Gudmund Skjåk-Bræk

Heparin-like properties of sulfated alginates with defined sequences and sulfation degrees.

Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar molecular weights were chemically sulfated and structurally characterized by the use of NMR and elemental analysis. These sulfated alginates were shown to bind and displace HGF from the surface of myeloma cells in a manner similar to heparin. We observed dependence on the sulfation degree (DS) as well as variation in efficacy based on the alginate monosaccharide sequence, relating to relative flexibility and charge density in the polysaccharide chains. Co-incubation with human plasma showed complement compatibility of the alginates and lowering of soluble terminal complement complex levels by sulfated alginates. The sulfated polyalternating (poly-MG) alginate proved to be the most reproducible in terms of precise sulfation degrees and showed the greatest relative degree of complement inhibition and HGF interaction, maintaining high activity at low DS values.

RGD-peptide modified alginate by a chemoenzymatic strategy for tissue engineering applications

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.

Sulfated Hydrogel Matrices Direct Mitogenicity and Maintenance of Chondrocyte Phenotype through Activation of FGF Signaling

Deciphering the roles of chemical and physical features of the extracellular matrix (ECM) is vital for developing biomimetic materials with desired cellular responses in regenerative medicine. Here, we demonstrate that sulfation of biopolymers, mimicking the proteoglycans in native tissues, induces mitogenicity, chondrogenic phenotype, and suppresses catabolic activity of chondrocytes, a cell type that resides in a highly sulfated tissue. We show through tunable modification of alginate that increased sulfation of the microenvironment promotes FGF signaling-mediated proliferation of chondrocytes in a three-dimensional (3D) matrix independent of stiffness, swelling, and porosity. Furthermore, we show for the first time that a biomimetic hydrogel acts as a 3D signaling matrix to mediate a heparan sulfate/heparin-like interaction between FGF and its receptor leading to signaling cascades inducing cell proliferation, cartilage matrix production, and suppression of de-differentiation markers. Collectively, this study reveals important insights on mimicking the ECM to guide self-renewal of cells via manipulation of distinct signaling mechanisms.

Analysis of G‑Block Distributions and Their Impact on Gel Properties of in Vitro Epimerized Mannuronan

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 Youngs 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.

Current and Future Perspectives on Alginate Encapsulated Pancreatic Islet

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 encapsulated insulin‐producing tissue is briefly discussed. Stem Cells Translational Medicine 2017;6:1053–1058

Single molecule investigation of the onset and minimum size of the calcium-mediated junction zone in alginate

One of the principal roles of alginate, both natively and in commercial applications, is gelation via Ca(2+)-mediated crosslinks between blocks of guluronic acid. In this work, single molecule measurements were carried out between well-characterised series of nearly monodisperse guluronic acid blocks (oligoGs) using dynamic force spectroscopy. The measurements provide evidence that for interaction times on the order of tens of milliseconds the maximum crosslink strength is achieved by pairs of oligoGs long enough to allow the coordination of 4Ca(2+) ions, with both shorter and longer oligomers forming weaker links. Extending the interaction time from tens to hundreds of milliseconds allows longer oligoGs to achieve much stronger crosslinks but does not change the strength of individual links between shorter oligoGs. These results are considered in light of extant models for the onset of cooperative crosslinking in polyelectrolytes and an anisotropic distribution of oligoGs on interacting surfaces and provide a timescale for the formation and relaxation of alginate gels at the single crosslink level.

The Impact of Chain Length and Flexibility in the Interaction between Sulfated Alginates and HGF and FGF-2.

Alginate is a promising polysaccharide for use in biomaterials as it is biologically inert. One way to functionalize alginate is by chemical sulfation to emulate sulfated glycosaminoglycans, which interact with a variety of proteins critical for tissue development and homeostasis. In the present work we studied the impact of chain length and flexibility of sulfated alginates for interactions with FGF-2 and HGF. Both growth factors interact with defined sequences of heparan sulfate (HS) at the cell surface or in the extracellular matrix. Whereas FGF-2 interacts with a pentasaccharide sequence containing a critical 2-O-sulfated iduronic acid, HGF has been suggested to require a highly sulfated HS/heparin octasaccharide. Here, oligosaccharides of alternating mannuronic and guluronic acid (MG) were sulfated and assessed by their relative efficacy at releasing growth factor bound to the surface of myeloma cells. 8-mers of sulfated MG (SMG) alginate showed significant HGF release compared to shorter fragments, while the maximum efficacy was achieved at a chain length average of 14 monosaccharides. FGF-2 release required a higher concentration of the SMG fragments, and the 14-mer was less potent compared to an equally sulfated high-molecular weight SMG. Sulfated mannuronan (SM) was subjected to periodate oxidation to increase chain flexibility. To assess the change in flexibility, the persistence length was estimated by SEC-MALLS analysis and the Bohdanecky approach to the worm-like chain model. A high degree of oxidation of SM resulted in approximately twice as potent HGF release compared to the nonoxidized SM alginate. The release of FGF-2 also increased with the degree of oxidation, but to a lower degree compared to that of HGF. It was found that the SM alginates were more efficient at releasing FGF-2 than the SMG alginates, indicating a greater dependence on monosaccharide identity and charge orientation over chain flexibility and charge density.

Sulfated alginate microspheres associate with factor H and dampen the inflammatory cytokine response.

UNLABELLEDnAlginate microspheres show promise for cell-encapsulation therapy but encounter challenges related to biocompatibility. In the present work we designed novel microbeads and microcapsules based on sulfated polyalternating MG alginate (SMG) and explored their inflammatory properties using a human whole blood model. SMG was either incorporated within the alginate microbeads or used as a secondary coat on poly-l-lysine (PLL)-containing microcapsules, resulting in reduction of the inflammatory cytokines (IL-1β, TNF, IL-6, IL-8, MIP-1α). The sulfated alginate microbeads exhibited a complement inert nature with no induction of terminal complement complex (TCC) above the values in freshly drawn blood and low surface accumulation of C3/C3b/iC3b. Conversely, SMG as a coating material lead to substantial TCC amounts and surface C3/C3b/iC3b. A common thread was an increased association of the complement inhibitor factor H to the alginate microbeads and microcapsules containing sulfated alginates. Factor H was also found to associate to non-sulfated alginate microbeads in lower amounts, indicating factor H binding as an inherent property of alginate. We conclude that the dampening effect on the cytokine response and increased factor H association points to sulfated alginate as a promising strategy for improving the biocompatibility of alginate microspheres.nnnSTATEMENT OF SIGNIFICANCEnAlginate microspheres are candidate devices for cell encapsulation therapy. The concept is challenged by the inflammatory host response, and modification strategies for improved biocompatibility are urgently needed. One potential strategy is using sulfated alginates, acting as versatile heparin analogues with similar anti-inflammatory properties. We designed novel alginate microspheres using sulfated alginate with an alternating sequence mimicking glycosominoglycans. Evaluation in a physiologically relevant human whole blood model revealed a reduction of inflammatory cytokines by a sulfated alginate coating, and sulfated alginate microbeads were complement inert. These effects were correlated with a strong factor H association, which may represent the mechanistic explanation. This novel approach could improve the biocompatibility of alginate microspheres in vivo and present a new strategy toward clinical use.

Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

Background: Alginate epimerases consist of catalytic and noncatalytic domains of yet unknown function. Results: The noncatalytic domains of AlgE4 and AlgE6 possess different alginate binding behavior despite highly similar structures. Conclusion: Noncatalytic subunits of AlgE6 and AlgE4 influence the product specificity of the catalytic domain. Significance: This work opens a new route to designing alginate epimerases producing tailored alginates. The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.

Alginate microsphere compositions dictate different mechanisms of complement activation with consequences for cytokine release and leukocyte activation

The inflammatory potential of 12 types of alginate-based microspheres was assessed in a human whole blood model. The inflammatory potential could be categorized from low to high based on the four main alginate microsphere types; alginate microbeads, liquefied core poly-l-ornithine (PLO)-containing microcapsules, liquefied core poly-l-lysine (PLL)-containing microcapsules, and solid core PLL-containing microcapsules. No complement or inflammatory cytokine activation was detected for the Ca/Ba alginate microbeads. Liquefied core PLO- and PLL-containing microcapsules induced significant fluid phase complement activation (TCC), but with low complement surface deposition (anti-C3c), and a low proinflammatory cytokine secretion, with exception of an elevated MCP-1(CCL2) secretion. The solid core PLL-containing microcapsules generated lower TCC but a marked complement surface deposition and significant induction of the proinflammatory cytokines interleukin (IL-1)β, TNF, IL-6, the chemokines IL-8 (CXCL8), and MIP-1α (CCL3) and MCP-1(CCL2). Inhibition with compstatin (C3 inhibitor) completely abolished complement surface deposition, leukocyte adhesion and the proinflammatory cytokines. The C5 inhibitions partly lead to a reduction of the proinflammatory cytokines. The leukocyte adhesion was abolished by inhibitory antibodies against CD18 and partly reduced by CD11b, but not by CD11c. Anti-CD18 significantly reduced the (IL-1)β, TNF, IL-6 and MIP-1α and anti-CD11b significantly reduced the IL-6 and VEGF secretion. MCP-1 was strongly activated by anti-CD18 and anti-CD11b. In conclusion the initial proinflammatory cytokine responses are driven by the microspheres potential to trigger complement C3 (C3b/iC3b) deposition, leukocyte activation and binding through complement receptor CR3 (CD11b/CD18). MCP-1 is one exception dependent on the fluid phase complement activation mediated through CR3.