EFFECTS OF COMBINED MECHANICAL AND PULSED ELECTROMAGNETIC FIELD STIMULATIONS ON THE OSTEOGENESIS OF BONE MARROW STEM CELLS NG KIAN SIANG NATIONAL UNIVERSITY OF SINGAPORE 2012 i EFFECTS OF COMBINED MECHANICAL AND PULSED ELECTROMAGNETIC FIELD STIMULATIONS ON THE OSTEOGENESIS OF BONE MARROW STEM CELLS NG KIAN SIANG (B Eng (Hons), NUS) A THESIS SUBMIITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 ii Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis The thesis has also not been submitted for any degree in any university previously Ng Kian Siang 16 October 2012 i Acknowledgements I would like to take this opportunity to express my deepest gratitude to the following people who had, in one way or another, helped me towards the completion of this research project First of all, I would like to thank God for His unmerited favour, sending great men to my aid and creating ways when there seemed to be none I would also like to thank my supervisors, Prof James Goh and A/Prof Toh Siew Lok for their patience and understanding, generous support, scientific and personal guidance throughout this journey of discovery and personal development I wish to thank all my colleagues in the Tissue Repair Lab who made the lab a pleasant place to work in Credit goes to the laboratory officer, Ms Lee Yee Wei, who ensured that the lab was in proper order and well stocked with the necessary research consumables I would also like to thank my fellow lab mates, Dr Zheng Ye, Dr Sambit Sahoo, Dr Teh Kok Hiong Thomas, He Pengfei, Chen Kelei, Tan Hwee Sim Pamela and Sujata Ravi We were always there for one another whenever problems arose in our research and our discussions often led to fruitful outcomes I also like to acknowledge my five undergraduate students, Mr Wong Xianrong, Ms Robina Ang, Ms Jasmin Seow, Ms Aishwarya and Mr Safwan for their great help in various parts of my research during their final year projects Besides my colleagues in Tissue Repair Lab, I am also grateful to the kind souls who extended their helping hands even though they were from different labs and departments These include: Prof Ashwin from the Electrical Engineering department for providing technical expertise; A/Prof Chen Nanguang for lending the function ii generators; Dr Susan Liao for providing copper wires; Mr Lam Kim Song from the Fabrication Support Lab for fabricating the solenoids; Mr Eric Wong for fabricating the bioreactors; Mr Ali Hasnain for helping to design the amplifying circuit and Mr Ang Beng Oon for helping with the porosity measurements Thanks are also due to the staff at the Animal Holding Unit, without which I would not have been able to collect bone marrow from the rabbits for my studies I am also grateful to my friends, especially those from my undergraduate course and went on to study for a Ph.D with me They are Dr Ong Peng Kai, Dr Chong Shau Poh, Dr Bong Jit Hon and Mr Poh Yong Cheng One thing I would certainly miss would be our regular fellowship over lunch Last but not least, I am extremely grateful to my family, in particular my parents for their love, understanding and encouragement Thanks especially to my mum, who believed in me even though she does not know exactly what I am doing, and always made sure that I had a hot sumptuous dinner despite coming late on most days iii Table of Contents Declaration i Acknowledgements ii Summary ix List of Tables xi List of Figures xii List of Symbols / Abbreviations xviii Chapter Introduction 1.1 Bone Biology 1.1.1 Functions of bone and the skeletal system 1.1.2 Structure and Composition of Bone 1.2 Bone defects 1.2.1 1.3 Bone Fracture Healing Current treatment and limitations 10 1.3.1 Bone tissue engineering 11 1.3.2 Biophysical stimulation – alternative options for bone healing 12 Chapter Literature Review 13 2.1 Bone tissue engineering strategy 13 2.2 Cellular sources for bone tissue engineering 15 2.2.1 Cell sources used for bone tissue engineering 15 2.2.2 Sources of MSCs 17 2.3 Scaffolds for Bone Tissue Engineering 18 2.3.1 Functions of scaffolds 18 2.3.2 Specifications for bone tissue engineering scaffolds 18 2.3.3 Selection of materials for scaffolds 20 2.3.4 Fabrication Methods 24 2.3.5 Freeze drying technique 27 iv 2.4 Bioreactors for Bone Tissue Engineering 29 2.4.1 Functions of bioreactors 30 2.4.2 Types of bioreactors 32 2.5 Biophysical Stimulations for Bone Tissue Engineering 36 2.5.1 Ultrasound 36 2.5.2 Electromagnetic Field 39 2.6 Hypothesis, Objectives and Scope 45 2.6.1 Summary of literature review 45 2.6.2 Significance and Originality of Work 47 2.6.3 Research Objectives 48 2.6.4 Hypotheses 48 2.6.5 Specific Aims 48 Chapter Stage I: Establish culture of BMSCs and Proof of concept of PEMF stimulation on BMSCs 51 3.1 Experimental Setup – Materials and Methods 51 3.1.1 Isolation and culture of BMSCs from rabbit model 51 3.1.2 Demonstration of BMSCs’ osteogenic potential 52 3.1.3 Pico green assay 53 3.1.4 ALP assay 53 3.1.5 Calcium content assay 54 3.1.6 RT-PCR 54 3.1.7 Alizarin Red Staining 55 3.1.8 PEMF Apparatus 55 3.1.9 Design and fabrication of solenoids 56 3.1.10 Magnetic field testing 59 3.1.11 Design and fabrication of amplifying circuit 60 3.1.12 PEMF Stimulation Parameters 63 3.1.13 Optimization of PEMF parameters 64 3.1.14 Statistical analysis 65 3.2 Results 66 3.2.1 Demonstration of osteogenic potential 66 3.2.2 Proof of concept of PEMF effects on BMSCs 71 v 3.2.3 3.3 Optimization of PEMF parameters 76 Discussion 84 3.3.1 Osteogenic potential of BMSCs 84 3.3.2 PEMF effects of BMSCs 86 3.3.3 Optimization of PEMF parameters 88 3.4 Concluding remarks 91 Chapter Stage II: Development of a scaffold for bone tissue engineering 92 4.1 Materials and Methods 92 4.1.1 Design of scaffold 92 4.1.2 Silk scaffold fabrication 93 4.1.3 Optimization of silk scaffold protocol 95 4.1.4 Scanning electron microscopy 95 4.1.5 Porosity 95 4.1.6 Static seeding technique 96 4.1.7 In vitro study 97 4.1.8 Cell seeding efficiency and Cell attachment observation 99 4.1.9 Pico green assay 100 4.1.10 ALP assay 101 4.1.11 Calcium content assay 101 4.1.12 RT-PCR 102 4.1.13 Histology 103 4.1.14 Statistical analysis 103 4.2 Results 103 4.2.1 Scanning electron microscopy 103 4.2.2 Mercury Porosimetry 106 4.2.3 Cell seeding efficiency and Cell attachment study 106 4.2.4 In-vitro study 106 4.3 Discussion 115 4.3.1 Optimization of scaffold architecture 115 4.3.2 Static cell seeding 118 4.3.3 In vitro study 118 vi 4.4 Concluding remarks 123 Chapter Stage III: Development of a perfusion bioreactor system 124 5.1 Materials and methods 124 5.1.1 Perfusion chamber design 124 5.1.2 Perfusion bioreactor system setup 125 5.1.3 Perfusion seeding of cells and medium change 127 5.1.4 Flow rate characterization 128 5.1.5 Cell seeding efficiency 129 5.1.6 Cell seeding distribution 129 5.1.7 Pico green assay 130 5.1.8 ALP assay 131 5.1.9 Calcium content assay 131 5.1.10 Statistical analysis 132 5.2 Results 132 5.2.1 Characterization of pump flow rate 132 5.2.2 Seeding efficiency 133 5.2.3 Seeding distribution 133 5.2.4 DNA content 137 5.2.5 ALP activity 137 5.2.6 Calcium content 138 5.3 Discussion 139 5.4 Concluding remarks 143 Chapter Stage IV: Integration of PEMF unit with flow perfusion bioreactor 144 6.1 Materials and methods 144 6.1.1 Integrated setup of PEMF stimulation with perfusion bioreactor system 144 6.1.2 In vitro culture of cell seeded construct within integrated setup 146 6.1.3 Pico green assay 147 6.1.4 ALP assay 148 6.1.5 Calcium content assay 148 vii 6.1.6 6.2 Statistical analysis 149 Results 149 6.2.1 DNA content 149 6.2.2 ALP activity 150 6.2.3 Calcium content 150 6.3 Discussion 151 6.4 Concluding remarks 157 Chapter Summary of Findings and Conclusion 158 7.1 Introduction 158 7.2 Objectives and Hypotheses 158 7.3 Summary of findings 159 7.3.1 MSCs Proof that PEMF stimulation affects the osteogenic development of 159 7.3.2 Silk sponge scaffold for seeding and culturing MSCs 160 7.3.3 Flow perfusion bioreactor as the in vitro culture system 161 7.3.4 Combining PEMF stimulation with flow perfusion bioreactor 163 7.4 Conclusion 164 Chapter Recommendation for Future Work 167 References 169 Appendix A List of publications 182 Appendix B Histology staining techniques 184 Appendix C Drawings 186 viii 69 Angle SR, Sena K, Sumner DR, Virdi AS Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound Ultrasonics Apr;51(3):281-288 70 Barnett SB, Rott HD, terHaar GR, Ziskin MC, Maeda K The sensitivity of biological tissue to ultrasound Ultrasound in Medicine and Biology 1997 1997;23(6):805-812 71 Zhang ZJ, Huckle J, Francomano CA, Spencer RGS The effects of pulsed lowintensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix 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computational modeling of media flow through scaffolds in a perfusion bioreactor Journal of Biomechanics 2005 Mar;38(3):543-549 180 153 Weinbaum S, Cowin SC, Zeng Y A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses Journal of Biomechanics 1994 Mar;27(3):339-360 154 Burger EH, Klein-Nulend J Mechanotransduction in bone - role of the lacunocanalicular network Faseb Journal 1999 1999;13:S101-S112 155 Wang LY, Fritton SP, Weinbaum S, Cowin SC On bone adaptation due to venous stasis Journal of Biomechanics 2003 Oct;36(10):1439-1451 156 Nauman EA, Satcher RL, Keaveny TM, Halloran BP, Bikle DD Osteoblasts respond to pulsatile fluid flow with shortterm increases in PGE(2) but no change in mineralization Journal of Applied Physiology 2001 May;90(5):1849-1854 157 Singh H, Ang ES, Lim TT, Hutmacher DW Flow modeling in a novel nonperfusion conical bioreactor Biotechnology and Bioengineering 2007 Aug 1;97(5):1291-1299 158 McCoy RJ, Jungreuthmayer C, O'Brien FJ Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor Biotechnology and Bioengineering Jun;109(6):1583-1594 159 Li D, Tang T, Lu J, Dai K Effects of Flow Shear Stress and Mass Transport on the Construction of a Large-Scale Tissue-Engineered Bone in a Perfusion Bioreactor Tissue Engineering Part A 2009 Oct;15(10):2773-2783 181 Appendix A List of publications Journals Ng KS, Toh SL, Goh JC Effects of pulsed electromagnetic field stimulation on the osteogenesis of bone marrow-derived mesenchymal stem cells seeded in three dimensional silk-fibroin scaffolds J Tissue Eng Regen Med 2012 (Submitted) Ng KS, Toh SL, Goh JC Effects of combined pulsed electromagnetic field and mechanical stimulation on the osteogenesis of bone marrow-derived mesenchymal stem cells seeded in three dimensional silk-fibroin scaffolds J Tissue Eng Regen Med 2012 (Submitted) Chen K, Ng KS, Ravi S, Goh JC, Toh SL In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells by employing a two chambered co-culture well design J Tissue Eng Regen Med 2012 He P, Ng KS, Toh SL, Goh JC In vitro ligament-bone interface regeneration using a trilineage coculture system on a hybrid silk scaffold Biomacromolecules 2012 Sep 10;13(9):2692-703 He P, Sahoo S, Ng KS, Chen K, Toh SL, Goh JC Enhanced osteoconductivity and osteoinductivity through hydroxyapatite coating of silk-based tissue engineered ligament scaffold J Biomed Mater Res Part A 2012 Chen K, Sahoo S, He P, Ng KS, Toh SL and Goh JC A hybrid silk/RADA-based fibrous scaffold with triple hierarchy for ligament regeneration Tissue Eng Part A 2012 Jul; 18(13-14):1399-409 182 CH Yeow, KS Ng, CH Cheong, Peter VS Lee and James CH Goh Repeated application of incremental landing impact loads to intact knee joints induces anterior cruciate ligament failure and tibiofemoral cartilage deformation and damage: a preliminary cadaveric investigation J Biomech 2009 May 29;42(8):972-81 CH Yeow, CH Cheong, KS Ng, PV Lee and JC Goh Anterior cruciate ligament failure and cartilage damage during knee joint compression: A preliminary study based on the porcine model Am J Sports Med 2008 May; 36(5):934-42 Conferences K.S Ng, J.C.H Goh, S.L Toh Design of a perfusion bioreactor system for the bone tissue engineering of a uniformly seeded large scaffold construct Presented at Tissue Engineering & Regenerative Medicine, Asia Pacific Meeting, – Aug 2011, Singapore K.S Ng, J.C.H Goh, S.L Toh Design of a perfusion bioreactor system for the bone tissue engineering of a uniformly seeded large scaffold construct Presented at Biomedical Engineering Society (Singapore) 5th Scientific Meeting of the Biomedical Engineering Society (Singapore), 28th May 2011, Singapore K.S Ng, M.L Seow, J.C.H Goh, and S.L Toh Effects of pulsed electromagnetic field on osteogenesis of mesenchymal stem cells Presented at 6th World Congress on Biomechanics, – Aug 2010, Singapore K.S Ng, X.R Wong, J.C.H Goh and S.L Toh ‘Development of a silk-chitosan blend scaffold for bone tissue engineering’ Presented at 13th International Conference on Biomedical Engineering, – December 2008, Singapore 183 Appendix B Histology staining techniques Hematoxylin & Eosin Staining Method De-paraffin sections in xylene for x OR until the wax is gone Blot excess xylene before going into ethanol Note: DO NOT touch the timer if there is xylene on gloves Clear sections in ethanol 100% x 95% x 70% x Wash in deionized water for Blot excess water before going into hematoxylin Stain in Mayer’s hematoxylin for 10 OR Harris’ hematoxylin for Rinse in water Optional: x in tap water Optional: Dip in 1% acid alcohol for second, rinse in water till water is clear Dip in alkaline solution for a few seconds Rinse in water Blot excess water before going into eosin 10 Counter-stain in eosin for to seconds ONLY 11 Rinse in water 12 Optional: View under microscope to check staining If unsatisfactory, de-stain in 1% acid alcohol Rinse in water 13 Dehydrate in ethanol 70% x 184 95% x 100% x 14 Clear in xylene x 15 Mount sections in mounting medium 16 Allow drying before viewing Results: Nuclei – blue to blue black (stained by hematoxylin) Cytoplasm – pink (stained by eosin) Von Kossa staining De-paraffin sections in xylene for x OR until the wax is gone Rinse in several changes of distilled water Incubate sections with 1% silver nitrate solution in a clear glass coplin jar placed under ultraviolet light for 20 minutes Rinse in several changes of distilled water Remove un-reacted silver with 5% sodium thiosulfate for minutes Rinse in distilled water Counter-stain with nuclear fast red for minutes Rinse in distilled water Dehydrate through graded alcohol and clear in xylene 10 Coverslip using mounting medium Wait for it to dry before viewing Results: Calcium salts – black or brown-black Nuclei – red Cytoplasm – pink 185 Appendix C Drawings Top compartment of bioreactor chamber 186 Bottom compartment of bioreactor chamber 187 Measurements for winding a solenoid 360 mm 330 mm (enameled copper wire, 0.85mm diameter, layers) mm (acrylic ring) 10 mm Acrylic tube ID = 70 mm OD = 80 mm 188 ... (cancellous) bone Cortical bone forms the dense outer shell of most bones and it accounts for 80% of the total bone mass Cortical bone is mostly found in the shaft portion of long bones and the outer.. .EFFECTS OF COMBINED MECHANICAL AND PULSED ELECTROMAGNETIC FIELD STIMULATIONS ON THE OSTEOGENESIS OF BONE MARROW STEM CELLS NG KIAN SIANG (B Eng (Hons), NUS) A THESIS SUBMIITED FOR THE DEGREE... cancellous bone On the other hand, cancellous bone consists of a network of struts surrounding interconnected spaces It is the spongy interior bone tissue which makes up 20% of the total bone mass