(15d) GAG-Augmented Chitosan Fibers for Mechanical Enhancement of Heart Valve Scaffolds

Several tissue engineering approaches are being developed to solve problems associated with the currently used heart valve replacement methods (mechanical and biological valves). Natural and synthetic polymers have been investigated as a potential scaffold for tissue-engineered heart valves (TEHVs). Chitosan is a promising, biocompatible, biodegradable, and non-toxic linear polymer that is currently used in a number of tissue engineering applications. It offers a wide variety of mechanical properties depending on the molecular weight (MW), degree of deacetylation (DD), and molecular architecture.

Objectives and Aims:

Current chitosan-based heart valve scaffolds have poor mechanical properties compared to the native valve. Our research effort focuses on improving the biomechanical properties of 3D porous chitosan scaffold for TEHVs. We have developed chitosan fibers with improved mechanical properties and have embedded them within chitosan scaffolds to effect property improvements. However, we observed swelling and partial dissolution of chitosan fibers in the scaffold due to the low pH during scaffold preparation. In order to prevent swelling-related loss of strength, we propose to crosslink chitosan fibers with different glycosaminoglycans (GAGs). This treatment has the added effect of introducing potentially desirable biological activity.

Materials and Methods:

Chitosan fibers were prepared using an extrusion and gelation technique. High molecular weight chitosan solution (1.5 wt% in 0.2M acetic acid) was extruded through a 24-gauge (0.045mm diameter) Teflon catheter directly into 10 vol% ammonia solution. The gelled fibers were then washed with distilled water and immersed in GAG solutions (10 mg/ml Heparin (HS), 5 mg/ml Dermatan sulfate (DS), and 5 mg/ml Chondroitin 4-sulfate (CSA)). GAGs were pre-activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for 15 minutes before immersion of fibers. The covalent crosslinking reaction was allowed to proceed overnight and then the chitosan-GAG fibers were dried at room temperature. Crosslinked fibers were immersed in 1% acetic acid solution and visualized over 24 hours to evaluate dissolution. Images of crosslinked fibers were captured using phase contrast microscopy and diameters were measured using image analysis software. Tensile testing was conducted in fully hydrated conditions to evaluate the ultimate strength, breaking strain and the modulus of elasticity.

Results and Discussion:

Phase contrast microscopy images showed a significant increase in diameters of the GAG-crosslinked fibers compared to the unmodified chitosan fibers (280, 180, 220 µm for CSA, DS, and HS respectively compared to 144 µm of control fibers, P<0.05). Mechanical testing results demonstrated a significant decrease in maximum stress (i.e. strength) and modulus of elasticity (i.e. stiffness) for the crosslinked fibers. HS-chitosan fibers had a 50% decrease in maximum stress (from 8Mpa to 4MPa) and a 75% decrease in modulus of elasticity (80MPa to 20MPa), however, the CSA-crosslinked fibers showed 2 fold improvements in maximum strain (i.e. elasticity) compared to the unmodified fibers (P<0.05), HS and DS maximum strain values were comparable to the unmodified fibers. Acetic acid dissolution studies showed an immediate dissolution of DS-crosslinked fibers, while CSA-crosslinked fibers dissolved over 1 hour. In contrast, HS-crosslinked fibers showed no dissolution up to 24 hours. These findings suggest that the covalent crosslinking of chitosan fibers with HS will prevent dissolution and associated strength loss for scaffold-embedded fibers, which may lead to greater improvement of the scaffold mechanical properties. The effect of embedded HS-chitosan fibers on the scaffold mechanical properties is currently under investigation.