The Material Properties of the Chordae Tendineae of the Mitral Valve: an in Vitro Investigation
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THE MATERIAL PROPERTIES OF THE CHORDAE TENDINEAE OF THE MITRAL VALVE: AN IN VITRO INVESTIGATION A Thesis Presented to The Academic Faculty By Jennifer Lynn Ritchie In Partial Fulfillment Of the Requirements for the Degree Master of Science in Bioengineering Georgia Institute of Technology August, 2004 THE MATERIAL PROPERTIES OF THE CHORDAE TENDINEAE OF THE MITRAL VALVE: AN IN VITRO INVESTIGATION Approved By: Dr. Ajit P. Yoganathan, Advisor Dr. Raymond Vito Dr. Robert Guldberg Dr. Michael Sacks Date Approved: August 19, 2004 For my family and husband, without whom I would not have survived. ACKNOWLEDGEMENTS First and foremost, I would like to thank my family for always being by my side. Without their continual support, I would not have survived. I would like to especially thank my husband Steve for being by my side the entire time, making all the trips to Atlanta to visit me, and not allowing me to give up. I love you more than you know. Special thanks goes to my advisor Dr. Ajit P. Yoganathan for his support and exceptional opportunities he gave me during my time at Georgia Tech. I would also like to thank Dr. Raymond Vito, Dr. Robert Guldberg, and Dr. Michael Sacks for being part of my thesis committee. I would like to thank all the members of the Cardiovascular Fluid Dynamics Laboratory for your support and friendship. Zhaoming and Jorge, thank you for everything you helped me to accomplish. Without you, I would not have been able to complete this work. Dennis, thank you for developing the autotracking software! James and Tracey, thanks for all your help with the histology and biochemical work. To Diane for her wonderful friendship, keep swimming. To Anna, Ashley, Helene, Yun, Suchitra, Leo, Dave, Hiroumi, Kartik, Kerem, Josie, Kelly, Lisa, and Mark, it was great working with you. I would also like to thank Dr. Guldberg’s lab for the use of the uniaxial testing machine. Special thanks goes to Angela Lin for all her help in getting the machine to work. To Jim McEntee and the Holifield Slaughter house, thank you for your contributions to my work. In addition, I would like to thank the National Heart, Lung, and Blood Institute for the support which made my work possible (grant # HL52009). iv TABLE OF CONTENTS Acknowledgements iv List of Tables viii List of Figures x Summary xviii Chapter 1 Introduction 1 Chapter 2 Background 4 2.1 Anatomy of the Heart 4 2.2 The Cardiac Cycle 7 2.3 Anatomy of the Mitral Valve 10 2.4 Anatomy of the Chordae Tendineae 12 2.5 Mechanical Properties of the Chordae Tendineae 17 2.6 Mitral Valve Pathologies 20 Chapter 3 Hypotheses and Specific Aims 26 3.1 Hypotheses 26 3.1.1 Hypothesis 1 26 3.1.2 Hypothesis 2 26 3.2 Specific Aims 26 3.2.1 Specific Aim 1 – Hypothesis 1 26 3.2.2 Specific Aim 2 – Hypothesis 1 27 3.2.3 Specific Aim 3 – Hypothesis 1 27 3.2.4 Specific Aim 4 – Hypothesis 2 28 Chapter 4 Materials and Methods 29 4.1 Mitral Valve Preparation 29 4.2 Chordae Tendineae Marker Technique 30 4.2.1 In Vitro Flow Loop 30 4.2.2 Left Ventricle Model 33 4.2.3 Annulus Chamber and Board 36 4.2.4 Instrument Calibrations 38 4.2.5 Graphite Markers 39 4.2.6 Dual Camera Stereo Photogrammetry 40 4.2.7 Extension Tubes 42 4.2.8 Experimental Protocol 43 v 4.2.9 Data Collection 45 4.2.10 Autotracking Code 45 4.2.11 Direct Linear Transformation Code 46 4.2.12 Strain Code 47 4.3 Uniaxial Test 48 4.3.1 Specimen Preparation 48 4.3.2 Experimental Protocol 51 4.3.3 Data Analysis 52 4.4 Chordae Force Marker Technique 53 4.5 Histology and Biochemical Composition 53 4.5.1 Tissue Processing 53 4.5.2 Hematoxylin and Eosin Stain 55 4.5.3 Verhoeff and van Gieson Stain 55 4.5.4 Verhoeff and Light Green Stain 56 4.5.5 Immunohistochemistry 56 4.5.6 DNA, Collagen, and Elastin Assays 57 4.6 Statistical Analysis 58 Chapter 5 Results 59 5.1 Overview 59 5.2 Anatomical Measurements 59 5.3 Flow Loop Experiments 61 5.4 Uniaxial Tests 71 5.5 Histology and Biochemical Composition 85 5.5.1 DNA Content 85 5.5.2 Collagen Content 87 5.5.3 Elastin Content 88 5.5.4 Hematoxylin and Eosin Stain 89 5.5.5 Verhoeff and Light Green Stain 93 5.5.6 Verhoeff and van Gieson Stain 94 5.5.7 Immunohistochemistry 97 Chapter 6 Discussion 99 Chapter 7 Conclusions 119 Chapter 8 Limitations and Recommendations 122 Appendix A: Valve Preparation and Harvesting Protocol 124 Appendix B: Engineering Drawings of the In Vitro Flow Loop 127 vi Appendix C: Data Analysis Algorithms 145 C.1 Autotracking Algorithm 146 C.1.1 Documentation for Autotracking 1.m and 146 Marker_check1.m C.1.2 Autotracking1.m 146 C.1.3 Marker_check1.m 154 C.2 3D Transformation Algorithm 156 C.2.1 Documentation for Matlab 3D Coordinate 156 Calculation C.2.2 SW3D6.m 159 C.2.3 DLTFU.m 153 C.2.4 RECONFU.m 165 C.3 Strain Calculation Algorithm 166 C.4 Lowpass Filter Algorithm 168 Appendix D: Histology and Biochemical Assay Protocols 171 D.1 Hematoxylin and Eosin Stain 172 D.2 Verhoeff and van Gieson Stain 172 D.3 Verhoeff and Light Green Stain 173 D.4 Immunhistochemistry 175 D.5 Biochemical Digestion 177 D.5.1 Protenaise K 177 D.5.2 Pepsin 177 D.6 Fastin Elastin Assay 178 D.7 Collagen Assay 178 D.8 Hoescht Dye DNA Assay 179 Appendix E: Biochemical Assay Results 180 E.1 DNA Assay Results 181 E.2 Collagen Assay Results 186 E.3 Elastin Assay Results 190 References 193 vii LIST OF TABLES Table 2.1 Average length, thickness, and insertion locations of chordae 16 tendineae (Lam et al.[7]). Table 2.2 Comparison of the mechanical properties of the chordae 18 according to their cross-sectional area (Liao et al.[8]). Table 2.3 Comparison of the mechanical properties of the chordae 18 according to chordal type (Liao et al.[8]). Table 2.4 Force distribution of the chordae tendineae (Jimenez et al.[13]). 19 Table 5.1 Diameter and length measurements for both strut chordae. It 61 was found that there was a significant difference (p<0.02) between the diameter and lengths of the two strut chordae. A p- value less than 0.05 was considered significant. Table 5.2 Maximum strain, loading, and unloading rates determined for 71 both strut chordae during the flow loop experiments. There was no significant difference in the strain, loading rates, or unloading rates between the two strut chordae. A p-value of less than 0.05 was considered significant. Table 5.3 Number of vessels per chordae. The strut chordae contain 97 significantly more vessels than the other chordae. Table 5.4 The number of vessels for each type of chord was compared 97 with the other 5 types of chordae using a t test for paired comparison. p-values are listed below. A p-value less than 0.05 was considered significant. The strut chord was significantly different than the other five chordae. Table E.1 DNA assay results for each chordae tendineae. The chordae 177 measured is listed at the top of each column of data. Table E.2 DNA content for each chordae tendineae. The average and 181 standard deviation for each chordae is given at the bottom of the table. viii Table E.3 p value between each chordae for the DNA assay results. A p 182 value less than 0.05 was considered significant. Table E.4 Collagen assay results for each chordae tendineae. The chordae 182 measured is listed at the top of each column of data. Table E.5 Collagen content for each chordae tendineae. The average and 185 standard deviation for each chordae is given at the bottom of the table. Table E.6 p value between each chordae for the collagen assay results. A 186 p value less than 0.05 was considered significant. Table E.7 Elastin assay results for each chordae tendineae. The chordae 186 measured is listed at the top of each column of data. Table E.8 Elastin content for each chordae tendineae. The average and 188 standard deviation for each chordae is given at the bottom of the table. Table E.9 p value between each chordae for the elastin assay results. A p 188 value less than 0.05 was considered significant. ix LIST OF FIGURES Figure 2.1 Diagram showing the anatomy of the heart. Illustrations copyright 5 2000 by Nucleus Communications, Inc. All rights reserved. http://www.nucleusinc.com. Figure 2.2 Pressure and volume relationships in the cardiac cycle. 8 Figure 2.3 Diagram representation of the mitral valve (Ranganathan et al.[4]). 11 Figure 2.4 Computer generated model of the microsection of the chordae 13 tendineae (Millington-Sanders et al.[6]). There are three layers: an inner collagen core, a middle elastin layer, and an outer endothelial layer. Figure 2.5 Diagram representation of the insertion locations of the chordae 17 tendineae. There are six primary chordae tendineae: anterior strut, anterior marginal, commissural, basal posterior, posterior intermediate, and posterior marginal. Figure 2.6 Diagram representation of a normal and prolapsed mitral valve. 20 Image copyright 2000 by Nucleus Communication, Inc. All rights reserved.