(430i) Optimizing Mechanical and Cell Adhesion Properties of Chitosan through Simultaneous Manipulation of Molecular Weight and Crosslinking

Introduction: Chitosan, obtained by N-deacetylation of the natural polymer chitin has sparked great interest in tissue engineering due to several desirable properties. These include: mild processing conditions, a minimal foreign body reaction, and the availability of side groups for attachment of other active molecules. Current difficulties with using chitosan include low strength and inconsistent behavior with cell adhesion. Covalent crosslinking of chitosan is one approach to modifying its mechanical and biological properties. The covalently stabilized chitosan obtained could have reduced crystallinity, and might thus produce a more amorphous network microstructure with the potential for elastomeric behavior. In this study, we examined the effects of both crosslinking density and chitosan molecular weight (MW) on the properties of crosslinked chitosans. Low molecular weight chitosans were crosslinked with a dicarboxylic acid (sebacic acid), and the mechanical and cell-interaction properties of the resultant materials were characterized. Specifically, monotonic tensile testing of cast chitosan-sebacic acid films was conducted and the adhesion and growth of lamb aortic smooth muscle cells (SMC) on these materials was examined.

Materials and Methods: 1.5 wt% Chitosan (85% deacetylated, 600 kDa, Fluka) was dissolved in 0.1 M HCl. Solution aliquots were reduced to molecular weights of 5 and 50 kDa using nitrous acid depolymerization [1]. Sebacic acid was activated in aqueous solution with 1-ethyl-3-(3-dimethylaminopropyl) carboiidide (EDC). The activated sebacic acid was then blended with reduced MW chitosan solutions for ½ hours to generate sebacate-crosslinked chitosan aggregates in solution. Activated sebacate was blended in at di-acid to chitosan-amine molar ratios of 0.05, 0.1, 0.2 and 0.5 Films of the crosslinked materials were prepared by casting the crosslinked solutions, air drying and then washing with 3% ammonia solution followed by PBS. Rectangular film samples were subjected to uniaxial tensile testing under hydrated conditions to determine tensile strength, elastic modulus (at 20% strain) and breaking strain for the crosslinked and non-crosslinked chitosan films. FTIR spectra, aqueous swelling, and contact angle studies done to characterize material properties. To evaluate cell interactions, films were cast into polystyrene tissue culture dishes, neutralized as described above, and sterilized with 70% isopropanol overnight. After washing with PBS, lamb aortic smooth muscle cells (SMC) were seeded onto the films in serum-supplemented MCDB 131 culture medium. The cells were seeded at a density of 5000 cells/cm2. Cell adhesion and spreading were characterized by quantitative image analysis.

Results and Discussion: Mechanical testing showed that sebacate crosslinking increased tensile strength for high MW chitosan. Furthermore, reducing the chitosan MW to 50 kDa or 10 kDa (from an initial value of 600 kDa) resulted in reduced tensile strengths of films at all crosslinking densities. Low MW chitosan (10kDa) exhibited a peak in strength, breaking strain and elastic modulus at crosslinking densities between 0.05 and 0.2. Membrane swelling characteristics exhibited an inversion for the reduced molecular weights. For 10 kDa and 50 kDa, swelling increased with crosslinking density in contrast to high MW chitosan, where swelling decreased with crosslinking density. Spreading and proliferation of vascular smooth muscle cells exhibited no substantial changes as a function of MW, but both were inhibited at high crosslinking densities. The results obtained are influenced by the presence of hydrophobic domains contributed by the sebacate crosslinker. In addition, changes in chitosan crystallinity due to the steric changes produced during solution crosslinking may also influence film microstructure and serum protein binding. In conclusion, sebacic acid crosslinking of reduced MW chitosans provides a mechanism for modulating both mechanical and cell adhesion/proliferation properties. Current work is aimed at more detailed characterization of the effects of MW, crosslinking density and membrane microstructure, and results will be reported.

References: 1. G Allan, M Peyron, Carbohydrate Research 277 (1995) 273-282.