Application of high voltage, high frequency pulsed electromagnetic field on cortical bone tissue

IV Pulsed Power Cortical bone High voltage, High frequency converter Positive Buck-Boost Converter Pulsed electromagnetic field Electrical stimulation Mechanical properties of bone Bone

Pulsed Electromagnetic Field on Cortical Bone

School of Biomedical Engineering and Medical Physics

Faculty of Science and Engineering Queensland University of Technology

Brisbane, Australia

June 2012

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The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made

Hajarossadat Asgarifar

 My supervisors, Prof Kunle Oloyede and Associate Prof Firuz Zare for their

invaluable guidance and support

 The many academic and technical staff and PhD students at IHIB for their

kind consultancies and assistances, in particular, Prof Christian Langton for ultrasound facilities and medical engineering laboratory technicians and research portfolio staff for their technical advices and continued helps

 My friends and colleagues for sharing knowledge and providing a warm

research environment

 The last but not the least, to my unique family, my beloved husband, Mehran,

for his most amazing support and great advices and my gorgeous favourite twins, Hossein and Mahdi, for their kindness and patience all through my study

 And to my dear parents for their infinite love, spiritual support and

encouragement during my life and study even when I was too far from them

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Pulsed Power

Cortical bone

High voltage, High frequency converter

Positive Buck-Boost Converter

Pulsed electromagnetic field

Electrical stimulation

Mechanical properties of bone

Bone functional behaviour

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Over the last few decades, electric and electromagnetic fields have achieved important role as stimulator and therapeutic facility in biology and medicine In particular, low magnitude, low frequency, pulsed electromagnetic field has shown significant positive effect on bone fracture healing and some bone diseases treatment Nevertheless, to date, little attention has been paid to investigate the possible effect of high frequency, high magnitude pulsed electromagnetic field (pulse power) on functional behaviour and biomechanical properties of bone tissue

Bone is a dynamic, complex organ, which is made of bone materials (consisting of organic components, inorganic mineral and water) known as extracellular matrix, and bone cells (live part) The cells give the bone the capability of self-repairing by adapting itself to its mechanical environment The specific bone material composite comprising of collagen matrix reinforced with mineral apatite provides the bone with particular biomechanical properties in an anisotropic, inhomogeneous structure

This project hypothesized to investigate the possible effect of pulse power signals on cortical bone characteristics through evaluating the fundamental mechanical properties of bone material A positive buck-boost converter was applied to generate adjustable high voltage, high frequency pulses up to 500 V and 10 kHz

Bone shows distinctive characteristics in different loading mode Thus, functional behaviour of bone in response to pulse power excitation were elucidated by using three different conventional mechanical tests applying three-point bending load in elastic region, tensile and compressive loading until failure Flexural stiffness, tensile and compressive

According to the results of non-destructive bending test, the flexural elasticity of cortical bone samples appeared to remain unchanged due to pulse power excitation Similar results were observed in the bone stiffness for all three orthogonal directions obtained from ultrasonic technique and in the bone stiffness from the compression test From tensile tests, no significant changes were found in tensile strength and total strain energy absorption of the bone samples exposed to pulse power compared with those of the control samples Also, the apparent microstructure of the fracture surfaces of PP-exposed samples (including porosity and microcracks diffusion) showed no significant variation due to pulse power stimulation Nevertheless, the compressive strength and toughness of millimetre-sized samples appeared to increase when the samples were exposed to 66 hours high power pulsed electromagnetic field through screws with small contact cross-section (increasing the pulsed electric field intensity) compare to the control samples This can show the different load-bearing characteristics of cortical bone tissue in response to pulse power excitation and effectiveness of this type of stimulation on smaller-sized samples These overall results may address that although, the pulse power stimulation can influence the arrangement or the quality of the collagen network causing the bone strength

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electromagnetic field at 500 V and 10 kHz through capacitive coupling method, was athermal and did not damage the bone tissue construction

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High power pulsed electromagnetic field (Pulse Power), has been applied recently in some fields of biology and medicine However, the effect of pulse power on physical characteristics of bone tissue has not yet been fully clarified On the other hand, according

to various studies during last century, electrical stimulation using both constant and pulsed electromagnetic field (PEMF) has had a drastic effect on bone growth and some bone diseases healing It was a good motivation for investigation of the possibility of applying pulse power signals for stimulating bone

The main contribution of the present thesis is to introduce a suitable, safe method with controlled parameters for application of high power, pulsed electromagnetic fields on bone tissue using capacitive coupling method The basic biomechanical properties of cortical bone material including stiffness, strength, toughness and brittleness have been investigated (considering just extracellular fraction of the bone) in response to high voltage, high frequency pulses up to 500V at 10 kHz These have been achieved by:

 The comparison and assessment of two pulse power application methods, direct

connection of bone with electrodes (which result in thermal effect and burning) and capacitive coupling method through electrodes isolation (Chapter 4)

 The determination and comparison of bone flexural elasticity before and after pulse

power excitation using the non-destructive three-point bending tests (in linear elastic region) on both whole long bone and cortical bone strips (Chapter 4)

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frequency pulsed electromagnetic field using tensile test until failure point by investigation of fracture energy, hysteresis energy and strength of the samples exposed to pulse power compared with those of the control samples and supplementary fractograph study via scanning electron microscopy of the fracture surfaces (Chapter 5)

 The evaluation of the compressive strength and fracture energy of the

millimetre-sized cortical bone samples exposed to pulse power signals compared with the control specimens (Chapter 6)

 The application of ultrasonic technique as an alternative, non-destructive method

with the capability of measurement in different orthogonal directions for determination and comparison of elastic property of cortical bone samples in response to pulse power excitation (Chapter 7)

To author’s knowledge, this project was the first research investigating the effect of

high voltage, high frequency pulsed electromagnetic field on fundamental properties of cortical bone structure

Providing a basic information about the effect of pulse power excitation on bone tissue structure, this study will contribute in further research on pulse power application on live bone, investigating the bone growth enhancement potential of this kind of stimulation for therapeutic purpose in musculoskeletal diseases

Some of the results of this research were presented as accepted international conference paper and item as below and other is going to submit as a journal paper:

 H Asgarifar, A Oloyede, F Zare, C M Langton “Evaluation of cortical bone

elasticity in response to pulse power excitation using ultrasonic technique” Ninth

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 H Asgarifar, A Oloyede, F Zare “Investigation of high frequency, high voltage

pulses application on bending properties of bone” EPSM-ABEC Conference, Aug

2011, Darwin Australia

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Statement of Originality II Acknowledgments III Keywords IV Abstract V Contribution VIII Table of Contents XI List of Figures XVIII List of Tables XXV List of Abbreviations and Symbols XXVII

Chapter 1: Introduction 1

Chapter 2: Physical Behaviour and Electrical Stimulation of Bone 8

2.1 Introduction 9

2.2 Hierarchical architecture of bone 10

2.3 Cortical bone structure 12

2.3.1 Bone cells 12

2.3.2 Extracellular matrix (ECM) architecture 15

Collagen fibrils arrangement 17

Mineral crystals structure 18

The water content 19

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2.4.1 The bone basic elements (molecular level) 22

Collagen fibrils 22

The mineral crystals 24

The bone water content 24

2.4.2 The mineralized collagen fibrils (nanoscale level) 26

2.4.3 The arrays of the collagen fibrils (mesoscale level) 26

2.4.4 The organization of the fibril arrays in lamellae and osteon (microscale level) 27

2.5 Bioelectric phenomena in bone 28

2.5.1 The origin of the stress generated potential (SGP) in bone 29

2.5.2 Electrical stimulation of bone with low intensity electromagnetic field 31 Application of direct contact method for bone tissue stimulation31 Application of the pulsed electromagnetic field stimulation on bone tissue 33

Inductive coupling 34

Capacitive coupling 36

2.5.3 Influential factors in electrical stimulation methods 38

2.5.4 Some of the hypothesized mechanisms involved in bone generation due to pulsed electromagnetic field 40

2.5.5 The effect of low intensity pulsed electromagnetic field on biomechanical properties of bone 41

2.5.6 Application of high intensity pulsed electromagnetic field on bone 42

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3.1 Introduction 45

3.2 Topology of pulse power generator 47

3.2.1 General configuration of positive Buck-Boost Converter 47

3.2.2 Switching Modes 49

First state: charging inductor (S1: on, S2: on) 49

Second state: circulating the inductor current (S1: off, S2: on) 49

Third state: charging the capacitor and load supplying (S1: off, S2: off) 50

3.3 The pulse power generators applied in this study 52

3.4 Load modeling 55

Chapter 4: Physical Characterisation of Bone Exposed to Pulse Power in Bending 57 4.1 Introduction 58

4.2 Factors influencing experimental measurement 59

4.3 Materials and Methods 62

4.3.1 Sample preparation 62

4.3.2 Three-point bending test 63

4.3.3 Data collection and calculation 65

4.3.4 Pulse Power excitation 68

4.4 Experimental procedure and Results 69 4.4.1 Pulse power excitation with voltage up to 180V and 100 Hz frequency 69

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4.4.2 Pulse power excitation with pulses up to 450 V magnitude at 10 kHz

frequency 73

Pulses up to 450 V at 340 Hz 73

Pulses up to 450 V at 10 kHz 75

4.5 Discussion 77

Chapter 5: Effect of Pulse Power Exposure on Functional Behaviour of Cortical Bone in Tension 80

5.1 Introduction 81

5.1.1 Fractographic study 83

5.2 Materials and Methods 84

5.2.1 Practical consideration for tensile testing 84

5.2.2 Sample preparation 85

5.2.3 Pulse Power excitation 88

5.2.4 Uniaxial quasi-static tensile test 90

5.2.5 Scanning electron fractograph 91

Sample preparation for SEM procedure 91

5.3 Experimental procedure and Results 92

5.3.1 Dumbbell shape tensile test samples with round junction versus those with sharp junction 92

5.3.2 Hysteresis energy absorption for PP-exposed samples versus the control samples 94

5.3.3 Tensile toughness and strength measurement 96

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5.4 Discussion 105

Chapter 6: Effect of Pulse Power Excitation on Basic Mechanical Properties of Cortical Bone in Compression 110

6.1 Introduction 111

6.2 Materials and Methods 112

6.2.1 Sample preparation 112

6.2.2 Experimental Procedure 113

Bone samples stimulation with pulse power signals 113

Compressive testing 115

6.3 Toughness and strength measurement (results) 116

6.4 Discussion 120

Chapter 7: Evaluation of Cortical Bone Elasticity in Response to Pulse Power Excitation Using Ultrasonic Technique 122

7.1 Introduction 123

7.2 The theoretical consideration 125

7.3 Materials and Methods 127

7.3.1 Sample preparation 127

7.3.2 Density measurement 128

7.3.3 Experimental Procedure 129

Ultrasound velocity measurement 129

Pulse Power excitation 132

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7.5 Discussion 135

Chapter 8: Effect of Pulse Power Stimulation on Functional and Physical Characteristics of Cortical Bone (Discussion and Conclusion) 138

8.1 Introduction 139

8.2 Research procedure description and justification 141

8.2.1 Introduction of a suitable pulse power application set up and evaluation of the flexural elasticity of cortical bone through non-destructive 3-point bending test 142

8.2.2 The effect of pulse power exposure on the tensile strength and total fracture energy accompanying the microstructure analysis of the test bone fracture surfaces 143

8.2.3 The effect of the pulse power excitation on the compressive strength and toughness of the small sized samples 144

8.2.4 Application of ultrasonic technique to evaluate the effect of pulse power on bone elasticity 144

8.3 The effect of pulse power stimulation on functional behaviour of cortical bone tissue 145 8.3.1 Results Interpretation 145

8.3.2 Final results 152

8.4 Discussion and Conclusion 152

8.5 Research limitations 155

8.6 Future work and recommendation 156

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Figure ‎ 2.1 Hierarchical structure of bone (a) Cortical and cancellous bone (b) Osteon

consist of haversian canal (c) Lamellae (d) Collagen fibers (e) Collagen molecules and mineral crystals23 11

Figure ‎ 2.2 Cross-section of a bone showing both cortical and cancellous bone

structure26 12

Figure ‎ 2.3 Response pattern of the bone cells to extrinsic/intrinsic applied load27 15

Figure ‎ 2.4 Multi scale of bone architecture (a) Amino acid building block (the

smallest scale of bone) (b) Tropocollgen molecules made from three polypeptide chains of over 1000 amino acid residues (c) Mineralized collagen fibrils consisting of mineral crystallites embedded within and between collagen fibrils (d) Fibrillar arrays, the arrangement of the mineralized collagen fibrils (e) Different organizations of fibrillar arrays in different bone types (f) The osteon which surrounds and protects the blood vessels (g) Bone tissue level (h) Whole bone level21 16

Figure ‎ 2.5 (a) Triple-helical structure of collagen molecule (tropocollagen molecule)

(b) The arrangement of the collagen molecules in the collagen fibrils , (the staggered arrays of tropocollagen molecules assembles in collagen fibrils which themselves organize into arrays The neighboring collagen molecules have the gap (G) of 40 nm and the overlap (O) of 27 nm relative to each other29.) 18

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Figure ‎ 2.7 Strain generated potential created on a femur under mechanical

deformation10 30

Figure ‎ 2.8 Four stimulatory techniques for application of electric current to the tissue by direct contact of the electrodes (A) The cathode in the target site and the anode on the skin (B) The cathode in target site and the anode in some distance with the cathode implanted in soft tissue (C) Non-invasive stimulation placing the electrodes on the skin (D) Both electrodes implanted in the soft tissue, away from the target site 87 32

Figure ‎ 2.9 Inductive coupling set up over a tibia fracture94 34

Figure ‎ 2.10 Capacitive coupling set up over the fracture site94 36

Figure ‎ 3.1 Conversion of low power, long time input waveform to high power, short time output waveform by a pulse power generator 45

Figure ‎ 3.2 Typical diagram for pulse power generators 46

Figure ‎ 3.3 A combination of current and voltage sources as a pulse power generator135 47

Figure ‎ 3.4 Circuit diagram of positive buck-boost converter 48

Figure ‎ 3.5 First switching state, charging the inductor 49

Figure ‎ 3.6 Second switching state, circulating the inductor current 50

Figure ‎ 3.7 Third switching state, charging the capacitor 51

Figure ‎ 3.8 Power delivery through the load switch 51

Figure ‎ 3.9 Pulse power generator A (PGA) with NEC microcontroller 53

forces along the surfaces of the loaded specimens[3] 60

Figure ‎ 4.2 Three-cycle bending load in linear elastic region on the bone strip sample64 Figure ‎ 4.3 A small drop on the first cycle of bending test in elastic region that was

removed on the further cycles 65

Figure ‎ 4.4 Three point bending test142 66

Figure ‎ 4.5 Assumed elliptical cross-section for whole bone 67 Figure ‎ 4.6 Cross-sectional area of whole long bone in ANSYS for determination the

area moment of inertia 67

Figure ‎ 4.7 Bone strip obtained from the cortical diaphysis 68 Figure ‎ 4.8 Variation of Young’s modulus of the ovine metatarsus exposed to 180V

and 100 Hz pulses over 5 days (PP-exposed sample) compared to that of the control sample 71

Figure ‎ 4.9 Sketch of experimental set-up for pulse power stimulation of the cortical

bone strip sample 72

Figure ‎ 4.10 Variation of the Young's modulus of femoral cortical strips exposed to

180V at 100 Hz pulse power over 9 days compared with that of the same samples without pulse power excitation 73

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applied on cortical bone samples 76

Figure ‎ 4.12 Elastic properties of the cortical bone samples exposed to pulse power

(450 V at 10 kHz) before and after excitation compared with those values of the control samples 76

Figure ‎ 5.1Typical macroscopic tensile test fracture (A) ductile shear fracture (B)

moderately ductile fracture (C) brittle fracture 155 84

Figure ‎ 5.2Dumbbell shape specimen with round junction (GL, GW and GT are gage

length, gage width and gage thickness respectively) 85

Figure ‎ 5.3 Sketch of partitioned tibia used for tensile test specimen preparation 87 Figure ‎ 5.4 Dumbbell shape specimen with sharp junction (GL, GW and GT are gage

length, gage width and gage thickness respectively) 87

Figure ‎ 5.5 Top view of a sketch of experimental set up for Pulse Power excitation of

the bone tensile test specimens between two isolated aluminium strips 89

Figure ‎ 5.6 Tensile testing of the cortical bone specimen 90 Figure ‎ 5.7 Cortical bone samples mounted on the SEM stubs, place for gold coating91 Figure ‎ 5.8 Tensile Stress-Strain responses until failure of dumbbell shape samples

with round junction ( ) versus those of dumbbell shape samples with sharp junction ( ) 93

Figure ‎ 5.9 Comparison of the strength and toughness of dumbbell shaped samples

with round junction and those of samples with sharp junction 93

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exposed to pulse power before and after 145 hours excitation 94

Figure ‎ 5.11 Hysteresis loops in the tensile loading-unloading cycle for a control

bone sample before and after 145 hours being in similar environmental condition as PP-exposed samples 95

Figure ‎ 5.12 Mean hysteresis energy of the control samples versus the samples

exposed to pulse power before and after 145 hours excitation 96

Figure ‎ 5.13 Tensile stress-strain graphs of the cortical bone samples in four groups

up to failure 97

Figure ‎ 5.14 SEM micrographs from the top and side views of the control samples

(unexposed to pulse power) with their corresponding stress-strain graphs 100

Figure ‎ 5.15 SEM micrographs from top and side views of cortical bone samples

exposed to 500Vand 10 kHz pulse power for 145 hours with their corresponding stress-strain graphs 101

Figure ‎ 5.16 SEM micrographs from top and side views of the cortical bone samples

exposed to pulse power, A and B for28 hours, C and D for 35 hours with their equivalent stress-strain graphs 102

Figure ‎ 5.17 Details of scanning electron micrographs of fracture surface in higher

magnification (A) Dimpled, irregular appearance of fracture surface (B) Microcrack diffusion (C) Microvoids (D) Crack bridging by collagen fibrils104

Figure ‎ 5.18 Higher magnification of scanning micrographs of the fracture surfaces

of the representative samples from each group (A) Control sample (B)

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Figure ‎ 6.1 Position and directions of the rectangular specimen obtained from the

tibial cortical dyaphysis 113

Figure ‎ 6.2 Sketch of experimental set-up for pulse power stimulation of

millimetre-sized cortical bone samples 115

Figure ‎ 6.3 Compressive testing of cortical bone specimen 116 Figure ‎ 6.4 Compressive stress-strain responses for the control specimens ( )

verse those for the samples exposed to pulse power ( ) 117

Figure ‎ 6.5 The total strain fracture energy of the samples exposed to 500V, 10 KHz

electromagnetic field compared to that of the control samples 118

Figure ‎ 6.6 The strength of the samples exposed to 500V, 10 KHz electromagnetic

field compared to that of the control samples 118

Figure ‎ 6.7 Comparison of the stiffness of the samples exposed to pulse power with

that of the control samples 119

Figure ‎ 7.1 Ultrasound wave propagation in a bone specimen142 125

Figure ‎ 7.2 Ultrasound velocity measurement set up inside water tank 130 Figure ‎ 7.3 Ultrasound wave propagation trough the sample and time delay

measurement on Lab view Signal Express 131

Figure ‎ 8.1 The elastic modulus of the normal specimens compared with the samples

exposed to pulse power for 144 hours obtained from ultrasonic technique146

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exposed samples before and after pulse power stimulation 147

Figure ‎ 8.3 Comparison of the bone mineral density of the control and PP-exposed

samples before and after pulse power excitation 148

Figure ‎ 8.4 Comparison of the hysteresis energy dissipated by the control and the

PP-exposed samples before and after excitation 149

Figure ‎ 8.5 Comparison of the tensile strength and total failure strain energy of the

samples exposed to pulse power for 145 hours with those of the control samples 150

Figure ‎ 8.6 The strength and total fracture energy absorption of the samples exposed

to pulse power for 66 hours compared with those parameters of the control samples 151

Figure ‎ 8.7 Comparison of the Young’s modulus of the samples exposed to pulse

power with that of the control samples obtained from compression tests 152

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Table ‎ 4.1 Comparison of the area moment of inertia of the whole bone samples

obtained from ANSYS and calculation 68

Table ‎4.2 Mean value± standard deviation for Young’s modulus of cortical bone

before and after pulse power excitation (450V at 340Hz) in three days 75

Table ‎ 5.1 Mean value ±standard deviation for the toughness and strength of the

tensile bone samples in four treated groups 98

Table ‎7.1 Comparison between the conventional mechanical tastings and the

ultrasonic technique161, 162 124

Table ‎7.2 Mean values ± standard deviation for the specimens’ dimensions 127

Table ‎7.3 Mean density ± standard deviation for cortical bone specimens before and

after pulse power excitation 129

Table ‎7.4 Mean value± standard deviation for ultrasound velocity and Young’s

modulus of PP-exposed samples before and after pulse power excitation in longitudinal, radial and tangential directions respectively 134

Table ‎7.5 Mean value± standard deviation for ultrasound velocity and Young’s

modulus of control samples before and after pulse power excitation period

in longitudinal, radial and tangential directions respectively 134

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modulus in PP-exposed groups after pulse power excitation compared with those of the control group in the same time 135

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CCPEF Capacitive Coupling Pulsed Electromagnetic Field

P value Probability, with a value ranging from zero to one

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Electrical phenomena play an important and effective role in biophysics, biology and medicine There is a strong evidence that human and animal bodies can generate endogenous electric signals with large and stable gradient1 Also, the research indicates that all organisms from bacteria to mammals respond to electromagnetic fields in different ways for example cell division, tissue growth, wound repair 2 Observation of the effect of this endogenous electrical current on the tissue growth and repair has induced interest in the study and application of exogenous electrical stimulation in the field of orthopaedics For instance, over the last four decades the application of time-varying, weak magnetic field, known as Pulsed Electromagnetic Field (PEMF), has opened a new, exciting gateway

to the connective tissue research and treatment for musculoskeletal disorders 3, 4 However, the first investigation in this field dates back to 160 years ago 5, 6

Bone is a dynamic tissue comprised of primarily cells including osteocyte, osteoblast and osteoclast ensconced in an extensive matrix called extracellular matrix (ECM) ECM is

a composite consists of both organic (mostly type I collagen fibrils) and inorganic material (mineral part, mostly hydroxyapatite) This particular composition results in a living, complicated hierarchical structure, which has different physical, solid-state and electro-mechanical properties7 These properties give the bone the capacity to respond to physical stimulation by generating a very small electric current relating to bone formation 4 A direct relationship between the mechanical deformation of bone and the generation of endogenous electrical currents (caused by Stress Generated Potential) in bone has been well indicated in different studies 7, 8

According to Wolf’s law, physical loadings on bone alter the bone structure and

leads to adapting the bone to its mechanical environment Although the mechanism under which bone responds to the applied physical loading is not fully understood, it has been

3

suggested that the stress generated potential in bone is related to the piezoelectric effect on collagen fibers with non-centrosymmetric structure in dry bone and deformation of fluid-flow in the small channels between osteocytes (canaliculi) in wet bone 2, 7 This endogenous electric signals and the mechanical strain in the cellular level generating through loading the bone are proposed as the possible stimuli that cause the cellular response leading to bone growth and osteogenesis

There are various in vivo studies which have reported the advantages of both direct current and PEMF stimulation on tissue growth 3, 4, 9 This effect led to the utilisation of electromagnetic fields stimulation for bone generation and as an accepted remedy for some bone disorders such as delayed-union bone fracture and failed joint 3, 10 In addition, a few in vitro experiments stated the beneficial effects of electrical stimulation on osteogenesis with both constant and pulsed electromagnetic field application 11, 12 However, a few studies reported the contradictory outcomes in the application of PEMF on cell proliferation and differentiation13 These diverse results are likely attributed to different PEMF parameters and experimental conditions

Three main parameters are involved in the application of any kind of electrical stimulation that influence the results: the magnitude of the applied energy, the amplitude of the stimulus and the frequency of application The tissues appear to respond differently to these factors To achieve the desired outcome, the choice of appropriate parameters in a suitable manner is essential

A review of the advantages of PEMF stimulation on connective tissue in both animal and clinical studies and on the other hand, the observation of the lack of studies in the application of high power, high frequency electromagnetic fields, spurred interest in the

it very quickly (in microseconds or less) which results in the delivery of larger amount of instantaneous power (several kilowatts) in a very short time, though the total energy is the same 14 To generate such electromagnetic fields, high voltage and high current sources are required For prevention of the thermal effect the pulse interval, needs to be very short 15

Pulse Power technology has been used variously in biology and medicine, especially

at intercellular scale Some of its established/demonstrated applications are controlling the ion transport processes across membranes, prevention of biofauling, bacterial decontamination of water and liquid food, delivery of chemotherapeutic drugs into tumour cells, gene therapy, transdermal drug delivery, programmed cell death which can be used for cancer treatment and intracellular electro manipulation for gene transfer into cell nuclei16 However, no published work has reported its utilization in skeletal system for stimulation purposes

The previous studies in the field of the electrical stimulation of the connective tissue have mostly considered the use of low energy, weak electromagnetic fields or high intensity electromagnetic field at low frequency Nevertheless, the physical characterestics

of bone at high-energy levels have not received adequate attention Additionally, there is a limited number of researches investigating the frequency dependence of the electrical properties of bone The motivation for this research project was to explore the safe and

on biomechanical properties of bone is crucial as a primary step for safe and controlled application of high voltage, high frequency pulsed electromagnetic field on musculoskeletal system

On the other hand, although there are many reported research regarding to the application of electrical stimulation of the bone cells proliferation, differentiation and some bone diseases treatment, very little studies investigated the effect of pulsed electromagnetic fields on biomechanical properties of bone tissue 17-19 For that reason, this research aims to investigate the possible effect of high-power pulses at high frequency relative to changes in the biomechanical/functional properties of the cortical bone samples Along this way, before animal or clinical study, assurance of the safe application of high power signals on bone tissue is necessary to prevent any thermal effect or extra loading which can disturb the quality of the bone composite material Therefore, this pilot study is established to investigate the controlled application of pulse power signals on bone tissue

Chapter 2 reviews the basic structure and the biomechanical properties and the electrical phenomenon of bone tissue and presents some of the previous in vitro and in vivo researches in the electrical stimulation of bone tissue

The pulse power generator was designed and fabricated based on the topology of the positive buck-boost converter (a subset of DC-DC converters) The output voltage was

6

adjustable in magnitude, frequency and duty cycle for determination of pulse power parameters The timing of pulse power stimulant in the experimental protocol was also considered in order to evaluate the possible effect of this factor on bone response This study establishes a new attempt in the field of pulse power technology to determine the safe application method and controlled limits of parameters (pulse width, magnitude, and frequency) for bone excitation The details of the principles of pulse power generators and the positive buck-boost converter topologies, which was utilized in this research, are presented in chapter 3

To evaluate the behaviour of bone in response to pulse power excitation, determination of the functional properties of bone is required The primary function of bone is to be stiff to bear the loads applied to it through both internal and external forces

In addition, it should be strong enough to resist breakage and remain stiff The effect of pulse power stimulation on bone stiffness, strength, the total strain failure energy absorbance (bone toughness) and the hysteresis energy and the ductility of cortical bone tissue were therefore evaluated These properties were determined conducting three conventional mechanical testing including three-point bending, tensile and compressive tests A non-invasive ultrasonic technique was also applied for evaluation of the cortical bone stiffness To investigate the effect of pulse power exposure on the microstructure of cortical bone tissue (its porosity and the diffusion of microcracks), the fracture surfaces of the bone specimens were evaluated using scanning electron microscopy (SEM) The results and analysis of these four methods were presented in chapters 4 to 7

The flowchart diagram presented in the next page, demonstrates the general research procedure that was traversed regarding to research hypothesis approach:

7

Stimulation of Bone

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2.1 Introduction

Bone is a rigid connective tissue with unique mechanical properties that forms the basis of the skeleton of vertebrates It acts as both a mechanically skeletal structure and a physiological unit with close relation With a complex structure, bones form the lightweight but hard and protective load-bearing framework for the body Bone has to bear

up a combination of different loading including compressive, tensile, bending and torsion during everyday activity, which strongly influences its structure and function For example,

the high continuous loading on the sport people’s bones have increased bone mass around

the muscle attachment points while significant reduction in bone mass was observed after long period of bed rest or for astronauts after prolonged space flights which is caused by decreased loading of bone20

From the biological aspect, bone is a connective tissue, which exists in different shape and size and provides a variety of mechanical, synthetic and metabolic functions in the body Beyond giving support and shape to the body, bones work in concert with the muscular system to assist the body with movement and enables sound transduction in the ear, serve as storage of minerals (calcium, phosphorous, etc.) and provide blood production and stem cells from bone marrow for healing and cell growth7, 21

From the structural aspect, bone is a dynamic, hierarchical structure, which has a unique capacity for self-repair, and adaptation to respond external mechanical loading with continuous remodeling Hence, an understanding of the micro and macro structure of bone from molecular level and the mechanical properties of its constituents and their relationship at different levels of the hierarchical structure is useful for realizing the biomechanical behaviour of bone tissue in response to pulse power stimulation

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This chapter firstly provides a brief description about structure and biomechanics of bone tissue (in particular cortical bone) and then reviews some past in vitro and in vivo researches on the use of electrical stimulation on connective tissue

2.2 Hierarchical architecture of bone

As mentioned earlier, bone has a complex and hierarchical structure which shows various physical, solid-state and electro-mechanical properties22 This special architecture makes the bone a highly anisotropic and inhomogeneous material differing in component distribution and spatial arrangement, which results in different mechanical properties in each direction The hierarchical organization of bone can be arranged in five levels with particular mechanical properties coherent to each level which are interrelating together 23,

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1) Macrostructure or tissue level, containing i) trabecular bone (also known as cancellous or spongy bone) which has unorganized lamellae arrangement with very high porosity in spongy nature and accounts for approximately 20% of the bone mass and fill the interior layer and two ends of long bones ii) cortical or compact bone which is a solid, dense material with less porous that makes up the hard outer layer of long bones and accounts for 80% of the bone mass25

2) Microstructure level (from 10 to 500 µm) including single osteons or trabeculae 3) Sub-microstructure (1-10 µm) lamellar level

4) Ultrastructure or nanostructure level (from a few hundred nanometres to 1 µm) consisting of collagen fibril and mineral components of bone

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5) sub-nanostructure, molecular level (less than one hundred nanometre) including

collagen and noncollagen protein molecules and mineral crystals 23, 24

Figure 2.1 shows a schematic diagram from this structural concept

Figure ‎2.1 Hierarchical structure of bone (a) Cortical and cancellous bone (b) Osteon consist of haversian

canal (c) Lamellae (d) Collagen fibers (e) Collagen molecules and mineral crystals23

The cortical bone which is the focus of this thesis, is comprised of dense osteons

The dense osteons themselves constitute of concentric lamellae in a layered structure with

porosities namely lacunae that are regularly diffused between layers and contain osteocytes

(a type of bone cells) The lacunas are connected with several canals containing osteocyte

fingers called canaliculi They carry nutrients to and waste from osteocytes to the blood

vessels embedded in the haversian canals As illustrated in Figure 2.2 the outer surface of

the bone cortex is the periosteum where the bone cells are laid down and act as the growth

source in the bone width The next layers are cortical and trabecular bones adjacent to

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marrow cavity The inner surface is endosteal which is covered by cells that remove the bone tissue20

Figure ‎2.2 Cross-section of a bone showing both cortical and cancellous bone structure26

2.3 Cortical bone structure

Compact bone primarily consists of 2% cells (by volume) ensconced in an extra cellular matrix (ECM) of organic (collagen fibers) and inorganic (hydroxyapatite) components 20 Although cells and the matrix are working separately, their functions interrelate to each other providing the bone growth and its dynamic behaviour and adaptation to different internal and external stimuli

2.3.1 Bone cells

There are three special types of cells that are found only in the bone: Osteoblasts, Osteoclasts and Osteocytes, which work continuously to maintain bone tissue through modeling and remodeling process These bone cells which are responsible for bone