The University of Maine DigitalCommons@UMaine Electronic Theses and Dissertations Fogler Library 12-2001 Characterization of Chitosan Films for Cell Culture Applications Michael Katalinich Follow this and additional works at: http://digitalcommons.library.umaine.edu/etd Part of the Complex Fluids Commons Recommended Citation Katalinich, Michael, "Characterization of Chitosan Films for Cell Culture Applications" (2001). Electronic Theses and Dissertations. 245. http://digitalcommons.library.umaine.edu/etd/245 This Open-Access Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of DigitalCommons@UMaine. CHARACTERIZATION OF CHITOSAN FILMS FOR CELL CULTURE APPLICATIONS I BY Michael Katalinich B.S. University of Colorado, Boulder, 1997 A THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science (in Chemical Engineering) The Graduate School The University of Maine December, 2001 Advisory Committee: Amy1 Ghanem, Assistant Professor of Chemical Engineering, Advisor Douglas Bousfield, Professor of Chemical Engineering Rebecca Van Beneden, Professor of Biochemistry and Marine Sciences CHARACTERIZATION OF CHITOSAN FILMS FOR CELL CULTURE APPLICATIONS By Michael Katalinich Thesis Adyisor: Dr. Amy1 Ghanem An Abstract of the Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science (in Chemical Engineering) December, 200 1 Chitosan (P-(l,4)-2-amino-2-deoxy-D-glucose)is a naturally occurring, abundant biopolymer exhibiting desirable biomaterial properties of biodegradability, low toxicity and good biocompatibility. These properties indicate the suitability of chitosan as a surface for mammalian cell growth. The goal of this thesis is to explore the potential for using chitosan as a surface for NIH 3T3 fibroblast attachment and growth. '4 standard, reproducible film-formation technique, based on other researchers techniques was established. The reproducibility of this technique. through characterization of the physical and chemical properties of these films, was good. The attachment and growth of NIH 3T3 fibroblasts on chitosan films and controls was measured. These films were modified by physical and chemical means to optimize the attachment and growth of the NIH 3T3 fibroblasts. Physical properties, including film tensile properties, surface roughness of films, and chemical properties including the degree of deacetylation (measure of number of acetylated amino groups in the chitosan polysaccharide) and surface free energy (SFE) estimated by contact angle measurements were performed to characterize the chitosan films. Chitosan films of 0.5, 1.5 and 3.0% (wlv) support the attachment and proliferation of NIH 3T3 fibroblasts at rates lower than polystyrene controls. The film tensile properties, surface roughness and surface free energies indicate that the film-formation technique gives films with reproducible physical and chemical properties. The sterilization of films with ultraviolet aAd infrared lamps (UV-IR) over time changes the water-in-air contact angle (WIA) and increases the overall SFE of the films. This is an important result because the WIA contact angle has been shown influential to the rate of cell attachment, and the SFE has been shown to affect the degree of fibroblast spreading. Our results indicate that UV-IR treatment of chitosan films can change the WIA contact angle and SFE of the films, and can potentially be used to optimize the attachment and spreading of fibroblasts on these films. The ability of these chitosan films to support cell attachment and growth indicates their potential use as biomedical surfaces. Good attachment and growth results in rapid and efficient wound repair. This research may result in the development of biodegradable tissue-engineering matrices. This development requires an understanding of the basic cell-chitosan surface interactions. DEDICATION This paper is dedicated to my father, David Katalinich. He was a significant driving force in the continuation of my education after high school. This work is a direct I reflection of his influence on my professional career. He is my real-life hero and a wonderful father. ACKNOWLEDGEMENTS The author would like to thank his advisor, Dr. Amy1 Ghanem, for her guidance, and support on all aspects of this project throughout the duration of this study. Dr. Ghanem's considerable effort towards fie review and correction of papers and presentations was both beneficial and enlightening. The author would also like to thank Dr. Douglas Bousfield for his input on various aspects of this project, especially advice concerning the characterization of the films utilized in this study. The author is pleased to thank Dr. Rebecca Van Beneden for her support of the author and the project over the last two years. Dr. Van Beneden's input regarding cell culture-related project aspects was very helpful. The author thankfully acknowledges all of those individuals who assisted with the completion of this project. Specifically, thanks to Manish Giri for his assistance with rheology testing of samples, Yang Xiang for help with image capture and analysis, Jong Tae Youn for help with tension tests of samples, Ben McCool for help with surface area and porosity measurements, and Brian Nimess for help with FTIR testing of samples. Additionally, thanks to Dr. Thompson for the donation of the UV-IR oven used to sterilze samples, Dr. Co and Amos Cline for IT support, and Mark Paradis for the use of vital testing equipment. Thanks to Angel Hildreth and Cathy Dum for help with department- related issues. Also, thanks to Dan Jolicoeur for help with obtaining supplies and with the fabrication and repair of tools integral to this project. Finally, a special thank you is extended to Scott Delcourt, Director of the Graduate School, and Chet Rock, Dean of the College of Engineering, for much-needed financial support towards the end of this project. TABLE OF CONTENTS .. DEDICATION .............................................................................................................11 ... ACKNOWLEDGEMENTS ... .... ...............................................................................ill LIST OF FIGURES ................ .... ............+ LIST OF TABLES .....................................................................................................xi LIST OF EQUATIONS ............................................................................................ xii 1. INTRODUCTION ..................................................................................................... 1 1.1. Project Objective .................................................................................................1 . 1.2. Chitin and Chitosan ............................................................................................. 1 1.3. Tissue Engineering and Biomaterials ................................................................. 5 1.4. Cell Culture . Use of NIH 3T3 Fibroblasts .........................................................6 2 . BACKGROUND .......................................................................................................8 2.1. Tissue Engineering and Biomaterials .................................................................8 2.1.1. Tissue Engineering: Applications and Approaches ....................................8 2.1.2. Biomaterials .............................................................................................. 12 2.2. Chitosan ............................................................................................................ 16 2.2.1. Applications of Chitosan as a Biomaterial ................................................ 16 2.2.1.1 Chitosan and Various Cell Types ......................................................... 18 2.2.1.1.1 Chitosan and Anchorage-Dependent Cells ..................................... 19 2.2.1.1.2 Chitosan, Immune Cells, and Blood-Related Tissues ..................... 20 2.2.1.1.3 Chitosan and Bone Tissues .............................................................22 2.2.1.2 Other Important Applications ............................................................... 24 2.2.1.3 Chitosan and Fibroblasts ....................................................................... 25 2.2.2 Film. Membrane and 3-D Matrix Formation Techniques for Chitosan .... 31 2.3. Cellular Function and the Wound Repair Process ........................................ 34 2.3.1. The Wound Repair Process .....................................................................34 3.3.2 . Cellular Interactions ...............................................................................40 I 2.3.2.1. Cell Attachment and Adhesion .........................................................40 2.3.2.2. Cell Spreading ................................................................................... 44 2.3.2.3. Cell Growth and Migration ...............................................................45 2.3.3. Extracellular Matrix ..................................................................................46 2.4. Fibroblasts .........................................................................................................50 2.5. Important Substrate Properties .......................................................................... 53 2.5.1. Surface Free Energy .................................................................................