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Beneficial effects of human serum albumin on stability and functionality of alginate microcapsules fabricated in different ways.

A key engineering challenge in designing microcapsules made from biocompatible alginate is maintaining adequate exchange of nutrients and oxygen between the entrapped cells and the environment, while simultaneously avoiding swelling and subsequent failure of the microcapsule. Approval for the use of alginate in pharmaceutical and/or biomedical applications also strictly requires that the components of the microcapsule material must meet the safety criteria of the ASTM and FDA. Incorporation of foetal calf serum (FCS) into the microcapsules for stabilization is not in accordance with the guidelines affirmed by these organizations. FCS should be substituted by microcapsule-stabilizing additives that are medically approved. In this communication, it is shown that 10% FCS can be replaced by 1% human serum albumin (i.e. by an agent for which medical approval is granted) without compromising effects on long-term in vitro stability. Furthermore, it is demonstrated that human serum albumin (HSA) significantly enhances cell survival and, particularly, insulin secretion of encapsulated rat islets over a time period of 3 weeks when incubated in culture medium. Thus, HSA-stabilized microcapsules made from UHV(Lam) alginate are apparently a promising system for immunoisolation of cells, particularly when alginate is cross-linked by injection of BaCl(2) crystals into the alginate droplets. Slight adjustments of the alginate concentration can tailor the microcapsule permeability to the released therapeutic factor.

Alginates↗

Novel solvent-based method for preparation of alginate beads with improved roundness and predictable size.

Attempts to determine conditions or processes within alginate gel beads often suffer from inaccuracies due to an improper roundness of the analysed beads. Therefore, a novel solvent-based method for the preparation of alginate beads with improved shape was developed: An aqueous solution of 2% (w/v) alginate in water was injected into a solvent layering consisting of hexane, n-butanol, n-butanol with 1% (w/v) CaCl2 and finally 2% (w/v) CaCl2 in water. Beads of up to 3.5 mm in diameter obtained with this method had a roundness which was approximately 5% better than comparable beads prepared by dropping an alginate solution into a CaCl2-hardening bath. This was determined by a software supported quantitative analysis of bead size and shape. Additionally, the novel solvent-based method allows for highly reproducible preparation of alginate beads with exactly predictable sizes. The biggest beads obtained with this method were 9 mm in diameter. Thus, with the solvent-based preparation of alginate beads it is now possible to easily obtain beads of exactly the type needed for a specific analytical purpose.

Alginates↗

In vitro and in vivo studies of ibuprofen-loaded biodegradable alginate beads.

The irritation effects of ibuprofen, a widely used non-steroidal anti-inflammatory drug (NSAID), were evaluated on mouse gastric and duodenal mucosa when suspended in 0.5% (w/v) sodiumcarboxymethylcellulose (NaCMC) solution and loaded in alginate beads. The ionotropic gelation method was used to prepare controlled release alginate beads of ibuprofen. The influence of various formulation factors on the encapsulation efficiency, as in vitro drug release and micromeritic properties, was investigated. Other variables included the alginate concentration, percentage drug loading and stirring speed during the microencapsulation process. Scanning electron micrographs of alginate beads loaded with ibuprofen showed rough surface morphology and particle sizes in the range of 1.15 +/- 0.4 - 3.15 +/- 0.6 mm. The yield of microspheres, as collected after drying, was generally 80-90%. Formulation code H showing t50% value of 3.5 h was chosen for in vivo trials because of the appropriate drug release properties. For in vivo trials, free ibuprofen (100 mg kg(-1)), blank and ibuprofen (100 mg kg(-1)) loaded alginate beads (formulation code H) were suspended in 0.5% (w/v) NaCMC solution and each group was given to six mice orally by gavage. NaCMC solution was used as a control in experimental studies. In vivo data showed that the administration of ibuprofen in alginate beads prevented the gastric lesions.

Alginates↗

Preparation and characterization of Mitomycin-C loaded chitosan-coated alginate microspheres for chemoembolization.

Mitomycin-C loaded and chitosan-coated alginate microspheres were prepared for use in chemoembolization studies. In this respect, first alginate microspheres were prepared by using a spraying method using an extrusion device with a small orifice and following suspension cross-linking in an oil phase. Chitosan-coating onto the alginate microspheres was achieved by polyionic complex formation between alginate and chitosan. CaCl(2) was used as a cross-linker for alginate microspheres. The obtained chitosan-coated alginate microspheres were spherical shaped and approximately 100-400 microm average size. The microspheres were evaluated based on their swellability and the swelling ratio was changed between 50-280%. CaCl(2) concentration, stirring rate, chitosan molecular weight, chitosan concentration and time for coating with chitosan were selected as the effective parameters on microsphere size and swelling ratio. Equilibrium swellings were achieved in approximately 30 min. On the other hand, chitosan molecular weight, chitosan concentration and time for coating with chitosan were found as the most effective parameters on both drug loading ratio and release studies. Maximum drug loading ratio of 65% was achieved with high molecular weight (HMW) chitosan, highest chitosan concentration (i.e. 1.0% v/v) and shortest time for coating with chitosan (i.e. 1 h) values.

Alginates↗

Ca-alginate microspheres encapsulated in chitosan beads.

Chitosan beads (CBs) incorporating Ca-alginate microspheres (CAMs), containing a drug, were prepared as an oral sustained delivery system. Stable and monodisperse Ca-alginate microspheres loaded with drug were obtained by a membrane emulsification method. The Ca-alginate microspheres were encapsulated in chitosan beads by the ionotropic gelation method with a polyelectrolyte complex reaction between two oppositely charged polyions. The surface and internal characteristics of the beads were improved by ionic cross-linking in tripolyphosphate (TPP) solution adjusted to pH 5.0. The release experiments were performed using lidocaine.HCl (cationic drug) and sodium salicylate (anionic drug) as model drugs. Initial release of drugs depended on the degree of swelling. Ca-alginate microspheres encapsulated in chitosan beads were superior to both drug-loaded CBs and CAMs beads for sustained release because they had a three-layer composition; a calcium alginate core bounded by an inter-phasic chitosan-alginate membrane, which itself was surrounded by a layer of chitosan-TPP.

Alginates↗

Effect of alginate composition and gelling cation on micro-bead swelling.

PURPOSE: The purpose of this study was to determine the roles of alginate composition and gelling cations on bead swelling, which affects its durability. METHOD: Using a 2-channel droplet generator, microspheres were generated with 1.5% solutions of low viscosity high-mannuronic acid (LVM), medium viscosity high-mannuronic acid (MVM), low viscosity high-guluronic acid (LVG) and medium viscosity high-guluronic acid (MVG) alginate. They were gelled by cross-linking with 1.1% solution of either BaCl2 or CaCl2. The diameters of the micro-beads were measured and recorded on day 0. The micro-beads were subsequently washed and incubated in saline at 37 degrees C for 2 weeks with size assessment every 2 days. The data were normalized by calculation of the percentage change from control (day 0) for all groups of micro-beads. RESULTS: Diameters of all beads were between 550 and 700 microm on day 0. Viscosity had no effect on swelling of Ba++- and Ca++-alginate micro-beads. Ca++-alginate micro-beads were more prone to swelling than the corresponding Ba++-alginate beads. High G-Ba++ beads had only a modest increase in size over time, in contrast to the high M-Ba++. CONCLUSION: Alginate composition and the gelling cation have significant effects on bead swelling.

Alginates↗

Effect of alginate composition and gelling cation on microbead swelling.

PURPOSE: The purpose of this study was to determine the roles of alginate composition and gelling cations on bead swelling, which affects its durability. METHOD: Using a 2-channel droplet generator, microspheres were generated with 1.5% solutions of low viscosity high-mannuronic acid (LVM), medium viscosity high-mannuronic acid (MVM), low viscosity high-guluronic acid (LVG) and medium viscosity high-guluronic acid (MVG) alginate. They were gelled by cross-linking with 1.1% solution of either BaCl2 or CaCl2. The diameters of the microbeads were measured and recorded on day 0. The microbeads were subsequently washed and incubated in saline at 37 degrees C for 2 weeks with size assessment every 2 days. The data were normalized by calculation of the percentage change from control (day 0) for all groups of microbeads. RESULTS: Diameters of all beads were between 550-700 microns on day 0. Viscosity had no effect on swelling of Ba++- and Ca++-alginate microbeads. Ca++-alginate microbeads were more prone to swelling than the corresponding Ba++-alginate beads. High G-Ba++ beads had only a modest increase in size over time, in contrast to the high M-Ba++. CONCLUSION: Alginate composition and the gelling cation have significant effects on bead swelling.

Alginates↗

Encapsulation of RIN-m5F cells within Ba2+ cross-linked alginate beads affects proliferation and insulin secretion.

The viability, proliferation and insulin production of RIN-m5F cells when loaded into alginate beads to form a 3D culture system has been investigated. The mechanism of alginate cross-linking (calcium ions vs barium ions), the addition of poly(L-lysine) (PLL) and poly(L-ornithine (PLO) and presence of different extra-cellular matrix proteins (ECM) influence the RIN-m5F cell behaviour. Cells in calcium alginate beads (CAB) proliferated and produced more insulin per cell than monolayer culture, but the physical properties of the beads were poor and they ruptured within a few days of culture. Barium alginate beads (BABs) provided a stable encapsulation method. Addition of PLL and PLO at concentrations above 0.1% w/v with the culture medium increased cell proliferation. With the addition of ECMs after bead formation there was a further increase in cell proliferation for certain combinations of ECM and PLO. It was concluded that RIN-m5F-loaded Ba-alginate beads (BABs), when incorporated with varying concentrations of poly (L) lysine (PLL), poly (L) ornithine (PLO) in the presence of extra-cellular matrix proteins (ECMs) were superior to both tissue culture and RIN-m5F-loaded Ca-alginate beads (CABs) in terms of physical stability, cell proliferation and insulin production.

Alginates↗

Calcium alginate microparticles for oral administration: II. Effect of formulation factors on drug release and drug entrapment efficiency.

The release rate of nicardipine HCl from various alginate microparticles was investigated. Manugel A7B618 which has a high guluronic acid content of 70% and a low polymerization degree of 60-400 was used as alginate. A 2(3) factorial design was utilized for the preparation of the alginate microparticles. The effect of drug:polymer weight ratio, CaCl2 concentration and curing time on parameters such as the time for 50% of the drug to be released (t50%) and the drug entrapment efficiency were evaluated with analysis of variance. The mean particle sizes and the swelling ratios of the microparticles were determined. The in vitro release studies were carried out with a flow-through cell apparatus at different media (pH 1.2, 2.5, 4.5, 7, 7.5 buffer solutions). Drug:polymer weight ratio and the concentration of the crosslinking agent were the influential factors on the release of NC from the alginate microparticles. The release of nicardipine was extended with alginate microparticles prepared in a ratio of 1:1 (drug:polymer weight ratio). The release of drug from alginate microparticles took place by both diffusion through the swollen matrix and relaxation of the polymer at pH: 1.2-4.5. However, the release was due to diffusion and erosion mechanisms at pH 7-7.5.

Administration, Oral↗

Degradation of dimethylphthalate by cells of Bacillus sp. immobilized in calcium alginate and polyurethane foam.

A Bacillus sp. which is capable of degrading dimethylphthalate (DMP) was immobilized in calcium alginate and polyurethane foam for efficient and long term degradation of DMP. Freely suspended cells (10(12) cfu ml-1) degraded a maximum of 20 mM DMP. Whereas, alginate-(10(12)cfu g-1 beads) and polyurethane foam-entrapped (0.34 x 10(6-9) cfu g-1 foam cubes) cells degraded a maximum of 40 mM DMP within 12-15 days of incubation. Polyurethane foam-entrapped cells degraded 30 mM of DMP at 4 days and alginate-entrapped cells degraded within 10 to 12 days of incubation irrespective of the cell population. When the initial concentration of DMP increased to 50 mM, the DMP degrading ability of the immobilized cells was not increased even after 20 days. Repeated batch cultures by alginate-entrapped cells with initial 35 mM DMP loading could be reused for a maximum of 20 cycles. However, the degradation rate was gradually decreased when the beads were reused for more than 15 cycles. On the other hand, the foam-entrapped cells, with the same initial DMP loading there was no decrease in DMP degrading ability and could be reused for more than 20 cycles. The packed bed reactor with alginate-entrapped cells (1 x 10(10-12) cfu g-1 bead) could be continuously operated for 7-8 days with an initial 25 mM DMP at a flow rate of 50 ml h-1. Whereas, the polyurethane foam-entrapped cells (1 x 10(6-9) cfu g-1 foam cubes) could be operated continuously for more than 90 days with the same initial DMP loading at a flow rate of 100 ml h-1. Thus the enhanced degradation of DMP could be achieved by immobilizing the cells of Bacillus sp. in calcium alginate and polyurethane foam as compared to that of freely suspended cells.

Alginates↗

Addition of fibronectin to alginate matrix improves peripheral nerve regeneration in tissue-engineered conduits.

Schwann cell (SC) transplantation has been proposed to encourage peripheral nerve regeneration, but an optimal SC-carrying matrix would be needed. The aim of this study was to characterize how the addition of fibronectin to alginate would affect the outcome of nerve regeneration promoted by Schwann cells embedded in this matrix. Genetically labeled rat SCs were obtained by lacZ gene transduction. SCs were suspended in alginate hydrogel matrix with/without addition of liquid fibronectin, and their viability and growth in the different types of matrices were assessed in vitro by AlamarBlue assay. In vivo assessment of SC transplantation in the matrix was carried out with poly-3-hydroxybutyrate (PHB) conduits to bridge a sciatic nerve gap. The grafted conduits were harvested at 2, 3, and 6 weeks and assessed for the presence of labeled SCs in relation to regrowing axons. The amount and rate of axonal regeneration were assessed by quantitative immunohistochemistry. Addition of fibronectin to alginate hydrogel improved SC viability and growth profile in vitro. X-Gal staining confirmed that SCs transplanted in PHB conduits were viable throughout the time course, and that the labeled SCs were clearly associated with regenerating axons. The regeneration rate was enhanced when liquid fibronectin was added to the alginate matrix. Furthermore, the presence of SCs also enhanced regeneration and there was an additive effect when both SCs and fibronectin were combined with alginate. In conclusion, the addition of fibronectin to alginate hydrogel matrix contributed to improve nerve regeneration, supporting SC viability and augmenting their effect on axonal growth when transplanted in a bioengineered nerve conduit.

Alginates↗

Alginate enhances elongation of early regenerating axons in spinal cord of young rats.

Freeze-dried alginate sponge cross-linked with covalent bonds has been demonstrated to enhance nerve regeneration in peripheral nerves and spinal cords. The present study examined, at early stages after surgery, the outgrowth of regenerating axons and reactions of astrocytes at the stump of transected spinal cord in young rats. Two segments (Th7-8) were resected, and alginate was implanted in the lesion. As controls, collagen gel was implanted in place of alginate or the lesion was left without implantation. Two and 4 weeks after surgery, nerve outgrowth and astrocyte reactions were examined. Many regenerating axons, some of which were accompanied by astrocytic processes, were found to extend from the stump into the alginate-implanted lesion. In the all nonimplanted animals, large cystic cavities were formed at both interfaces with no definite axonal outgrowth into the lesion. In collagen-implanted animals, cavity formation was found in some rats, and regenerating axons once formed at the stumps did not extend further into the lesion. Astrocytic processes extending into alginate-implanted lesion had no basal laminae, whereas those found in control experiments were covered by basal laminae. These findings suggest that alginate contributed to reducing the barrier composed of connective tissues and reactive astrocytic processes, and served as a scaffold for the outgrowth of regenerating axons and elongation of astrocytic processes.

Alginates↗

Adhesion and growth of bone marrow stromal cells on modified alginate hydrogels.

Alginate is a biodegradable, immunocompatible biopolymer that is capable of immobilizing viable cells and bioactive factors. Few investigations have analyzed the efficacy of alginate gels as substrata for cell attachment and proliferation. Here we have compared the adhesion and subsequent growth of human and rat bone marrow stromal fibroblastic cells on unmodified alginate hydrogel surfaces. It was found that, in contrast to rat cells, human cells did not readily attach or proliferate on unmodified alginates. In attempts to enhance these features, or collagen type I was incorporated into the gels, with no significant improvements in prolonged human cell adherence. However, alginate gels containing both collagen type I and beta-tricalcium phosphate were found to enhance human cell adherence and proliferation. Furthermore, interactions between the collagen and beta-tricalcium phosphate prevented loss of the protein from the hydrogels. These results indicate that alginate gels containing collagen have potential uses as vehicles for delivery of adherent cells to a tissue site. In addition, gels containing beta-tricalcium phosphate, with or without collagen type I incorporation, have potential to support cell growth and differentiation in vitro before implantation. This study emphasizes the limitations of the uses of cells derived from experimental animals in certain model studies relating to human tissue engineering.

Alginates↗

Effect on Pseudomonas aeruginosa alginate expression of direct plating and culture of fresh cystic fibrosis sputum on to pseudomonas isolation agar containing subinhibitory concentrations of roxithromycin and rifampicin.

The effects of subinhibitory concentrations of roxithromycin (16 mg/L) or rifampicin (16 mg/L) on alginate production by Pseudomanas aeruginosa were investigated. The weight of purified alginate from antibiotic-free cultures was significantly greater (52.5 +/- 24.0 mg, range 22.4-109.5), compared with alginate from cultures bacteria exposed to sub-MIC of roxithromycin (21.9 +/- 17.0, 0.0-42.1 (P < or = 0.037)) and to sub-MIC of rifampicin (28.6 +/- 15.0, 2.9-47.5 (P < or = 0.038)). Chromatographic analysis of hydrolysed and chemically transformed sub-units of alginate revealed that the presence and the molar ratio of D-mannuronic acid and L-guluronic acid were not affected in the remnant alginate exposed to sub-MIC of roxithromycin in contrast to that in the remnant alginate exposed to sub-MIC of rifampicin.

Alginates↗

Human septal chondrocyte redifferentiation in alginate, polyglycolic acid scaffold, and monolayer culture.

OBJECTIVES/HYPOTHESIS: Tissue engineering laboratories are attempting to create neocartilage that could serve as an implant material for structural support during reconstructive surgery. One approach to forming such tissue is to proliferate chondrocytes in monolayer culture and then seed the expanded cell population onto biodegradable scaffolds. However, chondrocytes are known to dedifferentiate after this type of monolayer growth and, as a result, decrease their production of cartilaginous extracellular matrix components such as sulfated glycosaminoglycans. The resultant tissue lacks the biomechanical properties characteristic of cartilage. The objective of the study was to determine whether different culture systems could induce monolayer-expanded human septal chondrocytes to redifferentiate and form extracellular matrix. STUDY DESIGN: Laboratory research. METHODS: Chondrocytes were isolated from human nasal septal cartilage of five donor patients (age, 35.8 +/- 9.3 y). Cell populations were seeded at low density (30,000 cells/cm2) into monolayer culture and expanded for 4 to 6 days. Following trypsin release, chondrocytes were placed into three different systems for neocartilage formation: alginate beads, polyglycolic acid scaffolds, and monolayer. After 7 and 14 days of growth, neocartilage was analyzed using histological and quantitative biochemical assessment of cellularity (Hoechst 33258 assay) and sulfated glycosaminoglycan content (dimethyl methylene blue assay). RESULTS: Histologically, alginate beads contained spherical chondrocytes surrounded by dense extracellular matrix, an appearance similar to that of native cartilage. In contrast, polyglycolic acid scaffolds and monolayer cultures contained elongated cells with scant staining for matrix sulfated glycosaminoglycans, which are features that are characteristic of dedifferentiated chondrocytes. Biochemical analysis demonstrated a lower level of cell proliferation (P <.001) in scaffolds (+52% over baseline) and alginate (+96% over baseline) than in monolayer (+366% over baseline), as well as a higher content of sulfated glycosaminoglycans per cell (P <.001), after 14 days of growth in alginate culture than in either polyglycolic acid scaffolds (19-fold difference) or monolayer (98-fold difference). CONCLUSIONS: Of the systems compared, monolayer-expanded human septal chondrocytes demonstrated the greatest accumulation of sulfated glycosaminoglycans per cell when grown in alginate beads. Future research on cartilage tissue engineering may use alginate culture for reverting dedifferentiated cells back to the chondrocytic phenotype.

Alginates↗

De novo cartilage generation using calcium alginate-chondrocyte constructs.

These studies investigated the utility of calcium alginate as a biocompatible polymer matrix within which large numbers of chondrocytes could be held successfully in a three-dimensional structure and implanted. Further, the ability of chondrocyte-calcium alginate constructs to engraft and generate new cartilage was examined. Chondrocytes isolated from calf shoulders were mixed with a 1.5% sodium alginate solution to generate cell suspensions with densities of 0, 1.0, 5.0, and 10.0 x 10(6) chondrocytes/ml. The cell suspensions were gelled to create disks that were placed in subcutaneous pockets on the dorsums of nude mice. The alginate concentration and CaCl2 concentration used to make the disks also were varied. A total of 20 mice were implanted with 67 bovine chondrocyte-calcium alginate constructs. Samples with an initial cellular density of at least 5.0 x 10(6) chondrocytes/ml demonstrated gross cartilage formation 12 weeks after implantation. Cartilage formation was observed microscopically in specimens with a cellular density as low as 1.0 x 10(6) chondrocytes/ml. The histoarchitecture of the new cartilage closely resembled that of native cartilage. Cartilage formation was independent of CaCl2 concentration (15 to 100 mM) or alginate concentration (0.5% to 4.0%) used in gel polymerization.

Alginates↗

Survival, proliferation, and functions of porcine hepatocytes encapsulated in coated alginate beads: a step toward a reliable bioartificial liver.

Orthotopic liver transplantation is the most effective treatment for fulminant hepatic failure. As an alternative treatment, an efficient extracorporeal bioartificial liver should contain a large yield of functional hepatocytes with an immunoprotective barrier, for providing temporary adequate metabolic support to allow spontaneous liver regeneration or for acting as a bridge toward transplantation. Survival, proliferation, and functions of porcine hepatocytes were evaluated in primary cultures and after embedding in alginate beads, which were subsequently coated with a membrane made by a transacylation reaction between propylene glycol alginate and human serum albumin. Disruption of total pig livers by collagenase perfusion/recirculation allowed the obtention of up to 10(11) hepatocytes with a viability greater than 95%. Hepatocytes in conventional cultures or embedded in coated alginate beads survived for about 10 days, secreted proteins, particularly albumin, and maintained several phase I and II enzymatic activities, namely ethoxyresorufin-O-deethylase, oxidation of nifedipine to pyridine, phenacetin deethylation to paracetamol, glucuroconjugation of paracetamol, and N-acetylation of procainamide. Typical features of mitosis and [3H]thymidine incorporation indicated that porcine hepatocytes proliferated in both conventional cultures and alginate beads. The efficacy of the membrane surrounding alginate beads for protecting cells from immunoglobulins was tested by embedding HLA-typed human lymphocytes, which were subsequently incubated with specific anti-HLA immunoglobulin G and complement. These data show that large yields of porcine hepatocytes that are embedded in coated alginate beads remain functional and are isolated from large molecular weight molecules, such as immunoglobulins. This system represents a promising tool for the design of an extracorporeal bioartificial liver, containing xenogeneic hepatocytes, to treat acute liver disease in humans.

Acetaminophen↗

Delivery of neurotrophin-3 to the cochlea using alginate beads.

OBJECTIVE: The aim of this study was to design a novel cochlear neurotrophin (NT) delivery system for the rescue of auditory neurons after ototoxicity-induced deafening. BACKGROUND: NT-3 is a trophic growth factor that promotes the survival of the auditory nerve and may have a potential therapeutic role in slowing neuron loss in progressive deafness, especially as an adjunct to the current cochlear implant. Beads made from alginate are biodegradable, slow release substances that can be placed at the round window or inside the cochlea. This study investigated the loading properties, release kinetics, and implantation potential of alginate beads loaded with NT-3. METHODS: Alginate beads were prepared using an ionic gelation technique and postloaded with NT-3. Release of NT-3 was measured using enzyme-linked immunosorbent assay over 5 days. Alginate beads were implanted into deafened guinea pigs for 28 days, after which survival of auditory neurons was assessed. RESULTS: Enzyme-linked immunosorbent assay studies demonstrated a 98% to 99% loading of NT-3 with a slow, partial release over 5 days in Ringer's solution. Furthermore, the addition of heparin to the delivery system modulated NT-3 release to a steadier pattern. Implantation of alginate-heparin beads in guinea pig cochleae produced minimal local tissue reaction. NT-3 loaded beads implanted at both the round window and within the scala tympani of the basal turn provided auditory neurons significant protection from degradation and apoptosis compared with unloaded beads or untreated cochleae. CONCLUSIONS: This study demonstrates alginate beads to be a safe, biodegradable and effective delivery system for NT-3 to the cochlea.

Alginates↗