Cell encapsulation in alginate microbeads is a promising approach to provide immune isolation in ... more 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.
Biologic scaffolds composed of extracellular matrix components have been proposed to repair and r... more Biologic scaffolds composed of extracellular matrix components have been proposed to repair and reconstruct a variety of tissues in clinical and pre-clinical studies. Injectable gels can fill and conform any three-dimensional shape and can be delivered to sites of interest by minimally invasive techniques. In this study, a biological gel was produced from a decellularized porcine urinary bladder by enzymatic digestion with pepsin. The enzymatic digestion was confirmed by visual inspection after dissolution in phosphate-buffered saline solution and Fourier-transform infrared spectroscopy. The rheological and biological properties of the gel were characterized and compared to those of the MatrigelTM chosen as a reference material. The storage modulus G’ reached 19.4 ± 3.7 Pa for the 30 mg/mL digested decellularized bladder gels after ca. 3 h at 37 °C. The results show that the gel formed of the porcine urinary bladder favored the spontaneous differentiation of human and rabbit adipose...
Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the m... more Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalli...
In this work, the mechanical properties and stability of alginate hydrogels containing functional... more In this work, the mechanical properties and stability of alginate hydrogels containing functionalized alginates (peptide and β-cyclodextrin) were studied. There is an increasing interest in the modification of alginates to add functions such as cell attachment and increased solubility of hydrophobic drugs, for better performance in tissue engineering and drug release, respectively. Functionalization was achieved in this study via periodate oxidation followed by reductive amination, previously shown to give a high and controllable degree of substitution. Young’s modulus and the stress at rupture of the hydrogels were in general lowered when exchanging native alginate with the modified alginate. Still, the gel strength could be adjusted by the fraction of modified alginate in the mixed hydrogels as well as the degree of oxidation. No notable difference in deformation at rupture was observed while syneresis was influenced by the degree of oxidation and possibly by the nature and amount...
Inks for 3D printing based on cellulose nanofibrils (CNFs) or mixtures of CNFs and either cellulo... more Inks for 3D printing based on cellulose nanofibrils (CNFs) or mixtures of CNFs and either cellulose nanocrystals (CNCs) or alginate were assessed by determining their viscoelastic properties i.e. complex viscosity and storage and loss moduli (G′ and G″). Two types of alginates were used, i.e. from Laminaria hyperborea stipe and Macrocystis pyrifera. Shape fidelity of 3D printed grids were qualitatively evaluated and compared to the viscoelastic properties of the inks. The biocomposite gels containing alginate were post stabilized by crosslinking with Ca2+. Mechanical properties of the crosslinked biocomposite gels were assessed. The complex viscosity, G′ and G″ of CNF suspensions increased when the solid content was increased from 3.5 to 4.0 wt%, but levelled off by further increase in CNF solid content. The complex viscosity at low angular frequency at 4 wt% was as high as 104 Pa·s. This seemed to be the necessary viscosity level for obtaining good shape fidelity of the printed structures for the studied systems. By replacing part of the CNFs with CNCs, the complex viscosity, G′ and G″ were reduced and so was also the shape fidelity of the printed grids. The changes in complex viscosity and moduli when CNFs was replaced with alginate depended on the relative amounts of CNFs/alginate. The type of alginate (from either L. hyp. stipe or M. pyr.) did not play a role for the viscoelastic properties of the inks, nor for the printed grids before post stabilization. Replacing CNFs with up to 1.5 wt% alginate gave satisfactory shape fidelity. The effect of adding alginate and subsequent crosslinking with Ca2+, strongly affected the strength properties of the gels. By appropriate choice of relative amounts of CNFs and alginate and type of alginate, the Young’s modulus and rupture strength could be controlled within the range of 30–150 kPa and 1.5–6 kg, respectively. The deformation at rupture was around 55%. The alginate from L. hyp. stipe yields higher Young’s modulus and lower syneresis compared to M. pyr. This shows that the choice of alginate plays a significant role for the mechanical properties of the final product, although it does not influence on the viscoelastic properties of the ink. The choice of alginate should be L. hyp. stipe if high strength is desired.Graphical abstract
Transplantation of pancreatic islets in immune protective capsules holds the promise as a functio... more Transplantation of pancreatic islets in immune protective capsules holds the promise as a functional cure for type 1 diabetes, also about 40 years after the first proof of principal study. The concept is simple in using semipermeable capsules that allow the ingress of oxygen and nutrients, but limit the access of the immune system. Encapsulated human islets have been evaluated in four small clinical trials where the procedure has been evaluated as safe, but lacking long-term efficacy. Host reactions toward the biomaterials used in the capsules may be one parameter limiting the long-term function of the graft in humans. The present article briefly discusses important capsule properties such as stability, permeability and biocompatibility, as well as possible strategies to overcome current challenges. Also, recent progress in capsule development as well as the production of insulin-producing cells from human stem cells that gives promising perspectives for the transplantation of encap...
Alginate-poly-L-lysine(PLL)-alginate capsules are the far most studied system for encapsulation o... more Alginate-poly-L-lysine(PLL)-alginate capsules are the far most studied system for encapsulation of living cells. Immobilisation in ionically cross-linked alginate provides a simple and safe encapsulation procedure at physiological conditions. The properties of alginate, a linear natural polysaccharide consisting of mannuronic acid (M) and guluronic acid (G), is very much dependent on the composition of monomers in the chain. Enzymatic modification of the alginate by in-chain conversion of M to G by mannuronan C-5 epimerases enables us to tailor alginate to specific properties. In this study, an epimerase that converts M-blocks to blocks of alternating M and G was used. By this conversion, more flexible segments are formed, but the molecular weight and the number and length of the G-blocks that determine the cross-linking sequences of the alginate is not altered. The enzymatic modification increased the elasticity of diffusion set Caalginate gels. Increased syneresis was also obtained that could be seen as reduction in size of the Ca-alginate gel beads. The osmotic stability was increased after epimerisation and reduced permeability to immunoglobulin G (IgG) and tumour necrosis factor (TNF) was obtained.We have developed an electrostatic droplet generator that enables large-scale production of small alginate gel beads with a narrow size distribution. Encapsulated islets of Langerhans preserved their function in the new system. We observed that the smaller beads were more vulnerable to the encapsulation procedure, but stable alginate PLLalginate capsules of 200μm in diameter that were impermeable to IgG could be made by small changes in the encapsulation procedure. As Ca-alginate beads of epimerised alginate are initially smaller and more resistant to swelling than those made from the original alginate, the use of epimerised alginate reduces the problems of making small capsules.Visualisation of the capsules in the confocal laser scanning microscopy (CLSM) by fluorescent-labelled alginate and PLL, revealed an easy method to assess the distribution of polymers in intact capsules. It thus makes it easier to characterise the final capsules also by means of polymer distribution. This has previously been shown to vary with different encapsulation procedures and is important for the capsule properties.PLL was shown to be the major factor causing overgrowth of implanted empty capsules in mice as the fibrotic reaction depended on the PLL exposure. Further studies revealed that at low concentrations, PLL induced TNF production in human monocytes. At higher concentrations, PLL was toxic, causing necrosis of the exposed cells. Both the TNF inducing and the toxic effect of PLL was reduced by addition of soluble alginate, but the study shows that for producing biocompatible capsules, low amounts of PLL should be used to prevent inflammation. The effect of PLL was further verified, as better coating with compositionally homogeneous alginates with MG as repeating unit reduced the overgrowth on the implanted capsules. This strictly alternating coating alginate was produced by modifying mannuronan with a recombinant mannuronan C-5 epimerase. No differences in biocompatibility was seen on alginate gel beads of high-G and epimerised high-G alginates.Hence, increasing the flexible segments in the alginate chain by epimerisation leads to capsules of improved properties as it comes to size, stability, permeability and biocompatibility. The use of specific mannuronan C-5 epimerases thus provides a novel method for tailoring alginates to specific uses, such as immunoisolation of living cells.
Biomineral formation in vivo is a complex process regulated by functional molecules. Highlighting... more Biomineral formation in vivo is a complex process regulated by functional molecules. Highlighting the mechanisms underlying biomineralization is necessary for a better understanding of in vivo processes and for enhanced in vitro model systems. Here, the effect of alginate and its well-defined oligomers with M- or G-block structure on brushite nucleation and growth is investigated by seeded and unseeded experiments. Growth kinetics were studied by seeded experiments, and it was shown that molecular weight and functionality of alginate additives affect the crystal growth rates and the growth mechanisms. Growth retardation was most prominent when G-block additives were present. Growth proceeded by surface nucleation when alginate and G-block oligomers were added in the crystallization medium, whereas in the presence of M-block oligomers parabolic rate laws were obtained. By decoupling the seeded and unseeded experiments, information was deduced on the effects of additives on brushite nucleation. In the prese...
Islet transplantation in diabetes is hampered by the need of life-long immunosuppression. Encapsu... more Islet transplantation in diabetes is hampered by the need of life-long immunosuppression. Encapsulation provides partial immunoprotection but could possibly limit oxygen supply, a factor that may enhance hypoxia-induced beta cell death in the early posttransplantation period. Here we tested susceptibility of alginate microencapsulated human islets to experimental hypoxia (0.1-0.3% O2 for 8 h, followed by reoxygenation) on viability and functional parameters. Hypoxia reduced viability as measured by MTT by 33.8 ± 3.5% in encapsulated and 42.9 ± 5.2% in nonencapsulated islets (P < 0.2). Nonencapsulated islets released 37.7% (median) more HMGB1 compared to encapsulated islets after hypoxic culture conditions (P < 0.001). Glucose-induced insulin release was marginally affected by hypoxia. Basal oxygen consumption was equally reduced in encapsulated and nonencapsulated islets, by 22.0 ± 6.1% versus 24.8 ± 5.7%. Among 27 tested cytokines/chemokines, hypoxia increased the secretion o...
Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic c... more Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.
ABSTRACT The whole blood model is a powerful method to determine the immediate inflammatory react... more ABSTRACT The whole blood model is a powerful method to determine the immediate inflammatory reactions towards foreign objects in general. This review focuses on the use of a lepirudin based whole blood model for evaluating microspheres relevant in cell transplantation applications. This whole blood model can be regarded as a holistic model with readouts from cross-talks between leukocytes, complement, most of the coagulation components and fibrinolysis. A major advantage of this model is the possibility of evaluating a panel of different microspheres under identical conditions, and also the possibility of comparing reaction patterns between species. This model is a valuable tool for gaining a mechanistic understanding by selected readouts (as complement and coagulation activation products, cytokines, cell-surface receptors, protein adsorption, cell-attachment), and by use of inflammatory blocking agents (inhibitors). The whole blood model is put in the context of today’s knowledge about inflammatory systems, discussed according to biocompatibility and biotolerability terms and finally discussed according to its ability to predict the outcome of transplanted microspheres in an in vivo environment.
Alginate-poly-L-lysine (PLL) microcapsules can be used for transplantation of insulin-producing c... more 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 microg/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 mo...
Cell encapsulation in alginate microbeads is a promising approach to provide immune isolation in ... more 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.
Biologic scaffolds composed of extracellular matrix components have been proposed to repair and r... more Biologic scaffolds composed of extracellular matrix components have been proposed to repair and reconstruct a variety of tissues in clinical and pre-clinical studies. Injectable gels can fill and conform any three-dimensional shape and can be delivered to sites of interest by minimally invasive techniques. In this study, a biological gel was produced from a decellularized porcine urinary bladder by enzymatic digestion with pepsin. The enzymatic digestion was confirmed by visual inspection after dissolution in phosphate-buffered saline solution and Fourier-transform infrared spectroscopy. The rheological and biological properties of the gel were characterized and compared to those of the MatrigelTM chosen as a reference material. The storage modulus G’ reached 19.4 ± 3.7 Pa for the 30 mg/mL digested decellularized bladder gels after ca. 3 h at 37 °C. The results show that the gel formed of the porcine urinary bladder favored the spontaneous differentiation of human and rabbit adipose...
Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the m... more Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalli...
In this work, the mechanical properties and stability of alginate hydrogels containing functional... more In this work, the mechanical properties and stability of alginate hydrogels containing functionalized alginates (peptide and β-cyclodextrin) were studied. There is an increasing interest in the modification of alginates to add functions such as cell attachment and increased solubility of hydrophobic drugs, for better performance in tissue engineering and drug release, respectively. Functionalization was achieved in this study via periodate oxidation followed by reductive amination, previously shown to give a high and controllable degree of substitution. Young’s modulus and the stress at rupture of the hydrogels were in general lowered when exchanging native alginate with the modified alginate. Still, the gel strength could be adjusted by the fraction of modified alginate in the mixed hydrogels as well as the degree of oxidation. No notable difference in deformation at rupture was observed while syneresis was influenced by the degree of oxidation and possibly by the nature and amount...
Inks for 3D printing based on cellulose nanofibrils (CNFs) or mixtures of CNFs and either cellulo... more Inks for 3D printing based on cellulose nanofibrils (CNFs) or mixtures of CNFs and either cellulose nanocrystals (CNCs) or alginate were assessed by determining their viscoelastic properties i.e. complex viscosity and storage and loss moduli (G′ and G″). Two types of alginates were used, i.e. from Laminaria hyperborea stipe and Macrocystis pyrifera. Shape fidelity of 3D printed grids were qualitatively evaluated and compared to the viscoelastic properties of the inks. The biocomposite gels containing alginate were post stabilized by crosslinking with Ca2+. Mechanical properties of the crosslinked biocomposite gels were assessed. The complex viscosity, G′ and G″ of CNF suspensions increased when the solid content was increased from 3.5 to 4.0 wt%, but levelled off by further increase in CNF solid content. The complex viscosity at low angular frequency at 4 wt% was as high as 104 Pa·s. This seemed to be the necessary viscosity level for obtaining good shape fidelity of the printed structures for the studied systems. By replacing part of the CNFs with CNCs, the complex viscosity, G′ and G″ were reduced and so was also the shape fidelity of the printed grids. The changes in complex viscosity and moduli when CNFs was replaced with alginate depended on the relative amounts of CNFs/alginate. The type of alginate (from either L. hyp. stipe or M. pyr.) did not play a role for the viscoelastic properties of the inks, nor for the printed grids before post stabilization. Replacing CNFs with up to 1.5 wt% alginate gave satisfactory shape fidelity. The effect of adding alginate and subsequent crosslinking with Ca2+, strongly affected the strength properties of the gels. By appropriate choice of relative amounts of CNFs and alginate and type of alginate, the Young’s modulus and rupture strength could be controlled within the range of 30–150 kPa and 1.5–6 kg, respectively. The deformation at rupture was around 55%. The alginate from L. hyp. stipe yields higher Young’s modulus and lower syneresis compared to M. pyr. This shows that the choice of alginate plays a significant role for the mechanical properties of the final product, although it does not influence on the viscoelastic properties of the ink. The choice of alginate should be L. hyp. stipe if high strength is desired.Graphical abstract
Transplantation of pancreatic islets in immune protective capsules holds the promise as a functio... more Transplantation of pancreatic islets in immune protective capsules holds the promise as a functional cure for type 1 diabetes, also about 40 years after the first proof of principal study. The concept is simple in using semipermeable capsules that allow the ingress of oxygen and nutrients, but limit the access of the immune system. Encapsulated human islets have been evaluated in four small clinical trials where the procedure has been evaluated as safe, but lacking long-term efficacy. Host reactions toward the biomaterials used in the capsules may be one parameter limiting the long-term function of the graft in humans. The present article briefly discusses important capsule properties such as stability, permeability and biocompatibility, as well as possible strategies to overcome current challenges. Also, recent progress in capsule development as well as the production of insulin-producing cells from human stem cells that gives promising perspectives for the transplantation of encap...
Alginate-poly-L-lysine(PLL)-alginate capsules are the far most studied system for encapsulation o... more Alginate-poly-L-lysine(PLL)-alginate capsules are the far most studied system for encapsulation of living cells. Immobilisation in ionically cross-linked alginate provides a simple and safe encapsulation procedure at physiological conditions. The properties of alginate, a linear natural polysaccharide consisting of mannuronic acid (M) and guluronic acid (G), is very much dependent on the composition of monomers in the chain. Enzymatic modification of the alginate by in-chain conversion of M to G by mannuronan C-5 epimerases enables us to tailor alginate to specific properties. In this study, an epimerase that converts M-blocks to blocks of alternating M and G was used. By this conversion, more flexible segments are formed, but the molecular weight and the number and length of the G-blocks that determine the cross-linking sequences of the alginate is not altered. The enzymatic modification increased the elasticity of diffusion set Caalginate gels. Increased syneresis was also obtained that could be seen as reduction in size of the Ca-alginate gel beads. The osmotic stability was increased after epimerisation and reduced permeability to immunoglobulin G (IgG) and tumour necrosis factor (TNF) was obtained.We have developed an electrostatic droplet generator that enables large-scale production of small alginate gel beads with a narrow size distribution. Encapsulated islets of Langerhans preserved their function in the new system. We observed that the smaller beads were more vulnerable to the encapsulation procedure, but stable alginate PLLalginate capsules of 200μm in diameter that were impermeable to IgG could be made by small changes in the encapsulation procedure. As Ca-alginate beads of epimerised alginate are initially smaller and more resistant to swelling than those made from the original alginate, the use of epimerised alginate reduces the problems of making small capsules.Visualisation of the capsules in the confocal laser scanning microscopy (CLSM) by fluorescent-labelled alginate and PLL, revealed an easy method to assess the distribution of polymers in intact capsules. It thus makes it easier to characterise the final capsules also by means of polymer distribution. This has previously been shown to vary with different encapsulation procedures and is important for the capsule properties.PLL was shown to be the major factor causing overgrowth of implanted empty capsules in mice as the fibrotic reaction depended on the PLL exposure. Further studies revealed that at low concentrations, PLL induced TNF production in human monocytes. At higher concentrations, PLL was toxic, causing necrosis of the exposed cells. Both the TNF inducing and the toxic effect of PLL was reduced by addition of soluble alginate, but the study shows that for producing biocompatible capsules, low amounts of PLL should be used to prevent inflammation. The effect of PLL was further verified, as better coating with compositionally homogeneous alginates with MG as repeating unit reduced the overgrowth on the implanted capsules. This strictly alternating coating alginate was produced by modifying mannuronan with a recombinant mannuronan C-5 epimerase. No differences in biocompatibility was seen on alginate gel beads of high-G and epimerised high-G alginates.Hence, increasing the flexible segments in the alginate chain by epimerisation leads to capsules of improved properties as it comes to size, stability, permeability and biocompatibility. The use of specific mannuronan C-5 epimerases thus provides a novel method for tailoring alginates to specific uses, such as immunoisolation of living cells.
Biomineral formation in vivo is a complex process regulated by functional molecules. Highlighting... more Biomineral formation in vivo is a complex process regulated by functional molecules. Highlighting the mechanisms underlying biomineralization is necessary for a better understanding of in vivo processes and for enhanced in vitro model systems. Here, the effect of alginate and its well-defined oligomers with M- or G-block structure on brushite nucleation and growth is investigated by seeded and unseeded experiments. Growth kinetics were studied by seeded experiments, and it was shown that molecular weight and functionality of alginate additives affect the crystal growth rates and the growth mechanisms. Growth retardation was most prominent when G-block additives were present. Growth proceeded by surface nucleation when alginate and G-block oligomers were added in the crystallization medium, whereas in the presence of M-block oligomers parabolic rate laws were obtained. By decoupling the seeded and unseeded experiments, information was deduced on the effects of additives on brushite nucleation. In the prese...
Islet transplantation in diabetes is hampered by the need of life-long immunosuppression. Encapsu... more Islet transplantation in diabetes is hampered by the need of life-long immunosuppression. Encapsulation provides partial immunoprotection but could possibly limit oxygen supply, a factor that may enhance hypoxia-induced beta cell death in the early posttransplantation period. Here we tested susceptibility of alginate microencapsulated human islets to experimental hypoxia (0.1-0.3% O2 for 8 h, followed by reoxygenation) on viability and functional parameters. Hypoxia reduced viability as measured by MTT by 33.8 ± 3.5% in encapsulated and 42.9 ± 5.2% in nonencapsulated islets (P < 0.2). Nonencapsulated islets released 37.7% (median) more HMGB1 compared to encapsulated islets after hypoxic culture conditions (P < 0.001). Glucose-induced insulin release was marginally affected by hypoxia. Basal oxygen consumption was equally reduced in encapsulated and nonencapsulated islets, by 22.0 ± 6.1% versus 24.8 ± 5.7%. Among 27 tested cytokines/chemokines, hypoxia increased the secretion o...
Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic c... more Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.
ABSTRACT The whole blood model is a powerful method to determine the immediate inflammatory react... more ABSTRACT The whole blood model is a powerful method to determine the immediate inflammatory reactions towards foreign objects in general. This review focuses on the use of a lepirudin based whole blood model for evaluating microspheres relevant in cell transplantation applications. This whole blood model can be regarded as a holistic model with readouts from cross-talks between leukocytes, complement, most of the coagulation components and fibrinolysis. A major advantage of this model is the possibility of evaluating a panel of different microspheres under identical conditions, and also the possibility of comparing reaction patterns between species. This model is a valuable tool for gaining a mechanistic understanding by selected readouts (as complement and coagulation activation products, cytokines, cell-surface receptors, protein adsorption, cell-attachment), and by use of inflammatory blocking agents (inhibitors). The whole blood model is put in the context of today’s knowledge about inflammatory systems, discussed according to biocompatibility and biotolerability terms and finally discussed according to its ability to predict the outcome of transplanted microspheres in an in vivo environment.
Alginate-poly-L-lysine (PLL) microcapsules can be used for transplantation of insulin-producing c... more 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 microg/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 mo...
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