Chitosan and chitosan derivatives in tissue engineering and stem cell biology

Abstract

Chitosan is a promising natural substances used in biomaterials research as it has several essential properties that can be applied in tissue engineering. This polymer can be easily combined with other biomaterials and it can be rapidly and economically processed to deliver growth factors and drugs. In the work presented in this thesis, the effect of natural, chitin-derived biomaterials on stem cell biology and osteogenic differentiation was determined and important properties of chitosan for tissue engineering applications were examined. Furthermore, it was evaluated how chitosan derivatives affect the expression and potentially regulate the chitinase-like protein YKL-40 in stem cells, which has been indicated to be involved in tissue remodeling, inflammation and disease pathogenesis. In paper I, we investigated the biological effects of the aminosugar glucosamine, which is the smallest, completely deacetylated subunit of chitin. Glucosamine is best known as a dietary supplement for chondro-protection, yet we were able to demonstrate that it upregulates the expression of osteogenic marker genes, which was strongly correlated to YKL-40 expression. This proposes a so far unknown role for YKL-40 in late-stage osteogenic differentiation. Chito-oligomers, derived from chitosan and chitin, are being increasingly studied owing to their bioactivity and water solubility. The biological potential is strongly dependent on the chemical properties and particularly hexamer and heptamer fractions are being considered most potent. The application of chito-oligomers is frequently limited to antitumor activity and inhibition of angiogenesis, but these chito-oligomers similarly affect gene expression and cytokine secretion, as described in paper II. The potency of hexamer fractions of chito-oligomers is strongly dependent on the degree of deacetylation, ultimately requiring the appropriate choice of chito-oligomer for any particular application. Endotoxin contamination is difficult to avoid during the handling of natural substances, and the biological effects of endotoxins on the body are extensive. Strict regulations are in place to reduce the risk of adverse health effects induced by medical devices, yet these recommendations remain inadequate and insufficiently specified. In paper III, we showed that endotoxin contamination in chitosan derivatives can result in false-positive results, completely altering product performance in vitro. In order to determine relevant properties of chitosan for tissue engineering applications, we prepared chitosan membranes as bioactive coatings. In paper IV, we compared chitosan membranes prepared from a wide range of degree of deacetylation and derived from different sources in terms of surface characteristics and bioactivity. This work resulted in paper V with the development of a standardized protocol for solution casting methods for chitosan membranes, in-house prediction of successful experimental outcome and long-term cell attachment comparable to commonly used tissue culture plastic.