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Modification of drug delivery materials with beta-cyclodextrins (β-CyD) is known to increase solubility of poorly water-soluble drugs, protect drugs from degradation and sustain release. In this study, we developed a hydrogel drug delivery system for local paclitaxel delivery using the natural polysaccharide alginate functionalized with β-CyD-moieties. Paclitaxel was chosen due to its ability to form inclusion complexes with cyclodextrins. The rheological and mechanical properties of the prepared hydro- gels were characterized, as well as in vitro release of the paclitaxel and in vitro activ- ity on PC-3 prostate cancer cells. Introduction of β-CyD-moieties into the hydrogel reduces the mechanical properties of the gels compared to nonmodified gels. How- ever, gelation kinetics were not markedly different. Furthermore, the β-CyD-modified alginate helped to reduce undesired crystallization of the paclitaxel in the gel and facilitated paclitaxel diffusion out of the gel network. Remarkably, the β-CyD grafted alginate showed increased capacity to complex paclitaxel compared to free HPβ- CyD. Release of both paclitaxel and degradation products were measured from the gels and were shown to have cytotoxic effects on the PC-3 cells. The results indicate that functionalized alginate with β-CyDs has potential as a material for drug delivery systems.

Engineered block polysaccharides is a relatively new class of biomacromolecules consisting of chemical assembly of separate block structures at the chain termini. In contrast to conventional, laterally substituted polysaccharide derivatives, the block arrangement allows for much higher preservation of inherent chain properties such as biodegradability and stimuli-responsive self-assembly, while at the same time inducing new macromolecular properties. Abundant, carbon neutral, and even recalcitrant biomass is an excellent source of blocks, opening for numerous new uses of biomass for a wide range of novel biomaterials. Among a limited range of methodologies available for block conjugation, bifunctional linkers allowing for oxyamine and hydrazide 'click' reactions have recently proven useful additions to the repertoire. This article focuses the chemistry and kinetics of these reactions. It also presents some new data with the aim to provide useful protocols and methods for general use towards new block polysaccharides.

Cell encapsulation in alginate microbeads is a promising approach to provide immune isolation in cell therapy without immunosuppression. However, the efficacy is hampered by pericapsular fibrotic overgrowth (PFO), causing encapsulated cells to lose function. Stability of the microbeads is important to maintain immune isolation in the long-term. Here, we report alginate microbeads with minimal PFO in immunocompetent C57BL/6JRj mice. Microbead formulations included either alginate with an intermediate (47 %) guluronate (G) content (IntG) or sulfated alginate (SA), gelled in Ca2+/Ba2+ or Sr2+. A screening panel of eleven microbead formulations were evaluated for PFO, yielding multiple promising microbeads. Two candidate formulations were evaluated for 112 days in vivo, exhibiting maintained stability and minimal PFO. Microbeads investigated in a human whole blood assay revealed low cytokine and complement responses, while SA microbeads activated coagulation. Protein deposition on microbeads explanted from mice investigated by confocal laser scanning microscopy (CLSM) showed minimal deposition of complement C3. Fibrinogen was positively associated with PFO, with a high deposition on microbeads of high G (68 %) alginate compared to IntG and SA microbeads. Overall, stable microbeads containing IntG or SA may serve in long-term therapeutic applications of cell encapsulation. Statement of significance Alginate-based hydrogels in the format of micrometer size beads is a promising approach for the immunoisolation of cells in cell therapy. Clinical trials in type 1 diabetes have so far had limited success due to fibrotic responses that hinder the diffusion of nutrients and oxygen to the encapsulated cells, resulting in graft failure. In this study, minimal fibrotic response towards micrometer size alginate beads was achieved by chemical modification of alginate with sulfate groups. Also, the use of alginate with intermediate guluronic acid content resulted in minimally fibrotic microbeads. Fibrinogen deposition was revealed to be a good indicator of fibrosis. This study points to both new microsphere developments and novel insight in the mechanisms behind the fibrotic responses.

Engineered block polysaccharides is a relatively new class of biomacromolecules consisting of chemical assembly of separate block structures at the chain termini. In contrast to conventional, laterally substituted polysaccharide derivatives, the block arrangement allows for much higher preservation of inherent chain properties such as biodegradability and stimuli-responsive self-assembly, while at the same time inducing new macromolecular properties. Abundant, carbon neutral, and even recalcitrant biomass is an excellent source of blocks, opening for numerous new uses of biomass for a wide range of novel biomaterials. Among a limited range of methodologies available for block conjugation, bifunctional linkers allowing for oxyamine and hydrazide ‘click’ reactions have recently proven useful additions to the repertoire. This article focuses the chemistry and kinetics of these reactions. It also presents some new data with the aim to provide useful protocols and methods for general use towards new block polysaccharides.

This work explores the largely unknown surface microstructure and elastic modulus of soft calcium-alginate hydrogels (E = 100–4500 Pa) in their hydrated state by atomic force microscopy (AFM) in quantitative imaging mode. Alginate concentration largely influenced the surface topography with surface roughness measured to be 101 ± 6 nm and 57 ± 1 nm for 0.5 and 2.0% (w/v) alginate, respectively. The calculated range of pore sizes increased with decreasing alginate concentration, from radius smaller than 360 nm, 570 nm and 1230 nm for 2.0%, 1.0% and 0.5% alginate, respectively. Small changes in calcium concentration (20 to 25 mM, 1.5% alginate) did not induce changes in surface microstructure, although it increased the elastic modulus mean values and distribution. Introducing oxidized or peptide-grafted alginate in the gels resulted in rougher surfaces, larger pore sizes and lower elasticity than the respective hydrogels with no modified alginate.

The effect of alginate-based scaffolds with added basement membrane proteins on the in vitro development of hESC-derived pancreatic progenitors was investigated. Cell clusters were encapsulated in scaffolds containing the basement membrane proteins collagen IV, laminin, fibronectin, or extracellular matrix-derived peptides, and maintained in culture for up to 46 days. The cells remained viable throughout the experiment with no signs of central necrosis. Whereas nonencapsulated cells aggregated into larger clusters, some of which showed signs of morphological changes and tissue organization, the alginate matrix stabilized the cluster size and displayed more homogeneous cell morphologies, allowing culture for long periods of time. For all conditions tested, a stable or declining expression of insulin and PDX1 and an increase in glucagon and somatostatin over time indicated a progressive reduction in beta cell-related gene expression. Alginate scaffolds can provide a chemically defined, xeno-free and easily scalable alternative for culture of pancreatic progenitors. Although no increase in insulin and PDX1 gene expression after alginate-immobilized cell culture was seen in this study, further optimization of the matrix physicochemical and biological properties and of the medium composition may still be a relevant strategy to promote the stabilization or maturation of stem cell-derived beta cells. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3717-3726, 2015.

Lyase-catalyzed degradation has been proposed as a more cell-friendly alternative to dissolution of alginate gels than using chelating agents. In this study, we investigated the effect of lyase specificity on degradation of alginate gels, including the effect of crosslinking ions with different affinity for the polymer. Degradation kinetics and products were analyzed. In particular, the degradation products were characterized using novel methods for alginate sequence determination by chromatography. Lyase-catalyzed gel disruption worked well for gels crosslinked with calcium, but was less effective when barium was included in the gel formulation. The importance of crosslinking of long G-blocks in maintaining the structural integrity of the gels was identified. The failure to degrade these long G-blocks, either due to protection of the G-blocks by strong ionic crosslinking or due to lack of lyase activity on G–G linkages, resulted in retained resistance to mechanical disruption of the gel.

One of the main challenges in tissue engineering and regenerative medicine is the ability to maintain optimal cell function and survival post-transplantation. Biomaterials such as alginates are commonly used for immunoisolation, while they may also provide structural support to the cell transplants by mimicking the extracellular matrix. In this study, arginine-glycine-aspartate (RGD)-peptide-coupled alginates of tailored composition were produced by adopting a unique chemoenzymatic strategy for substituting the nongelling mannuronic acid on the alginate. Alginates with and without RGD were produced with high and low content of G. Using carbodiimide chemistry 0.1-0.2% of the sugar units were substituted by peptide. Furthermore, the characterization by (1)H-nuclear magnetic resonance (NMR) revealed by-products from the coupling reaction that partly could be removed by coal filtration. Olfactory ensheathing cells (OECs) and myoblasts were grown in two-dimensional (2D) and 3D cultures of RGD-peptide modified or unmodified alginates obtained by the chemoenzymatically strategy and compared to native alginate. Both OECs and myoblasts adhered to the RGD-peptide modified alginates in 2D cultures, forming bipolar protrusions. OEC encapsulation resulted in cell survival for up to 9 days, thus demonstrating the potential for short-term 3D culture. Myoblasts showed long-term survival in 3D cultures, that is, up to 41 days post encapsulation. The RGD modifications did not result in marked changes in cell viability in 3D cultures. We demonstrate herein a unique technique for tailoring peptide substituted alginates with a precise and flexible composition, conserving the gel forming properties relevant for the use of alginate in tissue engineering.

Cell encapsulation has already shown its high potential and holds the promise for future cell therapies to enter the clinics as a large scale treatment option for various types of diseases. The advancement in cell biology towards this goal has to be complemented with functional biomaterials suitable for cell encapsulation. This cannot be achieved without understanding the close correlation between cell performance and properties of microspheres. The ongoing challenges in the field of cell encapsulation require a critical view on techniques and approaches currently utilized to characterize microspheres. This review deals with both principal subjects of microspheres characterization in the cell encapsulation field: physico-chemical characterization and biocompatibility. The up-to-day knowledge is summarized and discussed with the focus to identify missing knowledge and uncertainties, and to propose the mandatory next steps in characterization of microspheres for cell encapsulation. The primary conclusion of this review is that further success in development of microspheres for cell therapies cannot be accomplished without careful selection of characterization techniques, which are employed in conjunction with biological tests. (C) 2013 Elsevier B.V. All rights reserved.

This paper reports a study of the distribution and function of homopolymeric guluronic acid blocks (G-blocks) in enzymatically modified alginate. High molecular weight mannuronan was incubated with one native (AlgE6) and two engineered G-block generating mannuronan C-5 epimerases (AlgE64 and EM1). These samples were found to contain G-blocks with a DP ranging from 20 to approximately 50, lacking the extremely long G-blocks (DP > 100) found in algal alginates. Calcium gels from epimerized materials were highly compressible and exhibited higher syneresis and rupture strength but lower Youngs modulus than gels made from algal polymers of similar G-content. Addition of extremely long G-blocks to the epimerized alginate resulted in decreased syneresis and rupture strength and an increased Young’s modulus that can be explained by reinforcement of the cross-linking zones at the cost of length and/or numbers of elastic segments. The presence and impact of these extremely long G-blocks found in natural alginates suggest that alginate gels can be viewed as a nanocomposite material. © American Chemical Society 2013. This is the authors accepted and refereed manuscript to the article.

The cytokine-inducing potential of various microspheres were evaluated in a short-time screening assay of lepirudin-anticoagulated human whole blood utilizing the Bio-Plex Human cytokine 27-plex system. The inflammatory cytokines IL-1β, TNF and IL-6; the anti-inflammatory mediators IL-1ra and IL-10; the chemokines IL-8, MIP-1α and MCP-1; and the growth factor VEGF were induced by polycation (poly-l-lysine or poly(methylene-co-guanidine)) containing microspheres. Alginate microspheres without polycations did not induce the corresponding cytokine panel, nor did soluble alginate. By inhibiting complement C3 using compstatin analog CP20, a total inhibition of complement activation as well as the inflammatory mediators was achieved, indicating that complement activation alone was responsible for the induced cytokines. A strong deposition of C3c on the poly-l-lysine containing surface, while not on the microspheres lacking polycations, also points to the formation of C3 convertase as involved in the biomaterial-induced cytokine induction. These results show that complement is responsible for the induction of cytokines by polycation containing microspheres. We point to complement as an important initiator of inflammatory responses to biomaterials and the lepirudin anticoagulated whole blood assay as an important tool to identify the most tolerable and safe materials for implantation to humans.

Qi M, Morch Y, Lacik I, Formo K, Marchese E, Wang Y, Danielson KK, Kinzer K, Wang S, Barbaro B, Kollarikova G, Chorvat D Jr, Hunkeler D, Skjak-Braek G, Oberholzer J, Strand BL. Survival of human islets in microbeads containing high guluronic acid alginate crosslinked with Ca2+ and Ba2+. Xenotransplantation 2012; 19: 355–364. © 2012 John Wiley & Sons A/S. Abstract: Background: The main hurdles to the widespread use of islet transplantation for the treatment of type 1 diabetes continue to be the insufficient number of appropriate donors and the need for immunosuppression. Microencapsulation has been proposed as a means to protect transplanted islets from the host’s immune system. Methods: This study investigated the function of human pancreatic islets encapsulated in Ca2+/Ba2+–alginate microbeads intraperitoneally transplanted in diabetic Balb/c mice. Results: All mice transplanted with encapsulated human islets (n = 29), at a quantity of 3000 islet equivalent (IEQ), achieved normoglycemia 1 day after transplantation and retained normoglycemia for extended periods of time (mean graft survival 134 ± 17 days). In comparison, diabetic Balb/c mice transplanted with an equal amount of non-encapsulated human islets rejected the islets within 2 to 7 days after transplantation (n = 5). Microbeads retrieved after 232 days (n = 3) were found with little to no fibrotic overgrowth and contained viable insulin-positive islets. Immunofluorescent staining on the retrieved microbeads showed F4/80-positive macrophages and alpha smooth muscle actin–positive fibroblasts but no CD3-positive T lymphocytes. Conclusions: The Ca2+/Ba2+–alginate microbeads can protect human islets from xenogeneic rejection in immunocompetent mice without immunosuppression. However, grafts ultimately failed likely secondary to a macrophage-mediated foreign body reaction.

Microbeads of alginate cross-linked with Ca2+ and/or Ba2+ are popular matrices in cell-based therapy. The aim of this study was to quantify the binding of barium in alginate microbeads and its leakage under in vitro and accumulation under in vivo conditions. Low concentrations of barium (1 mM) in combination with calcium (50 mM) and high concentrations of barium (20 mM) in gelling solutions were used for preparation of microbeads made of high-G and high-M alginates. High-G microbeads accumulated barium from gelling solution and contained higher concentrations of divalent ions for both low- and high-Ba exposure compared to high-G microbeads exposed to calcium solely and to high-M microbeads for all gelling conditions. Although most of the unbound divalent ions were removed during the wash and culture steps, leakage of barium was still detected during storage. Barium accumulation in blood and femur bone of mice implanted with high-G beads was found to be dose-dependent. Estimated barium leakage relevant to transplantation to diabetic patients with islets in alginate microbeads showed that the leakage was 2.5 times lower than the tolerable intake value given by WHO for high-G microbeads made using low barium concentration. The similar estimate gave 1.5 times higher than is the tolerable intake value for the high-G microbeads made using high barium concentration. In order to reduce the risk of barium accumulation that may be of safety concern, the microbeads made of high-G alginate gelled with a combination of calcium and low concentration of barium ions is recommended for islet transplantation.

Alginate hydrogels have applications in biomedicine, ranging from delivery of cells and growth factors to wound management aids. However, they are mechanically soft and have shown little potential for the use in bone tissue engineering. Here, the viscoelastic properties of alginate hydrogel beads mineralized with calcium phosphate, both by a counter-diffusion (CD) and an enzymatic approach, are characterized by a micro-manipulation technique and mathematical modeling. Fabricated hydrogel materials have low mineral content (below 3 % of the total hydrogel mass, which corresponds to mineral content of up to 60 % of the dry mass) and low dry mass content (

Functional biominerals formed in living organisms require high control of inorganic phase crystal polymorphs, morphology, size, and orientation. Such control is often governed by both soluble and insoluble functional molecules, where the soluble ones usually are highly negatively charged proteins and polymers. Here, the effect of alginate and well-defined alginate oligomers on calcium carbonate crystallization is explored, by seeded and unseeded experiments, to separate the effects of the additive on the crystal growth process from the effect on nucleation. The experiments were done at well-defined activity-based levels of supersaturation, controlled pH and temperature. In the seeded experiments, crystal growth of calcite, aragonite, and vaterite were studied separately, and the observations were used to explain a switch in polymorphism when alginate oligomers were present during the crystallization process. Unseeded experiments showed that low concentrations of G-blocks (oligomers of α-l-guluronic acid) ...

A one-step method was used to make nanostructured composites from alginate and calcium carbonate or calcium phosphate. Nanometer-scale mineral phase was successfully formed within the gel network of alginate gel beads, and the composites were characterized. It was found that calcite was the dominating polymorph in the calcium carbonate mineralized beads, while stoichiometric hydroxyapatite was formed in the calcium phosphate mineralized beads. A combination of electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis and powder X-ray diffraction showed that alginate played an active role in controlling mineral size, morphology and polymorphy. For the calcium phosphate mineralized beads, alginate was shown to modulate stoichiometric hydroxyapatite with low crystallinity at room temperature, which may have important applications in tissue engineering. The results presented in this work demonstrate important aspects of alginate-controlled crystallization, which contributes to the understanding of composite material design.

Structural biocomposites found in nature often have a well-defined organization on the nanometer scale. For mineralized materials, interactions between organic and inorganic phases are important fo...

Bioencapsulation involves the envelopment of tissues or biological active substances in semipermeable membranes. Bioencapsulation has been shown to be efficacious in mimicking the cell's natural environment and thereby improves the efficiency of production of different metabolites and therapeutic agents. The field of application is broad. It is being applied in bioindustry and biomedicine. it is clinically applied for the treatment of a wide variety of endocrine diseases. During the past decades many procedures to fabricate capsules have been described. Unfortunately, most of these procedures lack an adequate documentation of the characterization of the biocapsules. As a result many procedures show an extreme lab-to-lab variation and many results cannot be adequately reproduced. The characterization of capsules can no longer be neglected, especially since new clinical trials with bioencapsulated therapeutic cells have been initiated and the industrial application of bioencapsulation is growing. In the present review we discuss novel Approached to produce and characterize biocapsules in view of clinical and industrial application. A dominant factor in bioencapsulation is selection and characterization of suitable polymers. We present the adequacy of using high-resolution NMR for characterizing polymers. These polymers are applied for producing semipermeable membranes. We present the pitfalls of the currently applied methods and provide recommendations for standardization to avoid lab-to-lab variations. Also, we compare and present methodologies to produce biocompatible biocapsules for specific fields of applications and we demonstrate how physico-chemical technologies such as FT-IR, XPS, and TOF-SIMS contribute to reproducibility and standardization of the bioencapsulation process. During recent years it has become more and more clear that bioencapsulation requires a multidisciplinary approach in which biomedical, physical, and chemical technologies are combined. For adequate reproducibility and for understanding variations in outcome of biocapsules it is advisable if not mandatory to include the characterization processes presented in this review in future studies. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

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.

Background A recent study from our laboratory documented significant improvements in post-transplant viability in an experimental model of intestinal transplantation when a novel, nutrient-rich preservation solution was used during cold storage. The current study investigated the relationship between energetic/oxidative stress responses and fundamental kinase signaling events during the period of organ storage. This relationship may be a key factor contributing to improved graft viability after storage in a nutrient-rich preservation solution. Methods Rat small intestine was harvested and flushed intraluminally with University of Wisconsin (UW) solution or an amino acid-rich (AA) solution as follows: Group 1, no luminal flush (clinical control); Group 2, luminal UW solution; Group 3, luminal AA solution. Energetics (ATP, total adenylates), oxidative stress (malondialdehyde), histology, and MAPK (P38, JNK, ERK)/AMPK/Caspase-3 were assessed throughout 12-hour cold storage. Results P38 and JNK were upregulated strongly in Group 2 after 1- and 12-hour storage. Group 3 exhibited a delayed activation and subsequent downregulation of these pre-apoptotic signals. Between 6 to 12 hours, a strong upregulation of ERK was observed in Group 3. AMPK downregulation correlated with a reduction in AMP/ATP ratio, ERK upregulation, and P38/JNK downregulation in Group 3. After 12-hour storage, histology indicated superior preservation of mucosal architecture in Group 3 tissues. Conclusions A nutrient-rich preservation solution abrogates pre-apoptotic signaling (JNK and P38) and upregulates cytoprotective signals (ERK). Our data support the concept of a concerted effort facilitating cellular protection in response to ischemic stress.

Microcapsules of alginate cross-linked with divalent ions are the most common system for cell immobilization. In this study, we wanted to characterize the effect of different alginates and cross-linking ions on important microcapsule properties. The dimensional stability and gel strength increased for high-G alginate gels when exchanging the traditional Ca2+ ions with Ba2+. The use of Ba2+ decreased the size of alginate beads and reduced the permeability to immunoglobulin G. Strontium gave gels with characteristics lying between calcium and barium. Interestingly, high-M alginate showed an opposite behavior in combination with barium and strontium as these beads were larger than beads of calcium−alginate and tended to swell more, also resulting in increased permeability. Binding studies revealed that different block structures in the alginate bind the ions to a different extent. More specifically, Ca2+ was found to bind to G- and MG-blocks, Ba2+ to G- and M-blocks, and Sr2+ to G-blocks solely.

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.

The role of capsule composition and biologic responses in the function of transplanted microencapsulated islets of langerhans

Improvement of the biocompatibility of alginate/poly-L-lysine/alginate microcapsules by the use of epimerized alginate as a coating

Alginate is widely used for encapsulation of cells. Alginate is a linear block copolymer consisting of mannuronic acid (M) and guluronic acid (G). It has been shown that enzymes known as C-5 epimerases convert M to G in the polymer chain, giving rise to novel alginates with tailored properties. One of these enzymes, AlgE4, converts M blocks into blocks of strictly alternating M and G. In this study we investigated how alginate epimerized by AlgE4 affected capsule properties such as stability and permeability. Inhomogeneous calcium-alginate gel beads were made with original and AlgE4-epimerized alginates of different origin. The epimerized alginates formed initially smaller alginate gels that showed increased resistance to osmotic swelling compared with the original nonmodified alginate samples. The permeability, measured as diffusion of immunoglobulin (Ig) G into Ca/Ba-alginate gel beads, was reduced by epimerization and further reduced by addition of poly-L-lysine (PLL). The osmotic stability of alginate-poly-D-lysine(PDL)-alginate capsules was enhanced by the use of epimerized alginate; indeed, stable capsules with low permeability to tumor necrosis factor (TNF) could be made with low PDL exposures. Finally, alginate with an alternating structure interacted more strongly with the alginate-PLL capsule than did alginate with a high content of M blocks or G blocks or than an alginate consisting mainly of M.

The effect of alginate-based scaffolds with added basement membrane proteins on the in vitro development of hESC-derived pancreatic progenitors was investigated. Cell clusters were encapsulated in scaffolds containing the basement membrane proteins collagen IV, laminin, fibronectin, or extracellular matrix-derived peptides, and maintained in culture for up to 46 days. The cells remained viable throughout the experiment with no signs of central necrosis. Whereas nonencapsulated cells aggregated into larger clusters, some of which showed signs of morphological changes and tissue organization, the alginate matrix stabilized the cluster size and displayed more homogeneous cell morphologies, allowing culture for long periods of time. For all conditions tested, a stable or declining expression of insulin and PDX1 and an increase in glucagon and somatostatin over time indicated a progressive reduction in beta cell-related gene expression. Alginate scaffolds can provide a chemically defined, xeno-free and easily scalable alternative for culture of pancreatic progenitors. Although no increase in insulin and PDX1 gene expression after alginate-immobilized cell culture was seen in this study, further optimization of the matrix physicochemical and biological properties and of the medium composition may still be a relevant strategy to promote the stabilization or maturation of stem cell-derived beta cells.

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 (Ca2+) 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.

Alginates comprise a rather broad family of polysaccharides found in brown seaweeds (Laminaria sp., Macrocystis sp., Lessonia sp. and others), from which they are produced industrially. The annual production is estimated to approximately 38.000 tons worldwide.1 In addition, a variety of different al...

Microencapsulation may allow for immunosuppression-free islet transplantation. Herein we investigated whether human islets can be shipped safely to a remote encapsulation core facility and maintain in vitro and in vivo functionality. In non-encapsulated islets before and encapsulated islets after shipment, viability was 88.3+/-2.5 and 87.5+/-2.7% (n=6, p=0.30). Stimulation index after static glucose incubation was 5.4+/-0.5 and 6.3+/-0.4 (n=6, p=0.18), respectively. After intraperitoneal transplantation, long-term normoglycemia was consistently achieved with 3,000, 5,000, and 10,000 IEQ encapsulated human islets. When transplanting 1,000 IEQ, mice returned to hyperglycemia after 30-55 (n=4/7) and 160 days (n=3/7). Transplanted mice showed human oral glucose tolerance with lower glucose levels than non-diabetic control mice. Capsules retrieved after transplantation were intact, with only minimal overgrowth. This study shows that human islets maintained the viability and in vitro function after encapsulation and the inhomogeneous alginate-Ca(2+)/Ba(2+) microbeads allow for long-term in vivo human islet graft function, despite long-distance shipment.

A chemoenzymatic strategy has been exploited to make covalently linked alginate beads with high stability. This was achieved by grafting mannuronan (alginate with 100% mannuronic acid (M)) with methacrylate moieties and then performing two enzymatic steps converting M to guluronic acid (G) in alternating sequences (MG-blocks) and in G-blocks. In this way a methacrylate grafted alginate with better gel-forming ability was achieved. Covalent bindings were introduced into the beads by using a photoinitiating system that initiated polymerization of the methacrylate moieties. The covalent links were demonstrated by beads remaining intact after treatment with EDTA. The new chemoenzymatic photocrosslinked (CEPC) beads were compatible with cells with low post-encapsulation ability like C2C12 myoblasts and human pancreatic islets. The islets continued secreting insulin after encapsulation. On contrary, cells with a high post-encapsulation proliferative ability like 293-endo cells died within 2-week post-encapsulation. The exceptional stability and the cell compatibility of the new CEPC beads make them interesting as bioreactors for delivering therapeutic proteins in future applications.

Microcapsules are referred to capsules of spherical shape with a diameter of about 100 to 1000 pm. Besides traditional capsules with a well defined shell and core structure, encapsulation in microbeads without a distinct membrane has also been successful in certain applications. For immobilisation of living cells such as bacteria, yeast or mammalian cells, the encapsulation needs to be performed under relative mild conditions, depending on the cell type. The capsules or beads should be semipermeable to allow diffusion of oxygen and nutrients into the cells inside the capsules and waste products out of the capsules. For immunoisolation purposes, the capsule must be impermeable to host cells and soluble components of the immune system.

Confocal laser scanning microscopy (CLSM) was used to study the distribution of polymers and cross-linking ions in alginate-poly-L-lysine (PLL) -alginate microcapsules made by fluorescent-labeled polymers. CLSM studies of Ca-alginate gel beads made in the presence and absence of non-gelling sodium ions revealed a more inhomogeneous distribution of alginate in beads formed in the absence of non-gelling ions. In the formation of alginate-PLL capsules, the polymer gradients in the preformed gel core were destabilized by the presence of non-gelling ions in the washing step and in the PLL solution. Ca-alginate gels preserved the inhomogeneous structure by exposure to ion-free solution in contrast to exposure to non-gelling ions (Na(+)). By exchanging Ca(2+) with Ba(2+) (10 mM), extremely inhomogeneous gel beads were formed that preserved their structure during the washing and exposure to PLL in saline. PLL was shown to bind at the very surface of the alginate core, forming a shell-like membrane. The thickness of the PLL-layer increased about 100% after 2 weeks of storage, but no further increase was seen after 2 years of storage. The coating alginate was shown to overlap the PLL layer. No difference in binding could be observed among coating alginates of different composition. This paper shows an easy and novel method to study the distribution of alginate and PLL in intact microcapsules. As the labeling procedures are easy to perform, the method can also be used for a variety of other polymers in other microencapsulation systems.

Alginate is widely used for encapsulation of cells. Alginate is a linear block copolymer consisting of mannuronic acid (M) and guluronic acid (G). It has been shown that enzymes known as C-5 epimerases convert M to G in the polymer chain, giving rise to novel alginates with tailored properties. One of these enzymes, AlgE4, converts M blocks into blocks of strictly alternating M and G. In this study we investigated how alginate epimerized by AlgE4 affected capsule properties such as stability and permeability. Inhomogeneous calcium-alginate gel beads were made with original and AlgE4-epimerized alginates of different origin. The epimerized alginates formed initially smaller alginate gels that showed increased resistance to osmotic swelling compared with the original nonmodified alginate samples. The permeability, measured as diffusion of immunoglobulin (Ig) G into Ca/Ba-alginate gel beads, was reduced by epimerization and further reduced by addition of poly-L-lysine (PLL). The osmotic stability of alginate-poly-D-lysine(PDL)-alginate capsules was enhanced by the use of epimerized alginate; indeed, stable capsules with low permeability to tumor necrosis factor (TNF) could be made with low PDL exposures. Finally, alginate with an alternating structure interacted more strongly with the alginate-PLL capsule than did alginate with a high content of M blocks or G blocks or than an alginate consisting mainly of M.

Alginate-polylysine-alginate capsules containing insulin-producing cells have been used as a bio-artificial pancreas in the treatment of diabetes mellitus. In a search for microcapsules with improved diffusion characteristics, a high voltage system was developed that produces 250,000 beads/min with a diameter of 160 microm +/- 3-5%. The diameter of the beads could be varied between 160-700 microm depending on the needle diameter and construction, the voltage, the distance between the electrodes and the flow of alginate solution. Ca-alginate beads with diameters of 200 and 500 microm were produced by the high voltage electrostatic system. The 200 microm beads were sensitive to poly-L-lysine (PLL) exposure and had to be washed in ion-free solution to avoid collapse. The 200 microm beads swelled more than the 500 microm beads in the washing and PLL treatment. Also, the porosity of the capsules changed with size, but capsules impermeable to tumour necrosis factor (TNF) could be made by exchanging PLL with poly-D-lysine (PDL) for the 500 microm beads. The 200 microm beads were impermeable to IgG after PLL exposure. Islets of Langerhans were encapsulated in alginate-PLL-alginate capsules and evaluated by measuring protruding islets and insulin production. Islets in microcapsules made by the high voltage electrostatic system did not function differently from islets in larger microcapsules made by an air jet system. In conclusion, alginate capsules made by a high voltage electrostatic system enable large-scale production of small capsules with a narrow size distribution that can meet the functional properties of larger capsules by small changes in the encapsulation procedure.

Alginate – poly-l-lysine (PLL) microcapsules can be used for transplantation of insulin-producing cells for treatment of type I diabetes. In this work we wanted to study the inflammatory reactions against implanted microcapsules due to PLL. We have seen that by reducing the PLL layer, less overgrowth of the capsule is obtained. By incubating different cell types with PLL and afterwards measuring cell viability with MTT, we found massive cell death at concentrations of PLL higher than 10 μg/ml. Staining with annexin V and propidium iodide showed that PLL induced necrosis but not apoptosis. The proinflammatory cytokine, tumor necrosis factor (TNF), was detected in supernatants from monocytes stimulated with PLL. The TNF response was partly inhibited with antibodies against CD14, which is a well-known receptor for lipopolysaccharide (LPS). Bactericidal permeability increasing protein (BPI) and a lipid A analogue (B-975), which both inhibit LPS, did not inhibit PLL from stimulating monocytes to TNF production. This indicates that PLL and LPS bind to different sites on monocytes, but because they both are inhibited by a p38 MAP kinase inhibitor, they seem to have a common element in the signal transducing pathway. These results suggest that PLL may provoke inflammatory responses either directly or indirectly through its necrosis-inducing abilities. By combining soluble PLL and alginate both the toxic and TNF-inducing effects of PLL were reduced. The implications of these data are to use alginate microcapsules with low amounts of PLL for transplantation purposes.