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PubMed2006

Development of mammalian cell-enclosing calcium-alginate hydrogel fibers in a co-flowing stream.

A jetting technique in a liquid-liquid co-flowing stream was applied to the preparation of mammalian cell-enclosing calcium-alginate (Ca-alg) hydrogel fibers of several hundred micrometers in cross-sectional diameter. One percent alginate aqueous solution was extruded from needles (270, 480, 940 microm inner diameter) into a co-flowing laminar stream of 100 mM aqueous calcium chloride solution. The extruded alginate solution was stretched by the CaCl(2) solution, which is known as a "jetting process", and the Ca-alg hydrogel fibers were formed by gelation of the alginate solution through the uptake of calcium ions in the CaCl(2) solution. The cross-sectional diameter of the hydrogel fibers could be controlled from approximately 100-800 microm by changing the velocities of the alginate and CaCl(2) solution, and the inner diameter of the needle. Approximately 95% of bovine carotid artery vascular endothelial cells remained alive after the process of preparing hydrogel fibers in a co-flowing stream, demonstrating that the cell-enclosing process scarcely influences the viability of the enclosed cells.

Alginates↗

PubMed2002

Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds.

Cardiac tissue engineering has evolved as a potential therapeutic approach to assist in cardiac regeneration. We have recently shown that tissue-engineered cardiac graft, constructed from cardiomyocytes seeded within an alginate scaffold, is capable of preventing the deterioration in cardiac function after myocardial infarction in rats. The present article addresses cell seeding within porous alginate scaffolds in an attempt to achieve 3D high-density cardiac constructs with a uniform cell distribution. Due to the hydrophilic nature of the alginate scaffold, its >90% porosity and interconnected pore structure, cell seeding onto the scaffold was efficient and short, up to 30 min. Application of a moderate centrifugal force during cell seeding resulted in a uniform cell distribution throughout the alginate scaffolds, consequently enabling the loading of a large number of cells onto the 3D scaffolds. The percent cell yield in the alginate scaffolds ranged between 60-90%, depending on cell density at seeding; it was 90% at seeding densities of up to 1 x 10(8) cells/cm(3) scaffold and decreased to 60% at higher densities. The highly dense cardiac constructs maintained high metabolic activity in culture. Scanning electron microscopy revealed that the cells aggregated within the scaffold pores. Some of the aggregates were contracting spontaneously within the matrix pores. Throughout the culture there was no indication of cardiomyocyte proliferation within the scaffolds, nor was it found in 3D cultures of cardiofibroblasts. This may enable the development of cardiac cocultures, without domination of cardiofibroblasts with time.

Alginates↗

PubMed2002

Chemistry and biocompatibility of alginate-PLL capsules for immunoprotection of mammalian cells.

Transplantation of encapsulated living cells is a promising approach for the treatment of a wide variety of diseases. Large-scale application of the technique, however, is hampered by insufficient biocompatibility of the capsules. In order to get means to study factors influencing the biocompatibility of capsule for encapsulation of living cells, we have correlated the chemical composition of the surface of commonly applied alginate-PLL capsules with the biological response in rats. Capsules prepared of alginates with an intermediate guluronic (G) acid content proved to be biocompatible, whereas capsules prepared of high-G alginates were overgrown by inflammatory cells. We applied X-ray photoelectron spectroscopy to correlate the biological responses with the chemical compositions of the capsule surfaces. High-G alginate capsules proved to have a higher PLL content but less surface binding sites for PLL than low-G alginates. This study, shows for the first time that biological responses against capsules can be successfully correlated to its chemical characteristics.

Alginates↗

PubMed2005

Chitosan-alginate as scaffolding material for cartilage tissue engineering.

Tissue compatibility of chitosan-alginate scaffolds was studied in vitro in terms of cell morphology, proliferation, and functionality using HTB-94 cells. The scaffold has an interconnected 3D porous structure, and was fabricated by thermally induced phase separation followed by freeze drying. Cell proliferation on the chitosan-alginate scaffold was found to be faster than on a pure chitosan scaffold. After cell culture for 2 weeks in vitro, the cells on the chitosan scaffold gradually assumed a fibroblast-like morphology while the cells on the chitosan-alginate scaffold retained their spherical morphology throughout the period of study. SDS-PAGE electrophoresis and Western blot assays for proteins extracted from cells grown on scaffolds indicated that production of cartilage-specific collagen type II, a marker for chondrocytic phenotype, increased from week 2 to week 3 on the chitosan-alginate scaffold but decreased on the chitosan scaffold. This study suggested that chitosan-alginate scaffolds promote cell proliferation, enhance phenotype expression of HTB-94 chondrocytes, and may potentially serve as an improved alternative to chitosan scaffolds for cartilage tissue engineering.

Alginates↗

PubMed2007

Modulation of peritendinous adhesion formation by alginate solution in a rabbit flexor tendon model.

To examine the antiadhesive effect of an alginate solution following tendon surgery, unilateral subtotal laceration of the flexor digitorum communis tendon was created in one hind limb while the other side was left intact in 32 Japanese white rabbits. The lesion was coated with alginate solution in 16 animals and not coated in the other 16. Degree of adhesion formation was assessed histologically and biomechanically by measuring the flexion angle of the first toe when the flexor digitorum tendon was pulled with a specified force at 4 weeks postoperatively. When compared with the control group, the alginate-treated group demonstrated significantly greater toe flexion, with less scar tissue formation at the repair site. Histologically, complete tendon healing with longitudinal remodeling of collagen fibers was observed in the alginate-treated group, while a random pattern of fibers was observed in the control group. Reduction in adhesion formation using alginate solution represents a novel strategy for the management of tendon injury and repair in the clinical setting.

Alginates↗

PubMed2006

Chondrocyte response to cyclic hydrostatic pressure in alginate versus pellet culture.

Cells are often cultured at high density (e.g., confluent monolayer and as pellets) to promote chondrogenic differentiation and to maintain the chondrocyte phenotype. They are also frequently suspended in hydrogels such as agarose or alginate for the same purposes. These culture techniques differ markedly with respect to frequency of direct contact between cells and overall intercellular spacing. Because these factors may significantly affect mechanotransduction, the purpose of this study was to determine if the response of articular chondrocytes to cyclic hydrostatic pressure would depend on the culture condition. Primary articular chondrocytes from young and mature pigs were cultured either as pellets or suspended in alginate beads. Both groups were exposed to dynamic hydrostatic pressure (4 MPa, 1 Hz, 5400 cycles per day) for 7 days. Cell proliferation was unaffected by pressure, but pressurized chondrocytes in pellet culture had significantly greater sGAG content and incorporated [3H]proline at a higher rate than nonpressurized controls. Electron microscopy revealed a fibrous extracellular matrix (ECM) surrounding pellets, but not cells in alginate. In addition, expression of Connexin 43 (Cx43) mRNA was slightly lower in alginate than in pellet cultures and was not significantly altered by loading. Thus, metabolic response of chondrocytes to dynamic hydrostatic pressure was affected by culture technique; chondrocytes cultured as pellets exhibited the classical anabolic response to dynamic hydrostatic pressure, but those in alginate did not. Although cell-ECM interaction could be important, the differential response is not likely attributable to differential expression of Cx43 mRNA.

Alginates↗

PubMed2001

Chitosan-alginate-CaCl(2) system for membrane coat application.

Water-based formulations are preferred for membrane coat application because they do not require the use of noxious solvents. A novel aqueous chitosan-alginate-CaCl(2) system was evaluated as a potential formulation to produce water-insoluble membranes of biodegradable polymers. Chitosan-alginate coacervates were prepared by controlled reaction of chitosan (0.25% w/v) and sodium alginate (0.25% w/v) solutions. Coherent membranes were obtained by casting and drying the coacervates suspended in aqueous CaCl(2) solutions (0.05-0.07% w/v). Increasing the calcium content did not modify membrane thickness (25-26 microm), but reduced the water vapor transmission rate from 658 to 566 g/m(2)/day, and improved the tensile strength of the membranes from 9.33 to 17.13 MPa. Differential scanning calorimetry, Fourier transform infrared spectroscopy, and elemental analyses of the chitosan-alginate coacervates indicated they were stable for up to 4 weeks of storage in distilled water at ambient temperature. Membranes of the stored coacervates required less calcium to attain maximum mechanical strength. They also had higher water vapor transmission rates than corresponding films prepared from fresh coacervates. On the basis of the properties of the cast film and its storage stability, the chitosan-alginate-CaCl(2) system can be considered for potential membrane coat application.

Alginates↗

PubMed1999

Mannuronan C-5-epimerases and their application for in vitro and in vivo design of new alginates useful in biotechnology.

The industrially important polysaccharide alginate is a linear copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). It is produced commercially by extraction from brown seaweeds, although some of the bacteria belonging to the genera Azotobacter and Pseudomonas also synthesize alginates. Alginates are synthesized as mannuronan, and varying amounts of the M residues in the polymer are then epimerized to G residues by mannuronan C-5-epimerases. The gel-forming, water-binding, and immunogenic properties of the polymer are dependent on the relative amount and sequence distribution of M and G residues. A family of seven calcium-dependent, secreted epimerases (AlgE1-7) from Azotobacter vinelandii have now been characterized, and in this paper the properties of all these enzymes are described. AlgE4 introduces alternating M and G residues into its substrate, while the remaining six enzymes introduce a mixture of continuous stretches of G residues and alternating sequences. Two of the enzymes, AlgE1 and AlgE3, are composed of two catalytically active domains, each introducing different G residue sequence patterns in alginate. These results indicate that the enzymes can be used for production of alginates with specialized properties.

Alginates↗

PubMed1994

Enhanced specific antibody productivity of calcium alginate-entrapped hybridoma is cell line-specific.

In order to determine whether the enhanced specific antibody productivity (qMAb) of calcium alginate-entrapped hybridoma is cell line-specific, calcium alginate-entrapped hybridomas (4A2 and DB9G8) were cultivated under the condition where we had previously observed significantly enhanced qMAb of calcium alginate-entrapped S3H5/gamma 2bA2 hybridoma. Unlike S3H5/gamma 2bA2 hybridoma, neither 4A2 nor DB9G8 hybridomas showed persistently enhanced qMAb when they were entrapped in calcium alginate beads. The enhanced qMAb of entrapped 4A2 and DB9G8 hybridomas, which was 2-3 times higher than the qMAb of free-suspended cells in a control experiment, was observed only during the early stage of the culture. During the early stage of the culture, the viable cell concentration decreased probably due to cell damage during the entrapment process. As cell growth resumed, the qMAb decreased to the similar level of qMAb of free-suspended cells within 5-7 days. Thus, we conclude that the enhanced qMAb of calcium alginate-entrapped hybridomas is cell line-specific.

Alginates↗

PubMed2000

[Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure].

One of the goals in the field of tissue engineering is the development of artificial cartilage for the treatment of cartilage defects. Therefore autologous chondrocytes are seeded on different artificial matrices to test their possible use as implants (resorption, antigenicity, toxicity and their integration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating large-scale defects or for comparing different matrices. An in-vitro-multiplication of the cells is needed which causes the chondrocytes to dedifferentiate and become fibroblast-like. Therefore parameters which induce a redifferentiation are of great interest. The objective of this study was to determine the influence of intermittent hydrostatic pressure and low oxygen partial pressure on the redifferentiation of dedifferentiated bovine articular chondrocytes in monolayer and three-dimensional alginate bead culture. The redifferentiation process was monitored by immunocytochemical detection of newly synthesized collagen type II. The viability of the cells was determined by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20%. After this they were subcultured in monolayer or three-dimensional alginate culture and subjected to three different stimuli for three weeks in order to redifferentiate: 1.) 20% O2 (= 20.26 kPa PO2) + 5% CO2 + 75% N2; 2.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2; 3.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer there was no detectable collagen type II found by immunocytochemistry under either of the three culture conditions. Therefore a redifferentiation of dedifferentiated chondrocytes was not possible in monolayer cultures with the tested parameters. In the three-dimensional alginate culture there was no immunocytochemical staining of collagen type II found in the beads cultured with 20% oxygen. With 5% oxygen we found a strong collagen type II-production by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5% oxygen lead to a decreased collagen type II-production compared to cells subjected to 5% oxygen only. Also chondrocytes closer to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chondrocytes in the alginate culture was close to 90% after three weeks. Our experiments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture.

Alginates↗

PubMed1991

Disinfection of alginate impression material using disinfectants as mixing and soak solutions.

Disinfectant solutions were used as water substitutes in the preparation of specimens of alginate impression material. Chlorhexidine gluconate and sodium peroxysulphate solutions were tested. The alginate specimens were infected by sucking, subsequently disinfected in the solution used in the alginate preparation, and then tested for infectivity by 24-h incubation in thioglycollate broth. Bacterial growth was measured by changes in turbidity using a turbidimeter. The results indicate that chlorhexidine gluconate is an effective alginate disinfectant, when it is used as the liquid for alginate preparation and post-setting disinfection solution.

Alginates↗

PubMed1993

Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product.

The alginate lyase-encoding gene (algL) of Pseudomonas aeruginosa was localized to a 1.7-kb EcoRI-XbaI fragment within the alginate biosynthetic gene cluster at 34 minutes on the chromosome. The nucleotide sequence of this DNA fragment revealed an ORF encoding a protein of M(r) 40,885 which is transcribed in the same orientation as the other alg genes within the biosynthetic gene cluster. The predicted protein has a potential N-terminal signal peptide which is consistent with its proposed periplasmic location. The AlgL protein was overproduced in Escherichia coli and purified. The purified protein was shown to have alginate lyase activity. In addition, an algL insertion mutant of the mucoid P. aeruginosa 8830 was constructed. This mutant (alm1) had a nonmucoid phenotype due to a polar effect on the transcription of an essential alg gene, algA. Thus, the algL gene is located within a region of the alginate biosynthetic gene cluster that appears to be non-essential for alginate production.

Alginates↗

PubMed1996

Effects of soluble sodium alginate on cholesterol excretion and glucose tolerance in rats.

We studied the effects of a natural sodium alginate (isolated from Laminaria angustata Kjellman var. longissima Miyabe, Phaeophyceae) (average molecular weight: 2700 kDa; AG-270) and three water-soluble low-molecular weight sodium alginates (average molecular weights, 10, 50 and 100 kDa; AG-1, AG 5, and AG-10, respectively) on cholesterol excretion and glucose tolerance in rats. AG-270, AG-5 and AG-10 enhanced cholesterol excretion into faeces. AG-270 and AG-10 inhibited blood glucose and insulin levels from rising 30 min after glucose administration. AG-5 inhibited the blood glucose level from rising 30 and 60 min after glucose administration, without affecting blood insulin levels. AG-1 had no effect on cholesterol excretion or glucose tolerance. These findings suggest that the effects of the natural sodium alginate and AG-5 and AG-10 on cholesterol excretion and glucose tolerance may be due to the inhibition of cholesterol and glucose absorption from the small intestine by the gelling of the free alginic acid converted in the stomach. These experimental results indicate that the low-molecular weight sodium alginates, AG-5 and AG-10, should be useful as dietary fibers for the prevention of obesity, hypercholesterolemia, and diabetes.

Administration, Oral↗

PubMed1989

Scavenging by alginate of free radicals released by macrophages.

Failure to eradicate mucoid forms of P. aeruginosa has implicated bacterial alginate in a local evasion of host defence mechanisms within the lung of Cystic Fibrosis (CF) patients. We have found that purified bacterial alginate scavenges free radicals released by triggered macrophages as detected by lucigenin amplified chemiluminescence (CL) and reduction of cytochrome c. In agreement with this, alginate was also able to scavenge radicals generated by a chemical system (hydrogen peroxide and copper; detected by benzoate hydroxylation and chemiluminescence), and by an enzymatic system (hypoxanthine and xanthine oxidase; detected by chemiluminescence). All inhibitions were dose-related. Oxygen consumption by neutrophils (unlike that of macrophages) could be detected in a Clark electrode, and was not reduced by alginate, confirming that scavenging of radicals was responsible for the earlier observations. These data suggest that bacterial alginate by scavenging free radicals, may favour the survival of mucoid forms of P. aeruginosa, particularly in the CF lung.

Alginates↗

PubMedDate not supplied

Development of alginate wound dressings linked with hybrid peptides derived from laminin and elastin.

We designed hybrid peptides, SIRVXVXPG (X: A or G), from a laminin-derived peptide, SIKVAV, and an elastin-derived peptide, VGVAPG, and tried to develop new alginate dressings linked covalently with the hybrid peptides. First, we examined the effectiveness of the hybrid peptides for cell attachment and proliferation using normal human dermal fibroblasts (NHDF) in vitro. The hybrid peptides promoted attachment of NHDF, whereas neither Ac-KSIKVAV nor Ac-KVGVAPG promoted attachment. Although all the peptides we examined promoted the proliferation of NHDF to some extent, the hybrid peptide-coated plates showed strong NHDF proliferative activity, compared with the other peptide. Next, we created alginate dressings linked with some of these peptides and examined their effectiveness in wound healing using a rabbit ear skin defect model in vivo. Nine days after operation, ears with the alginate dressings linked with the hybrid peptides showed significantly greater epithelialization and a larger volume of regenerated tissue compared to those treated with SIVAV-linked, VGVAPG-linked and unlinked alginate dressings. These new alginate dressings linked with the hybrid peptides could be promising dressings especially for wounds with impaired healing.

Alginates↗

PubMed2005

Chitosan-alginate hybrid scaffolds for bone tissue engineering.

A biodegradable scaffold in tissue engineering serves as a temporary skeleton to accommodate and stimulate new tissue growth. Here we report on the development of a biodegradable porous scaffold made from naturally derived chitosan and alginate polymers with significantly improved mechanical and biological properties as compared to its chitosan counterpart. Enhanced mechanical properties were attributable to the formation of a complex structure of chitosan and alginate. Bone-forming osteoblasts readily attached to the chitosan-alginate scaffold, proliferated well, and deposited calcified matrix. The in vivo study showed that the hybrid scaffold had a high degree of tissue compatibility. Calcium deposition occurred as early as the fourth week after implantation. The chitosan-alginate scaffold can be prepared from solutions of physiological pH, which may provide a favorable environment for incorporating proteins with less risk of denaturation. Coacervation of chitosan and alginate combined with liquid-solid separation provides a scaffold with high porosity, and mechanical and biological properties suitable for rapid advancement into clinical trials.

Alginates↗

PubMed2006

Reversed chitosan-alginate polyelectrolyte complex for stability improvement of alpha-amylase: optimization and physicochemical characterization.

The present work explores, using response surface methodology, the main and interaction effects of some process variables on the preparation of a reversed chitosan-alginate polyelectrolyte complex (PEC) with entrapped alpha-amylase for stability improvement. A 3(3) full factorial design was used to investigate the effect of the chitosan and alginate concentrations and hardening time on the percent entrapment, time required for 50% (T(50)) and 90% (T(90)) enzyme release, and particle size. The beads were prepared by dropping chitosan containing alpha-amylase into a sodium alginate solution without any salt. The in vitro enzyme release profile of the beads was fitted to various release kinetics models to study the release mechanism. A topographical characterization was carried out using scanning electron microscopy (SEM), and the entrapment was confirmed using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). Stability testing was carried out according to the International Conference on Harmonization (ICH) guidelines for zones III and IV. Beads prepared using 2.5%w/v chitosan and 3%w/v sodium alginate with a hardening time of 60 min had more than 90% entrapment and a T(90) value greater than 48 min. Moreover, the shelf-life of the enzyme-loaded beads was found to increase to 3.68 years, compared with 0.99 years for the conventional formulation. It can be inferred that the proposed methodology can be used to prepare a reversed PEC of chitosan and alginate with good mechanical strength, provided both the reactants are in a completely ionized form at the time of the reaction. Proper selection of the reaction pH, polymer concentration and hence charge density, and hardening time is important and determines the characteristics of the PEC.

Alginates↗

PubMed2006

Extended release of high pI proteins from alginate microspheres via a novel encapsulation technique.

Alginate has potential as a matrix for controlled delivery of protein-based drugs that require site-specific long-term delivery. In the current work albumin, lysozyme and chymotrypsin were encapsulated into alginate microspheres using a novel method that involved soaking the microspheres in a protein-containing NaCl solution. This was followed by recrosslinking with calcium chloride. High pI proteins also appeared to physically crosslink the sodium alginate which resulted in more sustained release. Release was affected by the nature of the releasate solution. In TRIS buffered saline, the high pI proteins chymotrypsin and lysozyme showed sustained release lasting over 150 h. Release into 0.15% NaCl led to relatively constant release of lysozyme and chymotrypsin over more than 2000 h; reduction of the releasate volume lengthened the lysozyme release to greater than 8 months. Released lysozyme was shown to remain active for at least 16 days, in some cases with activity greater than 100% of the active control. This encapsulation technique can therefore be used to rapidly load alginate microspheres with proteins, with high isoelectric point proteins showing particular promise. Furthermore, the interactions between the high pI proteins and the alginate gel could potentially be exploited to generate new protein delivery systems.

Alginates↗