SULFATED ALGINATES: Characterization and applications as biomaterials

Abstract

As natural constituents of the extracellular matrix, sulfated glycosaminoglycans (GAGs) are of great interest in the design of novel biomaterials for cell immobilization and tissue engineering. A substantial challenge in the characterization and application of sulfated GAGs lies in their structural heterogeneity, motivating the exploration of alternative functional analogs. Alginate shows promise for cell immobilization due to its gentle gelling conditions and good biocompatibility, but is limited by its inertness thus requiring functionalization to promote desired cell interactions. Throughout the work presented in this thesis alginates were chemically sulfated to create structural analogs to sulfated GAGs, exhibiting gel-forming capability and a customizable monosaccharide sequence and sulfation degree. Homogeneous sequences of alginate (poly-M, poly-G and poly-MG) were sulfated and shown to interact with hepatocyte growth factor (HGF) and fibroblast growth factor-2 (FGF- 2), where the interaction strength was influenced by both sulfation degree and alginate monosaccharide sequence. Conformational flexibility has been proposed to influence the protein-binding properties of heparin, and growth factor interaction with sulfated alginates was found to increase with chain flexibility introduced by periodate oxidation. Furthermore, the influence of chain length was studied using sulfated alginate oligosaccharides, revealing similarities to heparin/heparan sulfate (HS) interactions. Sulfated alginate was found to have a stimulating effect on the proliferation, spreading and deposition of extracellular matrix by encapsulated chondrocytes. The effect was shown to be mediated by increased FGF-2 signaling where the sulfated alginate scaffold interacts with the growth factor and the FGF cell receptor, analogous to heparan sulfate in native tissues. Sulfation has a derogatory effect on the gelling properties of alginate, resulting in decreased elastic modulus and increased osmotic swelling due to weakened ionic crosslinks. Mixtures containing non-sulfated alginate were found to generate more stable gels, allowing tuning of mechanical properties and inclusion of highly sulfated alginates to optimize biological activity. Encapsulation of chondrocytes in sulfated alginate gels resulted in reduced expression of inflammatory markers following stimulation with interleukin-1beta (IL-1β), while soluble sulfated alginates have been found to reduce complement activation in human plasma. The anti-inflammatory effects of sulfated alginates were further explored by incubation of alginate microspheres in human whole blood. Poly-L-lysine (PLL) coated alginate microcapsules were coated with an additional layer of sulfated poly-MG (SMG) alginate and were found to reduce the cytokine response whereas complement activation was increased. Non-coated microbeads of SMG mixed with unmodified alginate were found to be highly immunocompatible, reducing induction of complement and cytokines compared to pure alginate beads. Both alginate and to a greater extent sulfated alginate were found to bind factor H, potentially contributing to their anti-inflammatory nature.