Modular Tissue Engineering with GAG-Chitosan Complex Hollow Fibers
Alexander J. Gagliardi, and Howard W.T. Matthew
Wayne State University
Introduction: Modular tissue engineering is a promising approach to current regenerative medicine issues. Modules containing cells can be used as building blocks for tissue assembly. In general, a module must be able to induce an environment favorable for cell growth while retaining mechanical and architectural properties suitable for a specific application. Chitosan-polyanion ionic complex membranes can be used to encapsulate and culture a variety of cell types. Hollow fibers of these materials also have the potential to be used in a variety of tissue engineering applications both in vitro and in vivo. The chitosan, a naturally derived, structural polysaccharide, is biocompatible and can be modified for specific mechanical properties. Glycosaminoglycans (GAGs) provide an environment favorable for cell growth and matrix deposition because of their wide range of biological activities. In this study we investigated and developed procedures for continuous generation of chitosan-polyanion hollow fibers with cells and/or other biomaterials incorporated during fiber formation.
Materials and Methods: A polyanion solution, consisting of 1.5 wt% medium-viscosity carboxymethylcellulose and 4.0 wt% chondroitin 4-sulfate dissolved in sorbitol-HEPES buffer, was extruded into a solution of 0.6 wt% chitosan, 6.0 wt% sorbitol in 0.06 % acetic acid via a horizontal needle. An ionic complex fiber formed at the chitosan-polyanion solution interface. The fiber was pulled axially away from the needle and through the chitosan solution and was then either wrapped onto a rotating mandrel or pinched at opposite ends and lifted vertically from the chitosan solution. Fibers were surface stabilized by sequential washes with normal saline and saline-diluted polyanion solution. Non-stabilized fibers adhered to each other and could be wrapped, arranged, or stacked to form 3D structures. The ability to encapsulate materials within the fibers was examined by suspending these materials in the polyanion solution prior to fiber drawing. Materials investigated included suspensions of mesenchymal stem cells (MSCs), fixed hepatocytes, and microcarriers. MSCs-containing fibers were subsequently cultured in dishes with DMEM supplemented with 10% FBS.
Results: The polyanion solution was extruded into the chitosan solution at a rate of 95 µl/min in both manual and automatic setups. Fibers generated were observed to be decent candidates for encapsulation by the evidence of both microcarriers and cells enveloped within the complex fiber, with none of the designated materials seen outside of the fiber. Fiber diameter has been observed to be influenced by the ratio between the velocity of the GAG solution into the chitosan and the velocity of the pulling mechanism. An increasing ratio yielded thinner diameter fibers. Fibers with live cell suspensions placed in culture fractured and disintegrated within 24 hours in medium with normal FBS. This disintegration was found to be preventable through the use of heat inactivated serum. Fibers cultured in DMEM with heat-inactivated serum maintained integrity for at least 2 weeks. The mechanism behind fiber stability in heat inactivated serum and the potential roles of lysozyme or other serum enzymes is currently under investigation. Proliferation and osteogenic/chondrogenic differentiation of the fiber-encapsulated MSCs is also being evaluated and will be reported.
Conclusion: Results to date suggest that GAG-chitosan hollow fibers are promising candidates for modular tissue engineering applications due to their ease of preparation and assembly into complex 2D or 3D structures for engineered tissue assembly and analysis.
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