fatigue in different ways. This hypothesis was confirmed by oscillating experiments in which gel samples were subjected to repeated compressions below their fracture levels. Their resistance to compression clearly decreased with the number of oscillations. Fatigue is probably related to the development of microcracks and microfracture propagation within the material. We concluded that: (a) the use of tests based on bead rupture do not provide relevant information on the mechanical stability of gel beads to abrasion; and (b) abrasion of polymer beads is likely to be related to fatigue of the gel materials. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 517-529, 1997.     

Diffusivity of oxygen into carriers entrapping whole cells

The effective diffusivity of oxygen, D(e), in Ca-alginate and PVA-SbQ gels was measured using a two-chamber vessel with a membrane between the two chambers. The effect of cell density, C(c), on D(e) in Ca-alginate gels was studied. The effective diffusivity of oxygen decreased with increasing cell density, to C(c) = 170 kg dry cells/m(3) gel. The dependency of D(e) on cell density was discussed in terms of a random-pore model. The model correlated well with experimental data, i.e., kD(e)/D(0) = 0.86(1 - 1.47 x 10(-3) C(c))(2). Here, k is the partition coefficient, and D(0) is diffusivity in water.     

TEXTURE OF SWEET ORANGE GELS BY FREE-CHOICE PROFILING

Texture of orange gels prepared with 15% fruit pulp, sucrose up to 55° Brix and five different gelling agents — kappa-carrageenan, kappa-carrageenan plus locust bean gum, alginate, gellan gum, and gellan, xanthan and locust bean gums — was studied by Free-Choice Profile (FCP) analysis. Maximum rupture force and deformation at rupture were also determined by uniaxial compression in an Instron texturometer. Generalized Procrustes Analysis applied to FCP data permitted differentiation between samples and informed on the textural attributes responsible for the observed differences. Sensory differences were in general in accordance with mechanical differences. However, carrageenan and gellan gum gels were differentiated with the sensory method applied but not with mechanical tests.     

Barnacle Settlement on Hydrogels

Settlement of cultured Balanus amphitrite cyprid larvae was tested on different non-solid hydrogel surfaces. Gels consisting of alginate (highly anionic), chitosan (highly cationic), polyvinyl alcohol substituted with light-sensitive stilbazolium groups (PVA-SbQ; very low cationic) and agarose (neutral) were applied in cell culture multi-well plates. Polystyrene served as a solid surface reference. Preliminary experiments were performed to determine whether any substances leaching out of the gels could inhibit barnacle settlement. Whilst leachate from the gels revealed no toxicity towards Artemia salina nauplius larvae, PVA-SbQ in solution at and above a concentration of 0.4 ppm inhibited B. amphitrite cyprid settlement. Gels were therefore washed to avoid such effects during further testing, and toxicity and settlement tests with B. amphitrite nauplii and cyprids, respectively, applied to verify that washing was effective. Settlement was tested directly on the different test materials, followed by a quality test of non-settled larvae. All gels inhibited barnacle settlement compared to the polystyrene controls. Gels consisting of 2.5% PVA-SbQ or 0.5% agarose showed promising antifouling properties. Although some settlement occurred on 2.5% PVA-SbQ gels, metamorphosis was clearly inhibited. Only 10% of the larvae had settled on 0.5% agarose gels after 8 d. Less than 40% settlement occurred on alginate gels, as well as on 2% chitosan gels. Quality testing showed that the majority of remaining non-settled larvae in all gel experiments were able to settle when offered a suitable solid substratum.     

Anisotropic mechanical properties of magnetically aligned fibrin gels measured by magnetic resonance elastography

The anisotropic mechanical properties of magnetically aligned fibrin gels were measured by magnetic resonance elastography (MRE) and by a standard mechanical test: unconfined compression. Soft anisotropic biomaterials are notoriously difficult to characterize, especially in vivo. MRE is well-suited for efficient, non-invasive, and non-destructive assessment of shear modulus. Direction-dependent differences in shear modulus were found to be statistically significant for gels polymerized at magnetic fields of 11.7 and 4.7 T compared to control gels. Mechanical anisotropy was greater in the gels polymerized at the higher magnetic field. These observations were consistent with results from unconfined compression tests. Analysis of confocal microscopy images of gels showed measurable alignment of fibrils in gels polymerized at 11.7 T. This study provides direct, quantitative measurements of the anisotropy in mechanical properties that accompanies fibril alignment in fibrin gels.     

Relation between structural and release properties in a polysaccharide gel system

The potential utility of kappa-carrageenan gels for preparing drug release devices is here shown. Structural properties of kappa-carrageenan gels prepared with different salt composition and containing Ketoprofen sodium salt, as model drug, have been evaluated with static light scattering and rheological measurements. These properties have been correlated with release profiles in vitro at pH 5.5. Release properties from gelled matrices have been compared with those obtained by two commercial products containing the same drug. Results show that: i) in this system it is possible to easily control the gel texture by using different cationic concentration; ii) the kinetics of drug release by kappa-carrageenan gels are dependent on the structural properties of matrices; iii) in the typical interval time used in classical local applications, all gel samples release the loaded drug almost completely, at difference with the commercial products. All these findings can provide useful suggestions for the realization of classical topical release systems.     

Mixed photo-cross-linked polyvinyl alcohol and calcium-alginate gels for cell entrapment

Living cells may be immobilized by gel entrapment under very mild conditions. The ionotropic gelation of alginate with bivalent cations such as Ca²⁺, as well as photo-induced gelation of polyvinyl alcohol (PVA) bearing photosensitive stilbazolium (SbQ) groups, are procedures that are compatible with most bioactive materials. In the search for more stable and stronger alginate gel beads, experiments have been carried out to investigate mixed gels from alginate and PVA-SbQ. The swelling capacities, diffusion properties, and potential toxic effect of the binary gel beads have been evaluated. The gel beads of selected PVA-SbQ/alginate mixtures were applied successfully as carriers in a denitrification process with continuous feeding of unsterilized water medium. Under such conditions, the purely synthetic PVA-SbQ network is expected to have a longer lifespan than a natural biopolymer such as alginate.     

Kappa-carrageenan/gelatin gel beads for the co-immobilization of aerobic and anaerobic microbial communities degrading 2,4,6-trichlorophenol under air-limited conditions

Alginate and kappa-carrageenan gels were tested as bead materials for the co-immobilization of anaerobic and aerobic microorganisms for the mineralization of 2,4,6-trichlorophenol under air-limited conditions. Chemical, mechanical and thermal culture constraints were pre-defined and the gel resistances were established. Alginate was quickly eliminated because of its chemical instability in the culture media. In anaerobic conditions, the microorganisms transformed the substrates into CH4 and CO2. The kappa-carrageenan gel did not enable these gases to diffuse. They remained as bubbles in the core of the beads and made the beads float. Gelatin was added to kappa-carrageenan in order to change the carrier properties. No biogas bubbles appeared in the bead core during the cultures in anaerobic conditions and the beads reacted well to the culture conditions in the reactor. The co-immobilization of the anaerobic and aerobic microbial communities was successfully performed with the kappa-carrageenan/gelatin gel (2% (w/w) of each polymer). The biological activities, measured by the impedancemetry technique, were preserved in the beads.     

Elastic characterization of transversely isotropic soft materials by dynamic shear and asymmetric indentation

The mechanical characterization of soft anisotropic materials is a fundamental challenge because of difficulties in applying mechanical loads to soft matter and the need to combine information from multiple tests. A method to characterize the linear elastic properties of transversely isotropic soft materials is proposed, based on the combination of dynamic shear testing (DST) and asymmetric indentation. The procedure was demonstrated by characterizing a nearly incompressible transversely isotropic soft material. A soft gel with controlled anisotropy was obtained by polymerizing a mixture of fibrinogen and thrombin solutions in a high field magnet (B?=?11.7 T); fibrils in the resulting gel were predominantly aligned parallel to the magnetic field. Aligned fibrin gels were subject to dynamic (20-40 Hz) shear deformation in two orthogonal directions. The shear storage modulus was 1.08?±?0. 42 kPa (mean?±?std. dev.) for shear in a plane parallel to the dominant fiber direction, and 0.58?±?0.21 kPa for shear in the plane of isotropy. Gels were indented by a rectangular tip of a large aspect ratio, aligned either parallel or perpendicular to the normal to the plane of transverse isotropy. Aligned fibrin gels appeared stiffer when indented with the long axis of a rectangular tip perpendicular to the dominant fiber direction. Three-dimensional numerical simulations of asymmetric indentation were used to determine the relationship between direction-dependent differences in indentation stiffness and material parameters. This approach enables the estimation of a complete set of parameters for an incompressible, transversely isotropic, linear elastic material.     

Stereocontrolled Synthesis of an Octasaccharide Part of I-Active Glycolipids

The effects of glycerin and ethylene glycol on the elastic modulus and DSC thermograms of agarose and kappa-carrageenan gels were examined to clarify the relation between structure and properties. The elastic modulus of these gels as a function of the concentration of polyols increased up to a certain concentration and then decreased with increasing concentration of polyols. These polyols shifted the melting temperature of the gel to higher temperatures in kappa-carrageenan gels but to lower temperatures in agarose gels. The temperature dependence of elastic modulus was changed in opposite directions in agarose and kappa-carrageenan gels by the addition of polyols, and this is discussed on the basis of model consisting of junction zones which are connected by Langevin chains. It was suggested that the mean distance between junction zones became shorter in the presence of a small amount of polyols.     

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.     

Stabilization of kappa-carrageenan gel with polymeric amines: Use of immobilized cells as biocatalysts at elevated temperatures

Spherical beads of kappa-carrageenan containing entrapped cells were prepared in a two-step process. First, the beads were formed by dispersing a warm carrageenan cell suspension into stirring oil. After cooling (gelation) the beads were cured by treatment with amines. Ten amines of various sizes and structures were tested. We evaluated the mechanical strength and the applicability of aminetreated gels as immobilization matrices. The results of critical compression tests indicate that linear and branched polyethylenimines (PEI) are both good curing agents. PEI treated carrageenan beads also exhibited superior resistance to heat and abrasion. Furthermore, PEI polymers were demonstrated to be effective in stabilizing the lactase activity of the free and immobilized Bacillus stearothermophilus cells. The immobilized cell preparations of Saccharomyces cerevisiae, B. stearothermophilus, and Flavobacterium sp. were treated with branched PEI and evaluated for the activity of invertase (EC 3.2.1.26), lactase (EC 3.2.1.23), and glucose isomerase (EC 5.3.1.18), respectively, in a packed bed reactor at 60 degrees C. The apparent half-lives were 108, 39, and 64 days, respectively.     

New insight into deformation-dependent hydraulic permeability of gels and cartilage,and dynamic behavior of agarose gels in confined compression

Equilibrium, creep, and dynamic behaviors of agarose gels (2.0-14.8%) in confined compression were investigated in this study. The hydraulic permeabilities of gels were determined by curve-fitting creep data to the biphasic model (J. Biomech. Eng. 102 (1980) 73) and found to be similar in value to those published in the literature (AIChE J. 42 (1996) 1220). A new relationship between intrinsic permeability and volume fraction of water was found for agarose gel, capable of predicting deformation-dependent permeabilities of bovine articular cartilage and 2% agarose gel published in literature. This relationship is accurate for gels and cartilage over a wide range of permeabilities (four orders of magnitude variation). The dynamic stiffness of the gels increases with gel concentration and loading frequency (0.01-1.0Hz). The increase in dynamic stiffness with loading frequency is less pronounced for gels with higher concentrations. The results of this study provide a new insight into deformation-dependent permeability behavior of agarose gel and cartilage, and are important for understanding biological responses of cells to interstitial fluid flow in gel or in cartilage under dynamic mechanical loading.     

Effect of calcium concentration on textural properties of high and low acyl mixed gellan gels

Textural properties of 1% low and high acyl gellan gels and their mixtures were studied using compression tests and the microcentrifuge-microfiltration based water holding capacity (WHC) method. Low acyl (1% LA), high acyl (1% HA) gels and mixtures of 1% 25/75 LA/HA, 50/50 LA/HA, 75/25 LA/HA gels with calcium concentrations ranging from 2 to 80 mM were studied. HA or mixed gels with a lower LA/HA ratio had a greater WHC and failure strain than that of LA or mixed gels with a higher LA/HA ratio. Gellan gels with a higher LA/HA ratio had a larger initial Young's modulus. Our study also indicates that a higher LA/HA ratio does not necessarily result in a gel with a larger failure stress, although LA gels are generally firmer than HA gels. Gel strength and WHC of HA and LA/HA mixtures may reflect both stabilization and destabilization effects of glycerate groups positioned at one of the glucose residues in each repeating tetrasaccharide unit of HA gels.     

Rheological properties of mixtures of protein-polysaccharide-dynamic viscoelasticity of blend gels of acylated gelatin, kappa-carrageenan, and agarose

Complex Young's modulus of blend gels of gelatin and kappa-carrageenan or agarose has been measured in order to clarify the protein-polysaccharide interaction in biological systems. The mixture of gelatin and kappa-carrageenan showed phase separation in the intermediate volume fraction of gelatin, and it formed a homogeneous gel when the volume fraction of gelatin is very large or very small. Since the dynamic Young's modulus for blend gels of kappa-carrageenan and gelatin was larger than the calculated one from a theory for dispersed systems, some structural reinforcing must occur. The mixture of agarose and gelatin showed the inverse tendency. It was concluded that the role of electrolytic groups was dominant in dilute gels, while molecular entanglement became more important in concentrated gels.     

Characterization of kappa-carrageenan gels used for immobilization of Bacillus firmus.

In this study, aimed at a biochemical and physical characterization of kappa-carrageenan gels used for entrapment of Bacillus firmus NRS 783 (a superior producer of an alkaline protease), effects of carrageenan concentration, gelation temperature, initial cell loading, and strength of the curing agent (KCl) on the properties of cell-free and cell-laden gels were examined. The physical properties of the differently prepared gels that were examined included density, free volume fraction, mechanical strength, and change in gel volume during gel curing. The biochemical characteristics studied included viability of gel-entrapped cells, cell leakage from cell-laden gels, and cell penetration into cell-free gels. For the range of carrageenan contents investigated [between 2% and 5% (w/v)], the mechanical strength of the gels with/without KCl curing was observed to increase with an increase in carrageenan content of gels. The mechanical strength of each gel increased substantially upon extensive curing. Free volume fractions in excess of 0.8 were observed for all gels. Of cells that were viable prior to immobilization, 90-92% remained viable after formation and extensive curing of gels for cell-gel mixtures prepared at 45 degrees C. Attempts at prolonged storage of cell-laden gel beads at 0 degrees C as stock cultures of immobilized B. firmus were unsuccessful due to a significant decline in cell viability during such storage. On the basis of the cell leakage studies, the average pore sizes of 2%, 3%, 4%, and 5% gels were deduced to increase in the following order of carrageenan content (w/v): 4%, 3%, 2%, and 5%. Commensurate with the decrease in the average pore size (or the increased tightness of the gels) with increasing carrageenan content, both the extent of cell leakage and the extent of net cell penetration decreased with increasing carrageenan content for the first three gels. Owing to non-uniform distribution of free space and much larger pores, the extent of net cell penetration in 5% carrageenan gels was considerably low, while the extent of cell leakage in 5% carrageenan gels was an order of magnitude greater than the extents of cell leakage in the other three gels.     

The development of structural and mechanical anisotropy in fibroblast populated collagen gels

An in vitro model system was developed to study structure-function relationships and the development of structural and mechanical anisotropy in collagenous tissues. Fibroblast-populated collagen gels were constrained either biaxially or uniaxially. Gel remodeling, biaxial mechanical properties, and collagen orientation were determined after 72 h of culture. Collagen gels contracted spontaneously in the unconstrained direction, uniaxial mechanical constraints produced structural anisotropy, and this structural anisotropy was associated with mechanical anisotropy. Cardiac and tendon fibroblasts were compared to test the hypothesis that tendon fibroblasts should generate greater anisotropy in vitro. However, no differences were seen in either structure or mechanics of collagen gels populated with these two cell types, or between fibroblast populated gels and acellular gels. This study demonstrates our ability to control and measure the development of structural and mechanical anisotropy due to imposed mechanical constraints in a fibroblast-populated collagen gel model system. While imposed constraints were required for the development of anisotropy in this system, active remodeling of the gel by fibroblasts was not. This model system will provide a basis for investigating structure-function relationships in engineered constructs and for studying mechanisms underlying the development of anisotropy in collagenous tissues.     

Encapsulating chondrocytes in degrading PEG hydrogels with high modulus: engineering gel structural changes to facilitate cartilaginous tissue production

A major challenge when designing cell scaffolds for chondrocyte delivery in vivo is creating scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. To address this design challenge, degradable photocrosslinked hydrogels based on poly(ethylene glycol) were investigated. To alter the gel's initial mechanical properties, hydrogels were fabricated by varying the initial macromer concentration from 10% to 15% to 20%. A twofold increase in macromer concentration resulted in an eightfold increase in the initial compressive modulus from 60 to 500 kPa. Gel degradation was tailored by incorporating fast-degrading crosslinks that enable maximal extracellular matrix (ECM) diffusion with time and a minimal number of nondegrading (or slowly degrading) crosslinks to maintain scaffold integrity and prevent complete gel erosion during tissue formation. Chondrocytes encapsulated in these gels produced cartilaginous tissue rich in glycosaminoglycans and collagen as seen biochemically and histologically. Interestingly, mass loss appeared to more closely match tissue secretion in gels fabricated from a 15% macromer concentration. However, the spatial ECM distribution was grossly similar in all three gels. By tailoring gel degradation and controlling network evolution during degradation, gels with optimal properties can be fabricated to support initially physiologic compressive loads while simultaneously supporting the formation of a neotissue.     

Flexural and Creep Properties of Human Jaw Compact Bone for FEA Studies

The aim of this work was to improve the constitutive model of the human mandible and dentition system by taking into account the non-linear material properties of the structural boney matrix that forms the human jaw bone or mandible. Due to the specific structure of the jaw bone the time dependence of the mechanical properties also forms an important stage of the quantification process. The lack of specific experimental data of this type of material prevents the implementation of these properties into finite element simulations which results in poor quality modelling. Here an attempt was made to determine elastic and viscoelastic mechanical characteristics of the compact bone tissue forming the mandible. The elastic properties of compact bone were determined experimentally from 3 point bending tests and the viscoelastic properties were evaluated from creep tests in compression. A particular human jaw from this complex study was used to reconstruct a geometric model for further numerical experiments.     

Flexural and creep properties of human jaw compact bone for FEA studies

The aim of this work was to improve the constitutive model of the human mandible and dentition system by taking into account the non-linear material properties of the structural boney matrix that forms the human jaw bone or mandible. Due to the specific structure of the jaw bone the time dependence of the mechanical properties also forms an important stage of the quantification process. The lack of specific experimental data of this type of material prevents the implementation of these properties into finite element simulations which results in poor quality modelling. Here an attempt was made to determine elastic and viscoelastic mechanical characteristics of the compact bone tissue forming the mandible. The elastic properties of compact bone were determined experimentally from 3 point bending tests and the viscoelastic properties were evaluated from creep tests in compression. A particular human jaw from this complex study was used to reconstruct a geometric model for further numerical experiments.