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PubMed2000

Alginate hydrogel microspheres and microcapsules prepared by spinning disk atomization.

Our spinning disk atomization (SDA) can, relative to other existing techniques, produce micron-sized particles with very narrow size distribution. The aim of this work is to present this technology for the production of alginate microspheres and microcapsules. We atomized and gelled aqueous alginate solutions into very narrowly dispersed microspheres with sizes ranging from 300 to 600 microm. Here, the interest is to produce, at high rate, particles of a given size with a narrow size distribution and also to show a new method of encapsulation using SDA. The viscosity and flow rate contributions in the drop formation is qualitatively analyzed to show how they affect droplet size. In addition, a technique for high degree of encapsulation is presented in which yeast is used as a model system. The production of yeast-loaded microspheres by SDA shows the potential of the technique for biotechnology applications.

Biocompatible Materials↗
PubMed2002

Preparation and properties of alginate lyase modified with poly(ethylene glycol).

Modification of the enzyme alginate lyase (AL) with poly(ethylene glycol) (PEG) was attempted for the degradation and removal of alginate biofilms in infectious diseases. The modification of AL with PEG was attempted with three kinds of N-succinimidyl succinate PEG (SS-PEG), which differed in molecular weight (i.e., 2000, 5000 and 12,000 Da). The conjugation of PEG to free amino groups on AL was confirmed by gel permeation chromatography. Quantification of residual free amino groups revealed that PEG modification progressed further with a higher pH and a larger molar ratio of SS-PEG to AL. The reproducibility of the reaction was fairly good. The enzyme activity decreased with increasing PEG modification but the immunoreactivity toward anti-AL antibodies, as evaluated by an ELISA method, was much more remarkably reduced. The immunoreactivity was more reduced by the conjugated PEG with the larger molecular weight. In the reaction with PEG of molecular weight 12,000 Da, we obtained PEG-modified AL retaining approximately 40% enzyme activity but only 0.5% of the immunoreactivity of native AL.

Animals↗
PubMed1995

Oxygen consumption rates of free and alginate-entrapped beta TC3 mouse insulinoma cells.

Oxygen consumption rates of beta TC3 mouse insulinoma cells were measured in three different cell culture systems: monolayer, freshly trypsinized cell suspension, and trypsinized cells entrapped in alginate-poly-L-lysine-alginate (APA) polymer beads. The oxygen consumption rate for cells in the APA beads was similar to the rate for freshly trypsinized cells, indicating that cells can be entrapped without affecting their oxygen consumption rate. The cells in monolayers consumed oxygen at a higher rate than freshly trypsinized cells or cells in APA beads, suggesting that trypsinization may lower oxygen consumption capability. In addition, the oxygen consumption of beta TC3s during glucose-induced insulin secretion was not significantly different from the basal rate, in contrast to the increased oxygen demand of actively secreting islets. Our results suggest that beta TC3s may be better suited than islets for use in a bioartificial pancreas due to their stable oxygen requirements.

Animals↗
PubMed2005

Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures.

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Adult↗
PubMed2003

Effects of rhein on human articular chondrocytes in alginate beads.

This study was designed to investigate the effects of rhein, the active metabolite of diacerhein, on the metabolic functions of human chondrocytes cultured in alginate beads. Enzymatically isolated osteoarthritic (OA) chondrocytes were cultured in alginate beads in a well-defined culture medium for 12 days. Rhein was tested in a range of concentrations comprised between 10(-7) and 4 x 10(-5)M, in the presence or absence of 10(-10)M IL-1beta. Interleukin (IL)-6 and -8, macrophage inflammatory protein (MIP-1beta), stromelysin-1 (MMP-3), aggrecan (AGG), tissue inhibitor of metalloproteinases-1 (TIMP-1), prostaglandin E(2) (PGE(2)) and nitric oxide (NO) productions were assayed. Cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) mRNA steady-state levels were also quantified. In the basal condition, 10(-5)M rhein increased by 46.5% the production of AGG, decreased by 17-30% the production of IL-6, MMP-3, NO and MIP-1beta but enhanced by 50% the production of PGE(2). IL-1beta increased IL-6, IL-8, MIP-1beta, NO, PGE(2) and MMP-3 productions, but inhibited AGG and TIMP-1 synthesis. Rhein partially reversed the effect of IL-1beta on TIMP-1 and NO production, had no effect on AGG, IL-6 and MIP-1beta production, but up-regulated the IL-1beta stimulated PGE(2) production. The COX-2 and iNOS mRNA levels and IL-8 production were not modified by rhein.Overall, these results contribute to explain the clinical efficiency of rhein and give new information on its mechanisms of action.

Aggrecans↗
PubMed2001

Diffusion of (de)acylated antibiotic A40926 in alginate and carrageenan beads with or without cells and/or soybean meal.

Effective diffusion coefficients (D(e)) of antibiotic A40926 and its deacylated derivative were determined in Ca-alginate (2% wt/wt) and kappa-carrageenan (2.6% wt/wt) gel beads with or without immobilized Actinoplanes teichomyceticus cells and/or soybean meal (SBM). The method used was based on transient concentration changes in a well-stirred antibiotic solution in which gel beads, initially free of solute, were suspended. Unsteady-state diffusion in a sphere was applied and D(e) determined from the best fit of experimental data. A40926 showed markedly different diffusion characteristics than its deacylated derivative. Diffusivity of deacyl-A40926 in alginate or carrageenan gel beads was six to seven times that of A40926. Large differences in partition coefficients (Kp) were also found. In case of beads without additions, A40926, in contrast to deacyl-A40926, strongly partitioned to the liquid phase. Introduction of SBM and/or mycelium in the gel beads decreased the effective diffusivity of deacyl-A40926, but increased its partitioning to the solid phase. Our findings indicate that a relatively moderate structural change of a lipoglycopeptide molecule could lead to a major change in its diffusion/partition characteristics.

Journal Article↗
PubMed2000

Synergistic effect of active oxygen species and alginate on chitinase production by Wasabia japonica cells and its application.

The specific chitinase productivity of a Wasabia japonica cell suspension culture under pure oxygen aeration was 3.8 times higher than that of a suspension culture aerated with ordinary air. During aeration with pure oxygen, both oxygen consumption by the cells and the H2O2 concentration in the medium increased. Addition of H2O2 to the cultivation medium also promoted the specific chitinase productivity. H2O2 could pass freely through the cell membrane. It was assumed that the excess oxygen was converted into active oxygen species such as H2O2, and that the promotion of chitinase production was probably due to the generated active oxygen species. Addition of alginate oligomer (AO, an endogenous elicitor-like substance) to cultures aerated with pure oxygen or supplemented with H2O2 resulted in synergistic increases in chitinase production. Based on these results, the development of a simple and efficient chitinase production system was investigated. Cells were immobilized in alginate gel (instead of adding AO to the medium) and cultivated in a medium containing H2O2. The specific chitinase productivity increased to the levels observed in the suspension culture system. During repeated batch cultivation of immobilized cells, the chitinase production remained stable for three repeated batches. When immobilized protoplasts were cultivated in a medium containing H2O2, there was 7-fold increase in chitinase production compared with that of immobilized cells.

Journal Article↗
PubMed2004

A novel transfection method for mammalian cells using calcium alginate microbeads.

The direct transfer of genetic materials into mammalian cells is an indispensable technique. We have developed calcium alginate (CA) microbeads which can deliver plasmid DNAs and yeast artificial chromosomes into plant and yeast cells. In this paper, we demonstrate the effective transfection of mammalian cells by CA microbeads immobilizing plasmid DNAs. The transfection was performed using the pEGFP-C1 plasmid containing the cytomegalovirus (CMV) promoter and enhanced green fluorescent protein (EGFP) gene. The transient expression of EGFP was observed 24 h after transfection. The expression efficiency was maximum when the concentration of sodium alginate was 1% and the amount of plasmid DNA was increased to 100 microg. The expression efficiency of our method using CA microbeads is 2-10 times higher than that of the polyethylene glycol (PEG) method. Our results suggest that the CA microbead mediated transfection of mammalian cells effectively delivers genetic materials into mammalian suspension cells.

Journal Article↗
PubMed1999

Release of embryogenic carrot cells with high regeneration potency from immobilized alginate beads.

For the mass-production of regenerated carrot plantlets, embryogenic carrot callus immobilized in calcium alginate gel beads was cultivated in a growth medium and the regeneration frequency of cells released from alginate gel beads was compared with that in a suspension culture. Cells released in the immobilized culture were regenerated at a frequency which was about 1.5 times higher than that obtained in the suspension culture. When CaCl2 was added to the growth medium at 5 mM, repeated batch culture for plantlet production continued for 245 d with no significant decrease in the productivity (1.6 x 10(5) plantlets/l-medium/d).

Journal Article↗
PubMed2002

The effect of sodium alginate on the crystal growth of calcium carbonate.

The effect of sodium alginate in supersaturated solutions of calcium carbonate was investigated under plethostatic conditions. The rates of crystal growth measured in the presence of sodium alginate at concentrations as low as 0.83 x 10(-7) mol dm(-3) were drastically reduced. Kinetic analysis according to a Langmuir-type adsorption isotherm led to the calculation of an affinity constant Kaff = 999.8 x 10(-4) mol dm(-3). The apparent order found from kinetic data was 3.0+/-0.2 suggesting a surface nucleation mechanism.

Journal Article↗
PubMed1996

Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases.

A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni(2+)-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly.

Amino Acid Sequence↗
PubMedDate not supplied

Sustained release properties of alginate microspheres and tabletted microspheres of diclofenac sodium.

This study focused on the properties of diclofenac sodium (DNa) alginate (alg) microspheres and tabletted DNa alg microspheres using different polymers as additives. DNa alginate microspheres were prepared by the emulsification method and different polymers such as Eudragit (Eud) NE 30 D, Eudragit (Eud) RS 30 D and Aquacoat, which were incorporated into alg gel to control the release rate of drug. The release properties of DNa alg microspheres (1:1) were affected by the size, drug load of microspheres and also by the incorporated polymers, pH and ionic strength of dissolution medium. Tabletting of alg microspheres using carrageenan (carr), alg, pectin, NaCMC, tragacanth (trgh) and HPMC as additives in a (50:50) ratio produced tablets with good physical properties and also better controlled release of DNa. Dissolution studies were carried out in pH 7.2 phosphate buffer and phosphate buffers whose pH values were gradually changed from pH 3 to 7.4. The rank order of DNa release from tablets was carr < alg < pectin < NaCMC < trgh < HPMC which relates to the viscosity and swelling properties of polymers. The drug release was very slow from trgh and HPMC based tablets, but addition of carr or alg in different ratios could adjust the release rate of drug.

Capsules↗
PubMedDate not supplied

Cloning, sequence analysis and expression of gene alyVI encoding alginate lyase from marine bacterium Vibrio sp. QY101.

The gene (alyVI) encoding an alginate lyase of marine bacterium Vibrio sp. QY101, which was isolated from a decaying thallus of Laminaria, was cloned using a strategy of combined degenerate PCR and long range-inverse PCR (LR-IPCR), then sequenced and expressed in Escherichia coli. Gene alyVI was composed of a 1014 bp open reading frame (ORF) encoding 338 amino acid residues. The calculated molecular mass of alyVI product is 38.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 34 kDa. AlyVI was purified from culture supernatants to electrophoretic homogeneity using affinity chromatography. AlyVI was most active at pH 7.5 and 40 degrees C in the presence of 1 mM ZnCl2. A nine-amino-acid consensus region (YXRESLREM), which was only found in polyguluronate lyases, was also observed in the amino-terminal region of AlyVI. However, AlyVI could degrade both M block and G block. These results indicate that a novel alginate lyase-encoding gene has been cloned.

Amino Acid Sequence↗
PubMed2005

Exposure to pulsed low intensity ultrasound stimulates extracellular matrix metabolism of bovine intervertebral disc cells cultured in alginate beads.

STUDY DESIGN: In vitro study on the effects of pulsed low intensity ultrasound on the cellular metabolism of bovine intervertebral disc cells. OBJECTIVE: To determine whether pulsed low intensity ultrasound has effects on cell proliferation and extracellular matrix metabolism by bovine intervertebral disc cells. SUMMARY OF BACKGROUND DATA: The application of pulsed low intensity ultrasound is known to be effective in stimulating fracture and cartilage repair. However, the effects of pulsed low intensity ultrasound on intervertebral disc cells are not known. METHODS: Cells of the nucleus pulposus and inner and outer anulus fibrosus were enzymatically isolated from bovine coccygeal tissue and precultured in alginate beads for 14 days. In the ultrasound group, pulsed low intensity ultrasound was administered to the culture for 20 minutes daily for an additional 20 days. The control group was cultured in the same way but without administration of ultrasound. Cell viability, DNA content, proteoglycan and collagen synthesis, and proteoglycan content at days 10 and 20 after the initiation of treatment were evaluated. Characterization of newly synthesized collagen and proteoglycan was performed. RESULTS: No significant differences in cell viability and DNA content were observed between the two groups. On day 20, proteoglycan synthesis was increased by the application of pulsed low intensity ultrasound in nucleus pulposus and inner and outer anulus fibrosus cells (24%-26% increase, P < 0.001). The application of pulsed low intensity ultrasound increased proteoglycan content in alginate beads containing inner and outer anulus fibrosus cells (P < 0.05). Collagen synthesis by cells isolated from all three zones of the intervertebral disc was increased by the application of pulsed low intensity ultrasound (16%-19% increase, P < 0.05-0.0001). CONCLUSIONS: The application of pulsed low intensity ultrasound stimulated extracellular matrix metabolism in intervertebral disc cells. Pulsed low intensity ultrasound may prove useful for the physical stimulation of cell metabolism for tissue engineering of intervertebral disc tissue.

Animals↗
PubMed1992

A randomized study to compare calcium sodium alginate fibre with two commonly used materials for packing after nasal surgery.

A prospective randomized study was undertaken to compare the qualities of calcium sodium alginate (Kaltostat), trousered paraffin gauze, and glove finger packs as nasal packing material following the operation of partial inferior turbinectomy. All three types of packing material were found to be similarly effective in preventing bleeding whilst the packs were in situ. Calcium sodium alginate (Kaltostat) was associated with significantly less bleeding on pack removal than the other two packing materials. Further, irrespective of the material used, leaving the packs in situ for 48 hours produced significantly less bleeding than when they were removed after 24 hours.

Blood Loss, Surgical↗
PubMed1989

A double-blind study of hyposensitization with an alginate conjugated extract of D. pteronyssinus (Conjuvac) in patients with perennial rhinitis. 1. Clinical aspects.

The efficacy of hyposensitization with a standardised extract of Dermatophagoides pteronyssinus (D. pteronyssinus) conjugated to alginate and containing known amounts of antigen P1 (Conjuvac) was tested in a double blind, placebo controlled, multi-centre study in 66 adult patients with perennial rhinitis. Patients received 11 weekly injections of increasing concentrations of Conjuvac containing from 56 x 10(1) to 448 10(3) IU D. pteronyssinus or placebo injections of the alginate diluent to some of which 5 micrograms of histamine has been randomly added. This was followed by 15 monthly injections of Conjuvac or placebo. The severity of nasal blockage, sneezing and rhinorrhoea was recorded twice daily in a diary and visual analogue assessments (VAS) made at each clinic visit. Nasal provocation testing (NPT) was performed with increasing concentrations of the same extract of D. pteronyssinus as used in the hyposensitization injections, and changes in nasal airways resistance measured by passive anterior rhinomanometry. VAS was recorded and NPT was performed on entry to the study and after the fifth, ninth and final monthly injection. Conjuvac injections were well tolerated. Large local reactions (greater than 5 cm) occurred within 30 min in only 1% of patients but later in 23%. No systemic reactions or anaphylaxis occurred within 30 min of injections, but urticaria or worsening of asthma and rhinitis was reported later in 3% of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1992

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads.

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23 degrees C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO(3) was added to the growth media. There were strong interactions between CaCO(3) and media, CaCO(3) and pH level, and CaCO(3), media, and pH level. In media with CaCO(3), all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO(3) increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 x 10/g). In CaCO(3)-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.

Journal Article↗
PubMed1992

Characteristics of a hydrated, alginate-based delivery system for cultivation of the button mushroom.

The production of the button mushroom Agaricus bisporus with mycelium-colonized alginate pellets as an inoculant of the growing medium was investigated. Pellets having an irregular surface and porous internal structure were prepared by complexing a mixture of 1% sodium alginate, 2 to 6% vermiculite, 2% hygramer, and various concentrations of Nutrisoy (soy protein) with calcium chloride. The porous structure allowed the pellets to be formed septically and then inoculated and colonized with the fungus following sterilization. By using an enzyme-linked immunosorbent assay (ELISA) to estimate fungal biomass, the matrix components of the pellet were found to be of no nutritive value to A. bisporus. Pellets amended with Nutrisoy at a concentration of 0.5 to 8% supported extensive mycelial growth, as determined by significantly increased ELISA values, with a concentration of 4% being optimal and higher concentrations proving inhibitory. The addition of hydrated, mycelium-invaded pellets to the compost or casing layer supported the thorough colonization of the growing substrate and culminated in the formation of mushrooms that showed normal development and typical morphology. Yields and sizes of mushrooms were comparable from composts seeded with either colonized pellets or cereal grain spawn. Similarly, amending the casing layer with pelletized-mycelium-colonized compost resulted in a 2- to 3-day-earlier and more-synchronous emergence of mushrooms than with untreated casing. This technology shows the greatest potential as a pathogen-free inoculant of the casing layer in the commercial cultivation of mushrooms.

Journal Article↗