THE EFFECT OF GAMMA RADIATION STERILIZATION ON YIELD PROPERTIES AND MICROSCOPIC TISSUE DAMAGE IN DENSE CANCELLOUS BONE by STEPHANIE JOY DUX Submitted in partial fulfillment of the requirements For the degree of Masters of Science Thesis Advisor: Dr. Christopher Hernandez Department of Mechanical and Aerospace Engineering CASE WESTERN RESERVE UNIVERSITY January, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables ................................................................................................................................. iv List of Figures ................................................................................................................................. v Acknowledgements ....................................................................................................................... ix List of Abbreviations ..................................................................................................................... x Glossary ......................................................................................................................................... xi Abstract ......................................................................................................................................... xii Introduction .................................................................................................................................... 1 Bone Structure and Composition ................................................................................................. 2 Cortical Bone and Gamma Radiation Sterilization ..................................................................... 3 Cancellous Bone and Gamma Radiation Sterilization ................................................................ 5 Cancellous Bone Allograft vs. Cortical Bone Allograft ............................................................... 6 Damage in Cancellous Bone ........................................................................................................ 7 Objectives/Focus of Study ............................................................................................................ 9 Methods ......................................................................................................................................... 11 Specimen Preparation ................................................................................................................ 11 Mechanical Testing .................................................................................................................... 13 Histological Preparation ........................................................................................................... 17 Histomorphometry ..................................................................................................................... 18 Statistical analysis ..................................................................................................................... 23 Results ........................................................................................................................................... 25 Mechanical Properties ............................................................................................................... 25 Microscopic Tissue Damage ...................................................................................................... 28 Discussion ..................................................................................................................................... 31 Appendices .................................................................................................................................... 38 ii Appendix I: SOP – Techniques Using the Butcher Saw (The Biro Model 11) ........................... 38 Appendix II: SOP – Preparing and Slabbing Bovine Long Bones ............................................. 42 Appendix III: SOP – Templating and Cutting Trabecular Bone Parallelepipeds ..................... 47 Appendix IV: SOP – Coring Trabecular Bone Specimens ......................................................... 52 Appendix V: SOP – Polishing Trabecular Bone Specimens ...................................................... 58 Appendix VI: SOP – Marrow Removal for Trabecular Bone Specimens .................................. 61 Appendix VII: SOP – Gamma Radiation Sterilization of Trabecular Bone Specimens ............. 64 Appendix VIII: SOP – Staining Techniques for Trabecular Bone Cores ................................... 68 Appendix IX: SOP – Mechanical Testing of Trabecular Bone Using Platens ........................... 71 Appendix X: SOP – Calculating Modulus from Instron Data .................................................... 75 Appendix XI: SOP – Cutting Toe Region from Raw Data ......................................................... 77 Appendix XII: SOP – Clear Embedding Protocol for Cylindrical Bovine Cancellous Bone Specimens ................................................................................................................................... 79 Appendix XIII: SOP – Staining, Polishing and Mounting of Embedded Cylindrical Bovine Cancellous Bone Specimens ...................................................................................................... 84 Appendix XV: SOP – Microdamage Counting of Cancellous Bone ......................................... 89 Appendix XVI: Matlab Code for Plotting Stress – Strain Curves and Calculating Mechanical Properties (xltension2.m) ........................................................................................................... 96 Appendix XVII: Stress – Strain Curves for Control Specimens .............................................. 100 Appendix XVII: Stress – Strain Curves for Control Specimens .............................................. 100 Appendix XVIII: Stress – Strain Curves for Irradiated Specimens .......................................... 102 Appendix XIX: Mechanical Properties Data ........................................................................... 104 Appendix XX: Microscopic Tissue Damage Data .................................................................... 106 Works Cited ................................................................................................................................ 108 iii List of Tables Table 1. The mean values for bone volume fraction and mechanical properties are shown for both study groups along with the results of ANOVA comparisons. ………26 Table 2. Results of linear regression models between mechanical properties and bone volume fraction (BV/TV) are shown (y = a + b(BV/TV)). Standard error for each coefficient is listed in parentheses. ……………………….…………………………...27 Table 3. Average amounts of microscopic tissue damage in the region of interest are shown (standard deviation in parentheses) along with the results of ANOVA comparisons between the two groups. ………………………………………………………………………………………….29 Table 4. Average amounts of microscopic tissue damage are shown for our data along with data reported in literature (standard deviation in parentheses). Moore and Gibson loaded bovine proximal tibia to 1.3% strain and Wang et al. loaded bovine proximal tibia to 2% strain. (N.R. = not reported) …………………………………………………….35 iv List of Figures Figure 1. The mechanical testing arrangement is shown. Cylindrical bovine cancellous bone specimens were tested between platens with a 10mm extensometer attached directly to the specimen. The bottom platen pivots to adjust for slight differences in the specimen. The specimen is colored because it has been stained with xylenol orange to mark any pre-existing microscopic tissue damage. ……………………………………15 Figure 2. The figure above shows an example of stress – strain data gathered from compression testing. The zero point is found by extending a tangent line from the steepest slope on the stress – strain graph. The elastic modulus is found by fitting a quadratic fit to the first 0.2% of the strain data and the yield point is found using the 0.2% offset method. ………………………………………………………………………….17 Figure 3. Diffuse damage is the area of green stain in the center of the image indicated with the arrow. The blue areas represent bone and the black areas represent porous space. …………………………………………………….……………………..19 Figure 4. A region of cross hatched damage is present in this trabecula and indicated with the arrow. The blue areas represent bone and the black areas represent porous space. …………………………………………………………………………………...20 Figure 5. A microcrack is seen above as the thin, sharp, linear regions of green stain in the center of the image indicated with the arrow. The blue areas represent bone and the black areas represent porous space. .....………………………………………………...21 Figure 6. A microfracture is seen above in the center of the image as the fractured trabecula surrounded