Biomimetic sulphated alginate hydrogels suppress IL-1ß-induced inflammatory responses in human chondrocytes.

Loss of articular cartilage from ageing, injury or degenerative disease is commonly associated with inflammation, causing pain and accelerating degradation of the cartilage matrix. Sulphated glycosaminoglycans (GAGs) are involved in the regulation of immune responses in vivo, and analogous polysaccharides are currently being evaluated for tissue engineering matrices to form a biomimetic environment promoting tissue growth while suppressing inflammatory and catabolic activities. Here, we characterise physical properties of sulphated alginate (S-Alg) gels for use in cartilage engineering scaffolds, and study their anti-inflammatory effects on encapsulated chondrocytes stimulated with IL-1ß. Sulphation resulted in decreased storage modulus and increased swelling of alginate gels, whereas mixing highly sulphated alginate with unmodified alginate resulted in improved mechanical properties compared to gels from pure S-Alg. S-Alg gels showed extensive anti-inflammatory and anti-catabolic effects on encapsulated chondrocytes induced by IL-1ß. Cytokine-stimulated gene expression of pro-inflammatory markers IL-6, IL-8, COX-2 and aggrecanase ADAMTS-5 were significantly lower in the sulphated gels compared to unmodified alginate gels. Moreover, sulphation of the microenvironment suppressed the protein expression of COX-2 and NF-κB as well as the activation of NF-κB and p38-MAPK. The sulphated alginate matrices were found to interact with IL-1ß, and proposed to inhibit inflammatory induction by sequestering cytokines from their receptors. This study shows promising potential for sulphated alginates in biomimetic tissue engineering scaffolds, by reducing cytokine-mediated inflammation and providing a protective microenvironment for encapsulated cells.

Mechanical properties of C-5 epimerized alginates.

There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Young's modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.

Molecular engineering as an approach to design new functional properties of alginate.

Through enzymatic modification, we are now able to manipulate the composition and sequential nanostructures of alginate, one of the most versatile gelling polymers found in nature. Here we report the application of a set of processive polymer-modifying epimerases for the preparation of novel alginates with highly improved functional properties essential for numerous applications as gel matrices. Gels of enzymatically engineered alginate were found to be more elastic and compact, less permeable, and extremely stable under physiological conditions, offering significant advantages over native alginates. As a result, this study shows that, by controlling alginate nanostructure, its macroscopic properties can be highly controlled. The ability to tailor alginate has a great impact on the wide use of this biomaterial in industry and medicine. More importantly, this adds more knowledge to the link between polymer nanostructure and macroscopic properties and may serve as a model system for other polymer-based materials.

Evaluation of different types of alginate microcapsules as bioreactors for producing endostatin.

The use of nonautologous cell lines producing a therapeutic substance encapsulated within alginate microcapsules could be an alternative way of treating different diseases in a cost-effective way. Malignant brain tumors have been proposed to be treated locally using engineered cells secreting proteins with therapeutic potential encapsulated within alginate microcapsules. Optimization of the alginate capsule bioreactors is needed before this treatment can be a reality. Recently, we have demonstrated that alginate-poly-L-lysine microcapsules made with high-G alginate and a gelled core disintegrated as cells proliferated. In this study we examined the growth and endostatin secretion of 293-EBNA (293 endo) cells encapsulated in six different alginate microcapsules made with native high-G alginate or enzymatically tailored alginate. Stability studies using an osmotic pressure test showed that alginate-poly-L-lysine-alginate microcapsules made with enzymatically tailored alginate was mechanically stronger than alginate capsules made with native high-G alginate. Growth studies showed that the proliferation of 293 endo cells was diminished in microcapsules made with enzymatically tailored alginate and gelled in a barium solution. Secretion of endostatin was detected in lower amounts from the enzymatically tailored alginate microcapsules compared with the native alginate microcapsules. The stability of the alginate microcapsules diminished as the 293 endo cells grew inside the capsules, while empty alginate microcapsules remained stable. By using microcapsules made of fluorescenamine-labeled alginate it was clearly visualized that cells perforated the alginate microcapsules as they grew, destroying the alginate network. Soluble fluorescence-labeled alginate was taken up by the 293 endo cells, while alginate was not detected in live spheroids within fluorescence-labeled alginate microcapsules. Despite that increased stability was achieved by using enzymatically tailored alginate, the cell proliferation destroyed the alginate microcapsules with time. It is therefore necessary to use cell lines that have properties more suited for alginate encapsulation before this technology can be used for therapy.

Enzymatic epimerization of bacterial mannuronan and of C-6 oxidized, galactose-depleted guar: a circular dichroism and 1H NMR study.

Attention has been focused on two uronans, namely, mannuronan and galactose-depleted C-6 oxidized guar, the former of microbial origin and the latter of artificial nature, to provide original data on the extent of epimerization they can undergo in dilute aqueous solution using two C-5 mannuronic acid epimerizing enzymes, that is, AlgE-4 and AlgE-6, alone or in admixture. Original circular dichroism data coupled with 1H NMR spectra clearly point out that both uronans can be epimerized, depending on the enzyme or enzyme mixture used, to high levels yielding guluronic-rich alginate samples and guluronic-rich heteropolysaccharides, respectively. Mannuronan and its epimerization products can easily form clear, firm aqueous gels when an excess of HCl is added or when mixed with aqueous CaCl2, respectively. Depleted-guarox does not gel upon addition of excess HCl, while the heterouronan derived from it having a percent of epimerization nearly identical to that of epimerized mannuronan, that is, ca. 70%, can form gel in the presence of Ca(II) only at higher polymer and Ca(II) concentrations. With the latter, heterouronan alpha-D-galacturonic side groups exert hindrance to "junction zone" formation.

The catalytic activities of the bifunctional Azotobacter vinelandii mannuronan C-5-epimerase and alginate lyase AlgE7 probably originate from the same active site in the enzyme.

The Azotobacter vinelandii genome encodes a family of seven secreted Ca(2+)-dependent epimerases (AlgE1--7) catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. AlgE1--7 are composed of two types of protein modules, A and R, and the A-modules have previously been found to be sufficient for epimerization. AlgE7 is both an epimerase and an alginase, and here we show that the lyase activity is Ca(2+)-dependent and also responds similarly to the epimerases in the presence of other divalent cations. The AlgE7 lyase degraded M-rich alginates and a relatively G-rich alginate from the brown algae Macrocystis pyrifera most effectively, producing oligomers of 4 (mannuronan) to 7 units. The sequences cleaved were mainly G/MM and/or G/GM. Since G-moieties dominated at the reducing ends even when mannuronan was used as substrate, the AlgE7 epimerase probably stimulates the lyase pathway, indicating a complex interplay between the two activities. A truncated form of AlgE1 (AlgE1-1) was converted to a combined epimerase and lyase by replacing the 5'-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from algE7. Furthermore, substitution of an aspartic acid residue at position 152 with glycine in AlgE7A eliminated almost all of both the lyase and epimerase activities. Epimerization and lyase activity are believed to be mechanistically related, and the results reported here strongly support this hypothesis by suggesting that the same enzymatic site can catalyze both reactions.

Influence of environmental conditions on the activity of the recombinant mannuronan C-5-epimerase AlgE2.

The mannuronan C-5-epimerase AlgE2 is one of a family of Ca(2+)-dependent epimerases secreted by Azotobacter vinelandii. These enzymes catalyze the conversion of beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid residues (G) in alginate. AlgE2 had a pH optimum between 6.5 and 7 and a temperature optimum around 55 degrees C. Addition of low molecular weight organic compounds, including buffers, amino acids and osmoprotective compounds, affected the activity of the enzyme. The charge, size and stereochemistry of the added compounds were important. The activity of AlgE2, dissolved in various buffers (same pH), decreased with increasing fraction of positively charged buffer ions. Mono- and divalent metal ions also influenced the activity. When Ca(2+) was omitted only Sr(2+), of the metal ions tested, supported some activity of AlgE2. At high concentration of Ca(2+) (3.3 mM) these ions had a negative effect on the activity, whereas at low Ca(2+) concentration (0.58 mM) the activity was enhanced by addition of Sr(2+), and to some degree also by addition of Mg(2+) and Mn(2+). During epimerization AlgE2 occasionally causes cleavage of the alginate chain. These chain breaks could not be prevented by changes in the conditions during the epimerization. The composition and sequential structure of epimerized alginate was not altered by changes in the epimerization conditions.

Transplantation of alginate microcapsules with proliferating cells in mice: capsular overgrowth and survival of encapsulated cells of mice and human origin.

Alginate microcapsules may be used to encapsulate therapeutic cells and, thereby, to protect them from the host immune system. Both the biomaterial, as well as the therapeutic cells, may give rise to immunological reactions. We have developed methods that are useful in the study of capsule biocompatibility, as well as reactions against the grafts. These imply investigation of the survival of the encapsulated cells as well as fibrotic reactions against the microcapsules. Studies were performed in Balb/c mice with empty alginate-PLL-alginate microcapsules as well as microcapsules containing cells of human or mouse origin. Confocal laser scanning microscopy (CLSM) was used to visualize live and dead cells within the microcapsules and to define some of the cells involved in the fibrotic reaction against the microcapsules. In both grafts, live cells were detected seven days after transplantation. Minor fibrotic reactions were found against empty alginate-PLL-alginate microcapsules and to microcapsules containing mouse cells. An extensive fibrotic reaction was found one week after transplantation against microcapsules containing human cells, and the secretion of therapeutic protein endostatin had ceased. Fibroblasts and macrophages were involved in the fibrotic reaction against the xenograft.

Molecular weight dependency on the production of the TNF stimulated by fractions of rye (1-->3),(1-->4)-beta-D-glucan.

Mixed-linkage (1-->3),(1-->4)-beta-D-glucan with a weight average molecular weight varying between 79,800 and 13,900 was purified from rye. These fractions were used for stimulation of human monocytes to produce tumour necrosis factor (TNF). A mixed-linkage beta-glucan with a weight average molecular weight of 18,900 was found to be the most potent immunostimulator.

Involvement of CD14 and beta2-integrins in activating cells with soluble and particulate lipopolysaccharides and mannuronic acid polymers.

Lipopolysaccharide (LPS) and related bacterial products can be recognized by host inflammatory cells in a particulate, bacterium-bound form, as well as in various soluble, released forms. In the present study we have compared the mechanisms used by LPS, detoxified LPS (DLPS), and mannuronic acid polymers (M-polymers), in solution or covalently linked to particles, in stimulating monocytes to tumor necrosis factor (TNF) production. The addition of recombinant LPS binding protein (LBP) and/or soluble CD14 (sCD14) enhanced the production of TNF from monocytes stimulated with soluble LPS, DLPS, or M-polymer, but did not affect the response to M-polymer or DLPS attached to particles. Treatment of monocytes with antibody to CD14, CD18, or CD11b showed that CD14, but not CR3 (CD11b/CD18), mediated monocyte TNF production in response to the soluble antigens. In contrast, anti-CD14, anti-CD11b and anti-CD18 monoclonal antibodies all inhibited the response to the particulate stimuli. On the other hand, B975, a synthetic analog of Rhodobacter capsulatus lipid A, completely abrogated the monocyte TNF response induced by LPS but did not affect the TNF induction by DLPS or M-polymer, either in soluble or particulate forms. These data demonstrate that the engagement of immune receptors by bacterial products such as LPS, DLPS, and M-polymer is dependent upon the presentation form of their constituent carbohydrates, and that factors such as aggregation state, acylation, carbohydrate chain length, and solid versus liquid phase of bacterial ligands influence the mechanisms used by cells in mediating proinflammatory responses.

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

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

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

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

Epimerization of nonnatural uronans with mannuronan C-5-epimerases to obtain alginatelike polysaccharides.

Different polysaccharides containing D-mannose residues have been C-6-oxidized by a selective TEMPO-mediated hypohalite oxidation to obtain the corresponding uronans. These have been treated with various recombinant mannuronan C-5-epimerases and the resulting products were analyzed by 1H NMR spectroscopy. Oxidized konjac mannan could be epimerized to obtain a uronan with a content of about 12% alpha-L-gulopyranuronate (G) residues. On prolonged epimerization, beta-elimination was observed. The oxidized galactomannan locust bean gum could only be scarcely epimerized, probably due to steric effects exerted by its 26% alpha-D-galacturopyranosyl side groups. Oxidized, galactose-depleted guar gum with a alpha-D-galactosyl content of 11% could be epimerized to a G content of about 15%. With oxidized cellulose as a substrate, mainly beta-elimination was observed. It thus seems that the mannuronan C-5-epimerases employed recognize glucuronate residues and abstract proton-5 but are unable to perform the second epimerization step and instead yield beta-eliminated products.

Microcapsules of alginate-chitosan. II. A study of capsule stability and permeability.

The stability of alginate-chitosan capsules was shown to depend strongly on the amount of chitosan bound to the capsules. When the capsules were made by dropping a solution of sodium alginate into a chitosan solution (one-stage procedure), all the chitosan was located in a thin alginate-chitosan membrane on the surface. These capsules were much weaker than the capsules made by reacting calcium alginate beads in an aqueous solution of chitosan and calcium chloride (two-stage procedure). Capsules with high mechanical strength were obtained after shorter reaction times when the number-average molecular weight of the chitosan was reduced to around 15,000, when the capsules were made more homogeneous and when the capsule diameter was reduced to around 300 microm. When these capsules were treated with calcium sequestrant such as citrate under conditions where calcium alginate gels normally dissolve, they still had a gel core indicating the presence of chitosan throughout the capsule matrix. The permeability of the two-stage capsules was reduced when the chitosan molecular weight was increased and the degree of acetylation was increased, and when the capsules were made more inhomogeneous. The addition of another several layers of alginate and chitosan resulted in capsules virtually impermeable to IgG, suggesting an average capsule pore diameter less than 90 A.

Transplantation of alginate microcapsules: generation of antibodies against alginates and encapsulated porcine islet-like cell clusters.

BACKGROUND: Microencapsulation of islets of Langherhans in alginate poly-L-lysine capsules provides an effective protection against cell-mediated immune destruction, and ideally should allow the transplantation of islets in the absence of immunosuppression. It has previously been suggested that alginate rich in mannuronic acid (high M) is more immunogenic than alginate rich in guluronic acid (high G). The ability of these alginates to induce an antibody response in the recipient or act as an adjuvant to antibody responses against antigens leaked from the capsule was investigated in the present study. METHODS: Empty capsules made from these different types of alginate were transplanted intraperitoneally to Wistar rats or Balb/c mice. In addition, some animals were also injected with bovine serum albumin to assess the ability of the alginates to act as an adjuvant to this antigen. Antibody responses to intraperitoneally transplanted free and microencapsulated fetal porcine islet like cell clusters (ICC) were also evaluated, in animals treated with or without cyclosporine. RESULTS: Antibodies against high M-alginate capsules were detected in the sera of mice transplanted with this capsule type. However, this response was not seen after the transplantation of high G capsules. When Wistar rats were used as recipients, no antibody responses were detected against any type of alginate capsules. Neither type of capsule acted as an adjuvant. Antibodies against ICC were present, in rats transplanted with both nonencapsulated and encapsulated ICCs. Administration of cyclosporine could abolish this production of antibodies against ICC. CONCLUSIONS: High G-alginate capsules are less immunogenic than high M capsules. Because encapsulation did not protect against the generation of antibodies against ICC, it can be assumed that antigen leakage from the capsules occurs, as no evidence was found for capsules breaking in vivo.

The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism.

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.

Glucose metabolism in vitro of cultured and transplanted mouse pancreatic islets microencapsulated by means of a high-voltage electrostatic field.

The aim of this study was to assess the function of mouse pancreatic islets microencapsulated using a high-voltage electrostatic field. Islets were microencapsulated in alginate/poly-L-lysine/alginate (APA) capsules and maintained in tissue culture. Rates of glucose oxidation and insulin release were then assessed. Glucose metabolism was also measured in microencapsulated islets retrieved after transplantation to normal syngeneic mice. The high-voltage electrostatic system made possible the production of uniformly sized microcapsules, which were smaller than those produced by co-axial air-jet systems. Nonencapsulated islets were used as controls. Empty microcapsules or islet-containing microcapsules were transplanted intraperitoneally and retrieved after 2 weeks for assessment of foreign-body reactions and glucose oxidation rates. After 1 day and 2 weeks in tissue culture, both control islets and microencapsulated islets increased their rates of glucose oxidation and insulin release 7- to 10-fold in response to an increase in glucose concentration from 1.7 to 16.7 mmol/l. Both empty and islet-containing microcapsules, retrieved 2 weeks after transplantation, showed high rates of glucose oxidation at both low and high glucose concentrations, suggesting overgrowth with metabolically active fibroblasts. Morphological studies indicated a marked foreign-body reaction on the surface of all transplanted microcapsules. The islets in cultured microcapsules had a normal histological appearance, whereas the islets within transplanted microcapsules showed a range of morphological appearances, from intact islets to cell debris. In conclusion, this study shows that mouse pancreatic islets survive and remain functionally competent for at least 2 weeks in vitro after microencapsulation in APA capsules generated in an electrostatic field. However, a foreign-body reaction with cellular growth on the capsular surface was present after intraperitoneal syngeneic transplantation.

Cloning and expression of three new Aazotobacter vinelandii genes closely related to a previously described gene family encoding mannuronan C-5-epimerases.

The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously been reported. The corresponding proteins catalyze the Ca2+-dependent polymer-level epimerization of beta-D-mannuronic acid to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6, algE7, and algY. All three genes were sequenced and expressed in Escherichia coli. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5'-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase. Southern blot analysis of genomic A. vinelandii DNA, using the 5' part of algE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here.

[Treatment of diabetes mellitus with transplantation of immunoprotected pancreatic islet tissue]. / Behandling av diabetes mellitus med transplantasjon av immunbeskyttet øyvev fra pancreas.

Research in islet transplantation in the treatment of diabetes mellitus is making rapid progress. Vascularized pancreas allotransplantation is now an accepted treatment, but with major limitations. The shortage of human islets for allotransplantation requires new sources of insulin-producing tissue. Xenotransplantation is one such approach, though the procedure is limited by immunological rejection of the graft. Immunoisolation of insulin-producing graft in alginate capsules protects the transplant from immunological damage. The alginate capsule consists of a semipermeable membrane, which act as an immunological barrier between the graft and the host. In addition to exclude immunocells and humoral mediators of rejection, the membrane must be permeable to nutrients and toxic end products from the cell metabolism, and permit free diffusion of glucose and insulin. We may hope that this immunoisolating technique will establish prolonged survival of allo- or xenografted tissue without immunosuppressive treatment.

Microcapsules of alginate-chitosan--I. A quantitative study of the interaction between alginate and chitosan.

The binding of chitosan to alginate beads was studied quantitatively by using radioactive labelled fractions of chitosan. The alginate-chitosan capsules were made either by dropping a solution of sodium alginate into a solution containing chitosan or by incubating calcium alginate beads in a solution of chitosan. The first procedure yielded a binding of 0.015 microg chitosan per mm2 of capsule surface, while the latter procedure yielded over 2 microg mm(-2). The maximum obtained weight ratio of chitosan to alginate in a microcapsule after 24 h was 0.40. The binding of chitosan was markedly increased by reducing the number average molecular weight of chitosan below 20000 Da and by increasing the porosity of the alginate gel. The porosity was increased by producing homogeneous gels, and by adding calcium chloride to the chitosan solution during the membrane forming stage. The effect of calcium ions on the porosity of the gel was studied by experiments involving release of blue dextran from calcium alginate beads. The binding of chitosan was also found to increase with decreasing fraction of N-acetylations, FA, on chitosan in the range of FA = 0.3 to FA = 0, and with increasing pH in the range from pH 4 to 6. Capsules with a diameter of 500 microm had a higher weight ratio of chitosan to alginate after 24 h of binding than the capsules with the larger diameter of 1500 microm.