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Interaction of Electric Field with Vascular Cells

A dissertation submitted to the

Graduate School

Of the University of Cincinnati

In partial fulfillment of the

Requirements for the degree of

Doctor of Philosophy

in the Department of Physics

of the College of Arts and Science

by

Toloo Taghian

M.S., Isfahan University of Technology, Isfahan, Iran

July 2015

Dissertation Committee:

Andrei Kogan, Ph.D. (Chair)

Daria Narmoneva, Ph.D.

Margaret Hanson, Ph.D.

Howard Everett Jackson, Ph.D.

Rostislav Serota, Ph.D.


Abstract

Development of effective treatments for impaired healing of vascular tissues is essential

to address significant health care problems associated with these conditions, including

chronic ulcers. Clinical studies reveal that an electric field has the potential as a novel

vascular therapy to accelerate healing of hard-to-heal wounds. However, widespread

acceptance of electric field therapies for wound healing has been prevented by a lack of

standardized protocols, leading to variabilities in healing outcomes. External electric

fields can affect a variety of vascular cell responses through the manipulation of the

native electric field in the extracellular ionic environment and across the cell membrane.

Therefore, an electric field, together with the extracellular matrix and a milieu of

cytokines, represents a biophysical system that ultimately regulates vascular cell

function. Therapeutic modulation of this system requires advanced integration of

knowledge and technology from both physics and biomedical sciences. The long term

goal of this research is to create non-pharmacological, electric field-based therapies for

non- or slow-healing chronic ulcers. The studies in this research contribute to this goal

by developing a theoretical-experimental approach to elucidate the biophysical

mechanisms of cell- electric field interactions mediated by the extracellular matrix.

The numerical model of cell-electric field interaction determines the distribution of the

induced electric field in the cell and extracellular environment. The novelty of the model

is that it considers the cell attached to a substrate to represent cells within tissues. The

results show a striking difference in the (1) frequency dependence of electric field

penetration and (2) cell response between cells suspended in an electrolyte and cells

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attached to a substrate. The results demonstrate that, at low frequency, electric field is

confined in the cell membrane and is expected to regulate membrane-initiated

responses. At high frequency, electric field penetrates the cell and may directly activate

intracellular responses .Importantly, the sensitive dependence of electric field

distribution in the cell to the physical properties of the cell and its environment is

demonstrated. Additionally, the advantage of non- contact electric field application for

research and therapeutic purposes is discussed.

The experimental studies of cell– electric field interaction confirm the theoretical

predictions and show the frequency- specific cell responses and demonstrate the

intracellular pathway activation in response to high frequency electric field when electric

field is induced in the intracellular space. The results show that extracellular matrices

with different matrix properties can differentially regulate protein expression in response

to the high frequency electric field, confirming the importance of incorporating substrate

properties to study cell-field interactions. Importantly, cell responses that promote

wound healing process are activated following electrical stimulation.

Further, this study provides novel information about interaction of cells with surface

charges generated in DC regime. Finally, the deficiencies of endothelial cells in diabetic

condition are determined and the effect of electric field in improving the diabetic

endothelial cell response is demonstrated.

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These findings improve the mechanistic understanding of vascular cell interactions

within the complex biophysical system and can potentially contribute to development of

electric field-based therapies for vascular tissue regeneration.

iv
v
Acknowledgments

I would like to express my sincere appreciation to my advisors, Prof. Andrei Kogan and

Prof. Daria Narmoneva, for providing this great research opportunity, their endless

guidance and the time they have invested into my work.

I would like to thank my doctoral research committee members, Prof. Margaret Hanson,

Prof. Howard Jackson and Prof. Rostislav Serota for their kind support,

encouragement and assistance with this research.

I extend my appreciation to my group members in Physics Department Maryam Torabi,

Bryan Hemingway, Zhuting Sun and Tai-Min Liu and to my group members in

Biomedical Engineering Department Hongkwan Cho, Jenifer Hurley and Swathi Balaji

for being valued colleagues. In particular, I would like to thank Abdul Sheikh for his

patience in training me and his advice during my graduate studies.

The Physics Department at the University of Cincinnati, faculty and the staff has been

very influential in my life during these years. I would like to thank John Markus and John

Whitaker for their patience and assistance with the technical issues.

I would also like to thank my parents and my sister who patiently supported me in so

many ways during my graduate studies. This dissertation wouldn’t have been possible

without their loving support.

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Table of Contents

Abstract…………………………………………………………………….…………………....ii

Acknowledgments…………………………………………………………………………….vi

Table of contents……………………………………………………………………………..vii

List of tables and figures…………………………………………………………………...xiii

List of abbreviation………………………………………………………………………….xvi

1. Introduction and specific aims

1.1. Statement of Problem………………………………………………………………….1

1.2. Specific Aims…………………………………………………………………………...2

2. Background

2.1. Clinical problem………………………………………………………………………...4

2.1.1. Significance……………………………………………………………………....4

2.1.2. Factors contributing to the development of chronic diabetic wounds……...4

2.2. Endothelial cells and wound healing………………………………………………...6

2.3. Hydrogel scaffolds to mimic extracellular matrix properties……………………….6

2.4. Electric field as a biophysical stimulus to promote wound healing…………….....8

2.4.1. Charged Surfaces……………………………………………………………..…9

2.4.2. Conductive Polymer Scaffolds………………………………………………..12

2.4.3. Electrical Stimulation Devices………………………………………………...13

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3. Modulation of cell function by electric field: a high-resolution analysis

3.1. Objective………………………………………………………………………………17

3.2. Introduction……………………………………………………………………………17

3.3. Material and methods ……………………………………………………………….21

3.3.1. Model system…………………………………………………………………….24

3.3.2. Modelling the cell on a substrate………………………………………….……28

3.4. Results and discussion………………………………………………………………29

3.4.1. Electric field distribution in the cell: frequency dependence………………...29

3.4.2. Spatial variation of electric field…………………………………………….…..32

3.4.3. Conductivity of the cytoplasm: regulation of the cell cytoplasm screening in

response to electrical stimulation………………………………………………….…..36

3.4.4. Substrate and culture medium properties and field penetration into the

culture medium………………………………………………………………………..…38

3.4.5. Physiological implications…………………………………………………….…40

3.5. Conclusions…………………………………………………………………...………42

4. Extracellular matrix: A mediator of endothelial cell response to electromagnetic

stimulation

4.1. Objective………………………………………………………………………………45

4.2. Introduction……………………………………………………………………………45

4.3. Materials and Methods………………………………………………………….……53

4.3.1. High-frequency electric field set-up…………………………………………….53

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4.3.2. Microvascular endothelial cell isolation and culture………………………….56

4.3.3. Endothelial cell culture system…………………………………………………56

4.3.4. Immunohistochemistry…………………………………………………………..57

4.3.5. Vascular endothelial growth factor expression and nitric oxide release…...58

4.3.6. Focal adhesion kinase expression……………………………………………..58

4.3.7. Nitric oxide expression and inhibitor studies…………………….……………59

4.3.8. Statistical analysis……………………………………………………………….60

4.4. Results…………………………………………………………………………………60

4.4.1. Endothelial cell morphology differs on matrigel vs synthetic peptide………60

4.4.2. Hydrogel properties regulate expression of angiogenic growth factor in

endothelial cells……………………………………………………………………….…62

4.4.3. Effect of electrical stimulation on angiogenic growth factor expression is

mediated by hydrogel properties………………………………………………………62

4.4.4. Hydrogel properties modulate expression of focal adhesion kinase in

endothelial cells……………………………………………………………………….…65

4.4.5. Effect of electrical stimulation on focal adhesion kinase expression is

modulated by hydrogel properties………………………………………………….….68

4.4.6. Hydrogel properties regulates Vinculin expression…………………………..71

4.4.7. Effect of electrical stimulation on vinculin expression is modulated by

hydrogelproperties…………………………..………………………………….………71

4.4.8. Hydrogel properties regulate nitric oxide expression of endothelial cells….75

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4.4.9. Electrical stimulation enhances nitric oxide expression of endothelial cells

independent of hydrogel properties……………………………………………………75

4.4.10. Calcium regulates electric field mediated nitric oxide expression of

endothelial cells……………………………………………………………………….…75

4.5. Discussion………………………………………………….…………………….……79

4.6. Conclusions…………………………………………………………………………...83

5. Interaction of vascular cells with a non-oscillating electric field

5.1. Objective……………………………………………………………………………....84

5.2. Introduction……………………………………………………………………………84

5.3. Materials and Methods………………….……………………………………………85

5.3.1. In vitro EF exposure setup………………………………………………………85

5.3.2. EF exposure setup for live imaging of cells……………..…………………….88

5.3.3. Model system…………………………………………………………………….90

5.3.4. Fibroblast isolation and culture…………………………………………………93

5.3.5. Live-dead assay………………………………………………………………….93

5.3.6. Adhesion assay…………………………………………………………………..93

5.3.7. Measurement of angiogenic growth factor expression………………………94

5.3.8. Proliferation assay………………………...……………………………………..94

5.3.9. Statistical analysis…………..………………………………………………..….95

5.4. Results………………………………...……………………………………………....95

5.4.1. Capacitively coupled electric field maintain the cell viability…………….….95

5.4.2. Electric field stimulation increases the proliferation of fibroblasts………….97

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5.4.3. Electric field polarity regulates the adhesion of fibroblasts to the

substrate…………………………………………………………………………………99

5.4.4. Electric field stimulation enhances vascular endothelial growth factor

expression………………………………………………………………………………102

5.5. Discussion……………………………………………………………………………104

5.6. Conclusions……………………………………………...………………………..…108

6. Enhancement of diabetic deficiencies by biophysical stimuli and extracellular

environment

6.1. Objective……………………………………………………………………………..109

6.2. Introduction…………………………………………………………………………..109

6.3. Materials and Methods………………..……………………………………………110

6.3.1. Microvascular endothelial cell isolation and culture for electrical

stimulation………………………………………………………………………………110

6.3.2. Microvascular endothelial cell isolation and experimental groups for strain

experiments…………………………………………………………………………….111

6.3.3. Application of cyclic strain……………………………………………………..111

6.3.4. Endothelial cell culture system………..………………………………………113

6.3.5. Capillary morphogenesis………………………………………………………113

6.3.6. Vascular endothelial growth factor assay……………………………………114

6.3.7. Nitric oxide assay………………………………….…………………….……..114

6.4. Results……………………………………………………………………………………114

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6.4.1. Capillary morphogenesis is impaired in diabetic condition…………….…..114

6.4.2. Expression of angiogenic growth factor in endothelial cells is impaired in

diabetic condition………………………………………………………………..……..115

6.4.3. Nitric oxide expression of endothelial cells is impaired in diabetic

condition……………………………………………………….………………………..115

6.4.4. Electric field stimulation enhances nitric oxide expression of endothelial cells

in diabetic condition………………………………..……………………………….….117

6.4.5. Application of strain increases nitric oxide expression of endothelial cells in

diabetic condition………………………………………………………………………118

6.4.6. Extracellular environment stimulates nitric oxide expression of endothelial

cells in diabetic condition………………………………………….…….…………….119

6.5. Discussion and Conclusions………………………………………………………..….120

7. Conclusion and future directions…………………………….………………………122

Bibliography…………………………………………………………………………………127

Appendix……………………………………………………………………………………..146

xii
List of tables and figures

Chapter 2

Figure 2.1 Schematics of cutaneous wounds 5

Chapter 3

Table 3.1 Model parameters used in the simulations 23

Figure 3.1 Schematics of non-contact electrical stimulation system 25

Figure 3.2 Induced electric field in the culture medium as a function of 26

the applied field frequency in the non contact model

Figure 3.3 Schematic of a cell attached to the substrate 27

Figure 3.4 Induced electric field in the cell membrane, cytoplasm and 31

culture medium

Figure 3.5 Spatial distribution of induced electric field in the cell 33

Figure 3.6 Induced electric field as a function of frequency of the 35

applied field at different positions along the cell membrane

Figure 3.7 Effect of cytoplasm conductivity on the induced electric 37

field in different cell compartments

Figure 3.8 Effect of the geometrical and electrical parameters of the 39

stimulation system on induced electric field in the culture

medium

Chapter 4

Table 4.1 Effect of substrate properties on the endothelial cell 49

response to high frequency electric field

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Figure 4.1 Experimental set-up for microvascular endothelial cells 55

exposure to electric field

Figure 4.2 Morphology of ECs seeded on hydrogels 61

Figure 4.3 Regulation of vascular endothelial growth factor 64

expression by extracellular matrix properties and high

frequency electric field

Figure 4.4 Regulation of focal adhesion kinase expression by 66

extracellular matrix properties

Figure 4.5 Regulation of focal adhesion kinase expression by high 69

frequency electric field

Figure 4.6 Vinculin expression 73

Figure 4.7 Regulation of nitric oxide expression by extracellular matrix 77

properties and high frequency electric field

Chapter 5

Figure 5.1 The custom system for electrical stimulation of cells using 87

the capacitive coupling method

Figure 5.2 Custom system for live imaging of cells during electrical 89

stimulation

Table 5.1 Parametes used in the Model system 92

Figure 5.3 Percentage of live cells in electric field stimulated 96

Figure 5.4 Cell Proliferation following electrical stimulation 98

xiv
Figure 5.5 Delayed adhesion of electric field exposed cells to the 100

substrate influenced by negative surface charge deposited

on the substrate at different time points

Figure 5.6 Increased vascular endothelial growth factor expression 103

following electrical stimulation

Figure 5.7 Normalized induced voltage and surface charge in the 107

model system

Chapter 6

Figure 6.1 Flexcell tension system 113

Figure 6.2 Endothelial deficiencies in diabetic condition 116

Figure 6.3 High frequency electrical stimulation increases NO 117

expression in both wild type and diabetic endothelial cells

Figure 6.4 Application of strain significantly increases NO expression 118

in wild type, diabetic and high glucose groups

Figure 6.5 Nanofiber scaffold significantly increases NO expression in 119

wild type, diabetic and high glucose groups

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List of Abbreviation

EF electric field

DC EF direct current electric field

HF EF high frequency electric field

EC endothelial cell

VEGF vascular endothelial growth factor

NO Nitric oxide

FAK focal adhesion kinase

p-FAK Phosphorylated focal adhesion kinase

ECM extracellular matrix

Ca2+ Calcium ions

MAPK mitogen-activated protein kinases

ERK extracellular regulated kinase

eNOS endothelial nitric oxide synthase

FBS fetal bovine serum

ELISA enzyme-linked immunosorbent assay

PBS phosphate buffered saline

DCM diabetic cardiomyopathy

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Chapter 1

Introduction and specific aims

1.1. Statement of Problem

Chronic ulcers are a major concern for patients and healthcare professionals that affect

6.5 million patients in the United States and an excess of $25 billion is spent annually

on the wound care (1). Current pharmacological treatments including application of

moist dressings, antibiotic therapy of infected wounds, hyperbaric oxygen therapy, and

surgical vascular reconstruction to restore blood flow are often inadequate for timely

and complete healing (2). The mechanism responsible for failure of pharmacological

treatments in chronic wounds is insufficient blood supply in the wound caused by

impaired angiogenesis, defined as formation of new blood vessels from existing ones

(3, 4). Increased extracellular matrix degradation, impaired function of vascular cells

(endothelial cells) and cell death lead to impairment of angiogenesis. Non-

pharmacological treatments through biophysical manipulation of wound environment

may accelerate the wound healing process (5). To date, different approaches have

been used to enhance chronic wound healing. Electric field (EF) based therapy has

been shown to improve the blood vessel formation and accelerate wound healing but its

widespread acceptance has been prevented by variability in healing outcomes of clinical

trials. This stems from incomplete understanding of the biophysics principles that

govern electric field interactions with cells in the ionic extracellular environment of the

wound (6). In addition to electrical signals, use of nanofibers as extracellular matrix

scaffolds has been shown to stimulate angiogenesis and accelerate wound healing in

animal models (7).

1
The long term goal of this research is to create non-pharmacological electric field-

based therapies for non- or slow- healing chronic ulcers. In order to accelerate wound

healing, we need to characterize, control and enhance the external biophysical signals

that regulate the healing process and perform experiments in a controlled environment.

The objective of this dissertation is to perform a combined theoretical-experimental

investigation to elucidate the biophysical mechanisms of the interaction of an electric

field with vascular cells mediated by an extracellular matrix. The central hypothesis of

this dissertation is that stimulation of endothelial cells via electrical signals combined

with extracellular matrix can trigger angiogenic responses. The central hypothesis was

tested via the following four aims.

1.2. Specific Aims

AIM I: To develop a theoretical model to study the biophysics of EF interaction

with cells

Research aim 1.1: To determine the spatial distribution of EF in the cell membrane,

cytoplasm and extracellular medium.

Research aim 1.2: To determine whether the structure of induced EF in the cell

membrane is influenced by changing the EF frequency.

Research aim 1.3: To investigate whether the extracellular environment can affect the

structure of the induced EF in the cell.

2
AIM II: To quantify the regulatory effect of high frequency EF on the endothelial

cell function.

Research aim 2.1: To determine the effect of EF on nitric oxide expression of

endothelial cells and the mechanisms underlying this effect.

Research aim 2.2: To investigate the effect of different extracellular matrices (natural vs

synthetic hydrogel scaffolds) on endothelial cell response to EF.

Research aim 2.3: To validate the predictions of the model in high frequency range.

AIM III: To determine the effect of non-oscillating EF on the cell function.

Research aim 3.1: To design and build an exposure device that enables live study of

the interaction of non-oscillating EF with cells.

Research aim 3.2: To theoretically determine the EF distribution in the model system

and validate it experimentally.

Research aim 3.3: To investigate the effect of EF polarity on regulation of cell

responses to EF.

AIM IV. To determine the endothelial cell deficiencies in diabetic condition

Research aim 4.1: To quantify the deficiency of diabetic endothelial cells in nitric oxide

expression and capillary morphogenesis.

Research aim 4.2: To examine the role of external biophysical stimuli in improving the

diabetic cell response.

3
Chapter 2

Background

2.1. Clinical problem


2.1.1. Significance

Diabetes is a growing epidemic in the United States, affecting 26 million people, (8.3%

of population) and is responsible for $116 billion in direct medical cost. Chronic non-

healing ulcers represent one of the major complications of diabetes. They occur in

about 15% of the diabetic patients and are responsible for more than 27% of the annual

$116 billion diabetic health care cost in US. They are a major risk factor for lower

extremity amputations in these patients (8).

2.1.2. Factors contributing to the development of chronic diabetic wounds

Wound healing is a dynamic process that is orchestrated in an orderly and timely

manner. It consists of three overlapping, distinct phases including inflammation,

proliferation (new tissue formation), and remodeling (9). Normal healing involves proper

circulation, nutrition and balanced immune status to initiate cell migration to the wound

site, and regulated interaction of cells with the wound matrix and the response to

cytokines (10). In a diabetic wound, the healing process is disrupted and includes

altered cellular metabolism, increased extracellular matrix degradation and decreased

cell sensitivity to growth factor stimulation (Fig.2.1). Therefore, the diabetic wound gets

stuck in the first phase of healing (inflammatory phase) which impairs the recruitment of

endothelial cells in later phases (11).

4
Figure 2.1. Schematics of cutaneous wounds: normal (top) and diabetic (bottom).
The normal wound shows the presence of growth factors and extracellular matrix
laid down by fibroblast as well as formation of blood vessels to the wound site
(12). In contrast, the diabetic wound is stuck at the inflammatory phase with more
inflammatory cells, such as macrophages, which secret proteases in the wound,
resulting in excessive degradation of growth factors and extracellular matrix
molecules, leading to non-healing diabetic ulcers.

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2.2. Endothelial cells and wound healing
The inner monolayer of a blood vessel wall is lined by cells called endothelial cells

(ECs) (13). Independent of the kind of vessel, ECs are flat with a diameter of 10 − 20

μm. ECs play an important role in several physiological processes. They not only

regulate the nutrient exchange between tissue and blood, but also produce important

biochemical factors for the regulation of blood pressure and modulate the vascular tone

(14). Another crucial biological process, where ECs are involved is angiogenesis (15).

Angiogenesis is the formation of new blood capillaries from the pre-existing ones which

is of high importance during the wound healing process. New blood vessel formation is

based on the capacity of ECs to migrate, proliferate, elongate, and organize in three

dimensional tubules (16). Impaired angiogenesis in diabetic wounds leads to

exacerbated peripheral limb ischemia due to the lack of vessel development and

delayed healing. Delayed wound healing of the lower extremities can cause severe

infections, which go on to cause 86,000 lower limb amputations in the US per year (17).

2.3. Hydrogel scaffolds to mimic extracellular matrix properties

Biomaterials play a central role in modern strategies in regenerative medicine and

tissue engineering as designable biophysical and biochemical milieus that direct cellular

behavior and function (18). The guidance provided by biomaterials may facilitate

restoration of structure and function of damaged or dysfunctional tissues.

Both biologically-derived and synthetic materials have been extensively explored in

regenerative medicine and tissue engineering (19). In general, materials from natural

sources including collagen, matrigel and fibrin can provide cells with an in vivo-like

6
biochemical and biophysical microenvironment, such as the presentation of receptor-

binding ligands. Despite these advantages, the concerns associated with immunogenic

reactions, gel degradation which imped the long term application and animal virus

contamination of natural material, have urged the development of alternative materials

(20). Synthetic biomaterials that mimic advantageous properties of natural materials can

interact with their biological environment and participate actively in pathways of tissue

morphogenesis.

Self-assembling peptide gels are synthetic biomaterials with an extracellular matrix- like

microarchitecture (21). This class of 8–32 amino acid long oligopeptides is

characterized by having alternating hydrophilic and hydrophobic amino acid side chains.

The self-assembled peptides are arranged in stable β-sheet at low pH. Upon exposure

to physiological pH they can self-assemble into a 3D network of interweaving nanofibers

of 10 nm diameter with 5-200 nm pores (22). These gels have high water content (99%)

and their pore size is similar to native ECM allowing 3D cell interaction with the

surrounding microenvironment.

The RAD16-II peptide nanofibers are an attractive tissue-engineered biomaterial for

creating a suitable microenvironment for diabetic wound healing. RAD16-II scaffold

provides an angiogenic environment that inhibits endothelial cell apoptosis, enhances

cell proliferation and expression of the angiogenic factors in vitro and promotes

recruitment of endothelial cells and neovascularization in cardiac tissue in vivo (23).

Previous studies of the peptide injection in vivo in mouse cardiac tissue showed that

peptide was still present at 4 weeks after injection without causing detectable immune

reaction, while enabling cell infiltration and new matrix deposition (22). The peptide is a

7
fully synthetic material and enables attachment of several cell types due to its cell-native

amino acid composition.

2.4. Electric field as a biophysical stimulus to promote wound healing


Studies have revealed the importance of electrical characteristics as one of the native

regulators of cell functions. Electrical characteristics in the form of electrical fields,

currents or charges provide spatial and temporal regulation of cellular activities ranging

from embryonic development to regeneration of injured tissue (24-26). Electrical fields

are most important for the functioning of ion channels and pumps. These molecules are

expressed across the cell membrane and generate the electric field on the order of 107

mV/mm along the membrane (27). Electric fields are induced by the transport of ions

and separation of charges along the tissue and can regulate tissue physiological

response (28, 29). For example, in normal skin, there is a native trans-epithelial electric

potential difference of ~ 40-70 mV that forms across the epithelial tissue (30). When the

skin is injured, the trans-epithelial electric potential drops at the center of the wound

site. As a result, a potential difference develops between the center of the wound and

the surrounding tissues, giving rise to an electric field on the order of 100 mV/mm that

guides the cells to the site of injury and helps heal the wound (31, 32). Electrostatic

interactions are key regulators of major cell functions such as adhesion; cell interactions

with signals on the extracellular surfaces and may even participate in immune function

and infection prevention (26, 33-35). Therefore, modulation of native electrical signals is

a promising approach to regulate single cell function as well as cell-cell and cell-

extracellular interactions and activate appropriate cell signaling pathways. On a

practical level, this can be achieved by (a) using charged surfaces to regulate

8
electrostatic interactions, (b) delivering electrical signals through fabricated conductive

scaffolds, or (c) application of external electrical signals to cells and tissues using

custom made devices to locally induce an electric field or generate an electric field.

Here we review recent advances in the applications of external electrical signals as a

tool for tissue engineering.

2.4.1. Charged surfaces

Development of tissue from multicellular organisms during growth or repair relies on cell

adhesion process which can be influenced by electrostatic charges (26, 36). Therefore,

manipulation of the substrate charge, for example, by coating the implant to enhance

cell adhesion is a promising strategy to control cell movement, assembly and responses

in tissue engineering applications. One of the most effective materials that can be used

for surface coating is hydroxyapatite (HAp). It is polarized by the application of a DC

electric field at high temperature after deposition on the surface, which results in a high

charge storage capacity. The polarity of the induced surface charge depends on the

polarity of the applied DC electric field (37, 38). It has been shown that this surface

charge can accelerate or decelerate cell adhesion and growth on the charged surfaces

through attraction or repulsion of positive ions, specifically, divalent cations in the cell

culture medium (26, 39). These cations enable interactions between the surface and

negatively charged cell membrane and play an important role in formation of focal

adhesions (40). In practical applications, HAp coating is often applied on a titanium

substrate, which is commonly used in dental and orthopedic implants (41). Experiments

with bone cells demonstrated that on negatively charged HAp coated titanium, cell

9
proliferation and expression of vinculin (one of the major players in the cell adhesion

process) were enhanced meanwhile on positively charged titanium, these responses

were inhibited during the early stage of cell culture (42, 43). Thus, an improved

adhesive property accelerates the tissue growth on implants. On the other hand, the

decreased adhesion due to positive-charge coating can be used to regulate cell

morphology. Another example of regulation of cell behavior via control of the substrate

surface charge is polyion complex nanoparticles (PIC) coated polystyrene (44). PIC

nanoparticles are formed by mixing a cationic homopolymer (N,N-dimethylaminoethyl

methacrylate) with anionic plasmid DNA at various charge ratios which can be adjusted

to negative or positive coating (45). Studies showed that adipose-derived stromal

progenitor cells (ADSCs) cultured on PIC coated polystyrene change their morphology

by altering the charge of the coating (44). ADSCs show good adhesion with spindle-

spread shape on negatively charged PIC-coated surfaces, while on the positively

charged PIC surface, the cells change their morphology to form capillary-like networks.

This finding is especially important for tissue engineering applications, where

development of transplants with induced capillaries to introduce nutrient into

transplanted tissue is necessary to prevent ischaemia (46).

An important consideration in the development of the strategies to control cell behavior

via regulation of the surface charge is that changing the substrate charge may modify

other properties of the surface, such as rigidity or even chemistry, which in turn can

further affect the cell adhesion process (44). This consideration becomes critical for the

soft substrates like hydrogels, where several studies demonstrated that the substrate

10
charge density can strongly affect cell attachment. Thus, osteoblast and fibroblast

attachment and spreading on the positively charged HEMA or PEG hydrogels are

significantly higher than on the negatively charged hydrogels (47). Positively charged

hydrogels (i.e. oligo-(polyethylene glycol) fumarate (OPF)) also support attachment of

rat dorsal root ganglion explants and enhance the neurite outgrowth, in contrast to the

unmodified hydrogels (48). These results are encouraging and suggest that hydrogels

with incorporated charges can be used to manipulate cell attachment for engineering of

hard or soft tissue for clinical applications.

In addition to their use as a substrate to control cell adhesion, charged surfaces can

also be utilized as antibacterial and antibiofilm substrates. Bacterial infection can be the

critical factor in determining the outcome of a variety of implants (49, 50). Bacteria

surface is negatively charged (in a neutral medium); therefore, initial adhesion of

bacteria is expected to be prevented on negatively charged surfaces and promoted on

positively charged surfaces. Similarly, surface growth of bacteria can be minimized or

prevented by the charge of the surface (51). For example, it has been shown that E. coli

forms a spare and mushroom-like biofilm on negatively charged surface, whereas on a

positively charged surface, the E. coli biofilm is dense and homogeneous (52). Similarly,

positively charged poly(methacrylate) (PMMA/TMAEMA-Cl) has been shown to support

adhesion, but not bacterial growth, thus resulting in an antimicrobial effect on Gram-

negative bacteria (53). These results suggest a potential regulatory role for the surface

charge of implants in lowering the risk of infection and the prevention of implant failure,

as well as enhancing cell growth for a variety of applications.

11
2.4.2. Conductive polymer scaffolds

Electrically conductive polymers (i.e. polypyrrole (PPy), polyaniline (PANi),

polythiophene (PT), etc.) are biocompatible materials, which have physical and

chemical properties of organic polymers and electrical properties of metals. As a result,

these materials can deliver electrical signals directly to the cells attached to their

surface (54-56). Studies have shown that relatively small electrical field (50 mV/mm)

delivered by these conductive polymers can upregulate the growth and enhance viability

and cellular cytokine production of human fibroblasts (57, 58). Similarly, stimulation of

vascular smooth muscle cells cultured on these conductive polymers by a sinusoidal

electrical field (5, 500 HZ) leads to enhanced cell proliferation and protein expression

(59).

Among polymeric bio-substrates, nanofibrous scaffolds (i.e. scaffolds made from nano-

scale polymer fibers) provide the best support for cell survival and growth due to their

specific fiber size and alignment, as well as porous structure (60). However, conductive

polymer nanofibers may be especially effective in modulating cell functions, because

these materials are able to both mimic the three-dimensional architecture of natural

extracellular matrix and to deliver appropriate electrical signals to the cells. Indeed,

studies have reported that this combined stimulation results in significantly enhanced

rate of neurite outgrowth in dorsal root ganglia (61, 62). Similarly, conductive fibers have

been shown to stimulate myoblast differentiation (63, 64). In general, these findings

suggest that utilizing the complex bio-surfaces that are made from conductive polymers

12
may be the best approach to modulate substrate or scaffold properties for cell adhesion

and growth.

2.4.3. Electrical stimulation devices

Electrical stimulation devices are designed to apply external electrical fields to alter the

native cell electrical signals. Endogenous electrical fields plays an important role such

as directing cell migration during tissue repair and angiogenesis (blood vessel formation

and growth) (32, 65, 66). To mimic this electric field in vitro, a device has been designed

which applies direct current electrical fields (100-500 mV/mm) generated by two salt

bridges to cells cultured in a chamber filled with a cell culture medium (67). The direct

current electric field in the medium induces cell responses such as directional cell

migration, elongation and reorganization of actin cytoskeleton in a wide variety of

microvascular and macrovascular cells (68, 69). Application of this direct current electric

field has been shown to enhance growth factor expression, activate signaling pathways

and upregulate angiogenic factors in different cell types such as lens epithelial cells,

endothelial cells, keratinocytes and fibroblasts (68, 70-76).The electric field-directed cell

migration has been demonstrated in collective migration of epithelial monolayers as well

as 3D environment of spinal cord for neural progenitor cells (77, 78).

In addition to endogenous direct current electric field, electrical pluses are essential

regulators of organ function, specifically heart function. Cardiomyocytes are

continuously subjected to electrical signals that regulate their contractions (79). To

mimic the native pulse in vitro, a device has been designed that delivers rectangular-

wave pulses with millisecond pulse width to the culture medium (80). The application of

electrical stimulation enhances the contractile behavior and increases the amplitude of

13
contractions in cardiac myocytes (81). Electrical stimulation of cardiomyocytes also

increases expression of cardiac specific genes and transcripts. Importantly for

biophysical and engineering applications, electrical stimulation acts mechanisms that

differ from mechanical stretching (79). Cardiomyocytes are electrically coupled, and the

ionic wave (e.g. Ca2+, K+) between the cells acts as a regulator of their function.

Therefore, stimulation and monitoring of a pair of cardiomyocytes is required for cardiac

cell based therapy (82). Another approach is the use of multi-unit electrode arrays,

which allows localization of electrical stimulation on the single and multiple cell level to

investigate the propagation of ionic waves between adjacent cells in response to

electrical stimulation. These devices are composed of microfabricated PDMS

microchannels with embedded or patterned electrodes to deliver a millisecond pulsed

electric field to cells (83, 84).

In addition to short pulsed-based stimulation, radio frequency electric field has also

been employed to regulate cellular functions. In this method, cells are exposed to an

electric field at a frequency of 2.4 GHz. Radio electric asymmetric conveyer (REAC)

stimulates the cells through the cell culture medium(85). REAC exposure for 24-48 hrs

enhances the expression of cardiac, skeletal and neuronal lineage-restricted marker

proteins in mouse embryonic stem cells, with the increased gene expression retained

for 2-7 days after stimulus removal (85). Human dermal fibroblasts exposed to REAC

show increased transcription of tissue restricted genes for cardiac reprogramming,

skeletal myogenesis and neurogenesis (86). REAC exposed human adipose derived

stem cells (hASCs) show an increase in the expression of the lineage restricted genes

14
and proteins in both transcriptional and protein expression level (87). These findings

suggest a great promise of the REAC-based approach towards achievement of complex

lineages for regenerative medicine, with an important advantage of cell stimulation

without the use of chemical agonists, thus avoiding potentially significant side effects.

Another approach that has been introduced for electrical stimulation of mammalian cells

is based on a non-contact method of inducing electric field in cells and culture

medium(88). The stimulation device is composed of a cavity resonator fed by a coaxial

microwave line to stimulate cells with a low amplitude (~100mV/mm), high frequency

(7.5 GHz) electric field during cell culture on native or synthetic scaffolds or substrates.

The high frequency electric field enhances angiogenic endothelial cell responses,

including capillary morphogenesis, vascular endothelial growth factor expression and

MAPK/ERK intracellular pathway activation of endothelial cells (88). This method

induces electric fields in the cell membrane and cytoplasm without any physical contact

between electrodes and medium. The non-contact electric field-based technology may

present an attractive alternative or adjuvant therapy to standard treatments of chronic

wounds or vascular tissue regeneration by integrating engineering and biological

principles to stimulate cellular responses in the wound using extracellular signal with no

systemic drug-associated side effects (89-92).

The devices described above generate low amplitude electric fields to induce electric

fields in the cell membrane. The induced field is usually smaller than the natural electric

field due to the electric potential difference across the cell membrane and therefore

15
does not damage the cell. Electroporation is a technique of inducing a very high

magnitude electric fields in the cell membrane using pulse generators (93). Application

of a high magnitude pulsed electric field forms nanoscale pores in the membrane and

permeabilizes the cell (94). Electroporation could be reversible or irreversible depending

on the electric field amplitude and duration (95, 96). Reversible electroporation has

been used to introduce drugs and genes through the pores into the cells (97-100).

Irreversible electroporation opens permanent pores in the membrane of targeted cells

and can be used to obtain decellularized tissue scaffolds. This approach allows

preservation of the intact extracellular matrix (ECM) for subsequent use in the tissue

engineering applications. For example, the decellularized artery obtained using this

technique has no vascular muscle cell layer, but is able to support growth of endothelial

cells along the lumen, thus demonstrating that the ECM is not harmed following the

electrical treatment (101). In another study, an epithelial layer was formed on the

decellularized small intestine tissue following in vivo stimulation, indicating that ECM of

the intestine remains fully functional following electrical treatment (102).

16
Chapter 3

Modulation of Cell Function by Electric Field: A High-Resolution

Analysis

3.1. Objective

This study presents a numerical model of interaction of a non-contact electric field with

a cell. The high resolution distribution of electric field in the cell membrane, cytoplasm

and extracellular environment is determined in a wide frequency range. For the first

time, this physiologically relevant model considers the cell attached to a substrate to

represent cells in tissues. This model demonstrate the dependence of cell responses to

EF to (1) frequency of the electric field (2) substrate properties.

3.2. Introduction

Electric field (EF) can regulate a variety of cell functions, including growth, adhesion,

reorganization of cytoskeleton, contractility, differentiation, proliferation, activation of

intracellular pathways, secretion of proteins and gene expression (81, 103-108). The

regulatory effect of EF has been demonstrated on different cell types such as neurons,

osteoblasts, myoblasts, fibroblasts, endothelial cells, muscle cell, and epithelial cells

(77, 109-114). These effects result from manipulation of the native EF in the ionic

extracellular environment and across the cell membrane (115-117). The mechanisms

for the cell-EF interaction are not yet understood, which limits development of EF based

therapies.

17
Cells can interact with the surrounding environment through receptors and ion channels

embedded in the cell membranes, which transmit the chemical, mechanical and

electrical signals outside-in and inside-out the cells. Therefore, coupling of

electromagnetic fields with a live cell can occur via either field interaction with charged

molecules and proteins in the cell membrane that alters the flow of ions through the ion

channels or rearranges the distribution of the membrane receptors, or via direct field

penetration inside the cell and interaction with charged entities in the cytoplasm (26,

118, 119).

Experimental evidence suggests that one possible mechanism for the EF effects on cell

function may involve induced field in the cell membrane that may cause alterations in

the transmembrane potential (31, 32, 118, 120-122). The transmembrane potential is

established by the balance of intracellular and extracellular ionic concentrations that

generate the voltage difference across the cell membrane (120), and is maintained by

ion channels, pumps and transporter proteins embedded in the cell membrane (123). In

response to the external EF, the induced field in the cell membrane can cause

modulation of the cell membrane potential, which in turn can increase the activity of the

sodium, potassium or calcium channels and alter the enzyme activity of phosphates

containing the voltage sensor domain, as has been shown using genetic or

pharmacological cell manipulations (26, 124-126). Depending on the EF parameters,

the effects of EF can also include membrane electroporation in response to very high

amplitude EF (107 mV/mm) (mathematical model is explained in (96, 127, 128)) and can

be used in electrochemotherapy, drug delivery and gene therapy(129), as well as

18
redistribution of membrane channels and receptors in response to non-oscillating EF

with low, physiological amplitude (100 mV/mm)(32, 72).

Theoretical models indicate the possibility of the high frequency (1-10GHz), low

amplitude EF penetration into the cell via mechanisms other than electroporation,

although the exact conditions for this effect are not well established (119). Notably, this

frequency range is well outside the range that is likely to be encountered physiologically

(e.g., <1000Hz in nervous system) without external stimulation. Recent experimental

studies by us and others provide indirect support for this possibility by demonstrating

that such fields may trigger a variety of intracellular signaling events that involve charge

redistribution and ion flow, including signal propagation via Ca2+, gap junctions, or even

protein-protein interactions (85, 86, 104, 130).

In addition to the variation in EF parameters, the variability of methods of low-amplitude

EF delivery to cells can also contribute to the diversity in the observed cell responses.

EF can be delivered to cells by direct contact of electrodes with cells and the culture

medium (67, 74, 131-133) or through a non-contact approach, which isolates electrodes

from cells and the culture medium and capacitively couple EF to cells and culture

medium as reported by us (104) and others (134-137). Overall, experimental results

suggest that different combinations of EF intensity, frequency, and/or polarization with

respect to the cell, as well as methods of EF delivery, can elicit a variety of distinct cell

responses, making mechanistic studies of the EF-cell interactions quite difficult.

The complexity of mechanisms for EF-cell interaction calls for a detailed understanding

of the induced EF structure in cells subjected to electromagnetic field stimulation. Fricke

introduced an empirical equation for the potential induced in an ellipsoidal cell in

19
suspension exposed to external EF (138). Schwan developed the first theoretical

equation for the induced potential in a spherical cell in suspension exposed to external

EF by an analytical solution of the Laplace equation, where the cell is approximated by

a spherical shell representing the cell membrane (139). The model by Shawn treats the

cell as a non-conducting membrane subjected to a constant and alternating external EF

(140). Kotnik extended the Schwan’s theory by taking into account the conductivity

using constant, oscillating and pulsed EF (141). Other geometries - cylindrical,

spheroidal and ellipsoidal - of cells suspended in the medium have been investigated

later (142-145). Several studies have modeled the cell as multiple concentric shells to

determine the induced EF in the internal membranes (146, 147). The effect of surface

charge and electrical properties such as membrane conductivity on the induced

potential in spherical and non-spherical cell geometries has been examined (141, 148).

Numeric finite-element modeling (FEM) (149-152) and transport lattice (TLM) models

(153-155) and approaches based on equivalent circuits (94, 156) have been used to

examine complex cells of complex shapes immersed in electrolyte. However, in most in

vivo conditions, the cells are surrounded by and interact with the extracellular matrix,

rather than being suspended in an electrolyte medium. While the cell-matrix interactions

may play an important role in cell responses to the external EF, the effects of these

interactions on the electric field distribution within the cell compartments are not

understood, and the comprehensive analyses of cellular responses, to our knowledge,

have not been incorporated into the existing models.

The objective of this study, therefore, is to determine the effects of the EF frequency

and extracellular environment on cell responses to the external EF. The model is based

20
on the physiologically relevant configuration when parts of the cell membrane are in

close contact with the extracellular substrate. The cell is modeled as a semi-spherical

nonconducting shell separating two conducting regions, the culture medium and the

cytoplasm, in direct contact with a flat dielectric substrate. To recapitulate our

experimental configuration (104), the electrodes supplying the EF are isolated from the

medium. The EF is therefore coupled to the cell and its environment capacitively, which

eliminates electrochemical processes in the medium and reduces the electric current

and associated ionic flow effects on the cell membrane. We obtain a high resolution

distribution of induced EF in a wide frequency range (1 Hz-10 GHz) in the cell

membrane, cytoplasm and extracellular medium. We then examine the sensitive

dependence of the induced EF in the cell membrane and cytoplasm on cytoplasm

electric properties. The results demonstrate that the field distribution exhibits

physiologically important features that strongly depend on the EF frequency and differ

substantially when compared to the all-electrolyte environment. The presented model

and numerical method can be easily adapted to in-vivo arrangements.

3.3. Materials and Methods

High-frequency structure simulator (HFSS, version 14) software (ANSYS Corp, PA,

USA) was used for numeric solutions of Maxwell’s field equations. A variable-density

adaptive mesh was generated to enable field calculations over a wide range of length

scales, from nanometers for the membrane thickness to microns for the cytoplasm. The

mesh was refined until an acceptable accuracy for the calculated electric field was

attained at all characteristic dimensions of the model. The large-scale mesh accuracy

was tested by comparing the numeric results to the analytical solution (equation 1, given

21
in the section below). The fine-scale mesh for intra-cell and membrane field

calculations was refined to achieve a proper convergence of the path integrals of EF to

zero along small closed paths. We verified that the meshes used in the simulations

were sufficiently dense to produce mesh density-independent simulation output.

Dimensions used in the simulation and material parameters (94, 119, 157-159) are

given in Table 3.1.

22
Table 3.1. Model parameters used in the simulations.

Parameter Value used


Component in References
simulation
thickness 5 (nm) (67)
*
Cell membrane relative permittivity (εmembrane) 11.3 (20)

conductivity (σmembrane) 0 (20)

radius 5 (µm) (66)


*
Cell cytoplasm relative permittivity (εcytoplasm) 80 (68)

conductivity (σcytoplasm) 1.5 (S/m) (69)


*
relative permittivity (εmedium) 80 (68)

conductivity (σmedium) 1.5 (S/m) (69)


Culture
height 48 (µm)
medium
#
resistance (Rmedium) 3332 (Ω)
#
capacitance (Cmedium) 0.14 (pF)
*
relative permittivity (εsubstrate) 2.6

conductivity (σsubstrate) 0
Cell substrate
thickness 1 (µm)
#
capacitance (Csubstrate) 0.22 (pF)

Electrodes electrode separation 50 (µm)

( *relative permittivity: εmaterial /εair , # : values used in the simulation)

23
3.3.1. Model system

The model follows the design of our recent experimental in-vitro studies (104). The

electrodes used to generate the EF are isolated from the medium by dielectric spacers

(figure 3.1a) and modeled as infinitely conducting planes. These electrodes define the

simulation boundaries. The EF produced by the electrodes is oriented perpendicular to

the substrate and is influenced, at the cell location, by both the potential difference

applied to the electrodes and the substrate polarization. In the absence of cells, the

system is equivalent to a one-dimensional circuit (figure 3.1b). The upper and lower

capacitors represent the capacitance of the top dielectric and the substrate (C substrate).

The resistive (Rmedium) and capacitive (Cmedium) components of the culture medium

impedance are also shown. The potential difference between the electrodes created by

an external source (Vapplied) creates an EF in the culture medium given by equation 3.1,

𝜔 𝑅𝑚𝑒𝑑𝑖𝑢𝑚 𝐶𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
𝐸𝐹𝑖𝑛𝑑𝑢𝑐𝑒𝑑 = 1 𝑉𝑎𝑝𝑝𝑙𝑖𝑒𝑑 (3.1)
2 2
𝐶
𝑑×(4+(𝜔 𝑅𝑚𝑒𝑑𝑖𝑢𝑚 𝐶𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 (1+2 𝑚𝑒𝑑𝑖𝑢𝑚 )) )
𝑐𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒

where ω is the angular frequency of the applied field and d is the separation of the

dielectric coatings. The magnitude of the EF induced in the medium, EFinduced, is plotted

in figure 3.2 as a function of the applied field frequency. The plot is generated by Igor

Pro software (WaveMetrics, OR, USA) and the values used for parameters are listed in

Table 3.1. At low EF oscillation frequency, the EF is screened from the medium by the

redistribution of the ionic charges on the medium boundaries. The EF appears in the

medium when the frequency becomes comparable to or larger than the characteristic

inverse charging/discharging times, approximately 100 MHz for our geometry.

24
Figure 3.1. (a) Non-contact electrical stimulation of live cells. The electrodes

are isolated from the culture medium by the substrate layer (bottom) and the

coating layer (top). Electrodes are oriented to ensure that the resulting

electric field, EFinduced, is perpendicular to the cell substrate. Cells are placed

on the substrate surface which is in contact with the stimulation apparatus.

(b) Equivalent electric circuit with no cell present. Ccoating and Csubstrate

represent the capacitance of the dielectric insulating layers; Cmedium and

Rmedium are the capacitance and resistance associated with the electrolyte

(culture medium), respectively. The oscillating voltage V is applied to the top

and bottom electrodes.

25
Figure 3.2. Induced electric field in the culture medium as a function of

the applied field frequency in the non contact model. At low frequency of

the applied field, EFinduced is excluded from the culture medium. In

contrast, EFinduced in the culture medium rises dramatically as the

frequency approaches 100 MHz and reaches a constant value above 10

GHz. Electrode separation is 50 µm and V=10 v.

26
Figure 3.3. Top: Schematic of a cell attached to the substrate. Bottom:

Hemispherical cell model used for simulations. Cell radius is 5 µm and

membrane thickness is 5 nm (not drawn to scale). Polar coordinate

system (r, θ) is used to characterize the magnitude of the induced EF in

the cell membrane.

27
3.3.2. Modeling the cell on a substrate

The cell is modeled as a membrane-enclosed hemisphere with a realistic membrane

thickness and radius attached to a substrate (extracellular matrix) and surrounded by

cell culture medium (figure 3.3). The cell and the substrate are non-conductive

dielectrics with different dielectric permittivities (Table 3.1). The culture medium and the

cell cytoplasm are modeled as electrolyte with ionic mobilities and concentrations that

mimic those in vivo. The top and bottom electrodes are infinitely conducting metallic

layers. Each region is homogeneous and isotropic. A 10 V potential difference is applied

between the electrodes. We note that electric properties in the range of field intensities

relevant to the experiments under physiological conditions are nearly independent of the

field intensity; therefore, the problem is linear and describes the distribution of EF

produced by an arbitrary driving voltage with an appropriate rescaling of the simulation

results. We find the induced EF in the cell membrane, cytoplasm, extracellular medium

and cell substrate by solving a full set of Maxwell’s equations shown in equation 3.2,

𝜕𝐵 𝜕𝐷
∇×𝐸 = − ,∇× 𝐻 = 𝐽 + , ∇. 𝐷 = 𝜌 , ∇. 𝐵 = 0 (3.2)
𝜕𝑡 𝜕𝑡

Here, E, B, H=B/µ, J and D=εE are the position- and time-dependent vectors of the

electric field, magnetic field, magnetic field strength, electric current density and electric

displacement, µ and ε are the magnetic and electric permittivities, and ρ is the local

density of electric charge. The induced EF is found by solving the wave equation as

shown in equation 3.3 which is obtained by combining the first two Maxwell’s equations,

1
∇ × ( ∇ × 𝐸) − 𝑘02 𝜖𝑟 𝐸 = 0 (3.3)
µ𝑟

28
with

µ 𝜖
µ𝑟 = , 𝜖𝑟 = , 𝑘02 = 𝜔2 𝜖0 µ0 (3.4)
µ0 𝜖0

where µr and εr are the (generally complex-valued) relative permeability and permittivity,

k0 is the free space wave number and ω is the frequency of the applied electric field.

The boundary conditions are fixed potentials on the two electrode boundaries, Vtop and

Vbottom ,

𝑉𝑏𝑜𝑡𝑡𝑜𝑚 − 𝑉𝑡𝑜𝑝 = 𝑉 cos 𝜔𝑡, (3.5)

where V is the externally applied driving voltage and ω is the frequency of the applied

field (1Hz-10GHz).

3.4. Results and Discussion

3.4.1. EF distribution in the cell: frequency dependence.

We first present the results of simulations at four locations along the axis of the model

symmetry (figure 3.4): a site in the culture medium, a site inside the cytoplasm, the

“apex” and the “substrate side” of the membrane. For all four locations, three distinct

frequency regimes are identified. Region I (approx.1 Hz-1 MHz): In agreement with the

one-dimensional circuit model (figure 3.1), the EF is screened in the electrolyte

compartments (the cytoplasm and the culture medium). Importantly, a strong field is still

present in the cell membrane, both at the apex point and the substrate side. This is the

central result of this work. It shows that electrodes isolated from the medium can

produce an electric field even in free membranes, as long as a portion of the cell

membrane is attached to the substrate. This is an important difference between the

29
arrangement presented here and cells suspended in a culture medium electrolyte

entirely, where no field can be produced in the membrane unless there is a penetration

of the field into the electrolyte. An important implication of this finding is that even at DC

voltages (as well as frequencies up to 1 MHz), manipulation of the field strength in the

cell membrane can be achieved.

Region II (approx. 1MHz – 100MHz): The penetration of the field into the cytoplasm

and the culture medium develops. Both the cytoplasm and the culture medium exhibit

very similar behavior; the field in the membrane at the apex point rises and reaches

values comparable to those in the substrate side of the membrane.

Region III (approx. 100 MHz – 10 GHz): The penetration of the field into electrolyte is

fully developed and becomes frequency-independent again. The fields in the apex and

the substrate side of the membrane decrease slightly and reach comparable, frequency-

independent values at frequencies of 1 GHz and higher.

30
Figure 3.4. Frequency dependence of EFinduced in the the cell cytoplasm

and the culture medium (a), and the cell membrane at apex and

substrate side (b). The response to the applied electric field can be

divided into three regions. In region I, the electric field is induced in the

cell membrane and is excluded from the cytoplasm and culture medium.

In region II, EFinduced in the cell membrane increases at the cell apex up to
5
the maximum value (Emax ~ 12x10 V/m) and starts to penetrate into the

cytoplasm and culture medium, while it does not change significantly at

the cell membrane facing the substrate. In region III, the magnitudes of

EFinduced at the cell apex and the substrate side both decrease and

eventually reach the same plateau, while EFinduced in the cytoplasm and

culture medium both increase and reach plateau values at high

frequencies.

31
3.4.2. Spatial variation of EF

Having identified the characteristic frequency domains of the problem, we turn to the

spatial dependence of the induced field (figure 3.5). The Color plots (figure 3.5, left)

illustrate the EF distribution at three characteristic frequencies: 60 Hz, 10 MHz, and 1

GHz. At each frequency, the EF is present in the membrane and the EF penetration to

the electrolyte-filled regions is apparent as the frequency increases. In the membrane,

the field is nearly homogeneous along the substrate and varies strongly through the free

section of the membrane facing the medium (figure 3.5, right). We also note a rapid

change in the EF amplitude at points close to the cell edges.

A detailed calculation of the evolution of the angular distribution of the membrane field

with frequency (figure 3.6) reveals two characteristic behaviors. At the locations close to

the substrate (θ<300), there is a gradual decrease in the field magnitude with increasing

frequency. EF distribution at the points positioned further into the free membrane

section (θ>500) shows a characteristic rise to a plateau followed by the eventual

reduction of the field with increasing frequency. This is similar to the “Apex” response

presented in figure 3.4, which corresponds to θ=900. Interestingly, the results show that

the field in the substrate-facing section of the membrane close to the cell center (line

labeled “substrate side”), far from the cell edge, is significantly stronger than the field in

the low-lying points of the free membrane at all frequencies. This apparent discrepancy

is a real effect that reflects the rapid change of the membrane field near the cell edge in

the transition zone between the substrate-facing and the free medium facing parts of the

membrane (figure 3.5, left).

32
33
Figure 3.5. Left: Spatial distribution of induced electric field in a single cell

attached to a substrate and exposed to the external electric field that is

perpendicular to the substrate. The scale applies to all color plots, with
5
the maximum value of Emax ~ 12x10 V/m. At low frequency of the applied

field, the induced electric field is present only in the cell membrane and is

excluded from the cell cytoplasm and culture medium (left-top). As

frequency increases, electric field starts penetrating into the cell

cytoplasm and culture medium (left-middle). At high frequencies, strong

electric field is induced in all cell compartments and culture medium (left-

bottom), with the significant spatial variation in the values for EF induced

along the cell membrane. Right: Distribution of EFinduced is shown for the

cell membrane facing the medium (solid line) and for the cell membrane

facing to the substrate (dashed line) for the same frequency ranges,

demonstrating significant frequency dependence of the magnitude of the

EFinduced in the membrane at either side of the membrane.

34
Figure 3.6. EFinduced as a function of frequency of the applied field at

different positions along the cell membrane. Within the 10 6 - 107 Hz

range, EFinduced increases near the apex but decreases near the

substrate-facing side of the membrane.

35
3.4.3. Conductivity of the cytoplasm: regulation of the cell cytoplasm screening in

response to electrical stimulation

Figure 3.7 shows the sensitivity of the induced EF at the membrane apex and substrate

(figure 3.7a) and the cytoplasm (figure 3.7b) to the cytoplasm conductivity values. At

the apex, a higher cytoplasm conductivity results in larger fields. In the cytoplasm itself,

increasing the conductivity has the opposite effect: the higher the cytoplasm

conductivity, the lower is the magnitude of the induced EF. Effectively, increasing the

conductivity of the cytoplasm makes the screening of the field in the cytoplasm more

powerful, thus leading to increases in the in-membrane field and decreases in the

cytoplasm field. As expected, the field in the membrane facing the substrate is not

sensitive to the cytoplasm properties.

36
Figure 3.7. Effect of cytoplasm conductivity on the induced electric field in

different cell compartments. (a) The increase in the EFinduced at the cell

apex disappears with decreasing the cytoplasmic conductivity, while

EFinduced in the membrane facing the substrate side does not depend on

σcytoplasm. (b) Lower values of the cytoplasmic conductivity lead to a

weakened cytoplasm EF screening and a shift of the EF penetration to a

lower frequency.

37
3.4.4. Substrate and culture medium properties and field penetration into the

culture medium

Finally, we determine the effect of the substrate properties on the field penetration

(figure 3.8). The numeric results presented here are similar to the results obtained via

the one-dimensional model (figure 3.2). Increasing the capacitance of the spacer layers

reduces the corresponding voltage drops. As a result, the characteristic frequency at

which the field begins to penetrate the medium decreases. Therefore, the high

frequency boundary of Region I (figure 3.4) can be tuned. This observation creates an

interesting opportunity for experiments that focus on intra-cellular effects: a

measurement with a relatively low substrate capacitance at a given frequency can be

used as a control as the field will be effectively screened from the cytoplasm. Increasing

the substrate capacitance will introduce the field into the cytoplasm, so EF-mediated

effects could be quantified. A relatively weak but noticeable dependence of the Region I

width on the culture medium and substrate properties is presented in figure 8b, which

demonstrates that decreasing the conductivity of the medium or increasing the

permittivity of the substrate results in the shift in the characteristic frequency of field

penetration into the cell to the lower frequency values.

38
Figure 3.8. Effect of the geometrical and electrical parameters of the

stimulation system on EFinduced in the culture medium. (a) Thinner

substrate allows EF penetration into the medium at lower frequencies of

the applied field. (b) Decreasing the conductivity of the medium or

increasing the permittivity of the substrate shifts the penetration

frequency to lower frequencies.

39
3.4.5. Physiological implications

Our findings demonstrate pronounced differences in cell responses to non-contact

application of EF for two major configurations: cells freely suspended in the electrolyte

medium and cells in direct contact with a dielectric substrate. Importantly, the latter

configuration is more physiologically relevant and may describe, for example, vascular

endothelial cells attached to the basement membrane, or cells within the tissues in

contact with the extracellular matrix, as well as cells in contact with the implant surface.

Absence of the electrolyte along parts of the membrane permits the EF to enter the cell

membrane even at low frequencies and DC limit. In this regime (Region I in our

classification of observed field patterns with respect to frequency), this difference in cell

responses between suspended and adherent configurations is the most striking:

suspended cells experience no electric phenomena at low frequencies, while the cells

attached to a substrate will have significant, location-dependent fields in the cell

membrane. These fields result from the redistribution of charges in the cell culture

medium and in the cytoplasm. We have shown, therefore, that non-contact application

of the EF to cells attached to non-conducting surfaces can be used to manipulate the

EF in cellular membranes, which has important physiological implications. For example,

several studies have reported that the alteration of the cell membrane potential can lead

to the modification of the cellular structure like redistribution of actin cytoskeleton,

change in localization and expression of focal adhesion proteins (e.g. vinculin) (160-

164). The induced potential evoked by an applied EF shown to alter the adhesive

properties of cells and activation of adhesion related proteins (105, 165). Induced

membrane potential can activate signaling mechanisms (e.g. Rho/ROCK) involved in

40
the structural alteration and increase the level of membrane proteins that regulate

attachment of cytoskeleton to cell membrane (166-168). Induced EF can affect voltage-

gated channels embedded in the cell membrane, increase gene expression and

extracellular related proteins (134-137).

Interaction of a cell with EF changes as the field frequency increases. The EF in the

membrane is retained, as an increasingly larger EF appears in the cytoplasm. This

result is consistent with earlier reports that found penetration of the cytoplasm by high

frequency electric field in a free-floating configuration (119).We show that the

characteristic frequency at which the penetration of the field into the culture medium

occurs can be tuned by varying the capacitance of the substrate layers and can be

lowered substantially if materials with higher dielectric permittivities are used. An

important implication of this finding is that this effect can potentially be utilized in EF-

based therapy to stimulate intracellular cell activation and desired cell responses (for

example, migration along the surface of the implant) via appropriate combination of the

implant material and the applied EF field. Indeed, in our previous studies (104), we

discovered that high frequency EF stimulation results in activation of vascular

endothelial cells and angiogenic cell responses via the mechanism consistent with the

model presented above. The results of the present study, therefore, may lead to the

development of new approaches for vascular stents, where endothelial cell activation,

proliferation and migration can be the key factor that determines success or failure of

the stent (169, 170).

Our results demonstrate a close relationship between cytoplasm conductivity and

induced EF in the different cell compartments, which results from physiological cell

41
homeostasis. The cytoplasmic conductivity depends on the concentration of the

intracellular ions regulated by fluxes of ions into and out of the cell through ion channels

in the cell membrane (171, 172). Voltage gated ion channels can be opened in

response to changes in the membrane potential to let inorganic ions diffuse down their

electrochemical gradients across the cell membrane; therefore the cytoplasm

conductivity can be changed during electrical stimulation which in turn may affect the

induced EF (173-175).

In this study, we have not included any dispersive, frequency dependent terms in the

dielectric properties of the materials. While small at frequencies that we have

considered, these will become progressively larger as the frequency increases into tens

of GHz and would need to be included in higher-frequency calculations.

3.5. Conclusions

This study presents a novel model for a high-resolution microscopic analysis of the

induced EF in a single cell in a physiological configuration (i.e. in the electrolyte medium

and attached to the extracellular substrate) exposed to the external electric field. Our

findings demonstrate striking differences in cell responses between cells suspended in

a medium and cells attached to a dielectric substrate. We identify several characteristic

regimes and present their classification with respect to frequency, location, and the

electrical properties of the model components. These findings provide key information

for understanding the mechanisms of cell responses to the electrical stimulation, both in

42
the context of the data interpretation from recent in vitro experimental results by us

(104) and others (134-137), and for the future therapeutic applications.

Manipulation of cells via precisely applied EF can be used to trigger desired responses

at the cell and tissue level and to restore impaired cell functions (104, 107, 176-183),

suggesting a great potential for development of safe and effective EF-based clinical

treatments. To date, clinical application of EF has been reported for treatment of bone

fracture, pain relief and chronic wound healing (184-188). The majority of the stimulation

devices used in clinical studies for chronic wound healing and pain relief deliver EF

through generation of direct current (200-800 µA), monophasic or biphasic pulsed

current with voltage ranges between 20-500 V (186, 189-193), or using transcutaneous

electrical stimulation device (194, 195). The EF current is generated between the

electrodes that are placed on the skin. Although on average these therapies show a

beneficial effect on wound healing, the outcomes still vary significantly, which is likely

due to wide variability in the wound type, patient type, medical personnel training,

electrode placement, duration of stimulation and EF parameters used for stimulation. As

a result, EF-based stimulation is not currently used as a standard therapy (193, 196).

Important advantages of our approach for potential EF-based wound healing therapy is

in a non-contact application of EF that does not require placement of electrodes on the

skin, which may improve patient’s comfort and treatment compliance (193, 196), and

may eliminate the concerns related to application of direct current EF due to PH

changes in the stimulated area (189). Our numerical model demonstrates that by non-

contact application of EF, we can induce sufficient EF in cell membrane and cytoplasm

to manipulate protein expression and activate intracellular pathways as supported by

43
our experimental results (104). Such numerical modeling of EF-cell interactions is an

essential and necessary part of the clinical translation process; it helps to establish

therapeutic concepts and to advance approaches toward the development of non-

pharmacological EF-therapies (197-199), which can be applied locally and without direct

contact with tissue (92, 192, 196, 200-202). Importantly, our theoretical approach can

be generalized to investigate the interaction of EF with cells of an arbitrary shape.

These studies, as well as an investigation of EF induced in the cell nucleus or other

internal cell components and penetration of the field into tissue modeled in 3D cellular

arrays will be performed in the future.

44
Chapter 4

Extracellular matrix: A mediator of endothelial cell response

to electromagnetic stimulation

4.1. Objective

In this study the effect of a high frequency electric field on (1) improvement of the

endothelial cell function and (2) activation of intracellular pathways is determined. For

the first time, this study demonstrates the differential regulation of protein expression of

endothelial cells by extracellular matrix properties in response to a high frequency

electric field. The results presented in this chapter confirm our model prediction that a

high frequency electric field is induced in the intracellular space and can mediate the

intracellular pathway activation.

4.2. Introduction

Electrical stimulation has been used as a therapeutic approach in a variety of clinical

applications such as treatment of bone fracture, pain management, drug delivery and

cancer therapy (202-205). The rational to use EF is based on the existence of natural

EF across the cell membrane (107 mV/mm) at the single cell level and across the skin

(100-200 mV/mm) at the tissue level (27, 206). Therefore, externally-applied EF can

potentially manipulate natural EF and affect cell function (28). Electrical cues, along with

chemical, mechanical and structural cues are regulators of body function (123). There

are emerging evidences that wound healing is accelerated by electrical stimulation (32).

45
Wound healing impairment which occurs in chronic wounds represents a particularly

challenging clinical problem for which no efficient pharmacological treatment currently

exists (207). In chronic wounds, the process of angiogenesis is impaired, and wounds

fail to complete the process of healing (208).These wounds arise from a diverse number

of etiologies, including diabetes mellitus, atherosclerotic, vascular disease, radiation

injury, trauma, and persistent infection (209). Electrical stimulation used in animal

models and clinical trials has shown improved wound healing rates, indicating a critical

role for electrical signaling in wound healing. This suggests a promising role for

electrical stimulation in current strategies for improving tissue repair and regeneration

(186, 210). However, significant variations in the outcome of clinical trials impede its

clinical acceptance as a therapy (196). These variations could stem from different

methods of EF application, different EF parameters, patient type or wound type used in

each trial (6). To significantly improve the healing rate, we need to simultaneously

integrate and control the contribution of chemical, structural and electrical regulators of

healing. To fully harness the potential of EF for improvement of wound healing, a

systematic investigation into the individual and combined effects of these regulators is

needed.

Wound healing is a dynamic process that is orchestrated in an orderly and timely

manner with the goal of restoring tissue integrity (10). It requires the interactions of

many cell types as well as release of growth factors and cytokines to regulate cell-cell

and cell-matrix interactions during all stages of wound repair (211). Although protein-

type mediators are well-established players in this process, emerging evidence from

46
animal studies indicates that nitric oxide (NO) plays a key role in wound repair (212).

NO modulates cells and cytokines involved in all phases of the wound healing.

Therefore, NO may impact several distinct aspects of wound healing (213). Importantly,

NO regulates the function of ECs. EC activation is required for angiogenesis, the

process of forming new microvessels, which is an important component of normal

wound repair (16, 214). In addition to chemical regulation, wound healing is

simultaneously regulated electrically. Endogenous EF, a natural component of healing,

induces directional cell migration towards the wound site (215). This ionic current is

generated as a result of the potential difference between the intact and the disrupted

parts of the tissue barriers (216).

To date, EF with various parameters (i.e. amplitude and frequency) such as direct

current, pulsed, low- and high-frequency oscillating fields has been used to study cell-

EF interaction at the molecular level (217). Physiological direct current EF has been

employed to mimic EF between the cells and induce cell responses such as migration

(77, 215). High amplitude pulsed EF has been used to manipulate EF across the cell

membrane and increase membrane permeability (electroporation) for drug delivery and

gene therapy purposes (96, 127). High-frequency electric field (HF EF) has been

utilized to directly target the cell cytoplasm and affect intracellular interaction (85, 218).

The delivery of EF to cells is done via sending an EF through the culture medium by a

direct contact of electrodes with cells or via non-contact application of EF that

capacitively induces EF in cells and the culture medium (136, 218, 219). Various cell

47
types and substrates have been examined to investigate the effect of EF on cells as

summarized in Table 4.1.

48
Table 4.1. Effect of substrate properties on the endothelial cell response to high
frequency electric field

Electrical
Stimulation Cell Type Substrate Cell Response
Reference
Type

PCNA and ERK1/2


electric current adipose-
upregulation,
(50 µA/mm2, 400 derived plastic
enhanced cell (220)
kHz-450 kHz) stem cells
proliferation

direct current EF Neural organotypic


directed migration in
(DC EF) 500 progenitor spinal cord
the electric field (221)
mV/mm cell slice

direct current mRNA, VEGF165,


endothelial
EF(DC EF) 200 coverslip VEGF121, IL-8
cell (132)
mV/mm upregulation

increased focal
adhesion
direct current EF Human
glass kinase(FAK)
(DC EF) 150 trophoblastic
coverslip phosphorylation (105)
mV/mm cells
increased expression
levels of MMP-2

pulsatile meniscus
3-D fibrin enhanced migration
electrical cells
hydrogel enhanced integrative
stimulation(3 meniscus (222)
meniscus repair
V/cm, 0.1-10 Hz) explants
explants

enhanced the
contractile behavior
Neonatal rat
electrical pulses ( collagen elevated levels of
ventricular
5 V/cm, 1 Hz) sponges MHC, Cx-43, creatine (223)
myocytes
kinase-MM and
cardiac Tn-I

Radio Electric human


increased expression
Asymmetric adipose tissue culture
of the lineage
Conveyer (2.4 derived plates (224)
restricted genes
GHz) stem cells

49
endothelial enhanced VEGF
low amplitude
cell expression, capillary
electric field(7.5 Nanofibers
morphogenesis, (218)
GHz)
activation of
MAPK/ERK pathway
increased
phosphorylation of
biphasic square
AKT, FAK and
pulse(1.5 V/1.8 cardiac stem dish
glycogen synthase (225)
cm, 5 Hz) cells
kinase (GSK3b)

increased collagen
alternating polyacrylic
content,
electrical field smooth acid
enhanced matrix
(0.06 V/mm, muscle cells (PAA)/fibrin (226)
metalloproteinase
0.0167 Hz) hydrogel
(MMP)-2 activity

direct current
EF(DC EF) 0.1- Fibroblast collagen gel induced cell migration
(227)
V/cm

fivefold increase in
grown on fibroblast growth
gelatin factor β-2(FGF-2),
Pulsed microcarriers smaller increases in
electromagnetic HUVECs and angiogenic growth
(228)
fields (15 Hz) embedded in factors (angiopoietin-
a fibrin gel 2,
thrombopoietin, and
epidermal growth
factor)

a 3-D sponge-
sinusoidal
like network
electromagnetic
of
field enhancement of
denatured
(20Hz, EMF; 6 collagen type I
Human type I (229)
mT and 113 expression
osteoblastic collagen
mV/cm max)
cells fibers

50
To replicate wound environment in vitro, it is important to provide cells with the three

dimensional tissue architecture and its molecular cues. Cell stimulation in a tissue

engineered scaffold incorporated with molecular cues mimicking certain functions of the

extracellular microenvironment provides physical support and ECM like biophysical and

biochemical stimuli for cells residing in the scaffold (18). Biologically derived or synthetic

scaffolds functionalized with adhesion motifs of native ECM such as RAD or RGD

binding motifs have been shown to significantly support cell function (230, 231).

Attachment of cells with ECM is mediated via interaction between integrins expressed

on the surface of cells and adhesive proteins of ECM (232). The binding motifs used in

the scaffolds have different affinities and binding strengths. For example, the RAD motif

exhibits a low affinity interaction with integrin while the RGD motif shows a high affinity

interaction (233). It is well established that ECM properties as well as external chemical,

mechanical and electrical stimuli can regulate cell behavior (121, 234, 235). Importantly,

our recent numerical model of cell- EF interactions predicts that properties of

extracellular environment can control the magnitude of the induced EF in the cell (236).

Therefore, taken together, these suggest a role for ECM in mediating the cell response

to EF. Table 4.1 demonstrates the variation seen in the reported response of cells to

electrical stimulation. The difference in the extracellular matrices, EF parameters and

cell type used in each study makes it difficult to make a convincing conclusion about the

mechanisms of cell-EF interaction. Therefore, to investigate the mechanism of cell-EF

interaction there is a need to utilize a controlled environment with well-defined variables.

Application of such well-characterized external EF to cells residing in the in-vitro

scaffolds, functionalized with different types or concentrations of adhesive motifs

51
enables investigation of the mechanisms that ECM regulate EF-cell interaction in a

controlled environment.

We previously have shown a novel mechanism for activation of angiogenic response of

endothelial cells cultured in RAD 16-II nanofibers by electrical stimulation (218). We

showed that physiological amplitude, high frequency (7.5 GHz) electric field activates

MAPK/ERK pathway and increases VEGF release. Our results demonstrate a major

role for cRaf/MEK/ERK and Ca2+ pathway in EF mediated stimulation of angiogenic

responses. Therefore, our previous results demonstrate the stimulatory effect of HF EF

on activation of angiogenic pathways in ECs interacting with RAD 16-II nanofibers.

The EF employed in our experimental studies has the physiological amplitude and an

electric field frequency of 7.5 GHz. EF is applied without direct contact of electrodes and

cells and is oriented perpendicular to the cell plane. Non-contact application of EF

eliminates the risk of contamination of cells with electrochemical products. The

distribution of the induced EF in the cell has been determined using our developed

numerical model of cell-EF interaction which is previously described in Chapter 3. The

high frequency electric field that we use in our experiments is induced in the cell

cytoplasm as well as the cell membrane suggesting activation of intracellular pathways

in response to HF EF. The experimental studies by us and others have shown activation

of intracellular responses to HF EF in the regime that EF is induced in the cytoplasm

(85, 218).

In this work, we employed a 3D in-vitro model of wound healing which mimics in vivo

features of tissue to quantify cell response to HF EF (7.5 GHz) in a controlled manner.

Considering the demonstrated effect of HF EF in the activation of angiogenic pathways

52
and the importance of NO for promotion of angiogenesis in EC and tissue repair, first

we test the hypothesis that whether HF EF results in activation of the eNOS pathway.

Second, we quantify the effect of ECM properties on angiogenic EC response. Third we

test the hypothesis of whether different ECM properties can mediate cell response to

HF EF. We investigate the effect of synthetic ECM (nanofibers) with various RGD

concentrations and native ECM (matrigel) on mediating EC response to HF EF. Our

results provide new information regarding the stimulatory effect of HF EF on VEGF,

FAK and NO expression of ECs. This in-vitro model could provide insights into the

selection of EF parameters and biomaterials to translate the method to clinical practice.

4.3. Materials and Methods

4.3.1. High-frequency electric field set-up

A custom set-up was built that allowed EF exposure operating at 7.5 GHz frequency

(Figure 4.1). The high-frequency EF set-up operated in transverse magnetic mode

(TM010), where the dominant EF was normal to the plane of the cultured cells, and the

magnetic field at the location of the cells was approximately zero. The apparatus

consists of a cylindrical cavity resonator made from a copper waveguide with length of

31.9 mm and diameter of 31.7 mm. The cavity resonator was placed in a temperature-

controlled 5 per cent CO2 cell culture incubator and connected to a semirigid coax

(Microcoax, PA, USA) transmission line supplying 7.5 GHz EF from a vector network

analyser (Anritsu, CA, USA). Cells were seeded in 12 mm diameter culture insert

53
(Millipore, MA, USA), which was placed in a small plastic dish filled with the culture

medium (20 mm in diameter) located inside the cavity resonator. This dish was

connected to a large reservoir outside the resonator to ensure a constant medium level.

Once coupled, a frequency sweep of the reflected power showed a dip that occurred

when the frequency matched the resonant frequency of the cavity (7.5 GHz). Under

these critical coupling conditions, the reflected signal on resonance dropped, and more

than 90 per cent power supplied by the coaxial was used to support the oscillating

cavity mode (TM010). The measured quality factor (Q) of the cavity was 170, and the

calculated field intensity for the set-up with the insert without cells was 156 mV/ mm,

within physiological range (88).

54
High Freq. EF Setup : (~7.5GHz)

cavity resonator control EC sample

medium
reservoir

EC sample

Figure 4.1. Experimental set-up for microvascular endothelial cells


exposure to EF. High-frequency (7.5 GHz) EF set-up: an insert with
endothelial cells is placed in the small plastic dish inside the cavity
resonator to which EF is delivered from a vector network analyzer
(VNA) via a coaxial line. The resonator is placed inside a cell culture
incubator (378C, 5% CO2). Cell culture medium flowed continuously
between the cell insert and reservoir outside the resonator.

55
4.3.2. Microvascular endothelial cell isolation and culture

Primary microvascular endothelial cells (MVECs) were isolated from mouse (C57BL/6J,

Jackson Laboratory, ME, USA) lung tissues using collagenase digestion, as described

previously (88). Cells were doubly sorted using anti-CD-31 (BD Pharmingen, San Jose,

CA) antibody with appropriate secondary antibodies conjugated to the magnetic beads

(Dynabeads, Invitrogen Corporation, Carlsbad, CA). Cells were cultured in gelatin-

coated dishes in Medium 199 (HyClone, Logan, UT) supplemented with 10% FBS

(Atlanta Biologicals, Lawrenceville, GA, USA), 1% antibiotic/antimycotic (Atlanta

Biologicals, Lawrenceville, GA, USA), 10 µg ml-1 heparin (Sigma Aldrich, St Louis, MO,

USA), and 0.2 ng ml-1 growth supplement (Sigma Aldrich, St Louis, MO). Cell cultures

were maintained at 37 ºC in 100% humidified air containing 5% CO 2. Cells from

passages four to nine were used. All experiments were conducted in the culture

medium (medium M199, 10% FBS, 1% antibiotic/antimycotic and 1% heparin) without

additional growth factor supplementation. Cells from passages four to nine were used.

4.3.3. Endothelial cell culture system

ECs were embedded in (at the density of 250,000 cells/ insert) or cultured on (at the

density of 5000 cells/insert) the surface of the synthetic peptide nanofibers with varying

RGD concentration and matrigel. The hydrogels were loaded in culture plate inserts (13

mm diameter, 0.4 µm pore size; Millipore, Billerica, MA). Synthetic peptides were

obtained from RS Synthesis (Louisville, KY) and SynBioSci Corporation (Livermore, CA)

56
and included RADA16-II (AcN-RARADADARARADADA-CNH2), functionalized RAD16-

II (RGD-RAD16-II, AcN-RGDGG-RARADADARARADADA-CNH2). Peptides from stock

solution were diluted in water to a working concentration of 10 mg ml-1 and sonicated.

Matrigel was purchased from BD Biosciences (San Diego, CA).

4.3.4. Immunohistochemistry

ECs were seeded on the surface of synthetic peptides or matrigel at the density of 5000

cells/insert for 12 hours. At 12 hours samples were fixed in 2% paraformaldehyde and

permeabilized in 0.1% tritonX-100. Samples were incubated with phalloidin-TRITC

(Sigma-Aldrich, St. Louis, MO) to stain actin cytoskeleton, followed by DAPI nuclear

staining (Invitrogen, Carlsbad, CA). To stain vinculin, samples were blocked using a

blocking buffer (10% goat serum, 0.1% tritonX-100 in PBS), incubated with primary

antibody against vinculin (Sigma-Aldrich, St. Louis, MO) and subsequent goat anti-

rabbit biotinylated antibody (vector laboratories, Burlingame, CA) incubation as

secondary antibody followed by Vectastain Elite ABC reagent (vector laboratories,

Burlingame, CA) incubation and subsequent DAB substrate solution (vector

laboratories, Burlingame, CA) incubation. Fluorescence or bright field images of the

ECs on the surface of hydrogels were captured at 40X magnification using an inverted

fluorescent microscope (Olympus IX81; Olympus, PA, USA).

57
4.3.5. Vascular endothelial growth factor expression and nitric oxide release

MVECs at the density of 250000 cells/insert were embedded in 10 mg ml -1 peptide

nanofibers (100% RAD16-II, 50% RAD16-II 50% RGD-RAD16-II, 100% RGD-RAD16-II)

and matrigel in culture plate inserts (at least n = 5 separate experiments), and cultured

in medium without growth factor supplement for 12 hours. At 12 hours, medium of EF

exposed and control groups were collected. VEGF protein expression by MVECs was

quantified using Mouse VEGF Quantikine ELISA kit (R&D Systems, Minneapolis, MN,

USA) VEGF protein expression by MVECs was quantified using Mouse VEGF

Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA). Additional controls of the

cell culture medium without growth factor supplement (containing 10% serum) were

included to confirm that growth factors in the serum would not affect protein expression

and detection, with no differences observed between medium samples and the 0 pg ml-1

standard. All ELISA assays were performed in duplicates.

4.3.6. Focal adhesion kinase expression

The total and phosphorylated levels of FAK expressed by MVECs were quantified using

a total-FAK and phospho-FAK DouSet ELISA kit (R&D Systems, Minneapolis, MN,

USA). Briefly, MVECs at the density of 250000 cells/insert were embedded in 10 mg ml -


1
peptide nanofibers (100% RAD16-II, 50% RAD16-II 50% RGD-RAD16-II, 100% RGD-

RAD16-II) and matrigel in culture plate inserts (at least n = 5 separate experiments),

and cultured in medium without growth factor supplement for 12 hours. At 12 hours,

58
cells of EF exposed and control groups were lysed using a buffer containing 20 mM

Tris–HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1 per cent Triton, 2.5 mM

sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg ml21 leupeptin

and 1 mM phenylmethylsulphonyl fluoride. Total and phosphorylated levels of FAK were

determined using FAK ELISA and the manufacturer’s protocol. The total protein

concentration in each sample lysate was determined using the Coomassie plus assay

kit (Thermo Fisher Scientific, IL, USA). A 10 µg bolus of total protein was used for all

FAK pathway enzyme-linked immunosorbent assays (ELISAs). All ELISA assays were

performed in duplicates and the protein levels are presented in terms of optical

densities.

4.3.7. Nitric oxide expression and inhibitor studies

Nitric oxide expression of MVECs was quantified for the collected medium using nitric

oxide assay (Promega Corporation, WI, USA). To determine the role of calcium in EF

mediated NO expression, experiments were repeated in the presence of 250 mM 1,2-

bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA) (Ca2+ chelator). The

inhibitor was added to the culture supernatant and the hydrogel then pre-incubated for 1

h to equilibrate respective target blocking prior to EF exposure. All analyses were done

in triplicate, and all inhibitor experiments were repeated at least four times.

59
4.3.8. Statistical analysis

Statistical analyses were performed using single factor ANOVA to determine the effect

of EF on VEGF expression on nitric oxide release and total and phosphorylated

amounts of FAK protein. All experiments were repeated at least four times. Results

were considered statistically significant at p < 0.05.

4.4. Results:

4.4.1. Endothelial cell morphology differs on matrigel vs synthetic peptide

To compare the morphology of ECs on the selected ECMs, ECs were cultured on the

surface of the synthetic hydrogels and matrigel at the density of 5000 cells/insert and

were EF stimulated for 12 hours. Fluorescence images of ECs do not show a significant

difference in the morphology of the cells after HF electrical stimulation but the

morphology of cells on synthetic nanofibers differ from matrigel (Fig. 4.2). ECs on

synthetic nanofibers spread and show more pronounced stress fibers that span much of

the cytoplasm, while ECs cultured on matrigel show a rounded morphology.

Furthermore, ECs cultured on synthetic nanofibers displayed a different extent of

membrane projections (white arrows, Fig. 2), which increases with increasing RGD

concentration. Membrane projections of ECs on RAD16-II increased by the addition of

RGD sequence to nanofibers and ECs display more projections on nanofibers

containing 50% and 100% RGD respectively as demonstrated in Fig.4.2.

60
No-EF
B C D

EF

Figure 4.2. Morphology of ECs seeded on self-assembling nanofibers or matrigel with


three RGD concentrations after 12 hours incubation. ECs are cultured on the surface of
(A) 100% RAD16-II (B) 50% RAD16-II and 50% RGD-RAD16-II (C) 100% RGD-RAD16-
II and (D) matrigel. Top: Not EF stimulated ECs, Bottom: EF stimulated ECs. ECs on
functionalized RAD 16-II nanofibers spread and show more pronounced stress fibers.
ECs on matrigel showed less stress fiber and rounded morphology. The membrane
projections of ECs, indicated by arrows, on synthetic nanofibers increases with
increasing RGD concentration. Cell cytoskeleton is visualized with rhodamine-
phalloidin (red) and cell nuclear with dapi (blue). Scale bar is 25 µm, 40X magnification.

61
4.4.2. Hydrogel properties regulate expression of angiogenic growth factor in

endothelial cells

To investigate the effect of extracellular matrix on VEGF expression of ECs, the

regulatory effect of synthetic and natural hydrogels were quantified and compared. We

also examined whether VEGF expression is altered by RGD concentration of synthetic

peptide. ECs were embedded in the synthetic self-assembling nanofibers with 0%

(RAD16-II), 50% and 100% RGD containing nanofibers and matrigel. After 12 hours,

VEGF expression of ECs was assessed using ELISA. As illustrated in Fig.4.3-Top,

VEGF expression of ECs in synthetic nanofibers is enhanced by increasing the RGD

concentration. Functionalized nanofibers containing 50% RGD sequence released

130% more VEGF compared to RAD16-II with no RGD sequence. Our results

demonstrate that for nanofibers containing 100% RGD, the released VEGF is 440%

more than the VEGF level in RAD 16-II. Fig.4.3-Top shows that matrigel stimulates

VEGF expression of ECs and VEGF level is higher than the synthetic nanofibers.

4.4.3. Effect of electrical stimulation on angiogenic growth factor expression is

mediated by hydrogel properties

To examine the effect of EF stimulation on VEGF expression of ECs, the cells

embedded in the synthetic and natural hydrogels, were exposed to EF for 12 hours.

After 12 hours, VEGF expression was analyzed. As illustrated in Fig.4.3-bottom, EF

stimulation differentially regulates VEGF expression depending on the hydrogel. EF

stimulated ECs in synthetic nanofibers released significantly higher level of VEGF

62
compared to the no-EF synthetic group. In contrast, EF stimulation significantly

decreases VEGF release of ECs in matrigel compared to not stimulated matrigel.

Fig.4.3-bottom demonstrates that increasing RGD concentration results in an increase

in EF induced VEGF expression. Following HF EF stimulation, nanofibers containing

RGD show 100% increase in VEGF level compared to not stimulated RGD group while

ECs in RAD16-II nanofibers show 30% increase in VEGF level. In contrast, ECs in

matrigel expressed 20% less VEGF in compared to no-EF-matrigel group.

63
Figure 4.3.Top: Regulation of VEGF expression by extracellular matrix properties.
Increasing RGD concentration in synthetic nanofibers enhances VEGF expression.
VEGF release of ECs in matrigel is higher than synthetic nanofibers. Bottom:
Extracellular matrix regulated VEGF expression in response to high frequency electric
field. ECs in synthetic nanofibers released higher level of VEGF after electrical
stimulation compared to No-EF group. In contrast, VEFG expression of ECs in
matrigel group decreased in response to electrical stimulation (#: p<0.05, hydrogels
vs. RAD; *: p<0.05, EF vs. no-EF).
64
4.4.4. Hydrogel properties modulate expression of focal adhesion kinase in

endothelial cells

To determine the role of FAK in ECM mediated cell responses, FAK expression of ECs

in synthetic and natural hydrogels were quantified. After 12 hours, the total and

phosphorylated levels of FAK expressed by ECs were assessed using ELISA. No

significant difference was found in the total FAK levels expressed by ECs embedded in

the synthetic hydrogel. However, matrigel expressed significantly higher levels of total

FAK compared to synthetic hydrogel (Fig. 4.4.A). Phosphorylated FAK (p-FAK) levels

show a significant difference between groups (Fig. 4.4.B). In synthetic nanofibers,

release of the phosphorylated form of FAK increases significantly with increasing RGD

concentration. The p-FAK expressed in matrigel is significantly higher than the synthetic

nanofibers. Nanofibers containing 50% RGD sequence expressed 10% more p-FAK

compared to RAD16-II with no RGD sequence, while nanofibers containing 100% RGD

expressed 30% more p-FAK compared to RAD16-II. The ratio of the phosphorylated

FAK over total FAK does not differ significantly between RAD16-II and nanofibers with

50% RGD while this ratio is greater by 30% for nanofibers containing 100% RGD

compared to RAD16-II (Fig. 4.4.C). This ratio is significantly greater for matrigel

compared to the synthetic peptide.

65
A B

66
Figure 4.4. FAK expression of ECs on selected extracellular matrixes. A: Total

protein level of FAK does not differ among synthetic nanofibers while matrigel

expresses a higher level of total FAK compared to synthetic nanofibers. B:

Phosphorylated level of FAK increases significantly with RGD concentration in

synthetic nanofibers. ECS in matrigel express significantly higher level of

phosphorylated FAK compared to synthetic group. C: The ratio of the

phosphorylated to total FAK does not differ between RAD and RADRGD

group while this ratio is greater for RGD nanofibers compared to RAD and

RADRGD groups. This ratio is significantly greater for matrigel compared to

synthetic nanofibers (#: p<0.007 hydrogel vs. RAD).

67
4.4.5. Effect of electrical stimulation on focal adhesion kinase expression is

modulated by hydrogel properties

To investigate the effect of EF stimulation on FAK expression of ECs, cells embedded in

the synthetic and natural hydrogels, were exposed to EF for 12 hours. After 12 hours,

FAK expression was analyzed using ELISA. Electrical stimulation did not affect the total

FAK level compared to no-EF group in any of the hydrogels (Fig. 4.5.A).

Phosphorylated FAK levels for ECs embedded in synthetic nanofibers increased by

10% to 20% in response to HF EF compared to no-EF group while matrigel group

showed a 30% decrease compared to not-stimulated group (Fig. 4.5.B). In response to

HF EF stimulation, ECs in synthetic nanofibers group exhibited 20%-30% higher ratio of

phosphorylated FAK over total FAK. However this ratio showed a 20% decrease for

matrigel group compared to no-EF group (Fig. 4.5.C).

68
A

69
C

Figure 4.5. FAK expression of ECs on selected extracellular matrixes in

response to electrical stimulation. A: Electrical stimulation does not change the

total level of FAK compared to control. B: Electrical stimulation increases

phosphorylated level of FAK for ECs in synthetic nanofibers. In contrast, ECs in

matrigel group show a decrease in phosphorylated FAK level after electrical

stimulation. C: Ratio of phosphorylated to total FAK is significantly increased

after electrical stimulation in synthetic nanofibers. This ratio is significantly

decreased for ECs in matrigel after electrical stimulation (*: p<0.05, EF vs. no-

EF).

70
4.4.6. Hydrogel properties regulates Vinculin expression

To visualize the assembly of focal adhesions for ECs cultured on the synthetic

hydrogels and matrigel, ECs were seeded at the density of 5000 cells/insert on the

surface on the ECMs. After 12 hours, ECs were fixed and probed with anti vinculin

antibody to visualize focal adhesions (Fig. 4.6-left column). ECs on all ECMs expressed

vinculin throughout the area of the cell. ECs seeded on synthetic nanofibers displayed

RGD concentration-dependent expression of vinculin with lower expression for ECs on

RAD16-II and higher expression for 100% RGD nanofiber peptide. ECs grown on

matrigel showed a higher level of vinculin compared to synthetic peptide. No staining

was observed when cells were incubated with secondary antibodies alone (data not

shown).

4.4.7. Effect of electrical stimulation on vinculin expression is modulated by

hydrogel properties

To visualize the effect of EF stimulation on ECM-mediated vinculin expression, ECs

seeded on the surface of the synthetic hydrogels and matrigel were exposed to EF for

12 hours (Fig.4.6). EF stimulated cells on all the hydrogels presented vinculin

expression throughout the cellular span with more pronounced expression on the cell

nucleus area. In comparison to no-EF group, significantly higher levels of vinculin

expression were observed for EF stimulated ECs on synthetic peptide. However, the

intensity of the expressed vinculin on nanofibers varies depending on the peptide RGD

concentration. Vinculin expression of EF exposed ECs on RAD16-II increases with

incorporation of RGD sequence into the nanofibers; therefore EF exposed ECs on

71
nanofibers containing 50% RGD and 100% RGD display increased vinculin expression

respectively. EF stimulated ECs on matrigel showed decreased vinculin level in

comparison to no-EF group.

72
EF stimulated Not stimulated

A 100% RAD16-II 100% RAD16-II

B 50% RAD16-II and


50% RAD16-II and
50% RGD-RAD16-II 50% RGD-RAD16-II

C 100% RGD-RAD16-II 100% RGD-RAD16-II

D Matrigel Matrigel

73
Figure 4.6. Extracellular matrix mediated expression of focal adhesion

(vinculin) in response to high frequency electric field. Left: high frequency EF

stimulated ECs; Right: not stimulated ECs on (A) 100% RAD16-II (B) 50%

RAD16-II and 50% RGD-RAD16-II (C) 100% RGD-RAD16-II (D) matrigel.

High frequency electric field stimulation of ECs on synthetic peptide

increases vinculin expression compared to no-EF group. In response to high

frequency EF, ECs on matrigel express less vinculin compared to No-EF

group. Vinculin is visualized by immunofluorescence staining with anti-

vinculin antibody. Scale bar is 25 µm, 40X magnification.

74
4.4.8. Hydrogel properties regulate Nitric oxide expression of endothelial cells

To examine the effect of extracellular matrix on NO expression of ECs, NO release was

quantified after 12 hours of incubation for ECs embedded in synthetic nanofibers and

matrigel. Measurement of NO concentration indicates that synthetic nanofibers promote

NO expression in an RGD dependent manner (Fig. 4.7.A). Nanofibers containing 50%

and 100% RGD, express 60% and 160% more NO compared to RAD16-II, respectively.

ECs in matrigel express significantly a higher level of NO compared to synthetic

nanofibers.

4.4.9. Electrical stimulation enhances Nitric oxide expression of endothelial cells

independent of hydrogel properties

To investigate the effect of EF stimulation on NO expression, ECs embedded in

synthetic nanofibers and matrigel were stimulated for 12 hours. Following exposure, the

NO level was quantified. Electrical stimulation of ECs in both synthetic peptide and

matrigel group significantly enhanced NO expression compared to no-EF group. This

increase is more than 100% for ECs in synthetic nanofibers and 80% for ECs in matrigel

(Fig. 4.7.B).

4.4.10. Calcium regulates electric field mediated nitric oxide expression of

endothelial cells

To investigate the role of Ca2+ and ECM properties in EF mediated NO expression, ECs

in synthetic nanofibers and matrigel were treated with Ca2+ chelating agent in separate

experiments and stimulated for 12 hours. Following exposure, the NO level was

quantified. Treatment with Ca2+ chelating agent significantly reduced NO expression in

75
ECs in both EF stimulated and not stimulated group compared to no-inhibitor (control)

group as shown in Fig. 4.7.C. Application of HF EF increased the NO expression of ECs

more than 70% in synthetic nanofibers and matrigel compared to no-EF inhibitor group.

The NO level in EF stimulated Ca2+ chelated groups reaches the NO level of no-inhibitor

control group or higher. Electrical stimulation enhances NO release from 70% to 240%

depending on the ECM, where maximum enhancement occurs for RAD16-II nanofibers.

This percentage remains the same regardless of treatment with Ca2+ chelating agent.

76
A

77
C

Figure 4.7. A: Nitric oxide expression of ECs in selected extracellular matrixes.

Released nitric oxide for ECs in the synthetic group increases with RGD concentration

and matrigel expresses higher level of NO than synthetic nanofibers. B: Nitric oxide

expression of ECs on selected extracellular matrixes in response to electrical

stimulation. Release of NO is enhanced in all matrices after electrical stimulation. C:

Ca2+ chelating agent significantly reduces NO release of ECs in all ECMs. HF EF

stimulation increases the NO level and brings it to no-inhibitor values. (*: p<0.05, EF

vs. no-EF; ^: P<0.05, EF+ chelator vs. no-EF + chelator).

78
4.5. Discussion

The results of this study demonstrate that high frequency electric field can improve

function of endothelial cells. We have determined the pathways that mediate the effects

of HF-EF. Our results show that HF-EF can improve the function of ECs via a calcium

dependent activation of the eNOS pathway and stimulate protein expression of ECs

through an ECM regulated manner. In our 3D in-vitro model of wound healing HF-EF

enhances release of nitric oxide and regulates expression of VEGF and FAK.

Importantly our results show that VEGF and FAK expression of ECs are differentially

regulated in synthetic nanofibers versus matrigel. HF-EF in an RGD dependent manner

regulates VEGF and FAK expression in synthetic peptides. In our configuration, EF is

oriented perpendicular to the cell surface and is generated without direct contact with

the cells. HF-EF is induced in membrane as well as cytoplasm and can affect

intracellular interactions (236).

Our results demonstrate that increased NO expression is one of the ECs’ responses to

HF EF. Production of NO is part of the normal function of ECs (237). Reduced NO

bioavailability decreases the ability of endothelial cells to execute their functions in

regulating vascular tone and growth, immune cell responses, and vascular barrier

functions (238-240). We showed that HF-EF enhances NO expression of ECs in both

synthetic nanofibers and matrigel. Increased NO expression following electrical

stimulation has been reported previously. Pulsed electric field (15 Hz,10 ms) stimulated

human chondrocytes showed increased level of NO released in the media (241). In a

previous report a pulsed electric field (4 Hz, 0.2 ms) has been shown to stimulate the

79
release of endothelium derived NO in pulmonary artery (242).Nitric oxide plays many

important roles in wound healing. Recent findings demonstrate a significant increase of

eNOS protein expression after excisional wounding in normal mice (243). NO

modulates cytokines that in turn control the progress of each of these phases of wound

healing (e.g., chemoattraction, proliferation, collagen deposition, and angiogenesis)

(244). NO deficiency directly contributes to wound healing impairment (243). There are

strong correlations between reduced cutaneous NO levels and impaired wound healing

under disease conditions such as diabetes (245). Studies demonstrate that cutaneous

eNOS expression, and/or NO levels are significantly decreased in streptozotocin (STZ)-

induced type 1 diabetic animals (243, 246). In agreement with the above notion,

cutaneous gene therapy of eNOS restored eNOS protein and NO levels and

accelerated the wound healing rate in STZ-induced diabetic mice (243). Similarly, the

NO donor or NO releasing poly (vinyl alcohol) hydrogel dressings are also shown to

partially restore such healing impairment in STZ-induced diabetic rats (247, 248). Our

results demonstrate a significant increase in NO level by application of non-contact HF-

EF. Further we show that this EF stimulated increase is Ca2+ dependent. Our findings

are in agreement with previous studies on the role of Ca2+ in NO expression of

endothelial cells and rat thymocytes (249, 250). In our previous study we have

demonstrated the role of Ca2+ in MEK phosphorylation of ECs in response to HF EF

(7.5 GHz)(218).

In our configuration, HF EF stimulates phosphorylation of FAK and VEGF expression of

ECs. EF mediated FAK expression has been reported for DC EF of physiological

80
amplitude (150 mV/mm) that increases levels of phosphorylated FAK at Tyr-397 in

trophoblast cells cultured on glass coverslip while levels of total FAK remained

unchanged (105). Biphasic pulsed electric field (1.5V/1.8cm, 5HZ, 5ms) has been

shown to upregulate phosphorylation of FAK at Tyr-397, whereas total FAK is not

altered (105).The regulatory effect of EF on growth factor expression has been reported

in previous studies. DC EF of physiological amplitude (200 mV/mm) oriented parallel to

the cell plane enhances VEGF release and upregulates VEGF mRNA level of Human

Umbilical Vein Endothelial Cells (HUVEC) seeded on a coverslip (132). HUVECs grown

on gelatin microcarriers and embedded in a 3D fibrin gel exposed to pulsed

electromagnetic fields (15 Hz, 4.5ms) demonstrate an increase in level of fibroblast

growth factor β-2 level (251).

Our results demonstrate a difference in regulation of protein expression of ECs between

synthetic nanofibers and matrigel in response to HF-EF. Synthetic nanofibers enhance

expression of FAK and VEGF in ECs following HF-EF exposure. In contrast, matrigel

induces a decrease in release of FAK and VEGF in ECs in response to HF-EF. Our

results of reduced EC response mediated by matrigel are consistent with previous

reports, where total lengths of networks formed by human microvascular endothelial

cells were reduced following strain application (252).

As expected, expression of FAK and VEGF show similar trends in response to HF EF.

Focal adhesion kinase, a cytoplasmic tyrosine kinase activated by integrin, is an

upstream regulator of VEGF expression (253). While FAK signaling has been linked to

81
increase the basal levels of VEGF gene transcription, FAK inhibition reduces VEGF

expression (254). FAK activation, demonstrated by an increase in phosphorylation of

Tyr397 as well as other sites in the protein, creates a high-affinity binding site for other

proteins to initiate multiple downstream signaling pathways (255). In addition to FAK,

integrin binding to ECM recruits proteins including Raf, Ras and Rho GTPases to the

binding sites and activates intracellular pathways such as mitogen activated protein

kinase (MAPK) and extracellular-signal-regulated kinase (ERK). These in turn can

enhance angiogenic responses such as VEGF expression (256-258).

The result of this study demonstrates that in the synthetic group, protein expression and

NO release of ECs are enhanced by increasing the RGD concentration of the nanofiber.

This could be explained by higher strength of cell interaction with RGD motif (231).

Expression of bone-related markers of goat marrow stromal cells embedded in a 3D

hydrogel increase significantly as the RGD concentration increased (259). RGD-

modified surfaces have been reported to enhance attachment, spreading, and

proliferation of human and murine fibroblasts (260). Overall, our results support the

hypothesis that ECM properties regulate EC response to EF.

Stimulation of a wound with HF EF, combined with a suitable choice of hydrogel, will

provide the damaged tissue with improved integrity and biophysical stimuli needed for

promotion of healing. The combined effect of electrical stimulation and hydrogel has the

potential to meet the current needs of regenerative engineering and offer new

opportunities for generation of novel tissue substitutes which will direct cellular behavior,

resulting in the formation of functional tissue.

82
4.6. Conclusions

Development of effective treatments for impaired healing of vascular tissues is essential

to address significant health care problems associated with chronic ulcers. In this study,

we demonstrate that EF, together with the extracellular matrix, represents a biophysical

system that regulates vascular cell function. Our findings demonstrate the activation of

intracellular pathways in response to HF EF. This is consistent with the predictions of

the numerical model that HF EF penetrates into the cell and is induced in the

intracellular space. Our results show that HF EF can activate eNOS pathway in a Ca2+

dependent manner and increase NO release which is essential for promotion of wound

healing. Importantly, the differential protein expression of HF EF stimulated endothelial

cells in synthetic nanofibers versus natural hydrogel, demonstrates the effect of

extracellular matrix properties in regulation of cell response to HF EF. The results of this

study elucidate the mechanisms of interaction of cells with the electric field and fill a gap

in the current understanding of pathways involved in mediating the effect of electrical

signals. Our findings emphasize the beneficial use of electric field for development of

wound healing therapies and suggest that EF therapies with the proper choice of

implant material or wound dressing can increase the healing rate of chronic wounds.

83
Chapter 5

Interaction of vascular cells with a non-oscillating electric

field

5.1. Objective

The objective of this study is to investigate the interaction of a capacitively coupled non-

oscillating electric field with cells. First, the distribution of the electric field in the model

system is described analytically. Second, the interaction of cells with (1) induced

surface charges and (2) high magnitude electric field is quantified at different time

points.

5.2. Introduction

Direct current electric fields (EFs) have been shown to affect a variety of cell responses

in vitro including cell migration, differentiation, proliferation, redistribution of cell

membrane receptors and protein expression (32, 72, 73, 75). This EF mimics the

endogenous EF that cells experience in the body (216); therefore the externally applied

EF has the potential to manipulate this EF current and regulate the cell function. For in-

vitro studies, this EF (100-500 mV/mm) is generated using an electrotactic chamber

(67). In this chamber EFs are generated via Ag/AgCl electrodes and passed through the

medium using agar salt bridges. This chamber can be mounted on a microscope for live

cell imaging. In addition to the advantages of this method, there are a few

84
disadvantages such as changes in the PH of the medium, contamination with

electrochemical byproducts and the risk of heating the cells using this method of

stimulation.

To avoid these problems, electrical stimulation using capacitive coupling provides a

non-contact approach that has distinct advantages over using the electrotactic chamber

for application of a non-oscillating electric field. This study is carried out using the

capacitive coupling method which induces EF in the cells without direct contact of

electrodes with the cells. The present study characterizes the effect of capacitively

coupled non-oscillating electric fields on in-vitro fibroblast adhesion, proliferation and

protein expression. We analytically determine the coupling mechanisms of EF to this

model system and propose a device for live imaging of cells during electrical

stimulations.

5.3. Materials and Methods:

5.3.1. In vitro EF exposure setup:

A custom setup was built to allow cell exposure to EF by means of capacitive coupling

as shown in Fig.5.1.A. This method uses a parallel plate capacitor (135mm x 128mm,

26mm apart) connected to a voltage source and assembled in the cell culture incubator.

The cells are seeded in a petri dish filled with cell culture media and the petri dish is

placed between the capacitor plates (Fig.5.1.B); therefore cells do not have any contact

with the electrodes. The exposure system is capable of delivering 0-5KV voltage and

85
the delivered voltage is monitored using a digital multimeter. The electric field is normal

to the plane of the cells. Each experiment was performed for both EF polarities.

86
A

Figure 5.1. A. The custom system for electrical stimulation of cells using the

capacitive coupling method. B. Sketch of the cells in the petri dish placed between

plates of the capacitor. C. Simple sketch of the regions and boundaries in the

model system.

87
5.3.2. EF exposure setup for live imaging of cells

This set up was built for live observation and measurement of cell responses to

electrical stimulations (Fig.5.2.A). It consists of a parallel plate capacitor, the top

electrode is a circular plate made of brass and the bottom electrode is a grid which is

fabricated using photolithography (Fig.5.2.B). The gird has 30X30 (µm)2 openings to

allow cell observation during the stimulation (Fig.5.2.C). The two electrodes are

separated by a silicone sheet with a well in the middle and attached to a coverslip. For

the experiments, the cells are attached to the bottom coverslip, the cell culture medium

is added, and after which the well is covered by another coverslip. The body of the

capacitor is a cylindrical box made of Delrin. The diameter of the bottom piece is 1.98

inches and has an opening (D=1.18 inches) to allow the microscope lens to point up

through it. To provide the electrical connection for the lower plate of the capacitor, there

is a metal plate at the bottom of the box with an opening (D=1.18 inches) in it. It also

has two metal bars that are perpendicular to it and can be connected to the voltage

source. A horizontal bar provides the electrical connection between the top electrode

and the voltage source.

The grid electrode pattern was designed using AutoCAD and applied to a glass

coverslip (D=25 mm).The coverslip is first washed and then spin-coated with positive

photoresist (Shipley 1818), baked and then exposed to ultraviolet light through the

chrome-coated mask. After Nickel deposition on the coverslip, pattern was revealed by

lift off using acetone.

88
A B

C D

Figure 5.2. A. Custom system for live imaging of cells during exposure to EF. B. Sketch of

the capacitor mounted on the microscope stage during the stimulation. C. Picture of the

grid electrode placed on top of a mirror. The clear squares are the 30X30 (µm)2 openings

of the grid through which cells can be monitored. Magnification is 10X. D. Fluorescent

staining of the cells on the grid to visualize cell cytoskeleton (phalloidin) and nuclear (dapi).

Magnification is 20X.

89
5.3.3. Model system

Here we describe the behavior of the non-contact EF stimulation system analytically.

Electrodes are isolated from the medium by means of two layers of dielectrics on top

(coating ϵcoating) and bottom (substrate ϵsubstrate) (Fig.5.1.C). The boundary conditions are

fixed potentials on the two electrode boundaries, Vtop and Vbottom

Vtop –Vbottom= V (5.1)

where V is the externally applied voltage. The cell culture media between these

dielectrics is modeled as a layer with thickness of dmedium, conductivity σmedium and

permittivitty ϵmedium. Dimensions and material properties are given in Table 1.

The analysis to find the EF magnitude in this system is performed by solving the charge

conservation law at the boundaries with voltage constraint along three regions.

𝑑
n. [( J𝑚𝑒𝑑𝑖𝑢𝑚 − J𝑐𝑜𝑎𝑡𝑖𝑛𝑔 )+ (𝐷𝑚𝑒𝑑𝑖𝑢𝑚 − 𝐷𝐶𝑜𝑎𝑡𝑖𝑛𝑔 )] = 0 (5.2)
𝑑𝑡

𝑑𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 +𝑑𝑚𝑒𝑑𝑖𝑢𝑚 +𝑑𝑐𝑜𝑎𝑡𝑖𝑛𝑔


V=∫
0
𝐸. 𝑑𝑙 (5.3)

where E, J and D represent the electric field, current density and electric displacement

in each region respectively. The magnitude of the electric field normalized by the

external voltage in the media is given by

𝐸𝑚𝑒𝑑𝑖𝑢𝑚 𝜖𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
= 𝑒 −𝑡/𝜏 (5.4)
𝑉 𝜖𝑚𝑒𝑑𝑖𝑢𝑚 ×(2×𝑑𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 )+𝜖𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 ×(𝑑𝑚𝑒𝑑𝑖𝑢𝑚 )

where the time constant for the system is

90
𝜖𝑚𝑒𝑑𝑖𝑢𝑚 (𝑑𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 +𝑑𝑐𝑜𝑎𝑡𝑖𝑛𝑔 )+𝜖𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑑𝑚𝑒𝑑𝑖𝑢𝑚
𝜏= (5.5)
σ𝑚𝑒𝑑𝑖𝑢𝑚 (𝑑𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 +𝑑𝑐𝑜𝑎𝑡𝑖𝑛𝑔 )

This exponentially decaying EF in the medium indicates that application of a non-

oscillating EF using this non-contact method leads to surface charge accumulation on

the susbtrate where the normalized magnitude of this surface charge density is given by

𝜎𝑠𝑢𝑟𝑓𝑎𝑐𝑒 𝜖𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
= (𝑒 −𝑡/𝜏 − 1) (5.6)
𝑉 2 x 𝑑𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒

Using the parameters shown in Table 5.1 , the magnitude of the time constant is

Ƭ= 1.23 x 10-9 (s) (5.7)

The magnitude of the induced EF in the media and surface charge density versus time

is plotted in Figure 7 using Igor Pro software (WaveMatrics, OR, USA).

91
Table 5.1. Parametes used in the Model system

Parameter Symbol Simulating value

Thickness of the dielectric dcoating,


1 μm
layer dsubstrate

Thickness of the medium dmedium 98 μm

Relative permittivity of the εcoating ,


2.6
dielectric layer εsubstrate

Relative permittivity of the


εmedium 81
medium

Conductivity of the medium σmedium 1.5 (S/m)

92
5.3.4. Fibroblast isolation and culture

Cardiac Fibroblasts (FBs) were isolated from rat myocardial tissue cultured in Medium

199 (HyClone, UT, USA) containing 10% fetal bovine serum (FBS; Atlanta Biologicals,

GA, USA), 1% antibiotic/antimycotic (AB/AM; AtlantaBiologicals, USA), 1% heparin

(Sigma-Aldrich, MO, USA) and 0.2 ng/mL endothelial growth factor supplement (Sigma-

Aldrich, MO, USA). Cell cultures were maintained at 37ºC in 100% humidified air

containing 5% CO2. Cells from passages 6-10 were used. All experiments were

conducted in the culture medium (medium M199, 10% FBS, 1% antibiotic/antimycotic

and 1% heparin) without additional growth factor supplementation.

5.3.5. Live-dead assay

Cells were cultured at the density of 5X104 cell/cm2 in the silicon well attached to a

glass cover slip and incubated for 24 hours. Prior to assay, cells were washed with

PBS. Following incubation with live/dead reagent (Invitrogen) for 30 minutes at room

temperature, images of the labeled cells were taken using a fluorescent microscope

(Olympus IX81, Olympus, PA, USA) at 4X magnification and the percentage of live

(green) and dead (red) cells were calculated in each image.

5.3.6. Adhesion assay

FBs in suspension with the density of 5X104 cells/cm2 were added into wells of the 2

separate 24 well plates (EF and control (no EF) groups). One plate was immediately

exposed to EF using the custom setup. Exposure time ranged from 30 minutes to 1, 2, 3

hours and 3 days for both EF polarities. After exposure FBs were fixed by 2%

paraformaldehyde, stained with Dapi (Invitrogen, USA). Pictures were taken using a

93
fluorescent microscope (Olympus IX81, Olympus, PA, USA) at 4X magnification and the

number of adhered cells in each well was counted using Image Pro.

5.3.7. Measurement of angiogenic growth factor expression

To determine the effect of EF on the vascular endothelial growth factor (VEGF) release

by FBs into the medium, culture medium samples were collected from N=6 separate

experiments following EF exposure for both polarities. VEGF expression levels were

measured using ELISA kits (R&D Systems, MN, USA) according to the manufacturer’s

protocol. ELISA assay was performed in duplicates.

5.3.8. Proliferation assay

To determine the effect of EF on the FB proliferation, FBs were incubated with

bromodeoxyuridine (BrdU; Invitrogen, USA). After 12 hours, cells were fixed by 2%

formaldehyde and stained with anti-BrdU antibody (Invitrogen, USA) to identify

proliferating cells and DAPI nuclear staining (Invitrogen, USA). The images were taken

using a fluorescent microscope (Olympus IX81, Olympus, PA, USA) at 4X magnification

from N=6 separate experiments. Percentages of the proliferating cells were calculated

by counting the number of the proliferating and adhered cells in each well using Image

Pro.

94
5.3.9. Statistical analysis

A single factor ANOVA was used to test the effects of EF on cell adhesion, proliferation

and VEGF expression. Results were considered statistically significant at p<0.05.

5.4. Results

5.4.1. Capacitively coupled electric field maintains the cell viability

The viability assay was performed for 6 groups to test whether application of EF in

capacitive coupling approach and covering cells with coverslip during the stimulation

affect cell viability. Cells were cultured in the silicon wells attached on the glass

coverslip and 4 wells were covered with another coverslip on top. One group was used

for EF stimulation, one for sham control (placed in the device without stimulation), one

control was kept in an incubator and another kept at room temperature. Two wells (not-

covered) were kept in the incubator and room temperature. After 24 hours, the viability

of cells in all groups was close to 100% which indicates that this approach and setup

does not harm the cell viability (Fig.5.3.A-B). After stimulation, cells spread and

fluorescent staining show normal cell cytoskeleton and nuclear shape as well as

spreading of focal adhesion (vinculin) (Fig. 5.3.C).

95
A

B C

Figure 5.3. A. Percentage of live cells in electric field stimulated, controls

and sham group. B. Electrical stimulation of cells using the

microfabricated device did not affect cell viability (live cells, dead cells),

magnification 4X. C. Fluorescent staining of the stimulated cell (cell

cytoskeleton, cell nuclear, focal adhesion), Magnification 20X.

96
5.4.2. Electric field stimulation increases the proliferation of fibroblasts

To investigate the effect of EF on cell proliferation, the proliferation rate was assessed

using BrdU staining following 3 days of stimulation. Our results demonstrate a slight

increase in proliferation rate of EF stimulated samples compared to the control as

shown in Fig. 5.4. Regardless of the surface charge (EF polarity) EF stimulation

significantly increases cell proliferation after 3 days of stimulation (The data for the

substrate covered with positive surface charge is not shown).

97
A
EF NO-EF

Figure 5.4. A. Fluorescence staining of cells labeled with BrdU (proliferating)

and DAPI (nucleus), Magnification 4X. B. Cell Proliferation is significantly

enhanced following 3 days of electrical stimulation regardless of the negative

surface charge on the substrate.

98
5.4.3. Electric field polarity regulates the adhesion of fibroblasts to the substrate

We set up a method to investigate the effect of surface charge accumulation on FB

attachment to the substrate. Our results indicate that the number of the FBs attached to

the substrate covered with negative ions was significantly (p<0.05) less than the control

after 30 min, 1 hour and 2 hours of EF exposure. However, no statistically significant

difference with the control was observed after 3 hours (Fig.5.5.A-B). The number of FBs

attached on the surfaces covered with positive ions was not significantly different from

the control (Fig.5.5.C).

99
A

EF
30 min 1 hr 2 hrs 3 hrs

30 min 1 hr 2 hrs 3 hrs

No-EF

100
C

Figure 5.5. A. Delayed adhesion of EF exposed FBs to the substrate influenced by

negative surface charge deposited on the substrate at different time points,

Magnification 4X. B. Number of adhered cells to the surface covered with negative

charges is significantly less than the control up to 2 hours (*: p<0.05, #: p<0.005).

C. Reverse polarity EF which deposits positive surface charge did not significantly

affect the number of adhered cells in the EF exposed group in comparison to the

control.

101
5.4.4. Electric field stimulation enhances vascular endothelial growth factor

expression

To determine the effect of EF stimulation on VEGF expression of FBs, the media were

collected after 2 and 3 days of stimulation. Our results demonstrate that EF regardless

of its polarity significantly (p<0.05) enhances the VEGF expression of stimulated FBs

compared to the control (Fig. 5.6).

102
Figure 5.6. Exposure of FBs to DC EF for 2 and 3 days enhances VEGF

expression. Both EF polarities significantly enhance this effect. (*: p<0.05,

#: p<0.005).

103
5.5. Discussion

Our findings demonstrate that the application of non-oscillating EF through electrodes

isolated from the medium can regulate cell functions. This regulation happens while our

analytical results show there is no macroscopic EF in the medium (Fig.5.7.A); therefore

the effect of non-oscillating EF with cells in non-contact approach is mediated through

interaction of cells with accumulated surface charges on the boundaries (substrate) at

the microscopic level (Fig.5.7.B). This interaction resulted in the regulation of cell

adhesion to the substrate as well as protein expression of fibroblasts.

Our theoretical results demonstrate that, upon application of the voltage, the surface

charge starts to accumulate on the substrate and rapidly approaches its saturated value

(eqn. 5.6). Within a few time constants after application of the external voltage (t ~3 𝜏

~10-9 s) surface charge reaches to its saturated magnitude as shown in Figure 5.7.B.

This surface charge will screen EF from the media; therefore EF undergoes an

exponential decay (eqn. 5.4) and vanishies (Fig.5.7.B). Within a few time constants after

application of the external EF, the indcued EF in the model system reaches its steady-

state value (Fig.5.7.A). One of the advantages of the non contact application of EF is

that, by changing the physical properties of the system such as thickness or permittivity

of the susbstrate, we can control the time constant of the system (eqn. 5.5) and,

therefore, modulate the magnitude of the induced EF in different parts of the system

(eqn. 5.4).

For our experimental studies in the steady state regime, we quantify the interaction of

cells with EF in two regimes. First, within a few of hours of EF exposure, while cells are

104
still in suspension. Second, after one day of EF exposure, when the cells are attached

to the substrate during the EF exposure.

Our results demonstrate that, during the early stage (hours), polarity of the surface

charge can control fibroblast adhesion rate to the substrate. This finding is in agreement

with previous reports of cell adhesion as well as proliferation rate regulated by the

surface charge (42). Adhesion and proliferation of human osteoblast cells to polarized

hydroxyapatite (HAp) coated titanium was increased for a titanium surface covered with

positive ions and decreased with negative ions (43). These positive surface charges

enable interactions between the negatively charged cell membrane and the substrate

and play an important role in the formation of focal adhesions and cell attachment (40).

This suggests that induction of positive or negative surface charge on implants can

increase or decrease cell adhesion and growth.

Further, we demonstrate that non-oscilating EF can increase VEGF expression of FBs

which is quantifed during the second time scale (days). Previuosly in chappter 3 we

have numerically demonstrated that in low frequency regime very strong EF is induced

in the membrane of a cell attached to a substrate (236). Therefore in our experiment

when cells finally attach to the substrate, we expect to have induced EF and therefore

induced potential difference across their membranes. Previuos studies demonstrate that

chemically-induced changes in the membrane voltage triggers angiogenic responses of

vascular cells, such as altered expression of angiogenic growth factors at the

transcriptional level (261). In agreement with previuos studies, our EF induced changes

in the cell membrane potential increases VEGF expression of cells.

105
In this study, we demonstrate the advantageous features of the non contact method of

EF application to study cell-EF interaction. This method allows us to quantify the

interaction of cells with surface charge in the absence of macroscopic EF. Investigation

of cell- surface charge interaction is not possible using the current methods which EF is

passed through the medium around the cells. Additionally this method enables the

delivery of high magnitude EF to the system without induction of EF in the media and

heating of the cells. This allows us to induce sufficient EF in the cell membrane to

trigger the desired membrane initiated responses without any need for using the

chemical drugs.

106
A

Ƭ ~ 10-9 (s)

-9
Ƭ ~ 10 (s)

Figure 5.7. A. The applied voltage to the system is excluded from the culture

medium within a few time constants after applying the voltage. B. Surface charge

density deposited on the substrate reaches to its maximum magnitude within a few

time constants after applying the voltage. The values are normalized to the applied

voltage.

107
5.6. Conclusions
In this study, we analytically demonstrated the accumulation of surface charges on the

substrate and the absence of EF in the culture medium surrounding the cells during

non-contact method of EF application. For the first time, we experimentally showed that

the interaction of cells with the surface charge in a regime where EF is screened in the

medium is responsible for the regulation of the cell adhesion to the substrate. We

previously showed that the cell attachment to the substrate will result in the induction of

EF in the cell membrane and here we experimentally quantified the enhanced VEGF

expression of cells following electrical stimulation. This method of EF application

exhibited a positive influence on cell proliferation.

The approach used in this study can be used as a basis for further studies to elucidate

the mechanism of capacitive coupling of electric fields with cells and the resulting cell

response. This method of EF application is suitable for mechanistic studies of EF-cell

interaction because the cell response is mediated only by EF induced in the cell and is

not influenced by the flow of the medium around the cells.

108
Chapter 6

Enhancement of diabetic deficiencies by biophysical stimuli

and extracellular environment

6.1. Objective

In this chapter (1) the deficiencies of endothelial cells in diabetic condition are

determined and (2) the effect of biophysical stimuli, including high frequency electric

field and strain application as well as extracellular environment on improvement of the

diabetic cell function are quantified.

6.2. Introduction

Diabetes is one of the most common chronic illnesses in the world with 25.8 million

individuals (8.3% of the population) afflicted in the United States. Annual medical costs

associated with diabetic care were estimated at $174 billion in 2007 (262). While there

are many life-threatening complications associated with diabetes, development of

diabetic cardiomyopathy (DCM) and chronic ulcers are among the nation’s leading

cause of patient morbidity and mortality (263). DCM often results in damaged, ischemic

and/or fibrotic tissue caused by a loss of vascularization (264). DCM characterized by

structural changes in the myocardium of diabetic patients and eventually leads to left

ventricular hypertrophy, diastolic and systolic dysfunction, or a combination of these

conditions (264). Impaired angiogenesis in diabetic condition also results in

development of slow- or non-healing wounds in diabetic patients (265).

109
In this chapter, we aim to determine the deficiencies of ECs in diabetic conditions in

vitro and their response to an electric field and cyclic strain. In this study, diabetic ECs

were provided from two sources (1) from a rat/mouse model of DCM where cells were

exposed to the diabetic condition with subsequent cell harvest from tissue and cultured

in standard media and (2) in vitro acute hyperglycemic model, which included a short

time exposure of normal (wild type) cardiac ECs to high glucose media in vitro. We

apply electrical and mechanical stimuli and quantify the angiogenic responses of

endothelial cells.

6.3. Materials and Methods

6.3.1. Microvascular endothelial cell isolation and culture for electrical stimulation

Primary microvascular endothelial cells (MVECs) were isolated from diabetic mouse

(C57BL/6J, Jackson Laboratory, ME, USA) lung tissues using collagenase digestion, as

described previously (54). Cells were doubly sorted using anti-CD-31 (BD Pharmingen,

San Jose, CA) antibody with appropriate secondary antibodies conjugated to the

magnetic beads (Dynabeads, Invitrogen Corporation, Carlsbad, CA). Cells were

cultured in gelatin-coated dishes in Medium 199 (HyClone, Logan, UT) supplemented

with 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), 1% antibiotic/antimycotic

(Atlanta Biologicals, Lawrenceville, GA, USA), 10 μg ml-1 heparin (Sigma Aldrich, St

Louis, MO, USA), and 0.2 ng ml-1 growth supplement (Sigma Aldrich, St Louis, MO).

Cell cultures were maintained at 37 ºC in 100% humidified air containing 5% CO 2. Cells

from passages four to nine were used. All experiments were conducted in the culture

110
medium (medium M199, 10% FBS, 1% antibiotic/antimycotic and 1% heparin) without

additional growth factor supplementation.

6.3.2. Microvascular endothelial cell isolation and experimental groups for strain

experiments

Primary microvascular endothelial cells (MVECs) were isolated from Sprague-Dawley

rat (SAS SD Strain 400; Charles River, Wilmington, MA) heart tissues of wild type and

diabetic rats using collagenase digestion. Cells were doubly sorted using anti-CD-31

(BD Pharmingen, San Jose, CA) antibody with appropriate secondary antibodies

conjugated to the magnetic beads (Dynabeads, Invitrogen Corporation, Carlsbad,

CA).Three days prior to experiments, the cell culture media was changed to reflect the

appropriate experimental group. The first group was ‘‘normal’’ or wild type cells cultured

in standard low glucose media (wt; cell culture media supplemented with 5.5 mM D-

glucose, Sigma-Aldrich, St. Louis, MO). Two groups represented diabetic condition: wild

type cells cultured in high glucose media (wt HG; cell culture media supplemented with

22.2 mM D-glucose) and cells harvested from diabetic animals and cultured in standard

low glucose media (db; cell culture media supplemented with 5.5 mM D-glucose).

Diabetic cells were cultured in low glucose media to determine if the ‘‘diabetic’’

phenotype was retained, consistent with other studies investigating fibroblasts

harvested from diabetic animals.

6.3.3. Application of cyclic strain

A commercially available Flexcell system was used to determine biomechanical

response of endothelial cells. Cardiac endothelial cells were seeded onto 6-well

111
collagen I pre-coated UniFlex® Culture plates (Flexcell International Corp.,

Hillsborough, NC) at a seeding density of 200,000 cells/well in the appropriate

experimental group culture media. For static controls, cells (200,000 cells/well) were

cultured in six-well tissue culture plates coated with collagen I (PureCol®, Advanced

BioMatrix, San Diego, CA) and appropriate culture media (Figure 6.1) (266). Collagen I

was used as a culture substrate because it constitutes 80% of the total collagen in the

heart. All culture plates were incubated overnight prior to stimulation to allow for cell

attachment. UniFlex® Culture Plates were then loaded onto the Flexcell® FX-5000™

Tension System outfitted with ArcTangle™ Loading Stations™ (24 mm; Flexcell

International Corp.) for application of uniaxial strain and housed at 37ºC in 100%

humidified air containing 5% CO2. Strain parameters were set at 5% elongation at 1 Hz

and 60 cycles/minute for 24 hours. These strain parameters were selected based on

physiological in vivo cardiac parameters and matched those used in the previous

studies Static controls were maintained in the same incubator for the duration of the

experiments.

112
Figure 6.1. Left: 6 well UniFlex® Culture plates, Middle: Flexcell Tension

System outfitted with Loading Station. Right: Endothelial cells cultured on

culture plates.

6.3.4. Endothelial cell culture system

To perform VEGF and NO expression assays, ECs were embedded in the matrigel or

the synthetic peptide nanofibers (RAD16-II) at the density of 250,000 cells/ insert. For

capillary morphogenesis assay ECs cultured on the surface of matrigel or the synthetic

peptide nanofibers (RAD16-II) (at the density of 5000 cells/insert). The hydrogels were

loaded in culture plate inserts (13 mm diameter, 0.4 μm pore size; Millipore, Billerica,

MA). Synthetic peptides were obtained from RS Synthesis (Louisville, KY) and

SynBioSci Corporation (Livermore, CA). Matrigel was purchased from (BD Biosciences,

San Diego, CA).

6.3.5. Capillary morphogenesis

Endothelial cells were cultured on the surface of matrigel at a seeding density of 2500

cells/insert for 12 hours to allow capillary-like network formation. After 12 hours cell

113
samples were fixed with 2% paraformaldehyde and stained with phalloidin‐TRITC,

(Sigma‐Aldrich), followed by DAPI (Invitrogen Corporation). Images were taken (n=5 per

sample) using an inverted fluorescent microscope (Olympus IX81; Olympus, PA, USA).

The characteristic capillary size was determined using correlation analysis and a

custom-written Matlab code (TheMathWorks,MA, USA).

6.3.6. Vascular endothelial growth factor assay

Endothelial cells at the density of 250,000 cells/insert were embedded in matrigel and

cultured in medium without growth factor supplement in culture plate inserts. At 12

hours, the amount of VEGF released by cells into the medium were collected from at

least N=5 separate experiments. VEGF expression levels were measured using ELISA

kits (R&D Systems, MN, USA) according to manufacturer’s protocol. ELISA assays

were performed in duplicates.

6.3.7. Nitric oxide assay

Nitric oxide expression of ECs was quantified for the collected medium using nitric oxide

assay (Promega Corporation, WI, USA). All analyses were done in triplicate, and all

inhibitor experiments were repeated at least four times.

6.4. Results
6.4.1. Capillary morphogenesis is impaired in the diabetic condition

To investigate the effect of diabetes on capillary morphogenesis, wild type and diabetic

endothelial cells were cultured on the surface of matrigel at the density of 2500

cells/insert for 12 hours. Our results demonstrate that diabetes impaired the ability of

114
ECs to form capillary-like networks on the matrigel compared to the wild type group. As

shown in Fig. 6.2.A diabetic type ECs could not form the interconnected network like

wild type ECs and the characteristic network size of diabetic cells is smaller than the

network formed by the wild type ECs (Fig.6.2.B).

6.4.2. Expression of angiogenic growth factor in endothelial cells is impaired in

the diabetic condition

To examine the effect of diabetes on VEGF expression of ECs, wild type and diabetic

cells were embedded in matrigel. After 12 hours, VEGF expression was analyzed using

ELISA. As illustrated in Fig.6.2.C, diabetic cells released a lower amount of growth

factors (VEGF) compared to wild type ECs.

6.4.3. Nitric oxide expression of endothelial cells is impaired in the diabetic

condition

To investigate the effect of diabetes on NO expression, wild type and diabetic ECs were

embedded in matrigel. After 12 hours the NO level was quantified. Our results

demonstrate that diabetic ECs released significantly lower level of NO compared to wild

type ECs as plotted in Fig.6.2.D.

115
A Wild type Diabetic

B C

Figure 6.2. (A) In vitro capillary morphogenesis of wild type and diabetic
ECs cultured on matrigel; Magnification 4X. (B) Analysis of the size of the
capillaries demonstrates the impaired network formation of diabetic ECs
compared with the wild type ECs on matrigel. (C) Growth factor expression
of ECs is decreased in diabetic condition. (D) Nitric oxide expression of
diabetic ECs on matrigel is significantly less than wild type.

116
6.4.4. Electric field stimulation enhances nitric oxide expression of endothelial

cells in the diabetic condition

To investigate the effect of EF stimulation on NO expression, wild type and diabetic ECs

were embedded in matrigel and stimulated by high frequency electric field for 12 hours.

Following stimulation the NO level was quantified. Our results demonstrate that HF EF

stimulation increased NO expression of ECs in both wild type and diabetic group as

shown in Fig.6.3.

Figure 6.3. High frequency electrical stimulation increases NO expression in


both wild type and diabetic endothelial cells.

117
6.4.5. Application of strain increases nitric oxide expression of endothelial cells in

the diabetic condition

To examine the effect of strain on NO expression of ECs, cells from wild type, diabetic

and high glucose groups were subjected to strain. Application of strain resulted in an

increase of NO expression of wild type ECs as well as diabetic and high glucose group.

Strained ECs in diabetic and high glucose group released higher levels of NO compared

to ECs in wild type static (no strain) group as shown in Fig.6.4.

Figure 6.4. Application of strain significantly increases NO expression in wild

type, diabetic and high glucose groups (*: p<0.05).

118
6.4.6. Extracellular environment stimulates nitric oxide expression of endothelial

cells in the diabetic condition

To examine the effect of extracellular environment on NO expression of ECs, cells from

wild type, diabetic and high glucose groups were embedded in nanofibers. Nanofibers

are able to stimulate NO expression of diabetic and high glucose endothelial cells to

release higher levels of NO compared to wild type group (not stimulated by nanofibers)

as shown in Fig 6.5.

Figure 6.5. Nanofiber scaffold significantly increases NO expression in wild

type, diabetic and high glucose groups (*: p<0.05).

119
6.5. Discussion and Conclusions

In this chapter, we investigate the deficiencies in the function of endothelial cells in

diabetic condition. We demonstrate the positive effect of external biophysical stimuli and

extracellular matrix to improve the endothelial cell function through enhancing the

impaired NO expression of diabetic endothelial cells.

Our results demonstrate the impaired function of endothelial cells in NO expression. NO

expression is the indicator of the normal endothelial cell function (237). Reduced NO

bioavailability decreases the ability of endothelial cells to execute their functions in

regulating vascular tone and growth, immune cell responses, and vascular barrier

functions (238-240). Nitric oxide plays many important roles in wound healing. NO

modulates cytokines that in turn control the progress of each of these phases of wound

healing (e.g., chemoattraction, proliferation, collagen deposition, and angiogenesis)

(244). NO deficiency directly contributes to wound healing impairment (243). There are

strong correlations between reduced cutaneous NO levels and impaired wound healing

under disease conditions such as diabetes (245). Here we quantified the deficiency in

NO expression of diabetic endothelial cells in-vitro.

Our findings demonstrate that electrical stimulation and strain application enhances the

impaired NO expression of endothelial cells in diabetic condition. The NO concentration

is higher under strained conditions compared with static conditions for, diabetic and high

glucose groups as well as the wild type groups. These findings are consistent with

previous studies demonstrating the increase in the NO levels and NOS activity with

strain application in both bovine aortic endothelial cells and human microvascular

120
endothelial cells (262, 267, 268). In agreement with our results presented in chapter 4,

electrical stimulation and nanofiber scaffolds increases NO release of wild type

endothelial cells and importantly enhances it in the diabetic condition. Previous studies

have shown the positive role of strain application in increasing VEGF expression of

human microvascular endothelial cells and rat coronary microvascular endothelial cells

(269, 270).

In this study we used two models of diabetic cells, one from in vitro cell exposure to

high-glucose conditions and another group from in vivo diabetic animals (262). Previous

studies performed in our group suggest that the in vitro model may not be ideal for

mechanistic studies of the effect of diabetic on endothelial cells due to differential

responses observed between two groups (271). Therefore for electric field studies we

used cells derived from diabetic animals.

Overall these results suggest that combined effects of external biophysical stimuli and

extracellular environment have the potential to enhance impaired cell responses in

diabetic condition.

121
Chapter 7
Conclusion and future directions

The findings of this dissertation provide a basis for understanding the mechanisms

underlying the regulation of cell function through non-contact application of electric field.

Aim 1 of this dissertation was to develop a numerical model to study the biophysics of

EF interaction with cells. The goals of aim 1 were achieved and the results are

presented in Chapter 3. I have developed a high resolution numerical model of electric

field interaction with a cell that determines the spatial distribution of the electric field in a

cell, and extracellular environment in response to electrical stimulation in a wide

frequency range. For the first time, this model considers a cell attached to a substrate to

represent cells within tissues. My results show that EF structure in a cell is strongly

inhomogeneous and is sensitive to the physical properties of the cell (Research aim

1.1). I have demonstrated the frequency-sensitive cell response to EF that enables

targeting the cell compartments by EF frequency. The results indicate that EF is

induced in the cell membrane and excluded from the cell interior in low frequencies

while EF penetration into the cell increases with higher frequencies (Research aim 1.2).

This study shows the sensitivity of the EF distribution in the cell and extracellular

environment to substrate properties and the difference in cell responses between cells

suspended in an electrolyte and cells attached to a substrate (Research aim 1.3). This

numerical approach can be generalized to investigate the interaction of EF with cells of

any arbitrary shape. In future studies the EF induced in the cell nucleus or other internal

cell components can be investigated by incorporating the internal components to the

122
cell cytoplasm. Modeling the ion channels embedded in the cell membrane is a good

approach to determine local changes of EF induced in channels to study the response

of voltage-gated channels to electrical stimulations. This model can be extended to

multicellular systems to model three dimensional tissues and study the EF penetration

in the tissue.

Aim 2 of this study was to quantify the regulatory effect of high-frequency EF on the

endothelial cell function. The goals of aim 2 were achieved and the results are

presented in Chapter 4. My results show that high frequency EF can improve function of

endothelial cells and I determined the pathways that mediate the effect of high

frequency EF. I have demonstrated that high frequency EF increases nitric oxide

expression of endothelial cells in a Ca2+ dependent manner (Research aim 2.1). My

results demonstrate the differential regulation of protein expression in endothelial cells

by extracellular matrix properties (natural vs synthetic hydrogels) in response to high

frequency EF (Research aim 2.2). Experimental findings are in agreement with the

theoretical model predictions. Results show the activation of intracellular pathways in

response to high frequency electrical stimulations where EF is induced in the

intracellular space. The experimental results demonstrate the sensitivity of the EF

stimulated cell response to substrate properties (Research aim 2.3). Investigation of the

angiogenic intracellular pathway activation such as MAPK/ERK and RhoA/Rho and their

regulatory effect on eNOS pathway will be the subject of future work.

123
Aim 3 of this study was to determine the effect of non-oscillating EF on the cell function.

The goals of aim 3 were achieved and the results are presented in Chapter 5. An

electrophysiological device was designed and built which enables live measurement of

cell responses to a non-oscillating EF (Research aim 3.1). Theoretically, I have

determined how non oscillating EF is coupled to this model system for non-contact

electrical application (Research aim 3.2). My experimental results demonstrate the

interaction of cells with surface charge and differential cell response to different EF

polarities (Research aim 3.3). For future studies, the optical measurement of the cell

membrane potential in response to EF using the electrophysiological device is

suggested. Using this approach, we could experimentally quantify the magnitude of the

induced EF in the cell membrane and compare it with the cell responses, such as

protein expression, measured following electrical stimulation.

Aim 4 of this study was to determine the endothelial cell deficiencies in the diabetic

condition. The goals of aim 4 were achieved and the results are presented in Chapter 6.

My results confirm the impaired angiogenic endothelial cell responses in diabetic

condition (Research aim 4.1). My results show that application of the electrical,

mechanical stimulation and using nanofibers as the extracellular matrix improve the

diabetic cell responses (Research aim 4.2). To continue this research in-vitro,

mechanistic studies of EF interaction with diabetic cell are essential. Considering the

positive effect of nanofibers in improving the endothelial cell function, use of nanofibers

as the extracellular matrix combined with application of high frequency electrical

stimulation is suggested to continue in-vitro studies. Using this approach, we can test

124
whether the combined effect of EF and extracellular matrix can increase the

improvement of the endothelial cell function in diabetic condition. These Mechanistic

studies at the molecular level are essential for clinical translation of EF-therapies to treat

diabetic wounds.

125
126
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145
Appendix

This appendix describes how to create the model used in the numerical studies

presented in Chapter 3 using high frequency structure simulator (ANSYS HFSS). The

model consists of a parallel plate capacitor which is placed in the middle of an airbox. A

dish made of dielectric material is place between the plates of the capacitor. The dish is

filled with the electrolyte; therefore the setup represent non contact application of

electric field. The cell is modeled as a membrane enclosed hemisphere attached on the

bottom of the dish, surrounded by the electrolyte.

Opening a new file:

File/new/project menu/insert HFSS Design

HFSS/solution type/driven terminal

Creating the airbox:

Menu/ Draw/ Box

Set the material to vacuum

Assigning the radiation boundary:

Select the airbox: Edit/select/By name

Right-click on the selected box: Assign Boundary/radiation

146
Creating the parallel plate capacitor:

Create top and bottom electrodes: Menu/ Draw/ Box

Right-click on each box (electrode): assign material/ select a copper (metal) as

material

Creating the dish:

Create a hollow dish by drawing two boxes and then subtracting the smaller from the

bigger

Outer box: Menu/ Draw/ Box

Inner box: Menu/ Draw/ Box

Set the material for both of them to a dielectric

Subtract two layers:

Select both boxes: Edit/select/by name

Subtract both layers: 3D Modeler/ Boolean/ Subtract

Filling the dish with electrolyte:

Menu/ Draw/ Box

Set the material to electrolyte

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Creating the cell:

Create the cell by drawing a spherical shell and a box, then subtracting them to create

a hemispherical shell

Creating the spherical shell:

Draw two concentric sphere, then subtract them

Outer Sphere: Menu/ Draw/ Sphere

Inner Sphere: Menu/ Draw/ Sphere

Set the material for both of them to a dielectric

Subtract two spheres:

Select both spheres: Edit/select/by name

Subtract the spheres: 3D Modeler/ Boolean/ Subtract

Creating the hemispherical shell:

Menu/ Draw/ Box

Set the material to dielectric

Subtract the sphere and the box: 3D Modeler/ Boolean/ Subtract

Filling the cell with the electrolyte:

Select the inner volume of the cell: Edit/select/by name

Set the material to electrolyte


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Assigning mesh:

Select the hemispherical shell: Edit/select/by name

HFSS/ Mesh Operation/ Assign/ On Selection/ Length Based

Assigning excitation/voltage source:

Select one of the vertical faces of the dish: Edit/select/By name

Assign excitation: HFSS/ excitation/assign/voltage source

Hint: Assigning a voltage source requires drawing a vector that should be

perpendicular to the electrodes and starts and ends on the dielectric. It should not

touch the electrodes.

Creating an Analysis Setup

HFSS/ Analysis Setup/ Add Solution Setup/General/Solution Frequency

Creating a Frequency Sweep

HFSS/ Analysis Setup/ Add Sweep/Fast Sweep

Draw a sheet to plot the field data

Draw a sheet that passes through the cell: Menu/ Draw/ rectangle

Run the simulation

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HFSS/Analyze All

Plotting field data

Click on the sheet to select it

Right click on the sheet/ Plot E/Mag E

HFSS/ Fields / Plot Fields/ Mag_E

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