BMES 460/600 Biomaterials I Dr.

Elisabeth Papazoglou 12/08/2008

SOME SORT OF WITTY TITLE I HATE ALGINATE

OLGA FILIPPOVA, MUNIR NAHRI, AKASH PATEL, PREETHEM SPINATH

ABSTRACT

TABLE OF CONTENTS 1. Chemical and physical properties 1.1 Chemical structure 1.2 Molecular weight 1.3 Glass transition temperature 1.4 Properties in solution 1.4.1 Solubility 1.4.2 Thickening 1.4.3 Swelling 1.4.4 Film-forming properties 1.5 Crosslinking properties 1.6 Hydrogel strength 1.7 Hydrogel stiffness 1.8 Compositional variations (M/G) and effects on properties 1.9 Degradation 1.10 Physiological response 1.11 Alginate production 1.12 Major alginate producing companies 2. Current Applications 2.1 Biomedical applications 2.2 Consumer industrial applications 2.3 Advantages and disadvantages 3. Future Applications 3.1 Replacement materials 3.1.1 Dextran 3.1.2 Chitosan 3.1.3 Matrigel 4. References 2 2 3 4 5 5 6 6 7 7 8 10 11 13 14 14 16 17 18 20 21 22 24 25 25 25 26

CHEMICAL AND PHYSICAL PROPERTIES CHEMICAL STRUCTURE Alginate is a non-branched binary co-polymer composed of two 14 glycosidically linked monomers: -D-mannuronic acid (M) and -L-guluronic acid (G) (see Figure 1). The relative amounts of M and G, as well as their sequential arrangement along the polymer chain, is not fixed and varies greatly with the origin of the alginate, age of the algae, and the method of extraction. For example, in two common sources of alginate, Macrocystis pyrifera and Laminaria hyperborean, the M/G ratio varies from 1.6 to 0.45, respectively 1. In younger algae, the concentration of M is much higher than the concentration of G. In older plants, this is reversed, as enzyme C5 epimerase converts M monomers to G monomers2.

Figure 1. The molecular structure of the two monomers of alginate -D-mannuronic acid M (left) and -L-guluronic acid G (right)1. The arrangement of M and G monomers is limited, however, to three types of blocks: homopolymeric M block, homopolymeric G block, and heteropolymeric alternating M/G block (see Figure 2). M blocks are linear because they bind using the equatorial position and G blocks appear buckled because they bind using the axial position. The M blocks are considered flexible, as only the G blocks are involved in crosslinking, containing binding sites for cations. The M blocks determine the elasticity of the alginate, compared to the G blocks which influence rigidity2. The number and distribution of subunits present in the alginate determines the physical properties of that particular alginate. 3

Figure 2. The three types of blocks possible in the structure of alginate the M-exclusive block (top), and G-exclusive block (bottom), and the alternating M/G block (middle)3. MOLECULAR WEIGHT Commercial alginates are polydisperse with respect to molecular weight. The natural alginate polymer length is rather long, yet is decreased by the manufacturing process. Commercial alginate is usually sold with a molecular weight (Mn) of 500,000 g/mol or lower, with a degree polymerization of 25003. The most common method to calculate molecular weight is based upon intrinsic viscosity which can only give an estimate of the molecular weight. Most producers blend alginates to target a given viscosity specification product with the same intrinsic viscosity which may have different molecular weight populations. Therefore, in biomedical applications, equivalent products may have different molecular weight populations and therefore have differing biomaterial interactions3.

GLASS TRANSITION TEMPERATURE The glass transition temperature (Tg) is defined as the approximate midpoint of the temperature range at which a non-crystalline (amorphous) polymer changes from brittle, glass state to a rubbery, soft state. In Roger et al, powder samples of alginate exhibited Tg ranging from 95C to 136C. In the same experiment, no significant effect on T g was observed for different molecular weight samples4. There was, however, an increase in Tg with increase G content4 (see Figure 3). This effect can be explained by the presence of residual Ca2+ ions in the alginate powder, crosslinking oligomeric G-rich chains4. Roger et al also determined the Tg of sodium alginate films to be 158C. They mentioned that it was higher than the glass transition temperatures mentioned previously in literature for sodium alginate films, ranging from 95C to 110C4. The source of this discrepancy is hypothesized to be residual Ca 2+ content in the alginate sources used experimentally, highlighting the importance of source variability in determining the mechanical and thermal properties of alginates. Experimentally, substituting sodium with calcium in alginate films increases Tg concurrent with the theory that divalent cations have strong binding effects at junction points between chains4. Tg of alginate films also varies according to the crosslinking cation. Experimentally, the Tg of alginate films having various cations decreased in the order A13+ > Ca2+ > Fe2+ > Cu2+ > Na+ 5.

Figure 3. The relationship between Tg, ionic radius of crosslinking cation, and heat capacity in alginate films (Cp)5. Figure 3 above demonstrates the relationship between Tg, ionic radius of crosslinking cation, and heat capacity in alginate films5. The data demonstrates Tg of alginate film decreases and the heat capacity increases with increasing ionic radius of the cross-linking agent. PROPERTIES IN SOLUTION Solubility The solubility of alginate is related to the rate of dissociation of the alginate molecule 6. At low pH (pH<3), M-structures and G-structures will precipitate as alginic acid, while alternating M,G structures will remain in solution despite being fully protonated 6. This pH dependent physical property of alginates is used in pharmaceutical applications as an antireflux remedy which, when swallowed, forms an alginic acid raft on top of stomach contents7. Alginic acid is an insoluble polysaccharide1. Alginate salts are water soluble, creating a solution of considerable viscosity, and swell upon contact with moisture, without dissolving (standard hydrogel property)8. This swelling causes the alginate to become more porous, and thus more permeable9. Solubility and water retention are pH dependent, however, and when the pH

reaches below 4, alginate precipitates out of solution. Molecular weight also effects water retention, with lower molecular weight alginates having a higher water intake1. These rheological properties of alginate make is a useful thickening agent, colloidal stabilizer, and blood expander1, as well as make it best suitable for soft tissue applications8. Thickening The thickening properties of alginate are related to the molecular weight and conformation of the alginate molecule in solution. Alginates flow properties will be affected by interactions with other molecules as well as competition for water at high concentrations. Therefore, increasing alginate concentration corresponds to increased viscosity 10. Cross-linking materials, such as calcium, present in even small concentrations will induce artificial viscosities higher than real ones. Solutions like these have thixothropic flow properties. Sodium alginate is part of the hydrocolloids super family, which are sensitive to applied shear forces11. When alginate is simply added to an aqueous solution, the alginate chains become entangled, which increases the viscosity of the solution. Yet, when shear forces are applied, the alginate polymeric chains become aligned, oriented toward the force, decreasing the viscosity of the solution11. This behavior of shear thinning is called pseudoplasticity. Swelling Swellability is associated with the rate of hydration, and will strongly depend on the form of alginate. Cross-linked alginates will swell slower than pure sodium alginate. Swelling

properties of alginates have been used in dietary products and sustained release tablets 12. Furthermore, swelling properties of partially neutralized alginic acid has a long tradition as a tablet disintegrant.

Film-forming Properties The film forming properties of alginate are a result of entanglements of alginate molecules when water is removed. When cross-linked gels are dried, they exhibit this they exhibit this type of behavior. Therefore, the molecular weight of alginate needs to be above a lower limit to achieve film formation and avoid brittleness. In situ, alginate films can be formed by spraying alginate solution to a binding surface3. CROSS LINKING Dry alginate is usually sold in its sodium salt form, with a Na+ replacing the carboxylic acid hydrogen. Because the hydrogen from the carboxylic acid group can be easily removed, alginate is said to have a polyectronic nature, and is considered an anionic polymer8. Crosslinking of alginate occurs by the addition of divalent cations, which interact with the carboxylate groups on the guluronic acid13, most often replacing the sodium ion2, creating the egg box structures14 (see Figure 4). Crosslinking is most often achieved with the addition of Ca2+ (see Figure 5), however, the addition of other divalent cations is also possible, with ion selectivity decreasing from barium, to calcium, and stronium14. No gel forms after the addition of magnesium ions14. This failure to crosslink is directly related to the radius of magnesium, which is too small to ensure the proper stacking of the G blocks15.

Figure 4. The difference in crosslinking of alginate between an M-rich and a G-rich polymer. Also shown in the egg box structure of the crosslinked polymer (the black dots inside of the egg crate represent Ca2+ ions)3.

Figure 5. A close-up of the egg scare structure formed after alginate crosslinking. Clearly seen are the interacting G blocks and the Ca2+ ions17. HYDROGEL STRENGTH Gelling properties of alginate are related to both M/G composition and the sequential organization of M and G along the chain3. The binding of calcium to alginate is cooperative; the more monomers in a G block, the tighter the successive binding of calsium and the stronger the formed gel. Therefore, increases in G-content and molecular weight generally produce a stronger

gel17. Gel formation is reversible if the calcium is removed8. Crosslinking does not occur if the concentration of G blocks is less than 20%2. The gelling process is of particular importance to the gelling properties. Gelling can occur externally, through the diffusion of cross-linking agent from the outside, or internally, by homogeneously releasing a cross-linking agent from the inside. In internal gelling, ratio between the cross-linking agents and alginate is important in determination of gel strength. Furthermore, strength properties in high-G alginate is much different than in high-M alginate at a particular cross-linking agent/alginate ratio. Evidence of this is seen in the experimental graph below (Figure 6):

Figure 6. The affects of increasing concentrations of High-G and High-M alginates on gel strength at constant calcium and internal gelling conditions3. High-G alginates gels (G>60%) cross-linked with Ca2+, exhibit maximum gel strength, measured by the force needed (in grams) to rotate a metal plate immersed into the gel by 30, when the amount of calcium stoichiometrically matches the amount of calcium-binding guluronic acid blocks3. As seen in Figure 6, when the alginate concentration in the gelling solution is higher than the optimal ratio, calcium will not bind available G-sites optimally, and will have lower gel strength. Contrarily, high-M alginate have shorter and fewer G-blocks and

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therefore show a continuous increase in gel strength for higher alginate concentrations. This is because the addition of high-M alginate does not present a surplus of G-sites, but a relative increase in calcium ion binding G-sites below threshold that facilitate increased gel strength3. An important consideration when measuring gel strength is to differentiate between elastic and viscous moduli. In particular, the viscous modulus is often misinterpreted as gel strength in high viscosity solutions3. HYDROGEL STIFFNESS In general, the stiffness of alginate hydrogels can be enhanced by increasing alginate concentration10. However, this will also increase viscosity which may be unfavorable in particular processing applications. Therefore, to increase post-gel stiffness with minimal pre-gel viscosity increase, increasing quantities of low molecular weight (MW) alginate (M n ~ 3.3x104 g/mol) can be incorporated into 1% (w/w) alginate solutions composed of high MW alginate (Mn ~ 2.2x105 g/mol)10. The experimental results seen below are evidence of this method (Figure 7):

Figure 7. (Left) Effects of molecular weight distribution on stiffness of cross-linked hydrogels. (Right) The effects of molecular weight distribution on the viscosity of pre-gelled solution. (- unary system, - - binary system)10.

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COMPOSITIONAL VARIATIONS (M/G) AND EFFECTS ON PROPERTIES The properties of alginate are dictated by polymer composition, sequential structure, molecular weight, and conformation. Specifically, functional properties of alginate are a direct result of the amount of crosslinking, and are thus dependent on the G content, average number of monomers in a G block, and molecular weight. Composition and sequential structure of alginate can be determined by high resolution 1H and 13C nuclear magnetic resonance spectroscopy. From this scan, the M/G ratio and average M and G block lengths can be calculated. NMR scans were also used to show that alginate does not have a regular repeating unit3. Due to the highly varying M/G in alginates, the value of the compression modulus has seen to vary between 1kPa to 1MPa, and the shear modulus has been seen to vary from 0.02 to 40kPa18. The effect of the M/G ratio on the mechanical properties of alginate can be seen in Figures 8, 9 and 10. MVM represents an alginate high in M (M/G ratio of 0.85), MVG represents an alginate with a high G content (M/G ratio of 0.29), and FMC is an alginate with an even higher G content (M/G ratio of 0.26). Figure 8, it can be seen that an increase in G content increases the ultimate stress of the material. The tree bar colors represent different applied strains, with black representing 1% strain, light gray representing 10% strain, and dark grey representing 100% strain. From Figure 9, it can be seen that as the G content increases, the ultimate stress increases as well (the different bars within the alginate type represent different crosslinking conditions). For the elastic modulus, represented on Figure 10, the two alginates with a high G content (MVG and FMC) show similar modulus values, yet on average, the alginate with the highest G content, FMC, shows a higher modulus value18. From this data, it can be concluded that as the G content of the alginate increases, so do its mechanical properties.

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Figure 8. The relationship between alginate G content and ultimate stress (in mm/mm). Presented is the data for three alginate types MVM, with a high M content (M/G ratio 0.85), MVG, with a high G content (M/G ratio of 0.29) and FMC, with the higher G content (M/G ratio of 0.26). The different colors represent different applied strains black 1%, light gray 10%, and dark gray 100%18.

Figure 9. Relationship between alginate G content and ultimate stress (in kPa). The same three types of alginate are presented MVM, MVG, and FMC. The bars within the alginate types represent different crosslinking conditions18.

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. Figure 10. Relationship between alginate G content and its elastic modulus (in kPa). The same three types of alginate are presented MVM, MVG, and FMC. The bars within the alginate type once again represent different crosslinking conditions18. DEGRADATION To be degraded, the 14 linkage between monomer units needs to be degraded by alginate lyase. In the case of alginate, the body does not contain alginases, enzymes necessary to depolymerize alginate20. Because of this, the polymer chains of alginate will not degrade, especially if the molecular weight is high, and will thus not exit the body via normal absorption methods20,19,24. The divalent cation causing gelling may exit, however, resulting in the separation of the molecular chains and the loss of structural integrity19. In order to better control the degradation properties of alginate, it can be covalently crosslinked into poly(aldehyde guluronate) (PAG), which has been shown to provide control over its degradation based on crosslinking density. The higher the crosslinking density of PAG, the lower its mechanical properties (due to an increased number of network defects) and the lower the degradation rate of the polymer21.

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PHYSIOLOGICAL RESPONSE When using a natural material, such as those used for wound closure and dressings, the inflammatory response after implantation is always a concern as they tend to elicit a strong one. Subcutaneous implantation of alginate fibers into rats, showed only a slight foreign body response. The immune response to alginate does not involve T lymphocytes, which means that it produces less of an immunogenic response than proteins. Because T lymphocytes recognize small peptide fragments, polysaccharides are not capable of activating this immune response22. Alginate microspheres, however, have been shown to cause cellular and fibrous overgrowth at the site of implantation. Yet this reaction was greatly reduced when alginate with a high G content was used24. Mice pharmacokinetic studies of alginate have shown that most of the alginate is rapidly eliminated from the blood after intravenous injection, usually within 0 to 5 hours, with total elimination occurring slower over the next 5 to 48 hours. Thus, alginate demonstrates a primary half life of 4 hours, and a secondary half life of 22 hours. Intraperitoneal bolus injection mice studies have demonstrated alginate maximum absorption after 5 hours. After absorption, the serum concentration of alginate decreased, demonstrating a half life of 12.5 hours. It is also possible that biphasic elimination of intraperitoneal alginate administration is also possible, just as seen with intravenous injection. Alginate administered orally was not absorbed into the blood in the mice model3. ALGINATE PRODUCTION The primary source of algae is the Phaeophyta group of brown algae. From this group, the raw sources of algae include the following species: laminaria, alaria, ecklonia, eisenia, nereocystis, sargassum, cystoseiera, and fucus. Most of bulk production occurs by growing and

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harvesting Macrocystic pyrifera (giant kelp) and Ascopyyllum nodosum (Norwegian kelp). In brown algae, polyguluronic acid is present in the cell wall and polymannuronic acid is found in the extracellular space24. The main producers of alginate are China, United States, Norway and Scotland. China produces about 10,000 tons of alginate annually, from cultivated Laminaria japonica. The remaining countries combined produce about 15,000 tons of alginate annually, valued at approximately $120 million. Norway and Scotland use ascophyllum nodosum and laminaria hyperborean species to produce their alginate. Smaller alginate manufacturers include Japan, France and Chile. In brown algae, alginate is present in the cells wall as the sodium or calcium salt of alginic acid. The primary goal of the extraction process if to produce dry sodium alginate powder. Alginate is extracted based on the solubility of each different salt or form of alginate. Mainly, the fact that sodium alginate is soluble in water while calcium alginate is not. First, the algae is milled and sodium carbonate is added in order to draw the alginate into solution as sodium alginate. Next, the left over algae parts are removed, using flotation, centrifugation, sifting or filtration. The next steps involve the recover of the alginate from the aqueous solution. Then, acid is added to convert the sodium alginate back into alginic acid, in order to precipitate it from solution. Following, alcohol and sodium carbonate is added to the solution. Unlike in water, sodium alginate does not dissolve in alcohol, thus it can be separated from the mixture, dried and milled into the desired fine powder. An extra step can be added in the beginning of the recovery procedure by adding calcium chloride. This forms insoluble calcium alginate fibers, which can be separated. This extra step adds another level of separation for the alginate, increasing the purity1.

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For application purposes, two types of alginate exist commodity and ultrapure. Commodity alginate is traditionally used for topical wound treatment, anti-reflux remedy, and tablet excipient. Because of the low purity level obtained after manufacturing, it is unlikely that commodity alginates ever be used in implantable devices or parenteral drugs. Commodity alginates usually contain an excess of salt or free anginic acid, as a direct consequence of the dry blending technique. Ultrapure alginates, however, which contain a much lower level of endotoxin, protein, polyphenol, ash, and heavy metal contamination since they are manufactured to GMP/ISO 9000 guideline, are used for implant applications. Several implantable alginate products are currently being clinically evaluated. The main difference between commodity and ultrapure alginate, is that ultrapure alginate contains a stochiometric amount of ion, thus eliminating free acid3. MAJOR ALGINATE PRODUCING COMPANIES There are several major alginate-producing companies around the world. ISP Alginates Ltd, located in the United Kingdom, manufactures a broad range of products, including sodium, potassium, and ammonium alginate. FMC Biopolyer, based in Philadelphia, PA, manufactures alginate under the tradenames Protanal and Protacid3. Another company is NovaMatrix, based in Olso, Norway, produce two main types of alginate G rich (65%+) Pronova LVG sodium alginate and M rich (50%+) Pronova LVM sodium alginate (LV = low viscosity, between 20 and 200 mPa). These companies sell bulk alginate products, and require individual quotes based on product, amount, and shipping, however, the general price for alginate is $10 per ounce. Individual alginate products can be purchased with ease, some even over the counter at drug stores. For example, Medline Industries cells a box of ten would dressings for $45 and CM3 Inc cells a box of 50 weight-loss capsules for $100.

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CURRENT APPLICATIONS Sodium alginate is the main form of alginate used6. Other types of alginate include alginic acid, calcium, ammonium and potassium salts, and propylene glycol alginate, an ester of alginic acid. Alginic acid and calcium alginate can be removed during the calcium alginate process for making sodium alginate, and dried and milled to appropriate particle size. Ammonium and potassium salts are made by neutralization of moist alginic acid with ammonium hydroxide and potassium carbonate6. Propylene glycol alginate can be produced in a pressure vessel by treating moist alginic acid, partially reacted with sodium carbonate, with liquid propylene oxide6. Majority of the applications of alginates are based on three intrinsic properties. The first property is an ability to increase the viscosity of an aqueous solution when dissolved. The second property is alginates ability to form gels. This gelation process occurs when calcium salt is added to a solution of sodium alginate in water. Chemically, calcium displaces the sodium from the alginate, and holds the long alginate molecules together. This gelling process is independent of a heat requirement which contrasts traditional agar gels which require heating water to approximately 80C before the gel forms when cooled below 40C 6 The third important property is the ability to form films of sodium/calcium alginate and calcium alginates fibres 6. The effect of alginate properties on its use in applications in outlines on Table 1. Due to the large variability of alginates, sellers offer a range of alginates with different viscosities and mechanical properties3. The arrangement and overall M/G ratio of alginate chains varies from one species to another. Therefore, different alginates produce a range of viscosity values when dissolved in water3.

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Property Characteristic Molecule Weight Viscosity

Composition and Cross-linking Immobilization, Sequence encapsulation Dissociation, Soluble at pH, Solutions, Solutions/pastes, scaffolds, pKa precipitate at pH, fibers, films, dressings, membranes, table swelling capability absorption disintegration Polyanion Cation affinity Chelation Gelation, drug/metal bonding Table 1. Characteristics of alginate and their effect on alginate applications3. BIOMEDICAL APPLICATIONS Before, the use of alginate in pharmaceutical and/or biomedical applications (where is it mostly used for cell immobilization and drug deliver applications, respectively 25), they must have approval from regulatory agencies like United States Food and Drug Administration (FDA). Each product must go through the safety regulatory process separately, yet overall it has been estimated that alginate will be non-toxic to substances in vivo25. To assist demonstrating the safety of alginate in an application, the American Society for Testing and Materials has written a guide for the characterization and testing of alginates that are intended to be used for biomedical applications3. Good quality stable fibers made from mixed salts of sodium and calcium alginate and processed into non-woven fabric are used in wound dressings6. These dressing have good wound healing and haemostatic properties6. Furthermore, they can be absorbed by body fluids when the calcium in the fibers is exchanged by sodium from body fluids converting it into a soluble sodium alginate. This property makes it easy to remove from wounds as well 6. Allergic and inflammatory reactions to alginate dressings are rare26. Pharmaceutically, the swelling properties of alginic acid powder in water has led to its use as tablet disintegrant6. Alginic acid has also been used in dietary products and foods to give

Attribute Thickening, film-forming Gelling

Application Suspensions, coatings

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an artificial full feeling after swelling. To reduce irritating reflux, alginic acid has also been used to keep gastric contents in place6. Alginate is also widely used for the controlled release of medicinal drugs and chemicals. In certain applications, the chemicals of interest are placed in calcium alginate beads, with tight control of size distributions, and are slowly released when exposed to the appropriate environment. Alginate microspheres, coated with chitosan to improve mechanical strength, have been used in oral controlled drug release systems as well. A successful example of this application is the use of alginate microspheres, prepared by an emulsion based process, for oral insulin delivery in rats27. In tissue engineering, alginate is a biomaterial that can be used in scaffolds. Alginate has been used to encapsulate cells in the solid freeform fabrication of tissue constructs27. Successful examples of this application include the ability of hepatocytes to grow, proliferate, and maintain hepatocyte specific function in porous alginate scaffolds and tissue constructs27,29. Furthermore, the slow gelation kinetics of alginate allow it to be used as an injectable cell delivery vehicle30. This method minimizes tissue damage during implantation and could therefore theoretically decrease a negative immune response by the host30. Scaffolds of covalently crosslinked poly(aldehyde guluronate) (PAG), a polymer derived from alginate, presents a controllable degradation model dependent on crosslinking density21. Furthermore, the time dependent degradation of alginate microspheres has been used to better tissue engineering processes. Calcium alginate microcapsules are used to encapsulate signaling molecules, nucleotides, and biofluids in specific scaffolds31. Here, they offer the advantage of controlled release kinetics32.

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The table below (Table 2) summarizes the different biomedical and health related issues addressed by alginate applications. Matricies and Scaffolds Directed Drug Delivery Bone regeneration Covalent attachment Nerve regeneration Endostatin producing cells Bulking agent Hormones and growth Anti-reflux Parkinson's disease Soft tissue implant Central nervous system Table 2. Biomedical applications of alginate3. CONSUMER INDUSTRIAL APPLICATIONS In the food industry, the thickening properties of alginate are used in syrups, sauces, and ice cream6. However, sodium alginate is not useful when the emulsion is acidic due to the formation of insoluble alginic acid forms. Propylene glycol alginate is used instead because of its stability in mild acidic conditions. Sodium alginates improve the texture and body of yogurt, but propylene glycol alginate is used in the stabilization of milk proteins under acidic conditions. In ice cream, alginates are used as stabilizers, reducing the formation of ice crystals during freezing and producing a smoother product. Edible instant dessert jellies can be made from alginatecalcium mixtures because they are formed by simply mixing powders with water or milk without the addition of heat. Alginate gels, formed from powders composed of sodium alginate, calcium carbonate, lactic acid and calcium lactate are used to in re-formed/re-structured food products including meats. Calcium alginate coatings are used to preserve meats including fish to prevent oxidation and bacterial contamination6. Industrially, beads of calcium alginate are used to immobilize biocatalysts and active whole cells that are responsible industrially for the conversion of glucose to fructose, starch to ethanol, and the continuous production of yogurt. The material entraps the enzyme and is still penetrable enough to allow for the diffusion of the substance that needs to be converted. In Tissue Engineering Pancreas Liver Kidney

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textiles, alginates are used to thicken for pastes containing dyes. Advantages of using alginates of traditional starch include lower reactivity with the dyes and easier removal out of finished textiles6. Textiles account for a significant portion of the global alginate market (approx: 50%)6. The paper industry uses the excellent film forming properties of alginate for surface sizing. The oil resistance of alginate improves the oil resistance and greaseproof properties of paper surfaces. In the welding industry, alginate coatings are applied in the immediate vicinity of the weld to control temperature, oxygen, and hydrogen availability. Alginates intrinsic properties also make them very useful in the molding industry. The poor adhesion of alginate films to many surfaces in combination with insolubility in non-aqueous environments have made them ideal candidates for mold-release agents6. The extremely accurate, flexible, and fast setting medium properties of alginate molding compounds have led to their common use in dentistry, prosthetics, and life casting6. In dentistry, the ability of alginates to undergo transformation from sol to gel through ionotropic gelation is used for dental molding1. ADVANTAGES AND DISSADVANTAGES One of the biggest advantages of alginates includes their relatively low cost, abundance, and renewable source. Another advantage commonly utilized in biomedical applications is the time dependent gel formation under proper conditions, which allows for the dispension of alginate as a liquid and the formation of a gel in situ. Furthermore, the mechanical properties of alginate can be tailored to specific applications by varying the sequential structure and ratio of D-mannuronic acid (M) and -L-guluronic acid (G), molecular weight, cross-linking agent, alginate source, and complexed polycations including synthetic polymers, proteins, and polypeptides. All of these factors will also affect hydrogel porosity allowing for adjustable control of porosity tailored to a specific application. Alginate is also biocompatible and does not

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illicit a negative immunogenic response. Furthermore, its time dependent degradation properties can be used in the time-controlled release of substances within alginate capsules. Some of the disadvantages include significant source variability in chemical composition. Because so much of alginates physical properties are directly related to chemical composition, the significant source variability reduces the reliability of producing highly specific alginate structures with specifically controlled properties. The low mechanical properties of alginate may limit its use mechanically demanding applications. For the purposes of maintaining cell viability, the chemical crosslinking of alginates may be detrimental. Despite the fact that alginate degradation is time dependent, its true degradation characteristics cannot be tailored as well as they can be in other synthetic polymers. Other disadvantages will be related to its specific applications, and may involve one or a number of factors seen above. FUTURE APPLICATIONS Most of the new application possibilities for alginate are in the areas of drug delivery, cell therapy, and tissue engineering, owing to some of its unique and highly desirable properties. The previously described crosslinking properties of alginate, which occur by the addition of divalent cations, involve a highly cell-friendly mechanism. Because of this, alginate has been used has been used extensively for encapsulating cells. Also, since it is possible to have timecontrolled gelation of Alginate, by slowing down calcium binding kinetics, it is possible to use alginate as an injectable polymer that will form a hydrogel post injection. Most recently, alginate has been used as an injectable hydrogel scaffold for the regeneration of heart myoblasts at the infarct site after myocardial infarction33. A solution of 30-50 kDa alginate and calcium gluconate was prepared with a low apparent viscosity of 10-50 cP, making it an easily injectable solution. This solution was injected into infarct sites in rat

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hearts. The alginate hydrogel formed in situ at the infarct site, providing a scaffold for cardiac remodeling. Interestingly, the crosslinking of alginate was supplemented further by the inherent presence of calcium ions in heart tissue. The scaffold degraded almost completely over a period of 6 weeks (see Figure 11), and the infarct sites showed signs of myoblast formation, indicating that cardiac remodeling indeed took place33. This shows the successful application of an injectable cell-seeded alginate.

Figure 11: 4 left images: the degradation of Alginate over a period of 6 weeks in the injection site. This is characterized by a decrease in the amount of biotin staining (brown). 4 right images: myoblast formation at the alginate injection site, which was found to be more effective than even by injection of neonatal cardiomyocytesPZ5. Alginate has also been used for islet cell encapsulation for treatment of type-I diabetes in various test studies34, with extremely promising results. The encapsulated cells release insulin, and eliminate the need for external supplementation. Many studies are underway with numerous patent applications for extending results of this study for human application. This encapsulation procedure can also be extended for other cell types, such as fibroblasts and other genetically modified cells for secretion of biologically relevant macromolecules such as hormones. Implantation of such capsules containing fibroblasts secreting Brain-Derived Neurotrophic Factor (BDNF) at sites of injury can be used for regeneration of neurons in

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spinal cord injury35. Embryonic stem cells have been encapsulated in alginate and grown in a rotating microgravity bioreactor for bone tissue engineering37. Further, the presence of numerous carboxyl groups on each monomer of alginate greatly facilitates its covalent modification and linking to other biomaterials, such as peptides, growth factors, and extracellular matrix components37. This type of covalent modification can also be utilized to modify the type of crosslinking within alginate itself, thus eliciting greater control over mechanical and chemical properties of Alginate hydrogels. Alginate is also finding newer applications in biosensor design, by forming the immobilization surface for enzymes that can be used in detection of biologically relevant substances and microorganisms. Biotinylated alginate, which is covalently modified alginate with biotin, has been used to immobilize glucose oxidase onto the surface of an amperometric transducer for glucose sensing38. REPLACEMENT MATERIALS Alginate has clear advantages over other materials that have similar properties, including its highly cost efficiency, easy availability, ease of crosslinking, and high biocompatibility. It is mainly used instead of synthetic polymers and collagen because of the low immunogenic response after implantation. However, there are other naturally derived polymers that are sometimes used instead of alginate, mainly because of its low cellular interaction, making it a poor matrix for cells that require high extracellular matrix interactions (such as hepatocytes). Due to significant source variability that creates a wide range of alginate properties, it is sometimes desired to use a biomaterial that can be more tailed to the specific application.

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Dextran Dextran is a polymer of D-glucopyranose, a sucrose derivative of bacterial origin. It is a neutral, branched polysaccharide physically or chemically crosslinked through its numerous hydroxyl groups. It can have mechanical properties similar to alginate, depending on the type and extent of crosslinking, and is biodegradable. It has the potential to replace alginate in scaffold manufacture for tissue engineering, as a material for the manufacture of microcarriers, and as an encapsulation polymer for drug delivery39. Chitosan Chitosan is a linear polymer of 14 linked D-glucosamine and N-acetyl glucosamine. It is derived from chitin, the major constituent of the exoskeleton of sea crustaceans, making it a highly abundant and renewable natural biopolymer. It is easily processable, highly biocompatible and dissolves by enzymatic degradation. Its intrinsic antibacterial activity and minimum foreign body reaction make it a highly suitable material for tissue engineering scaffolds, would dressings, and drug delivery vehicles, all of which are current alginate applications as well40. Matrigel A relatively new material as compared to those previously described, Matrigel is a natural hydrogel derived from the basement membrane of mouse EHS sarcoma, the cancer of connective tissue. It is composed mainly of laminin and type IV collagen, and various growth factors, which are removed for most applications in order to better control experimental conditions41. Matrigel is thermally crosslinked, with a transition temperature of 4 oC, thus it is a gel at the physiological temperature of 37oC 42. Matrigel finds various applications in in vitro tissue engineering owing to its biocompatibility and ability to promote cellular differentiation.

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However, because of its cancerous source, Matrigel cannot be approved for in vivo applications, and thus only competes with alginate for in vitro uses. Also, Matrigels low mechanical integrity and little-promoted cell proliferation poses challenges for use41. REFERENCES 1. Deb, S. Chapter 15: Synthetic and Natural Degradable Polymeric Biomaterials. Biomaterials Fabrication and Processing Handbook. Ed. Chu, P.K. and Liu, X. New York: CRCP LLC, 2008. 469-70. 2. Khotimchenko, Y.S., Kovalev, V.V., Savchenko, O.V., and Ziganshina, O.A. Physical Chemical Properties Physiological Activity and Usage of Alginates the Polysaccharides of Brown Algae. Marine Pharmacology 27, 2001, S53. 3. Dornish, M. and Rauh, F. Chapter 15: Alginate. An Introduction to Biomaterials. Ed. Guelcher, S.A. and Hollinger, J.O. New York: CRCP LLC, 2005. 261-72. 4. Roger, S., Bee, A., Balnois, E., Bourmaud, A., Le Deit, H., Grohens, Y., Cabuil, V. Physical and mechanical properties of alginate films containing calcium cations and ferrofluids. Fifth International Conference Polymer-Solvent Complexes & Intercalates. July 11-14, 2004. Lorient, France. 5. Nakamura, K., Nishimura, Y., Hatakeyama, T., and Hatakeyama, H. Thermal properties of water insoluble alginate films containing di- and trivalent cations. Thermochimica Acta 267, 1995, 343. 6. McHugh, D.J. A Guide to the Seaweed Industry. New York: Food and Agriculture Organization of the United Nations, 2003. 7. Mandel, K.G., et al. Review: alginate-raft formulations in the treatment of heartburn and acid reflux. Ailment Pharm Therap, 14, 2000, 669. 8. Temenoff, J.S., and Mikos, A.G. Biomaterials : The Intersection of Biology and Materials Science. Upper Saddle River: Prentice Hall, 2007. 10-11. 9. Chen, W., Ji, B., and Lu, D.R. Chapter 20: Biomaterial Implants for Treatment of Central Nervous System Disease. Biomaterials Engineering and Devices, Human Applications: Volume 1, Fundamentals and Vascular and Carrier Applications. Ed. Wise, D.L., Trantolo, D.J., Lewandrowski, K.U., Gresser, J.D., Cattaneo, M.V., and Yaszemski, M.J. New York: Humana P, 2000. 309, 314. 10. Kong, H., Mooney, D.J. Controlling material properties of ionically cross-linked alginate hydrogels by varying molecular weight distribution. Mat Res Soc Symp Proc 711, 2002, 227.

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