Graduate School
Doctor of Philosophy
by
Toloo Taghian
July 2015
Dissertation Committee:
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
acceptance of electric field therapies for wound healing has been prevented by a lack of
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
knowledge and technology from both physics and biomedical sciences. The long term
non- or slow-healing chronic ulcers. The studies in this research contribute to this goal
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
ii
attached to a substrate. The results demonstrate that, at low frequency, electric field is
responses. At high frequency, electric field penetrates the cell and may directly activate
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
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
Further, this study provides novel information about interaction of cells with surface
condition are determined and the effect of electric field in improving the diabetic
iii
These findings improve the mechanistic understanding of vascular cell interactions
within the complex biophysical system and can potentially contribute to development of
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
I would like to thank my doctoral research committee members, Prof. Margaret Hanson,
Prof. Howard Jackson and Prof. Rostislav Serota for their kind support,
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
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
vi
Table of Contents
Abstract…………………………………………………………………….…………………....ii
Acknowledgments…………………………………………………………………………….vi
Table of contents……………………………………………………………………………..vii
List of abbreviation………………………………………………………………………….xvi
2. Background
2.1.1. Significance……………………………………………………………………....4
vii
3. Modulation of cell function by electric field: a high-resolution analysis
3.1. Objective………………………………………………………………………………17
3.2. Introduction……………………………………………………………………………17
3.4.4. Substrate and culture medium properties and field penetration into the
culture medium………………………………………………………………………..…38
3.5. Conclusions…………………………………………………………………...………42
stimulation
4.1. Objective………………………………………………………………………………45
4.2. Introduction……………………………………………………………………………45
viii
4.3.2. Microvascular endothelial cell isolation and culture………………………….56
4.3.4. Immunohistochemistry…………………………………………………………..57
4.3.5. Vascular endothelial growth factor expression and nitric oxide release…...58
4.4. Results…………………………………………………………………………………60
endothelial cells……………………………………………………………………….…62
endothelial cells……………………………………………………………………….…65
hydrogelproperties…………………………..………………………………….………71
ix
4.4.9. Electrical stimulation enhances nitric oxide expression of endothelial cells
endothelial cells……………………………………………………………………….…75
4.5. Discussion………………………………………………….…………………….……79
4.6. Conclusions…………………………………………………………………………...83
5.1. Objective……………………………………………………………………………....84
5.2. Introduction……………………………………………………………………………84
5.4. Results………………………………...……………………………………………....95
x
5.4.3. Electric field polarity regulates the adhesion of fibroblasts to the
substrate…………………………………………………………………………………99
expression………………………………………………………………………………102
5.5. Discussion……………………………………………………………………………104
5.6. Conclusions……………………………………………...………………………..…108
environment
6.1. Objective……………………………………………………………………………..109
6.2. Introduction…………………………………………………………………………..109
stimulation………………………………………………………………………………110
6.3.2. Microvascular endothelial cell isolation and experimental groups for strain
experiments…………………………………………………………………………….111
6.4. Results……………………………………………………………………………………114
xi
6.4.1. Capillary morphogenesis is impaired in diabetic condition…………….…..114
diabetic condition………………………………………………………………..……..115
condition……………………………………………………….………………………..115
6.4.4. Electric field stimulation enhances nitric oxide expression of endothelial cells
in diabetic condition………………………………..……………………………….….117
diabetic condition………………………………………………………………………118
Bibliography…………………………………………………………………………………127
Appendix……………………………………………………………………………………..146
xii
List of tables and figures
Chapter 2
Chapter 3
Figure 3.4 Induced electric field in the cell membrane, cytoplasm and 31
culture medium
medium
Chapter 4
xiii
Figure 4.1 Experimental set-up for microvascular endothelial cells 55
Chapter 5
Figure 5.1 The custom system for electrical stimulation of cells using 87
Figure 5.2 Custom system for live imaging of cells during electrical 89
stimulation
xiv
Figure 5.5 Delayed adhesion of electric field exposed cells to the 100
Figure 5.7 Normalized induced voltage and surface charge in the 107
model system
Chapter 6
xv
List of Abbreviation
EF electric field
EC endothelial cell
NO Nitric oxide
xvi
Chapter 1
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
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
impaired angiogenesis, defined as formation of new blood vessels from existing ones
(3, 4). Increased extracellular matrix degradation, impaired function of vascular cells
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
trials. This stems from incomplete understanding of the biophysics principles that
govern electric field interactions with cells in the ionic extracellular environment of the
scaffolds has been shown to stimulate angiogenesis and accelerate wound healing in
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.
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
with cells
Research aim 1.1: To determine the spatial distribution of EF in the cell membrane,
Research aim 1.2: To determine whether the structure of induced EF in the cell
Research aim 1.3: To investigate whether the extracellular environment can affect the
2
AIM II: To quantify the regulatory effect of high frequency EF on the endothelial
cell function.
Research aim 2.2: To investigate the effect of different extracellular matrices (natural vs
Research aim 2.3: To validate the predictions of the model in high frequency range.
Research aim 3.1: To design and build an exposure device that enables live study of
Research aim 3.2: To theoretically determine the EF distribution in the model system
responses to EF.
Research aim 4.1: To quantify the deficiency of diabetic endothelial cells in nitric oxide
Research aim 4.2: To examine the role of external biophysical stimuli in improving the
3
Chapter 2
Background
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
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
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
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.
5
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
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).
tissue engineering as designable biophysical and biochemical milieus that direct cellular
behavior and function (18). The guidance provided by biomaterials may facilitate
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
(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
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
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.
cell proliferation and expression of the angiogenic factors in vitro and promotes
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
currents or charges provide spatial and temporal regulation of cellular activities ranging
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-
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.
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
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
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
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
morphology. Another example of regulation of cell behavior via control of the substrate
surface charge is polyion complex nanoparticles (PIC) coated polystyrene (44). PIC
methacrylate) with anionic plasmid DNA at various charge ratios which can be adjusted
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-
charged PIC surface, the cells change their morphology to form capillary-like networks.
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
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
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
prevented by the charge of the surface (51). For example, it has been shown that E. coli
positively charged surface, the E. coli biofilm is dense and homogeneous (52). Similarly,
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,
11
2.4.2. Conductive polymer scaffolds
polythiophene (PT), etc.) are biocompatible materials, which have physical and
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
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
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.
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
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
In addition to endogenous direct current electric field, electrical pluses are essential
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
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.
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
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
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
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
without the use of chemical agonists, thus avoiding potentially significant side effects.
Another approach that has been introduced for electrical stimulation of mammalian cells
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,
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
principles to stimulate cellular responses in the wound using extracellular signal with no
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
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).
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
16
Chapter 3
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
3.2. Introduction
Electric field (EF) can regulate a variety of cell functions, including growth, adhesion,
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
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
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
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
18
redistribution of membrane channels and receptors in response to non-oscillating EF
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
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
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
respect to the cell, as well as methods of EF delivery, can elicit a variety of distinct cell
The complexity of mechanisms for EF-cell interaction calls for a detailed understanding
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
a spherical shell representing the cell membrane (139). The model by Shawn treats the
(140). Kotnik extended the Schwan’s theory by taking into account the conductivity
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
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
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
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
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
electric properties. The results demonstrate that the field distribution exhibits
physiologically important features that strongly depend on the EF frequency and differ
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
zero along small closed paths. We verified that the meshes used in the simulations
Dimensions used in the simulation and material parameters (94, 119, 157-159) are
22
Table 3.1. Model parameters used in the simulations.
conductivity (σsubstrate) 0
Cell substrate
thickness 1 (µm)
#
capacitance (Csubstrate) 0.22 (pF)
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
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
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
(b) Equivalent electric circuit with no cell present. Ccoating and Csubstrate
Rmedium are the capacitance and resistance associated with the electrolyte
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
26
Figure 3.3. Top: Schematic of a cell attached to the substrate. Bottom:
27
3.3.2. Modeling the cell on a substrate
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
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
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 ,
where V is the externally applied driving voltage and ω is the frequency of the applied
field (1Hz-10GHz).
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
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
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
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
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-
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
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
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)
GHz. At each frequency, the EF is present in the membrane and the EF penetration to
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
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
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
32
33
Figure 3.5. Left: Spatial distribution of induced electric field in a single cell
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
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,
34
Figure 3.6. EFinduced as a function of frequency of the applied field at
range, EFinduced increases near the apex but decreases near the
35
3.4.3. Conductivity of the cytoplasm: regulation of the cell cytoplasm screening in
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
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
EFinduced in the membrane facing the substrate side does not depend on
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
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
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
permittivity of the substrate results in the shift in the characteristic frequency of field
38
Figure 3.8. Effect of the geometrical and electrical parameters of the
39
3.4.5. Physiological implications
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
suspended cells experience no electric phenomena at low frequencies, while the cells
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
several studies have reported that the alteration of the cell membrane potential can lead
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
40
the structural alteration and increase the level of membrane proteins that regulate
gated channels embedded in the cell membrane, increase gene expression and
Interaction of a cell with EF changes as the field frequency increases. The EF in the
result is consistent with earlier reports that found penetration of the cytoplasm by high
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
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
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
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
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
considered, these will become progressively larger as the frequency increases into tens
3.5. Conclusions
This study presents a novel model for a high-resolution microscopic analysis of the
and attached to the extracellular substrate) exposed to the external electric field. Our
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
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,
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
skin, which may improve patient’s comfort and treatment compliance (193, 196), and
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
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
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
internal cell components and penetration of the field into tissue modeled in 3D cellular
44
Chapter 4
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
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
4.2. Introduction
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
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
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
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
systematic investigation into the individual and combined effects of these regulators is
needed.
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,
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
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
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
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
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
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,
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
cell type used in each study makes it difficult to make a convincing conclusion about the
51
enables investigation of the mechanisms that ECM regulate EF-cell interaction in a
controlled environment.
showed that physiological amplitude, high frequency (7.5 GHz) electric field activates
MAPK/ERK pathway and increases VEGF release. Our results demonstrate a major
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
distribution of the induced EF in the cell has been determined using our developed
high frequency electric field that we use in our experiments is induced in the cell
in response to HF EF. The experimental studies by us and others have shown activation
(85, 218).
In this work, we employed a 3D in-vitro model of wound healing which mimics in vivo
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.
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
FAK and NO expression of ECs. This in-vitro model could provide insights into the
A custom set-up was built that allowed EF exposure operating at 7.5 GHz frequency
(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,
54
High Freq. EF Setup : (~7.5GHz)
medium
reservoir
EC sample
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
coated dishes in Medium 199 (HyClone, Logan, UT) supplemented with 10% FBS
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
passages four to nine were used. All experiments were conducted in the culture
additional growth factor supplementation. Cells from passages four to nine were used.
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-
4.3.4. Immunohistochemistry
ECs were seeded on the surface of synthetic peptides or matrigel at the density of 5000
(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-
ECs on the surface of hydrogels were captured at 40X magnification using an inverted
57
4.3.5. Vascular endothelial growth factor expression and nitric oxide release
and matrigel in culture plate inserts (at least n = 5 separate experiments), and cultured
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
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,
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
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.
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
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
amounts of FAK protein. All experiments were repeated at least four times. Results
4.4. Results:
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
membrane projections (white arrows, Fig. 2), which increases with increasing RGD
60
No-EF
B C D
EF
61
4.4.2. Hydrogel properties regulate expression of angiogenic growth factor in
endothelial cells
regulatory effect of synthetic and natural hydrogels were quantified and compared. We
(RAD16-II), 50% and 100% RGD containing nanofibers and matrigel. After 12 hours,
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.
embedded in the synthetic and natural hydrogels, were exposed to EF for 12 hours.
62
compared to the no-EF synthetic group. In contrast, EF stimulation significantly
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
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
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
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
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
phosphorylated to total FAK does not differ between RAD and RADRGD
group while this ratio is greater for RGD nanofibers compared to RAD and
67
4.4.5. Effect of electrical stimulation on focal adhesion kinase expression is
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 over total FAK. However this ratio showed a 20% decrease for
68
A
69
C
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
RAD16-II and higher expression for 100% RGD nanofiber peptide. ECs grown on
was observed when cells were incubated with secondary antibodies alone (data not
shown).
hydrogel properties
seeded on the surface of the synthetic hydrogels and matrigel were exposed to EF for
expression throughout the cellular span with more pronounced expression on the cell
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
71
nanofibers containing 50% RGD and 100% RGD display increased vinculin expression
72
EF stimulated Not stimulated
D Matrigel Matrigel
73
Figure 4.6. Extracellular matrix mediated expression of focal adhesion
stimulated ECs; Right: not stimulated ECs on (A) 100% RAD16-II (B) 50%
74
4.4.8. Hydrogel properties regulate Nitric oxide expression of endothelial cells
quantified after 12 hours of incubation for ECs embedded in synthetic nanofibers and
and 100% RGD, express 60% and 160% more NO compared to RAD16-II, respectively.
nanofibers.
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
increase is more than 100% for ECs in synthetic nanofibers and 80% for ECs in matrigel
(Fig. 4.7.B).
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
75
ECs in both EF stimulated and not stimulated group compared to no-inhibitor (control)
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
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
stimulation increases the NO level and brings it to no-inhibitor values. (*: p<0.05, EF
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
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
Our results demonstrate that increased NO expression is one of the ECs’ responses to
regulating vascular tone and growth, immune cell responses, and vascular barrier
stimulation has been reported previously. Pulsed electric field (15 Hz,10 ms) stimulated
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
modulates cytokines that in turn control the progress of each of these phases of wound
(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
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
EF. Further we show that this EF stimulated increase is Ca2+ dependent. Our findings
endothelial cells and rat thymocytes (249, 250). In our previous study we have
(7.5 GHz)(218).
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
altered (105).The regulatory effect of EF on growth factor expression has been reported
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
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
As expected, expression of FAK and VEGF show similar trends in response to HF EF.
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
Tyr397 as well as other sites in the protein, creates a high-affinity binding site for other
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
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).
proliferation of human and murine fibroblasts (260). Overall, our results support the
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,
82
4.6. Conclusions
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
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
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
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
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
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-
(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.
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
model system and propose a device for live imaging of cells during electrical
stimulations.
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
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
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
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
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
where V is the externally applied voltage. The cell culture media between these
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
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
𝐸𝑚𝑒𝑑𝑖𝑢𝑚 𝜖𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
= 𝑒 −𝑡/𝜏 (5.4)
𝑉 𝜖𝑚𝑒𝑑𝑖𝑢𝑚 ×(2×𝑑𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 )+𝜖𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 ×(𝑑𝑚𝑒𝑑𝑖𝑢𝑚 )
90
𝜖𝑚𝑒𝑑𝑖𝑢𝑚 (𝑑𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 +𝑑𝑐𝑜𝑎𝑡𝑖𝑛𝑔 )+𝜖𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑑𝑚𝑒𝑑𝑖𝑢𝑚
𝜏= (5.5)
σ𝑚𝑒𝑑𝑖𝑢𝑚 (𝑑𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 +𝑑𝑐𝑜𝑎𝑡𝑖𝑛𝑔 )
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
The magnitude of the induced EF in the media and surface charge density versus time
91
Table 5.1. Parametes used in the Model system
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,
(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
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
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.
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
proliferating cells and DAPI nuclear staining (Invitrogen, USA). The images were taken
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
5.4. Results
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
95
A
B C
microfabricated device did not affect cell viability (live cells, dead cells),
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
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
97
A
EF NO-EF
98
5.4.3. Electric field polarity regulates the adhesion of fibroblasts to the substrate
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
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
99
A
EF
30 min 1 hr 2 hrs 3 hrs
No-EF
100
C
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
102
Figure 5.6. Exposure of FBs to DC EF for 2 and 3 days enhances VEGF
#: p<0.005).
103
5.5. Discussion
isolated from the medium can regulate cell functions. This regulation happens while our
the microscopic level (Fig.5.7.B). This interaction resulted in the regulation of cell
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
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
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
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
transcriptional level (261). In agreement with previuos studies, our EF induced changes
105
In this study, we demonstrate the advantageous features of the non contact method of
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
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
interaction because the cell response is mediated only by EF induced in the cell and is
108
Chapter 6
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
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
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
structural changes in the myocardium of diabetic patients and eventually leads to left
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.
6.3.1. 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
Louis, MO, USA), and 0.2 ng ml-1 growth supplement (Sigma Aldrich, St Louis, MO).
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
6.3.2. Microvascular endothelial cell isolation and experimental groups for strain
experiments
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
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’’
response of endothelial cells. Cardiac endothelial cells were seeded onto 6-well
111
collagen I pre-coated UniFlex® Culture plates (Flexcell International Corp.,
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%
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
culture plates.
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,
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
Endothelial cells at the density of 250,000 cells/insert were embedded in matrigel and
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
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
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
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
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
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
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.
117
6.4.5. Application of strain increases nitric oxide expression of endothelial cells in
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
118
6.4.6. Extracellular environment stimulates nitric oxide expression of endothelial
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)
119
6.5. Discussion and Conclusions
diabetic condition. We demonstrate the positive effect of external biophysical stimuli and
extracellular matrix to improve the endothelial cell function through enhancing the
expression is the indicator of the normal endothelial cell function (237). Reduced NO
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
(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
Our findings demonstrate that electrical stimulation and strain application enhances the
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,
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
responses observed between two groups (271). Therefore for electric field studies we
Overall these results suggest that combined effects of external biophysical stimuli and
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
field interaction with a cell that determines the spatial distribution of the electric field in a
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
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
any arbitrary shape. In future studies the EF induced in the cell nucleus or other internal
122
cell cytoplasm. Modeling the ion channels embedded in the cell membrane is a good
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
frequency EF (Research aim 2.2). Experimental findings are in agreement with the
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
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
determined how non oscillating EF is coupled to this model system for non-contact
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
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
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.
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
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
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
146
Creating the parallel plate capacitor:
material
Create a hollow dish by drawing two boxes and then subtracting the smaller from the
bigger
147
Creating the cell:
Create the cell by drawing a spherical shell and a box, then subtracting them to create
a hemispherical shell
perpendicular to the electrodes and starts and ends on the dielectric. It should not
Draw a sheet that passes through the cell: Menu/ Draw/ rectangle
149
HFSS/Analyze All
150