High Mannuronic Acid Content Research Articles

Biomedical-grade, high mannuronic acid content (BioMVM) alginate enhances the proteoglycan production of primary human meniscal fibrochondrocytes in a 3-D microenvironment.

Alginates are important hydrogels for meniscus tissue engineering as they support the meniscal fibrochondrocyte phenotype and proteoglycan production, the extracellular matrix (ECM) component chiefly responsible for its viscoelastic properties. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to provide the best three-dimensional (3-D) microenvironment and to support proteoglycan synthesis of encapsulated human meniscal fibrochondrocytes in vitro. Biomedical-grade, high mannuronic acid alginate spheres (BioLVM, BioMVM) were the most uniform in size, indicating an effect of the purity of alginate on the shape of the spheres. Interestingly, the purity of alginates did not affect cell viability. Of note, only fibrochondrocytes encapsulated in BioMVM alginate produced and retained significant amounts of proteoglycans. Following transplantation in an explant culture model, the alginate spheres containing fibrochondrocytes remained in close proximity with the meniscal tissue adjacent to the defect. The results reveal a promising role of BioMVM alginate to enhance the proteoglycan production of primary human meniscal fibrochondrocytes in a 3-D hydrogel microenvironment. These findings have significant implications for cell-based translational studies aiming at restoring lost meniscal tissue in regions containing high amounts of proteoglycans.

The effects of alginate composition on the behavior of encapsulated HepG2 cells

The purpose of this study was to evaluate the effects of alginate composition within microcapsules on the viability, gene expression, and albumin secretion of encapsulated hepatocarcinoma cells (HepG2). Two compositions were tested: a high guluronic acid content (64% in G) and a high mannuronic acid content (67% in M). In both alginates, HepG2 cells formed multi-cellular aggregates which exhibited a high degree of cell–cell contacts. Compared with cells encapsulated in alginates with a high M content, HepG2 cells encapsulated in alginates with a high G content experienced a low speed of proliferation and viability due to the growth inhibition of the alginate matrix. Our data also demonstrated that an increasing in the content of high G content reduced the albumin secretion and genes expression involved in liver-specific functions. These observations indicated that alginates with a high M content were favorable matrix used for liver tissue engineering.

In VitroandIn VivoCharacterization of Nonbiomedical- and Biomedical-Grade Alginates for Articular Chondrocyte Transplantation

Alginate is a key hydrogel for cartilage tissue engineering. Here, we systematically evaluated four biomedical- and two nonbiomedical-grade alginates for their capacity to support the in vitro culture and in vivo transplantation of articular chondrocytes. Chondrocytes in all ultrapure alginates maintained high cell viability. Spheres composed of biomedical-grade, low-viscosity, high-mannuronic acid content alginate showed the lowest decrease in size over time. Biomedical-grade, low-viscosity, high-guluronic acid content alginate allowed for optimal cell proliferation. Biomedical-grade, medium-viscosity, high-mannuronic acid content alginate promoted the highest production of proteoglycans. When transplanted into osteochondral defects in the knee joint of sheep in vivo, empty spheres were progressively surrounded by a granulation tissue. In marked contrast with these observations, all alginate spheres carrying allogeneic chondrocytes were gradually invaded by a granulation tissue containing multinucleated giant cells, lymphocytes, and fibroblasts, regardless whether they were based on biomedical- or nonbiomedical-grade alginates. After 21 days in vivo, transplanted chondrocytes were either viable or underwent necrosis, and apoptosis played a minor role in their early fate. The individual characteristics of these alginates may be valuable to tailor specific experimental and clinical strategies for cartilage tissue engineering.

Multilayered microcapsules for the sustained-release of angiogenic proteins from encapsulated cells

BackgroundMultilayered alginate microcapsules with a permselective poly-L-ornithine membrane can be used for the dual purpose of encapsulating cells in the inner core and sustained release of angiogenic proteins from the outer layer. The aim of this study was to examine the encapsulation and release of a novel chimeric form of fibroblast growth factor–1 (FGF-1) from the outer layer of alginate microcapsules. MethodsHeparin-binding growth-associated molecule bound to FGF-1 (HB-GAM/FGF-1) was encapsulated in the outer layer of multilayered alginate microbeads constructed using varying alginate conditions. The encapsulation and release of the chimera was quantified. ResultsThe outer layer was able to encapsulate and release HB-GAM/FGF-1 for up to 30 days. The outer layer made with 1% alginate of high mannuronic acid content provided the fastest release, while 1.25% high guluronic acid content alginate displayed the longest duration of release. ConclusionsThe outer layer of multilayered alginate microbeads can be used for the encapsulation and long-term release of HB-GAM/FGF-1.

Development of polysaccharide gel-coated pellets for oral administration: 2. Calcium alginate

Calcium alginate gel-coated pellets were developed by forming an insoluble gel coat on extruded–spheronized pellets by interfacial complexation. Experiments were designed to investigate the effect of pellet size, alginate type, alginate concentration, and dissolution medium on swelling and drug release behavior. Low swelling in acidic media was related to proton–calcium ion exchange forming insoluble acid gels. In contrast, partial formation of soluble sodium alginate in 0.1 M NaCl induced water uptake, resulting in greater swelling. Drug release from coated pellets showed a lag time when the gel coat hydrated and swelled, followed by a zero-order release. Significantly slower release was observed when either the pellet size or the alginate concentration was increased. Alginate with high guluronic acid content gave the slowest release. Different types of alginate with high mannuronic acid content showed different release behaviors that are probably due to the different monomer sequences and botanical sources. The faster drug release in acidic media and 0.1 M NaCl compared to water is probably due to reduced calcium cross-linking in the gel. These results suggest that the pellet size, alginate type and concentration and dissolution medium influenced the swelling and drug release behavior of calcium alginate gel-coated pellets.

The role of the CaCl 2–guluronic acid interaction on alginate encapsulated βTC3 cells

Previously we demonstrated that alginate composition has a significant effect on the growth of encapsulated βTC3 cells and consequently on the overall metabolic and secretory activities of the encapsulated cultures. Based on these results we postulated that the mechanical properties of alginate were not responsible for the observed effects but rather, changes in the strength of the alginate gel network caused by changes in the number of alginate strands held together in the “egg-box” model are responsible for the observed effects. In this study we address this hypothesis with a series of experiments in which the strength of this interaction is manipulated by varying the calcium concentration either at the time of gelation or during culture maintenance. Our data show that increasing the concentration of the CaCl 2 solution used at the time of gelation, thus increasing the strength of the alginate gel network, impedes the growth characteristics of βTC3 cells encapsulated in a high guluronic acid content alginate. This effect is amplified by maintaining a constant supply of calcium ions during culture thus sustaining the interaction between guluronic acid residues and calcium ions. However, preparations of βTC3 cells encapsulated in an alginate with high mannuronic acid content are not affected by changes in CaCl 2 concentration due to the low percentage of consecutive guluronic acid residues. Therefore, the present data show that the strength of the alginate gel network is an important factor that influences the growth characteristics of encapsulated cell preparations.

The effects of alginate composition on encapsulated βTC3 cells

The effects of alginate composition on the growth of murine insulinoma βTC3 cells encapsulated in alginate/poly- l-lysine/alginate (APA) beads, and on the overall metabolic and secretory characteristics of the encapsulated cell system, were investigated for four different types of alginate. Two of the alginates used had a high guluronic acid content (73% in guluronic acid residues) with varying molecular weight, while the other two had a high mannuronic acid content (68% in mannuronic acid residues) with varying molecular weight. Each composition was tested using two different polymer concentrations. Our data show that βTC3 cells encapsulated in alginates with a high guluronic acid content experienced a transient hindrance in their metabolic and secretory activity because of growth inhibition. Conversely, βTC3 cells encapsulated in alginates with a high mannuronic acid content experienced a rapid increase in metabolic and secretory activity as a result of rapid cell growth. Our data also demonstrate that an increase in either molecular weight or concentration of high mannuronic acid alginates did not alter the behavior of the encapsulated βTC3 cells. Conversely, an increase in molecular weight and concentration of high guluronic acid alginates prolonged the hindrance of glucose metabolism, insulin secretion and cell growth. These observations can be best interpreted by changes in the microstructure of the alginate matrix, i.e., interaction between the contiguous guluronic acid residues and the Ca 2+ ions, as a result of the different compositions.

The effects of microencapsulation on pancreatic islet osmotically induced volumetric response.

Microencapsulation of pancreatic islets has been proposed as a means to prevent allograft rejection and to protect islets during cryopreservation. The aim of this study was to investigate: 1) the effects of the cryoprotectants (CPAs) dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the volume of Ca2+ alginate microcapsules, and 2) the effects of microencapsulation on the volumetric response of human and canine pancreatic islets during CPA equilibration. Stock sodium alginate with a high mannuronic acid content (HM) or a high guluronic acid content (HG) was used to generate empty capsules (mean diameter 200 microm) with an electrostatic generator. The capsules were held in place by a holding pipette system and videotaped during the addition of 2 or 3 M CPA at 22 degrees C. Islets (isolated from human cadaveric donors and mongrel dogs and then cultured overnight at 37 degrees C) were encapsulated in alginate (HM), loaded into a microperfusion chamber, and the change in islet volume was videotaped after exposure to the same CPAs and concentrations. These were compared to the volume responses of nonencapsulated islets. Images were analyzed using a computerized image analysis system and the data were analyzed using ANOVA. HG microcapsules showed a significant (p < 0.05) increase in volume following exposure to EG but not to DMSO. HM microcapsule volume did not change significantly following exposure to either EG or DMSO and was therefore chosen as the substrate for islet encapsulation. Free, nonencapsulated canine and human islets responded to the osmotic challenge of the 2 M DMSO by shrinking to 70.00 +/- 1.04% (mean +/- SEM) and 70.11 +/- 1.05%, and in 2 M EG to 72.89 +/- 1.93% and 69.33 +/- 1.38%, respectively, of the isotonic volume before returning to the original cell volume. Exposure to 3 M DMSO or EG resulted in a further dehydration to 65.89 +/- 0.91% and 67.67 +/- 1.91% for canine and 62.22 +/- 0.66.% or 65.89 +/- 1.30% for human islets. Minimum volumes were reached within 30-40 s after exposure to the cryoprotectant. Encapsulated human islets reached 86.88 +/- 1.47% of their original volume in 2 M and 80.33 +/- 0.89% in 3 M DMSO, and 87.33 +/- 1.86% in 2 M and 82.80 +/- 1.57% in 3 M EG. This volume change was significantly less (p < 0.01) than that observed in corresponding free islets. Encapsulated canine islets reached 83.67 +/- 2.13% of their original volume in 2 M and 78.22 +/- 0.95% in 3 M DMSO, and 85.44 +/- 1.92% in 2 M and 78.11 +/- 2.01% in 3 M EG. As with human islets, this was significantly different than free islets (p < 0.01). These minimal volumes were reached within 30-50 s. These results demonstrate that there are cryoprotectant and alginate-specific interactions and that microencapsulation modulates the degree of osmotically induced shrinkage of islets. The development or modification of existing cryopreservation protocols to improve postcryopreservation recovery or function must account for these factors.

Alginate polylysine microcapsules as immune barrier: permeability of cytokines and immunoglobulins over the capsule membrane.

Transplantation of pancreatic islets in alginate polylysine microcapsules is a potential useful method for treating type I diabetes. In this study, the permeability for alginate-polylysine microcapsules to cytokines an immunoglobulines has been investigated by a newly developed method. Magnetic monodisperse polymer particles (Dynabeads) coated with antibodies against selected proteins were encapsulated in 0.7 mm alginate polylysine microcapsules. The capsule membrane permeability to IgG (150 kDa), Transferrin (81 kDa), Tumor necrosis factor (TNF, 51 kDa), Interleukin-1 beta (IL-1 beta, 17.5 kDa), and insulin (5.8 kDa) was estimated by measuring the binding of 125I-labeled proteins to the encapsulated antibody coated Dynabeads. Capsules with an inhomogeneous solid gel core were made of alginates with high guluronic or high mannuronic acid content and poly-L (PLL)- or poly-D-lysine (PDL) of concentrations varied from 0.05-0.2%. The various capsules examined were all impermeable to IgG. The capsules made with a PLL-, but not PDL-membranes were permeable for transferrin. IL-1 beta was found to penetrate all of the different capsule types. The high-G capsules, however, could be made impermeable to TNF and still allowed transferrin to pass. The permeability of these capsules to IL-1 beta, but not to TNF was confirmed in an assay where mouse islets of Langerhans were incubated with TNF and IL-1 beta, and comparing the IL-6 for encapsulated and non-encapsulated islets.

Alginate polylysine microcapsules as immune barrier: Permeability of cytokines and immunoglobulins over the capsule membrane

Transplantation of pancreatic islets in alginate polylysine microcapsules is a potential useful method for treating type I diabetes. In this study, the permeability for alginate-polylysine microcapsules to cytokines an immunoglobulines has been investigated by a newly developed method. Magnetic monodisperse polymer particles (Dynabeads) coated with antibodies against selected proteins were encapsulated in 0.7 mm alginate polylysine microcapsules. The capsule membrane permeability to IgG (150 kDa), Transferrin (81 kDa), Tumor necrosis factor (TNF, 51 kDa), Interleukin-1β (IL-1β, 17.5 kDa), and insulin (5.8 kDa) was estimated by measuring the binding of 125I-labeled proteins to the encapsulated antibody coated Dynabeads. Capsules with an inhomogeneous solid gel core were made of alginates with high guluronic or high mannuronic acid content and poly- l (PLL)- or poly- d-lysine (PDL) of concentrations varied from 0.05–0.2%. The various capsules examined were all impermeable to IgG. The capsules made with a PLL-, but not PDL-membranes were permeable for transferrin. IL-1β was found to penetrate all of the different capsule types. The high-G capsules, however, could be made impermeable to TNF and still allowed transferrin to pass. The permeability of these capsules to IL-1β, but not to TNF was confirmed in an assay where mouse islets of Langerhans were incubated with TNF and IL-1β, and comparing the IL-6 for encapsulated and nonencapsulated islets.

Biocompatibility of mannuronic acid-rich alginates

Highly purified algin preparations free of adverse contaminants with endotoxins and other mitogens recently became available by a new purification process (Klöck et al., Appl. Microbiol. Biotechnol., 1994, 40, 638–643). An advantage of this purification protocol is that it can be applied to alginates with various ratios of mannuronic acid to guluronic acid. High mannuronic acid alginate capsules are of particular practical interest for cell transplantation and for biohybrid organs, because mannuronate-rich alginates are usually less viscous, allowing one to make gels with a higher alginate content. This will increase their stability and reduce the diffusion permeability and could therefore protect immobilized cells more efficiently against the host immune system. Here we report the biocompatibility of purified, mannuronic acid-rich alginate (68% mannuronate residues) in a series of in vitro, as well as in vivo, assays. In contrast to raw alginate extracts, the purified product showed no mitogenic activity towards murine lymphocytes in vitro. Its endotoxin content was reduced to the level of the solvent. Animal studies with these new, purified algin formulations revealed the absence of a mitogen-induced foreign body reaction, even when the purified material (after cross-linking with Ba2+ ions) is implanted into animal models with elevated macrophage activity (diabetes-prone BB/OK rat). Thus, alginate capsules with high mannuronic acid content become available for applications such as implantation. In addition to the utilization as implantable cell reactors in therapy and biotechnology, these purified algins have broad application potential as ocular fillings, tissue replacements, microencapsulated growth factors and/or interleukins or slow-release dosage forms of antibodies, surface coatings of sensors and other invasive medical devices, and in encapsulation of genetically engineered cells for gene therapy.

Influence of Alginate Polysaccharide Composition and Culture Conditions on Chondrocytes in Three-Dimensional Culture

Alginate-embedded chondrocytes have been used for experimental analysis of cartilage matrix metabolism and as a potential bioartificial system for repairing cartilage defects. Alginates are linear copolymers composed of 1,4-linked beta-n-mannuronic acid and 1,4 linked alpha-L-guluronic acid, which occur as regions made up exclusively of one unit or the other, or as regions in which the monomers approximate an alternating sequence. Data presented here demonstrate that the mannuronic to guluronic acid (M/G) ratio and molecular weight of the alginate utilized effects the tissue-culture handling properties of the resultant gel and the activity of embedded chondrocytes. In experiments comparing chondrocyte survival and matrix synthesis, optimal conditions were obtained with an alginate of both high mannuronic acid content and high molecular weight. Chondrocytes survived and proliferated when maintained in unsupplemented media, in media supplemented with fetal calf serum, and in media supplemented with the defined serum replacement ITS+ (6.25 microg/ml insulin, 6.25 microg/ml transferrin, 6.25 ng/ml selenous acid, 1.25 mg/ml bovine serum albumin, 5.35 microg/ml linoleic acid). High cell survival rate and increase in cell number was obtained in the absence of serum. In contrast, long-term matrix synthesis and deposition required media supplementation as indicated by uptake of [(35)S]sulfate into glycosaminoglycans and by immunofluorescence using antibodies specific for cartilage matrix molecules.

Alginases from Azotobacter species

Alginate lyases (alginases) have been prepared from strains of Azotobacter vinelandii and Azotobacter chroococcum in which they were located in the periplasm. The enzymes are present in wild-type strains of each species and in mutants failing to encyst or produce bacterial alginate. The lyases have been partially purified by ion exchange chromatography and by affinity chromatography on a matrix prepared from poly-D-mannuronic acid. Although several bacterial and algal alginate preparations were degraded by the enzymes, highest activity was found on poly-D-mannuronic acid or on algal alginates with high mannuronic acid content. The major product from enzymes of either bacterium was an unsaturated uronic acid, when either alginates or poly-D-mannuronic acid were used as substrates. When tested against a series of algal alginates of increasing D-mannuronic acid content, the enzyme activity was highest against alginates of high D-mannuronic acid content, indicating that the enzymes are endo-D-mannurono-lyases. The alginases from the two bacterial species are not identical in their substrate specificity although both show the same generalized type of action.