UNIVERSITY OF CALIFORNIA, SAN DIEGO Understanding calcification in bone regeneration through a synthetic biomineralization-based approach A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Bioengineering by Ameya Phadke Committee in charge: Professor Shyni Varghese, Chair Professor Ratneshwar Lal Professor Koichi Masuda Professor Paul Price Professor Robert Sah 2012 Copyright Ameya Phadke, 2012 All rights reserved. The dissertation of Ameya Phadke is approved, and it is acceptable in quality and form for publication on microfilm: ___________________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ ____________________________________________________ Chair University of California, San Diego 2012 iii Table of Contents Signature page……………………………………………………………………………iii Table of Contents ............................................................................................................... iv List of Figures ..................................................................................................................... x List of Tables .................................................................................................................... xii Acknowledgements .......................................................................................................... xiii Vita ................................................................................................................................. xviii Abstract of the Dissertation ............................................................................................. xxi CHAPTER 1: Introduction ................................................................................................. 1 1.1. Biomineralization ................................................................................................. 1 1.2. Bone ..................................................................................................................... 1 1.2.1. Composition of bone ..................................................................................... 2 1.2.2. Cellular components of bone ........................................................................ 3 1.3. Bone grafting ........................................................................................................ 5 1.3.1. Current bone grafting strategies .................................................................... 6 1.3.2. Calcium phosphate-based bone graft substitutes .......................................... 8 1.3.3. Synthetic composite matrices ..................................................................... 12 1.3.4. Biomineralization of organic matrices ........................................................ 14 1.4. Figures ................................................................................................................ 21 1.5. References .......................................................................................................... 22 CHAPTER 2: Templated Mineralization of Synthetic Hydrogels for Bone-like Composite Materials: Effect of Matrix Hydrophobicity ..................................................................... 29 2.1. Abstract .............................................................................................................. 29 2.2. Introduction ........................................................................................................ 30 2.3. Results ................................................................................................................ 33 2.3.1. Mineralization with m-SBF ........................................................................ 33 2.3.2. Mineralization with serum-supplemented m-SBF ...................................... 34 2+ 2- 2.3.3. Mineralization with 40 mM Ca /24 mM HPO4 ....................................... 35 iv 2.4. Discussion .......................................................................................................... 37 2.5. Conclusion .......................................................................................................... 40 2.6. Acknowledgements ............................................................................................ 41 2.7. Experimental Section ......................................................................................... 42 2.7.1. Synthesis of monomers ............................................................................... 42 2.7.2. Synthesis of PEGDA-6K ............................................................................ 42 2.7.3. Preparation of simulated body fluid (SBF) ................................................. 42 2.6.4. Preparation of serum-supplemented SBF ................................................... 42 2+ 2- 2.6.5. Preparation of 40 mM Ca /24 mM HPO4 solution .................................. 43 2.6.6. Synthesis of hydrogels ................................................................................ 43 2.6.7. Contact angle measurement ........................................................................ 44 2.6.8. Mineralization of hydrogels by in protein-supplemented SBF ................... 44 2+ 2- 2.6.9. Mineralization of hydrogels in 40 mM Ca /24 mM HPO4 solution ....... 44 2.6.10. Adsorption of serum proteins on hydrogel surfaces................................ 45 2.6.11. SEM-EDS ................................................................................................ 45 2.6.12. X-Ray Diffraction ................................................................................... 45 2.6.13. Measurement of Ca2+ content in mineralized phase ................................ 46 2.6.14. Statistical analysis ................................................................................... 46 2.7. Figures ................................................................................................................ 47 2.8. Tables ................................................................................................................. 57 2.9. References .......................................................................................................... 60 CHAPTER 3: Mineralized synthetic matrices as an instructive microenvironment for osteogenic differentiation of human mesenchymal stem cells ......................................... 64 3.1. Abstract .............................................................................................................. 64 3.2. Introduction ........................................................................................................ 65 3.3. Results and Discussion ....................................................................................... 68 3.3.1. Material characterization ............................................................................ 68 3.3.2. Cell attachment ........................................................................................... 69 3.3.3. Osteogenic differentiation ........................................................................... 71 3.4. Conclusion .......................................................................................................... 75 v 3.5. Experimental Section ......................................................................................... 75 3.5.1. Hydrogel synthesis ...................................................................................... 75 3.5.2. Preparation of Hydrogel-Mineral composites ............................................. 76 3.5.3. Scanning Electron Microscopy ................................................................... 77 3.5.4. Measurement of rigidity .............................................................................. 77 3.5.5. Sterilization of substrates for cell culture ................................................... 77 3.5.6. Cell seeding and imaging ............................................................................ 78 3.5.7. Proliferation analysis .................................................................................. 78 3.5.8. Immunofluorescent staining........................................................................ 79 3.5.9. Quantitative RT-PCR .................................................................................. 79 3.5.10. Alkaline phosphatase assay ..................................................................... 80 3.5.11. Dissolution studies .................................................................................. 81 2+ 3- 3.5.12. Effect of soluble Ca and PO4 on hMSC differentiation ..................... 82 3.5.13. Statistical analyses................................................................................... 82 3.6. Acknowledgements ............................................................................................ 83 3.7. Figures ................................................................................................................ 84 3.8. Tables ................................................................................................................. 90 3.9. References .......................................................................................................... 91 CHAPTER 4: Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells ...................................................................................................