CSE C-S Style
Team Research Proposal
ELECTRODE:
Evaluating Low Electrical Currents for Tissue Repair and
Optimizing a Device for Experimentation
March 4, 2012
I pledge on my honor that I have not given or received any unauthorized assistance on this
assignment/ examination."
Sagah Ahmed __________________________
Natalie Anzures _________________________
Zach Bosley ____________________________
Brendan Bui ____________________________
Ariana Feizi ____________________________
Sudi Jawahery __________________________
Coutrney Koenig ________________________
Katie Lakomy __________________________
Research Proposal
Megan Lin _____________________________
Poorna Natarajan ________________________
Eisha Nathan ___________________________
Hiba Sayed _____________________________
Eduardo Solano __________________________
Mentor: Dr. John Fisher – Yes
Librarian: Jim Miller - Yes
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TABLE OF CONTENTS
I. Abstract………………………………………………………………………………………....3
II. Specific Aims……………………………………………………………………………….…4
III. Background & Significance……………………………………………………………….….6
IV. Experimental Design………………………………………………………………………..14
V. Timeline……………………………………………………………………………………...24
VI. Vertebrate Animals…………………………………………………………………………..24
VII. Budget…………………………………………………………………………...……....….27
VIII. Appendix A: Methods…………………………………………………….………………..28
IX. Appendix B: Glossary………………………………………………………………….…….32
X. References…………………………………………………………………………………….34
Research Proposal
ABSTRACT
We propose to examine the effects of electrical stimulation on the overall healing of
diabetic ulcers in a three-part experimental design. Diabetes is a debilitating condition that can
lead to non-healing wounds called ulcers. This formation of ulcers is caused by excessive
inflammation and lack of nutrients to the wound site. We propose to test both a linearly and
radially applied electric field in vitro, and then design a device based on most effective
application and apply this to an in vivo diabetic ulcers model in rats. Future research directions
will be to build devices for application to human patients that are afflicted by diabetic ulcers.
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SPECIFIC AIMS
The goal of this project is to develop a new treatment for diabetic ulcers. Diabetic ulcers
are chronic, non-healing wounds in people afflicted with diabetes. Unfortunately, there are few
effective treatments for the condition, creating a problem for those afflicted with these wounds.
It is known that in diabetic ulcers, the increased inflammatory response increases proteolytic
activity, or the degradation of proteins. This inhibits target pathways and thereby reduces the
production of intracellular growth factors including vascular endothelial growth factor (VEGF)
and basic fibroblast growth factor (bFGF)1.* VEGF and bFGF cause capillary formation and are
key in the initiation of the process of angiogenesis2. Angiogenesis, or vascular tissue formation,
is a crucial process in tissue repair and wound-healing, and is stimulated by both the production
of these intracellular growth factors, and cell migration towards the wound site3. This angiogenic
response that occurs immediately following wound formation is caused by an endogenous
electric field that develops across tissue layers and triggers wound healing cellular pathways4.
Our experiment will use an external electrical stimulus to activate these cellular pathways
associated with tissue repair, therefore promoting the rapid healing of the ulcer. Our motivation
for the use of electrical stimulation is due to its positive interactions with the human body’s
endogenous bioelectric healing system to heal injuries. We believe that electrical stimulation will
activate these cellular healing pathways because previous studies have shown that applied
current enhances ion transport through the wound and increases cell migration5. Other studies
have also shown that sensory electrical stimulation is able to release more levels of VEGF in
skin, which will improve the healing pathways in diabetic ulcers6.
We plan to explore a new aspect of electrical stimulation by varying the shape of the
applied electric field on angiogenesis. We propose that the inflammation of damaged tissue is
*
See glossary for explanation of terms
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the major factor suppressing angiogenesis in diabetic ulcers and that this electric field will
allow us to overcome the effects of chronic, excessive inflammation and promote the repair of
diabetic ulcers.
To establish the correlation between electrical stimulation and inflammation, we will
explore the relationship between electrical stimulation and angiogenic factors that are suppressed
by inflammation. Previous studies have demonstrated that excessive inflammation decreases the
expression of key angiogenic growth factors1 such as VEGF and bFGF and inhibits the migration
of cells involved in wound healing2,6. Based upon these findings, we hypothesize that the
application of an electrical stimulus will alleviate the effects of the inflammatory tissue
response in wounds, increasing levels of angiogenesis and reducing the healing time of
chronic diabetic ulcers. Specifically, we propose the following three hypotheses and their
associated specific aims:
1)
Growth factors that promote the proliferation of endothelial cells are highly correlated
with angiogenesis. The migration of these endothelial cells is of key importance. We aim
to examine the effects of an optimized linearly applied electric field on cell
proliferation, VEGF expression, bFGF expression and cell migration rates.
2)
The intensity and direction of the applied electric field has been shown to change levels
of angiogenesis and wound healing6. While the linearly applied electric field is
unidirectional and homogenous, a radially applied electric field would result in nonconstant electric field intensities in different regions of the area of interest. The electrical
gradient would align with the oxygen concentration gradient in the wound bed, applying
more electrical stimulus to the center of the wound where oxygen concentration is
lowest7. We aim to examine the effects of an optimized radially applied electric field
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on cell proliferation, the VEGF expression, bFGF expression and cell migration
rates. We will then compare the results of the second aim to the results of the first
aim and build a device based on the preferred electrical field, either linear or radial.
3)
The preferred shape of the electric field will increase levels of angiogenic growth factors,
increase cell migration rates, and cell proliferation, thereby promoting wound repair and
decreasing chronic inflammation. We aim to demonstrate that the application of a
radial or linear electric field in a designed prototype will reduce chronic
inflammation in diabetic ulcers with a diabetic rat model.
BACKGROUND & SIGNIFICANCE
Diabetes and Diabetic Ulcers
In 2010, 1.9 million Americans over the age of 20 were diagnosed with either Type I
diabetes, an autoimmune disorder that attacks the cells in the insulin-producing pancreas, or
Type II diabetes, an acquired disease where the body develops insulin resistance and is unable to
use insulin to absorb glucose from the bloodstream8. Diabetes is a disorder of the metabolism
that alters the way that the body breaks down food for growth and energy, which leads to
symptoms such as unusual weight loss, and extreme hunger and thirst. In addition, it can lead to
a number of debilitating complications that affect the lower extremities, such as peripheral
arterial disease, neuropathy and chronic diabetic ulcers, and non-healing wounds. Diabetic ulcers
in particular occur in approximately 15% of patients with diabetes and frequently require lower
limb amputation, which constitutes the leading cause of hospitalizations of diabetics9. Ulcers
tend to develop because of poor circulation and vasoconstriction, the narrowing of blood vessels,
in the foot or leg due to excessive inflammation. This causes neuropathy, which lessens the
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body’s ability to feel pain and other sensations, and leads to high risks of infection and slow
healing rates of ulcers8.
Existing treatments for diabetic ulcers do help diabetic ulcers recover, such as negative
pressure wound therapy, a treatment where a vacuum decreases tissue pressure at the wound site,
resulting in vasodilatation, easier blood flow to the wound site, and perfusion, the delivery of
nutrients to the wound site. However, many treatments carry adverse side effects, such as
improper drainage, long recovery periods, allergic reactions to ingredients, and an inability to
target the wound site directly9. They also require constant application, and their success rates are
unreliable. It can cost 8,000 USD for treatment of a single typical ulcer, 17,000 USD for an
infected ulcer, and up to 45,000 USD for an ulcer that requires amputation9.
Inflammation and Angiogenesis
Naturally occurring wounds
Naturally occurring wound healing has three stages: inflammation, proliferation, and
tissue remodeling. It is believed that the inflammatory response following an alteration to the
skin plays a major role in achieving proper tissue homeostasis during wound healing2. The
process of inflammation involves a balance between a network of immune cells and a plethora of
pro- and anti-inflammatory mediator molecules.
During the three stages mentioned above, especially inflammation, a cascade of
molecular and cellular events stimulates angiogenesis and subsequent overall wound healing8,14.
Macrophages that are recruited to the wound site synthesize numerous potent growth factors,
such as basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF),
which promote cell proliferation, vascularization, endothelial progenitor cell recruitment, and the
reformation of the extracellular matrix molecules, such as collagen10. This in turn stimulates
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angiogenesis, providing blood flow to the peripheral nerves of diabetic ulcers to reverse the
neuropathy commonly associated with diabetes3. In addition, leukocytes or white blood cells, a
group of immune cells that primarily fight infection, along with platelets, form and aggregate in
the blood clot of a wound, releasing a series of chemical factors that amplify the inflammatory
response and initiate the process of wound healing11. Neutrophils also intensify inflammation
and help by releasing highly active molecules, like proteases, that break down infectious
pathogens through the process of phagocytosis 12. Following inflammation, the complex repair
process of scarring finishes wound healing and restores the natural make up of the skin.
Non-healing chronic wounds
Health factors that alter the body’s homeostasis* and metabolism, such as Diabetes
mellitus can stimulate an accelerated inflammatory response. In excess, the constant
inflammatory response leads to the development of ulcers. Thus, although the acute
inflammatory response plays a major role in naturally occurring wound healing, a chronic
inflammatory response actually inhibits the natural healing process, causing these diabetic
ulcers13.
First, diabetes hinders the migration of leukocytes to the wound site. The lack of
leukocytes leaves the wound open to infection, thereby slowing the healing process. The same
wound healing deficiency is caused by additional factors, which include lack of collagen
accumulation for vascularization due to impaired growth factor (VEGF and bFGF) production
from weakened macrophage function and lack of upper epidermis healing due to less
keratinocyte and fibroblast migration1. Finally, the excessive inflammation creates a defect in the
normal angiogenesis process3.
*
See glossary for explanation of term
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The excessive inflammation increases hydrostatic pressure, which distorts the capillaries
in the wound region, and hampers angiogenesis2,3,13. Other studies have shown that this
persistent inflammatory response is characterized by an excess of proteolytic activity, which
directly breaks down crucial growth factors vital in the angiogenesis process. Therefore, due to
the lack of production of VEGF and bFGF and their subsequent decrease in interaction with the
extracellular matrix, the angiogenesis process, a foundation for these chronic wounds, is
significantly hampered16.
Body’s Natural Bioelectric Healing System
Human epithelial tissue maintains a natural trans-epithelial potential (TEP) ranging
anywhere between 10mV to 60mV; this naturally occurring electric potential across skin layers
makes wound sites positive with respect to their surroundings14. This phenomenon implies that
electric fields may be responsible for the directionality of cell migration upon wounding and
might play a key role in natural wound healing.
It has been shown that endogenous electric fields (EFs) develop in wounds in the
epithelial tissue4. Studies reveal that upon wounding, an electric field develops across the wound
site, which triggers cell migration into the wounded area. Once a wound occurs, the change in
electric potential creates an electric field that is maintained by the action of ion channels and
pumps from the cells surrounding the wound. The direction of this electric field dictates the
direction of migration of cells to the wound. Although it has been shown that cells start the
migration process once they recognize the EFs, the cell membrane receptors that detect such
changes are unknown. What is known, however, is the intracellular response that eventually
leads to cell migration. Studies show that once a cell recognizes the endogenous electric field, a
series of chemical pathways that lead to cell polarization occur, which then lead to cell
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migration. These pathways vary from cell to cell depending on the tissue, but it is known that in
epithelial cells the Golgi apparatus plays a role in the direction of the cell by reorganizing the
cytoskeleton of the cell, thus, allowing the cell to physically move5. Moreover, it is known that
among the signaling mediator pathways that occur inside the cells are the phosphoinositide 3kinase signaling pathway and Cyclic AMP-mediated pathways15. The combined effect of these
pathways leads to directional cell polarization (cells polarize in the same direction of the
endogenous electric field), cell migration and wound healing.
Physiological Response to External Electrical Stimulation
Previous studies involving applied electrical stimulation
Previous research establishes the validity of electrical stimulation on bodily wound
repair. The general theory involves the transfer of an electric current to the skin near the wound
edge via two electrodes. This current delivery creates a flow of ions through the wound tissue,
enhancing capillary density and perfusion, increasing oxygen flow to the wound site, boosting
directed cell migration to the wound site5,15, stimulating granulation formulation and fibroblast
activity, and increasing the production of VEGF – all factors that have a positive correlation with
wound repair16. Moreover, it has been shown that electrical stimulation induces the migration of
keratinocytes, which contribute to the skin’s first line of defense against pathogens, a key process
in wound healing17-19.The application of electrical stimulation has been shown to increase the
rate of healing by more than 50 percent20.
A study performed on 48 male Sprague-Dawley rats applied direct current electrical field
with an intensity equal to 600µA for one hour every other day for seven days to a wound,
inducing cell growth and migration of cells towards the wound perimeter21. This is due to
changes in membrane potentials of cells which release VEGF as well as other growth factors,
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which modifies the cell’s Rho-ROCK and PI3K-Akt signal transduction pathways21. Activation
of these signal transduction pathways leads to cell reorientation and directional movement of
cells towards the damaged tissue, and speeds up the rate angiogenesis and wound repair21.
Research on human subjects includes a study that used gastric electrical stimulation by
implanting a device that administered electrical stimulation to the large intestine. In a ten yearlong study performed on thirty-three individuals with severe symptoms of nausea, vomiting, and
gastric emptying caused by gastroparesis, patients had a device implanted in the smooth muscle
of the great curvature of their gastric antrum that administered electrical stimuli22. High
frequency electrical stimulation parameters were applied for a period of six months, and the
severity of symptoms and overall quality of life were measured. Many patients receiving the
treatment that previously experienced intractable nausea and vomiting noticed a significant
decrease of these symptoms and a significant increase in the Quality of Life 22.
Preliminary studies have experimented with electrical stimulation on diabetic ulcers.
They have shown that exposure to heat in addition to the administration of electrical stimulation
to the ulcer site significantly accelerated healing time23. The electrical stimulation in one study
was applied to 29 male and female patients with diabetic ulcers at a micro amperage of 20µA for
thirty minutes three times per week over a period of four weeks23. Patient exposure to global
heat, being placed in a thirty-two degree Celsius room, had a higher synergistic effect when used
alongside electrical stimulation in regeneration of tissue than application of local heat, raising the
temperature of the wound area to thirty-seven degrees Celsius23.
Investigating the Current of Electrical Stimulation Further
Studies that involve an external electrical stimulus have also investigated the effects of
various types of electrical stimulation on wound healing. It has been established that electrical
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stimulation is a valid means for wound repair. This following study conducted tests to see which
was more appropriate for wound repair: anodal or cathodal microamperage direct current
electrical stimulation. Application of continuous microamperage direct current is a plausible
method of treatment due to the inherent potential difference between a wound and its
surrounding intact skin. The study concluded that anodal microamperage direct current is more
effective than cathodal microamperage direct current in healing skin wounds because it decreases
the wound surface area faster, allowing for faster wound healing than cathodal electrical
stimulation14.
Different experiments have been conducted to test the viability of different types of
current on wound repair and healing. There exist many types of current that could be tested in
wound repair. Direct current has the electrons continuously flow in one direction, from the
negative pole to the positive pole. On the opposite spectrum to continuous current is pulsed
current. Pulsed current may be split into monophasic and biphasic. Monophasic currents are also
uni-directional but have periods with noncurrent flow. Pulsed biphasic current, unlike
monophasic currents, do not have only one phase; the electrons flow in from both the positive
and negative direction, as seen in Figure 1 below.
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An experiment was conducted to observe the effect of direct current on cell directionality,
where it was concluded that where cells are already migrating to the site of injury, direct current
may not speed up cell migration24. Pulsed current studies, however have shown that high-voltage
pulsed current is a “useful addition” to preventing limb amputation that may occur as a result of
diabetic ulcers of the “lower extremity”7. Pulsed electric current has also been shown to increase
wound healing by increasing FGF-2 release25.
Significance
Our research will be novel because we will be testing the effect of a radially applied
electric field on an angiogenesis in vitro model. Previous research has only tested the effect of a
linearly applied electric field on the healing of chronic wounds. The basis for both types of
electrical stimulation is rooted in excessive inflammation, inhibiting production of cellular
growth factors, cell migration, and cell proliferation, therefore also decreasing angiogenesis.
Because of the response of the endogenous electric field in normal wound healing, we believe
the application of an electrical field will assuage chronic inflammation to acute inflammation,
which promotes wound healing26.
We believe this study would be of interest to three groups: people with diabetes,
healthcare professionals, and medical researchers. Diabetics will have access to knowledge about
an alternative ulcer treatment that is non-invasive and effective, healthcare professionals can then
provide treatment to the patients, and medical researchers can develop technologies that will
provide electrical stimulation to diabetics with chronic ulcers.
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EXPERIMENTAL DESIGN
Overall Concept
The overall purpose of our experimental design is to test two different applications of an
electrical stimulus using a pulsed monophasic square wave to determine which design has the
greatest effect on the wound healing of a diabetic ulcer through the measurements of cell
proliferation, VEGF, bFGF, and cell migration. The pulsed monophasic square wave will be
used because it has been shown to accelerate wound healing in previous studies7,24,27. In one
study, the application of a pulsed monophasic current increased FGF expression in diabetic
wounds25. Another study found that wounds in a rat model that were subjected to pulsed
electromagnetic fields showed a statistically significant acceleration of wound healing due to the
manifestation of connective tissue, formation of capillaries, increased re-epithelization, and
structuration of collagen28. Cell proliferation, VEGF, bFGF, and cell migration have shown to be
factors increasing angiogenesis and decreasing chronic inflammation and therefore leading to the
healing of diabetic ulcers as the background section dictated3. The designs will first be tested in
an in vitro model and then the best design will be the basis for a device prototype, which will
used in a more applicable in vivo model.
SPECIFIC AIM ONE: LINEARLY APPLIED ELECTRIC FIELD
Hypothesis & Objective
For our first specific aim, we hypothesize that applying an electrical stimulation to an in
vitro model of diabetic ulceration, will allow for increased expression and migration of
angiogenic factors that are involved in wound healing2. These growth factors promote the
proliferation of endothelial cells and their migration is of key importance to the process of
wound healing. The objective for our first specific aim is to examine the effects of a linearly
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applied electric field on VEGF expression, bFGF expression, and cell migration rates in a
monolayer of rat endothelial cells that will serve as our in vitro model.2
Experimental Plan
Our model for examining the effects of linearly applied electrical stimulation on
angiogenesis will utilize rat aortic endothelial cells (RAOEC) that our team will culture in
DMEM (Dulbecco’s Modified Eagle Medium) media supplemented by standard 10% fetal
bovine serum. The RAOEC will simulate our proposed in-vivo model as closely as possible. The
endothelial cells are appropriate for our study because they express growth factors associated
with cell proliferation and migration, and serve to successfully model angiogenesis. The
angiogenesis endothelial model was chosen because of the importance of increased angiogenesis
in wound healing29 and the role of endothelial cells in the healing of open wounds including
diabetic ulcers, and their observed reactions to electrical stimulation1,30-32.
Additionally, endothelial cells are optimal for modeling and demonstrating angiogenesis,
as evidenced by previous studies conducted in which growth factors such as VEG-F and EGF
(vascular and epidermal growth factors, respectively) were applied to RAOEC, and found to
stimulate angiogenic behavior in the cultured cells33. The endothelial cells are directly impacted
by the incidence of diabetic ulcers in diabetic patients; studies have shown that endothelial cells
of diabetic rats produce markedly less endogenous levels of immunoreactive nerve growth
factor34.
We will have three experimental groups consisting of one control group, one applied with
a linear electric field, and one applied with a radial electric field. Each group will have five cell
cultures, and the control group will not receive any electrical stimulation.
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The chamber containing the growing cells will be modified in order to evenly apply the
electrical current to the culture. A waveform generator will allow us to apply a pulsed
monophasic current at .1 V and current of 15 µA with a frequency of 50 Hz to our experimental
cultures while allowing us to control other electrical factors35. Specifically, low-voltage
electrical stimulation has been correlated with the increased expression of angiogenic growth
factors, such as VEGF, in targeted endothelial cells in an experiment that used an unipolar square
wave36. The chamber will use titanium covered electrodes in direct contact with the cultures and
a waveform generator will be connected to the electrodes to produce the desired pulsed current
with the specified parameters. The titanium will coat the glass electrodes through the process of
vacuum evaporation titanium deposition using a Magnetron. This purpose of this initial round of
testing is to assess the effects of pulsed current electrical stimulation on the growth and
proliferation of endothelial cells when electrodes applying the current are parallel to each other,
therefore creating a linear electric field.
We will use rat aortic endothelial cells for our cell line. Every monolayer culture will be
5-cm in diameter. Titanium electrodes will be laid over the cells in the modified cell chamber so
that 1-cm of each electrode is in direct contact with the seeded endothelial culture. The
placement of the electrodes will be such that both are centrally in the culture with the cathode 1
cm away from the anode. The electrical current applied via the electrodes will have a set voltage,
current and frequency as stated above. The stimulation will be applied for 30 minute periods,
once per day for 5 days straight. The control groups will not receive any electrical stimulation.
After the electrical stimulation is complete, various tests will be completed on our control
and experimental groups to analyze our results. To measure cell proliferation, we will use a
hemocytometer to count the cells between the electrode plates in the culture and compare this
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number to the original number of cells in the culture. To measure cell migration, we will use a
Transwell assay. We will be administering a known volume of cells onto a specified area of our
petri dish, applying electrical stimulation onto the cells, and taking a picture of the cells after
stimulation under a microscope to track the distance of the migration of the cells. For the
angiogenic growth factors, we will examine gene expression through mRNA using the reverse
transcriptase polymerase chain reaction (rtPCR). For the actual growth factors, VEGF and
bGFG, we will use Western blotting, Immunohistochemistry, and Enzyme-linked
immunosorbent assay (ELISA) to measure the concentration of the protein. See the appendices
for lab protocols.
Our results will be compared with those of our second specific aim using success criterion to
know how we will progress to the final stages of in vivo testing.
Alternatives
If the results from the experimental group do not show statistically significant increases
in healing parameters when compared to the control group, an alternative method must be
sought. We will return to the beginning of the experiment and change the voltage and frequency
of the electrical stimulation when applying to a single culture and solely test this culture for cell
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proliferation. If there is significant proliferation, we will continue the experiment to completion
with the new parameters.
Statistical Analysis
After collecting data, the results will need to be analyzed by conducting statistical tests.
For our experiment, we will primarily use the T-test, because we are only comparing two groups:
the control and the electrically stimulated experimental. The null hypothesis will be that the
difference between the groups is due to chance. Our alpha level will be 0.05, and if our t-statistic
is below the alpha value, the difference between our groups will be statistically significant and
we can conclude that the electrical stimulus had a considerable effect on the angiogenic factors.
There are a number of confounding variables that may arise in the experimental design
that must be taken into consideration. Observable expression of angiogenic factors may be due to
natural healing processes and not the application of an electrical stimulus. This is a threat to
internal validity, as it is a potential variation within each group. To control for this threat to
internal validity, we will include a control in vitro ulcer model that is not subjected to electrical
stimulation. Additionally, in the case that expression of an angiogenic factor in the in vitro model
of the wound does not increase with the application of electrical stimulation, we will focus on
observing other angiogenic factors and/or cell migration in the in vivo portion of our
experimental design.
Success Criterion
The in vitro model should demonstrate the effects of electrical stimulation on angiogenic
factors of endothelial cells, most importantly cell migration. If there is a strong correlation
between the electrical stimulation and cell migration, the stimulus will have proved superior to
the control. If the in vitro model establishes statistically significant increases in cell proliferation
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and levels of growth factors associated with directing cell migration, VEGF and bFGF, it would
confirm the results of the cell migration rates after electrical stimulation26.
SPECIFIC AIM TWO: RADIALLY APPLIED ELECTRIC FIELD
Hypothesis and Objective
We hypothesize that the application of a radial electric field to an in vitro angiogenesis
model will alter growth factor expression and cell migration rates proximal to the center and
proximal to the edges of the treated area. The objective of this study is to determine whether or
not a radial electric field is superior to a linear electric field in targeting a specific region for
angiogenesis. Results from this study will be compared to results from our first specific aim. If a
radially applied electric field is shown to more effectively promote angiogenic factors than a
linearly applied electric field, our results will provide a rationale for the in vivo testing of a radial
electrode set-up.
Experimental Plan
In an in vivo application of linear electrical stimulation, an active electrode is placed
directly over the wound, while a passive electrode is placed some distance away. In this in vitro
model of a radially applied electric field, two electrodes will be used: one will be cylindrically
shaped, while the other will be a circular hoop.
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The electrodes will be inserted into the cell culture using the procedures described in our
first specific aim. The negatively charged cylindrical electrode will be placed in the center of the
outer hoop electrode and the cell culture. Each electrode will be connected to the arbitrary
waveform generator, completing the circuit, and an electrical stimulus with the same parameters
as the linear electric field will be applied to five experimental cell cultures for 30 minutes for a
period of 5 days. The control will be the same as the one used in specific aim one.
Previous studies have demonstrated the effect of electric field intensity on wound cell
proliferation37. In the linear electric field applied in the first specific aim, the intensity of the
electric field is more or less constant. In a radial electric field, however, the intensity of the
electric field varies by a factor of (r2)-1. Therefore, the intensity of the electric field will be
greater closer to the inner cylinder (see Figure 3).
If the intensity of the applied electric field correlates with better promotion of angiogenic
factors, we expect to find greater cell migration and angiogenic growth factor expression at the
center of the culture than the edges. At the closure of the study, the same cell analysis techniques
in specific aim one will be used to measure cell migration rates and growth factor concentration
in the culture regions closer to the outer hoop and the inner cylinder respectively. Data from
these measurements will be compared to each other, as well as data from our first specific aim
and our control angiogenesis model.
Alternatives
If the results from the experimental group do not show statistically significant increases
in the healing parameters when compared to the control group, an alternative solution must be
sought. We will return to the beginning of the experiment and change the voltage and frequency
of the electrical stimulation when applying to a single culture and solely test this culture for cell
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proliferation. If there is significant proliferation, we will continue the experiment to completion
with the new parameters. The intensity of the electric field is different in this aim as compared to
aim one, therefore there is a strong possibility of different parameters being optimal for specific
aim two.
If our findings indicate that the intensity of the electric field is not correlated with better
promotion of angiogenic factors even after different parameters have been tested, we will
conclude that a linearly applied electric field is the more effective design for an electrical
stimulus targeting angiogenesis in diabetic ulcers.
Statistical Analysis
We will use the ANOVA method of statistical analysis to assess the effects of a radially
applied electrical stimulus on cell migration rates, bFGF expression and VEGF expression in the
two specified regions of the cell culture. The -value will be set at the standard 0.05. P-values
will be calculated for each of the specified groups (cell migration, bFGF expression, and VEGF
expression). If the p-value for a group is less than 0.05, we will conclude (do we have to explain
this? Because in the comments heather asked about it) that there is a correlation between the
electrical stimulus and the angiogenic factor. Otherwise, we will conclude that the electrical
stimulus has no relationship with the angiogenic factor.
There are some confounding variables that may arise in the experimental design that
should be addressed. Observable expression of angiogenic factors may be caused by natural
healing processes instead of electrical stimulation. This is a threat to internal validity, as it is a
potential variation within each group. To address this variance, we will include a control in vitro
ulcer model that is not subjected to electrical stimulation. Additionally, in the case that
expression of an angiogenic factor in the in vitro model of the wound does not increase with the
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application of electrical stimulation, we will focus on observing other angiogenic factors and/or
cell migration in the in vivo portion of our experimental design.
Success Criterion
In order for the electrical stimulus to have an effect on angiogenesis, cell migration must
take place. Therefore, a correlation between cell migration and the electrical stimulus must be
observed in order for the experiment to be deemed successful. If a correlation is also observed
between the electrical stimulus and VEGF or bFGF expression, this will increase the success of
the experiment. Comparison of differences in angiogenic factors of endothelial cells after
application of a radial or linear electric field in vitro will determine the optimal method and
parameters of electrical stimulation for further testing and confirmation in vivo.
SPECIFIC AIM THREE: EVALUATING DEVICE ON IN VIVO MODEL
Hypothesis & Objective
For our third specific aim we hypothesize that the application of the optimal electrical
field determined in vitro, either a linearly applied electrical field or a radially applied electrical
field applied by our own electrical stimulation device will increase healing in diabetic ulcers by
decreasing inflammation. We will quantify a decrease in inflammation by an increase in cell
proliferation, angiogenic growth factors, and cell migration. The objective of our third specific
aim is to determine if the results found in the in vitro study can be replicated in vivo. Taking the
in vitro testing and successfully translating it into in vivo testing will provide significant
academic rationale for human studies of electrical stimulation on diabetic ulcers.
Experimental Plan
We will conduct our experiment on 4-8 week old male Sprague-Dawley rats that are
relatively the same size and weight. Ideally, we will use 10 rats for each experimental and
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control group38. We will induce wounds in the experimental group by scalding the rats briefly.
We will use a round iron with a diameter of approximately 2 cm to burn a section of skin on the
back for 30 seconds in order to make a deep scald. The layer of scald will then be removed 3
days later to simulate the ulcer39. In between electrical treatments, induced wounds will be
covered with sterile dressings40*.
The electrical stimulation will be applied to the wound either 1-cm apart, if using the
linear electrode set-up, or directly on the wound if using the radial electrode set up. The
parameters for electrical stimulation will be the ones optimized in the first two specific aims
although the current will still be a pulsed monophasic square wave41. The electrical stimulation
will be applied for 30 minutes, five days a week for a period of two weeks. Before each trial, the
electrodes will be placed on sterile pads cleaned with saline solution. The electrodes will then be
strapped tightly onto the subject in such a way as to both prevent electrode displacement14,42. The
control rats will not receive any electrical stimulation.
The ulcer area will be excised and used for cell analysis testing. The same tests as in the
first two specific aims will be used to measure cell proliferation, cell migration, and angiogenic
growth factors.
Alternatives
If for some reason, such as cost, we cannot use Immunohistochemistry, quantitative
reverse transcriptase PCR and Western Blotting techniques to measuring inflammation and
thereby measure healing, we could measure angiogenesis directly through a Matrigel plug assay.
Matrigel is a soluble basement membrane extract that allows endothelial cells to penetrate and
grow within the plug. This allows vascular tissue to take up root in the Matrigel plug. The
Matrigel is injected into the subject at the desired site, and after time the vascular tissue that has
*
See the Vertebrate Animal section for further detail.
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taken up in the Matrigel can be quantified by ultrasound43. Our original hypothesis stated that we
are measuring VEGF, FGF and cell migration because these are pre-angiogenic factors, and we
hypothesized that angiogenesis decreases inflammation and increases healing. We were using
VEGF, FGF and cell migration essentially as a measurement of angiogenesis and by using a
Matrigel plug assay, we can measure angiogenesis directly in the in vivo model.
Statistical Analysis
After collecting data, the results will need to be analyzed by conducting statistic tests.
For our experiment, we will primarily use the T-test, because we are only comparing two groups:
the control and the electrically stimulated experimental. The null hypothesis will be that the
difference between the groups is due to chance. Our alpha level will be 0.05 and if our t-value is
below the alpha value, the difference between our groups will be statistically significant and we
can conclude that the electrical stimulus had a considerable effect on the healing of the diabetic
ulcer.
Success Criterion
We will consider our experiments for our third specific aim successful if the experimental
group is observed to have significantly increased cell migration, levels of VEGF, bFGF and cell
proliferation. All these factors increase angiogenesis, thereby counteracting the effects of chronic
inflammation, which is prevalent in diabetic ulcers.
Timeline
Fall 2011
Winter 2011-2012
Finish Thesis Proposal Draft
Revise Thesis Proposal
Finalize Thesis Proposal
Spring 2012
Present Thesis Proposal (March)
Research Proposal
Submit HHMI, NSF, and ACCIAC grant applications
Complete laboratory training
Create team website
Lab work for Specific Aim 1
Fall 2012
Data analysis for Specific Aim 1
Present at Junior Colloquia
Write up for Specific Aim 1
Lab work for Specific Aim 2
Data analysis for Specific Aim 2
Spring 2013
Outline Team Thesis
Present at Undergraduate Research Day
Submit IACUC application
Assemble device
Summer 2013
Start writing team thesis
Lab work for Specific Aim 3
Data analysis for Specific Aim 3
Fall 2013
Finish analyzing data
Complete writing team thesis
Revise team thesis
Winter 2013-2014
Prepare thesis presentation
Prepare for thesis conference
Spring 2014
Present at thesis conference
26
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27
VERTEBRATE ANIMALS
Our experiment utilizing animal subjects will be conducted after University of
Maryland’s IACUC approval.
Species: Our in vivo experiment will be conducted on 1-year old male Sprague-Dawley rats of
the same size and weight. The rats will be housed in the Central Animal Resources Center at the
University of Maryland.
Approximate Number: Required sample size for each experimental animal model group was
calculated using a power analysis. First, the assay discriminating between the smallest significant
differences was identified. Previous studies identified this as the screening process for cell
migration using a Transwell (Boyden Chamber) Assay. In these studies, a permeable membrane
is used to measure cell movement through an extracellular matrix of endothelial cells. For this
analysis, a determination of sample size was conducted for the comparison of two independent
groups. (Note that independent groups are compared since different animals are used for each
time point studied.) Sample size for the comparison of two means from independent groups is
calculated using the formula n  2K2/2, where n is the sample size required in each group, K is
a constant based on significance level and power for comparison, 2 is the variance in groups
being compared, and  is the minimum difference in means that the study is required to detect.
Previous studies indicate that typical standard deviations () between stained areas were of the
magnitude of 3%44. The minimum difference detected was 5% since the experiment is designed
to detect significant changes, rather than small fluctuations in cell migration rates. The K
constant, based upon a two-sided significance test at a 5% confidence interval and with a power
to detect a treatment effect of 95%, is 13.0. This results in a sample size greater than or equal to
9.36, which was rounded up to 10. The experiments described are therefore based upon a sample
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size of 10 per time point. Please see Table 1 below for the allocation of the animal subjects for
the particular experiments.
Table 1: Number of Animal Subjects Required in the Proposed Research Program
Experimental
Control
Specific Aim
3
Required Animal
Time Points
Groups
Groups
1
1
Replicates
Subjects
1
10
20
Procedure: For the in vivo portion of our research we will be inducing diabetes in the rats to
analyze the effects of electrical stimulation on the rats as a diabetic ulcer model. To induce
diabetes in the experimental rats, 100 µL of streptozotocin in sterilized phosphate buffered saline
(PBS) will be injected. Rats will be anaesthetized with 80 mg/kg of Ketamine before injection.
Four days after injection, blood sugar test strips will be used to measure blood glucose levels,
and rats with a blood glucose level of at least 300 mg/dL will be considered as diabetic. In order
to minimize nutritional variables, the rats will be given standard rodent feed and tap water during
this process40. To induce the wound for formation of the diabetic ulcer after two months on the
experimental group, a round iron with a 2-cm diameter will be used to scald a part of the skin on
the back for 30 seconds. This layer of scald will be removed three days later to simulate the
ulcer40.
Personnel: All team members will be given proper laboratory training before beginning any in
vivo lab work. All lab work will be performed in our mentor, Dr. Fisher’s lab.
Assurance of Minimum Discomfort: All rats will be anaesthetized with approximately 80
mg/kg of Ketamine before a layer of skin is removed for streptozotocin injection. Rats will be
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kept in well-maintained cages, with their bedding changed twice a week. The room will be under
22-25 degrees Celsius temperature with a 12-hour light-dark cycle. Also, in between electrical
stimulation treatments, all wounds will be covered in new sterile dressing39.
PROJECTED BUDGET
Cell Culture (Rat aortic endothelial cells)
1000
Electrode Materials
274
Circuitry
180
In Vivo Model (Sprague-Dawley Rats)
873
Housing and Care for Rats
177
Cell Assay Supplies
1993
Projected Total
4497
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Appendix A: Methods
Creating angiogenic model:
In two-dimensional models, first, endothelial cells are seeded onto a plastic culture dish that has
been coated with adhesive proteins or loaded on top of a gel containing collagen, fibrin or
Matrigel. These two ways of seeding endothelial cells depend on the desired time length of the
angiogenesis assay—short term versus long term. While both models form the target CLS, shortterm models, with the benefit of the gel, can form CLS within 1 to 3 days of culture.
Determining parameters in the short-term model include number of cells seeded, cell
proliferation, and concentration and composition of the gel. A limitation of the short-term
models is that they do not take into account the proliferation and migration steps, so it is difficult
to maintain CLS over long periods of times. In long-term models, CLS develop on top of a
confluent monolayer of cells. CLS are not systematically observed in long-term models, and are
less reproducible than short-term models.
Creating radial electrodes:
A cylindrical glass shape will be cut from a 5-centimeter diameter block of glass. To mold a
hollow circular glass shape, lithography will be used. Prior to the process, a mask of the depth of
a microscope slide will be created from a 5-centimeter diameter block of glass. The metal mask
used to create the hoop electrode will cover circular sections in the center on both sides of the
block of glass. The circular glass block will be covered with liquid photoresist and then inserted
into the masks. At this point, UV light will be applied to all sides of the glass. The photoresist on
exposed areas of the glass will harden, whereas photoresist on unexposed areas will not. The
block of glass will then be removed from its mask and submerged in an acid. Areas of glass not
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coated with hardened photoresist will dissolve, creating a hoop shaped piece of glass. The
electrodes will be coated in titanium using the procedures described in our first specific aim.
Cell scraping:
To analyze angiogenic growth factor expression and cell migration rates in different areas of the
endothelial cell culture, the petri dish will be divided, before the application of an electric field,
into three different regions. The regions will be marked as concentric circles, dividing the culture
into an outer area, a middle area, and an inner area. The radii of the circles will be such that the
outer, middle and inner areas are equal. The outermost area will correspond to the least intense
electric field, whereas the inner area will correspond to the most intense electric field. The
average intensity of electric field in the middle area will be in between the least and most intense
electric fields.
A standard cell scraper, sterilized by irradiation, with a blade width of
approximately 1.75 cm, will be used to separate the cells from different regions after application
of the electric field.
Inducing Diabetic Ulcer:
We can induce a diabetic ulcer through two general processes. First, we can induce by way of a
burn. To do this we sterilize and shave the skin. Then we water boil a round iron and apply to the
skin for 2 – 30 s. The wound is then sterilized and covered with sterile dressing. The burn is
allowed to scab, then 1-3 days later the scab/dead skin is removed. Diabetic ulcers can also be
induced by removing a section of skin. First, we sterilize and shave the skin. Then we cut out a
section of skin of standard volume and on the same spot on each animal, and sterilize and cover
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the wound. We sterilize wounds by applying Hydrogen Peroxide or Povidone Iodide to the
wound and cover it with sterile dressing. Dressings should be changed once a day.
Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): Quantitative, reverse
transcriptase polymerase chain reaction will be performed using appropriate oligonucleotide
primers and probes, a TaqMan EZ RT-PCR kit, and sequence detector system (Applied
Biosystems) available in the PI’s Biomaterials Laboratory.45 Appropriate oligonucleotide primers
and probes for VEGF and bFGF will be developed following standard Applied Biosystems
protocols. Furthermore, the proper working concentrations for the forward primer, reverse
primer, and probe for each protein of interest will be determined following standard methods.
Briefly, a 3x3 factorial study of forward primer and reverse primer concentrations (with constant
probe concentration) is carried out, using 0.05, 0.30, and 0.90 μmol concentrations. The data is
then inspected for the forward and reverse primer concentration which results in the earliest
crossing of the threshold concentration and lowest variability. This procedure will be carried out
for all proteins of interest.
To carry RT-PCR studies, a primer and probe solution of predetermined concentration is
first fabricated. A master mix of buffer, manganese acetate, dATP, dCTP, dGTP, dUTP, DNA
polymerase, and amperase is also fabricated following standard methods. The RNA sample is
then added to the master mix. The master mix + RNA sample is the added to the appropriate
wells of a 96 well plate, followed by the addition of the primer – probe solution. This procedure
is carried out for each protein of interest. The RT-PCR reaction will be carried out on a ABI
Prism 7000 sequence detector, using thermal cycling conditions of 2 min at 50°C, 30 min at
60°C, 5 min at 95°C, and 40 cycles of 20 sec at 94°C and 1 min at 62°C. All RNA samples will
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be studied in triplicate. All qRT-PCR studies will be carried out in reference to GAPDH.
Results will be expressed as mean fold change and propagated errors.
Western Blotting: The concentration of the various proteins of interest (VEGF and bFGF) within
the tissue sample will be assessed by Western blotting. Initially, experimental samples will be
collected and, if in vitro, the hydrogel matrix physically disrupted. Cell culture media will be
added to the disrupted sample, and the suspension centrifuged for 10 min at 1000 rpm. The
supernate will be removed by aspiration and the procedure will be repeated twice so as to isolate
the maximum amount of the sample’s protein content. After the final centrifugation, lysis buffer
is added and the sample is homogenized by 5 passes through a syringe. A buffer solution
containing β-mercaptoethanol is added to the sample, and the solution boiled for 5 min at 95°C.
The samples, along with size markers, are then run in a polyacrylamide gel for 75 min at 160 V.
The gel is then blotted onto a PVDF membrane for 90 min at 25 V. The membrane is rinsed and
the proteins of interest realized either by Coomassie Blue or antibody staining.
Euthanasia and Harvesting of Tissue: All animals are sedated and sacrificed following an
approved protocol. Briefly, a dose of a ketamine, acepromazine cocktail is first given for deep
sedation. After sedation, an intravenous injection of an overdose of a pentobarbital preparation
is given to sacrifice the animal. The sample and surrounding tissue are then dissected intact
using a scalpel.
Immunohistochemistry: Tissue sections are immunostained following a standard protocol.(46,
See Appendix) The slides are prepared by encircling the sections with a hydrophobic ink. The
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slides are first incubated in 95% ethanol (300 µl) for 3 min and then rinsed with distilled water (3
ml). Next, the endogenous peroxidase activity is blocked with a H2O2/CH3OH solution (300 µl)
incubation for 1 hr and then rinsed twice with immunohistochemical (IHC) buffer (3 ml).
Random secondary antibody binding is then blocked with a normal blocking serum (300 µl)
incubation for 1 hr. (The normal blocking serum is from the same species as the secondary
antibody to be used later.) The primary antibody (300 µl) is then bound for 2 hr and followed by
two rinses of IHC buffer (3 ml). The biotinylated secondary antibody (300 µl) is then incubated
for 1 hr and followed by two rinses of IHC buffer (3 ml). The ABC reagent (300 µl) is incubated
next for 1 hr, followed by two rinses of IHC buffer (3 ml). The DAB developing reagent (300
µl) is incubated for 10 min and then followed by a distilled water (3 ml) rinse. The sections are
then stained with hematoxylin (300 µl) for 8 min, rinsed with distilled water (3 ml), and clarified
with acid alcohol (300 µl) for 3 min. Finally, the sections are rinsed twice with distilled water (3
ml) and then dehydrated with 2 x 95% ethanol (300 µl for 3 min) and 2 x 100% ethanol (300 µl
for 3 min) rinses. The slides are rinsed in xylene and mounted. Negative controls are obtained
by incubating the sections with 0.01M PBS in place of the primary antibody. A final group of
sections undergo conventional hematoxylin and eosin staining.
Transwell Assay:
Check tissue culture flasks to ensure endothelial cell cultures are viable and have achieved
confluence. Before passage of cells, prepare 0.1% gelatin mixture to place on transwell insert.
Place mixture into glass flask and place on hot plate with stir bar. Heat and stir mixture gently
for 4-5 minutes, or until solution clears. Pass mixture through filtration system. Remove
transwell inserts from 24 well plates and place them inverted into a 12 well plate. Pipette 100 μL
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of 0.1% gelatin solution over each inverted transwell insert. Place plate into incubator at 37°C
for 2 hours. Passage cells as described in manufacturer’s protocol. Place cells into 50cc conical
tube and centrifuge at 250 x g, 4°C, 5 minutes. Discard supernatant. Add 1 ml culture medium
and count live cells with 0.02% Trypan Blue. Resuspend cells at a concentration of
7.5×105 cells/ml. Remove plate from incubator and remove liquid gelatin overlying insert with
pipette, being careful not to scratch or puncture the membrane. Place 100 μl of resuspended cells
(7.5×104 cells) on inverted gelatin covered transwell insert. Replace into incubator for 3 days.
Remove culture plate from incubator. Remove a single transwell insert from the culture plate and
replace plate into incubator. Fix transwell in methanol for 30 seconds. Place sample in
Hematoxylin component stain for 30 seconds. Transfer sample to Eosin component stain for 30
seconds. Rinse in water for 5 seconds. Remove mesh membrane with endothelial cells from
transwell insert with sharp forceps, being careful not disrupt the cell monolayer. Place on slide
with tissue fixative and cover with slide cover. Assess for evenly confluent cell layer. If
endothelial cells have reached confluence, may proceed to transendothelial migration assay.
After assessing confluence as per directions above, remove 12-well plates with inverted
transwell inserts from incubator. Remove bead of medium overlying inverted transwell inserts
with 200 μl pipette, being careful not to scratch the surface of the membrane. Remove inverted
transwell inserts from 12 well plates and replace in proper position into 24 well plates. Place 600
μl of complete medium with appropriate chemotactic signal (e.g., chemokine) in bottom of well.
Add 100 μl of previously prepared T cells (5×105 cells) to upper chamber of transwell. Set up
wells in triplicate for each condition. Incubate, 37°C, 4 hours. Remove plate from incubator.
Remove transwell inserts and gently agitate remaining unmigrated cells in upper chamber with a
pipette and remove for quantification. Membranes with endothelial monolayers can be removed
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with sharp forceps and stained per local laboratory protocol to asses cells within endothelium.
Resuspend cells in lower well in 1 ml medium and count using hemacytometer to quantify
migration.
ELISA
Antigen Coating
Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer or
PBS. Pipette 0.2 ml of the above solution to each well of the microtiter plate. Incubate at 37 °C
for 30 min., or incubate (covered) overnight at 4 °C. Remove the coating solution. Wash three
times with PBS-T.
Primary Antibody Reaction
Dilute the monoclonal primary antibody in PBS-T. The optimal dilution should be determined
using a titration assay. Add 0.2 ml of the diluted monoclonal antibody to each well. The
negative control should be species- and isotype-matched, non-specific immunoglobulin diluted
in PBS-T. Incubate at room temperature for 2 hours. Wash as in step 4 of Antigen Coating.
Application of Secondary Antibody
Dilute the enzyme-conjugated secondary antibody in PBS-T. Add 0.2 ml of this solution to each
well. The optimal dilution should be determined using a titration assay. Incubate at room
temperature for 2 hours. Wash as in step 4 of Antigen Coating.
Substrate Preparation
During the last incubation and immediately before use, prepare the enzyme substrate or bring the
premade liquid substrate to room temperature.
Development
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Add 0.2 ml of the freshly prepared substrate to each well. Color should develop in positive wells
after 30 minutes (yellow or orange, for pNPP or OPD, respectively). Absorbance may be read
directly in a microplate reader (at 405 nm or 450 nm, for pNPP or OPD, respectively) or the
reaction may be stopped with 50 µl per well of the appropriate stopping reagent and absorbance
read later (at 405 nm or 492 nm, for pNPP or OPD, respectively).
Hemocytometer
Place the coverslip over the hemocytometer counting chamber and using a Pasteur pipette, place
a drop of the cell suspension at the edge of the “V” shape of the chamber. Allow the suspension
to be drawn into the chamber by capillary action. Care should be taken not to overfill or underfill
the chamber. Fill the opposite chamber in the same manner. Place the chamber on the
microscope stage. The hemocytometer consists of nine 1 mm squares divided into smaller
squares. One of
the 1 mm squares represents a volume of 0.1 mm^3 or 10^-4 ml. Using the 10X
objective, count the number of cells in a 1 mm square area (see figure 1). If there are fewer than
100 cells in a square mm, 2 or more 1-mm square areas should be counted and the results
averaged. Use the same procedure to count the cells on the other side of the hemocytometer. To
calculate the concentration of the cells, first calculate the average of all 1mm^2 areas counted
and apply this formula:
c=n/v where: c = cell concentration in cells/ml, n = avg. number of cells/mm^2 area, v = volume
counted = 10^-4 and thus c = n x 10^-4.
Appendix B: Glossary
Actin tail polymerization: creation of the end of the actin protein
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Angiogenesis: the generation of new blood-vessels, involved in the process of wound healing
Anode: terminal where the current flows into a polarized electrical device from the outside
Arbitrary waveform generator (AWG): electronic test equipment that generates repetitive or
single-shot waveforms for devices under testing. It allows for complete control over voltage,
pulse, and frequency settings.
Cathode: terminal where the current flows out of a polarized electrical device
Capillary density: the number of capillaries within a given area
Cell migration: cell processes in multicellular organisms involved in development of tissue
Endogenous electric field: electric field within a cell
Fibroblast: cell that makes the extracellular matrix and collagen
Granulation: perfused, fibrous connective tissue that replaces clots
Golgi apparatus: membrane-bound organelle that packages, sorts, and sends proteins to
different parts of the cell
Hemocytometer: device used to count cells
Hydrostatic: pressure created by a fluid at rest
Immunohistochemistry: a laboratory technique used to detect presence of specific proteins in a
certain tissue sample by binding to protein specific antibodies. These bound proteins are visible
through fluorescent tagging, and can help us determine the concentration of VEGF.
Inflammatory response: The first stage of wound healing in which body defends against
harmful substances, disposes of dead or dying tissue and promotes the renewal of normal tissue
Keratinocyte: cell in the upper layer of skin
Lithography: method used to structure materials such as glass
Macrophage: defense cells
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Matrigel: gelatinous protein mixture that resembles the extracellular matrix of a cell
Metabolism: chemical reactions that sustain life
Negative Pressure Wound Therapy: a diabetic ulcer treatment, where a vacuum creates a
decreased amount of tissue pressure at the pore site, resulting in vasodilatation, easier blood flow
to the wound site and increased perfusion
Neuropathy: damage to nerves which causes diminished ability to feel pain or other sensations
Perfusion: supplying nutrients to a particular tissue or organ through blood vessels
Peripheral arterial disease: condition where plaque builds up in the arteries
Phagocytosis: a cellular process where the cellular membrane engulfs solid particles
Photoresist: a light-sensitive material that forms a pattern coating on a surface
Proteolytic activity: the degradation of proteins
Pulsed biphasic current: on-and-off current in two directions
Pulsed monophasic current: on-and-off current in only one direction
Quality of Life: term used to evaluate the well-being of an individual
Streptozotocin: chemical toxic to insulin-producing beta cells of the pancreas, sometimes used
to treat severe inoperable cases of pancreatic cancer
Reverse Transcriptase PCR: laboratory technique used to make many copies of mRNA
Type I Diabetes: an autoimmune disorder that attacks the cells in the insulin-producing pancreas
Type II Diabetes: acquired disease in which the body develops insulin resistance and is unable
to use insulin to absorb glucose from the bloodstream
Vacuum evaporation titanium deposition: a technique used to deposit a thin-layer of coating
on a surface
Vascularization: the formation of new blood vessels in tissues
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Vasoconstriction: narrowing of blood vessels
Western Blotting: detects specific proteins in a sample of tissue, employs gel electrophoresis to
separate native and/or denatures proteins and then uses antibodies specific to a given protein for
identification
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References
1.
Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes. J
Clin Invest 2007;117(5):1219-22.
2.
Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: molecular and
cellular mechanisms. J Invest Dermatol 2007;127(3):514-25.
3.
Li WWM, Li VWM, Tsakayannis DM. Angiogenesis in Wound Healing. Contemporary
Surgery 2003.
4.
Zhao M. Electrical fields in wound healing-An overriding signal that directs cell
migration. Semin Cell Dev Biol 2009;20(6):674-82.
5.
Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT,
Horwitz AR. Cell migration: integrating signals from front to back. Science
2003;302(5651):1704-9.
6.
Asadi MR, Torkaman G, Hedayati M. The Effect of Electrical Stimulation Intensity on
VEGF Expression and Biomechanical Properties during Wound Journal of
Rehabilitation Research and Development 2011;48:195-202.
7.
Kloth LC, Feedar JA. Acceleration of wound healing with high voltage, monophasic,
pulsed current. Phys Ther 1988;68(4):503-8.
8.
Boulton AJ, Kirsner RS, Vileikyte L. Clinical practice. Neuropathic diabetic foot
ulcers. N Engl J Med 2004;351(1):48-55.
9.
Edwards J. Debridement of diabetic foot ulcers. The Cochrane Library 2009.
10.
Koh TJ, DiPietro LA. Inflammation and wound healing: the role of the macrophage.
Expert Rev Mol Med 2011;13:e23.
Research Proposal
11.
42
Szpaderska AM, Egozi EI, Gamelli RL, DiPietro LA. The effect of thrombocytopenia on
dermal wound healing. J Invest Dermatol 2003;120(6):1130-7.
12.
Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing. J Leukoc Biol
2001;69(4):513-21.
13.
Loots MA, Lamme EN, Zeegelaar J, Mekkes JR, Bos JD, Middelkoop E. Differences in
cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers
versus acute wounds. J Invest Dermatol 1998;111(5):850-7.
14.
Talebi G, Torkaman G, Firoozabadi M, Shariat S. Effect of anodal and cathodal
microamperage direct current electrical stimulation on injury potential and wound
size in guinea pigs. J Rehabil Res Dev 2008;45(1):153-9.
15.
Pullar CE, Isseroff RR. Cyclic AMP mediates keratinocyte directional migration in an
electric field. J Cell Sci 2005;118(Pt 9):2023-34.
16.
Asadi MR, Torkaman G, Hedayati M. Effect of sensory and motor electrical
stimulation in vascular endothelial growth factor expression of muscle and skin in
full-thickness wound. Journal of Rehabilitation Research & Development
2011;48(3):195-201.
17.
Pullar CE. The physiology of bioelectricity in development, tissue regeneration, and
cancer. Boca Raton: CRC Press; 2011. xiv, 304 p. p.
18.
Hampton S, King L. Healing an intractable wound using bio-electrical stimulation
therapy. British Journal of Nursing (BJN) 2005;14(15):S30-S32.
19.
R B, G S. The Embryo at the Wound, The Sign of the Miracle, Life’s Potentials, The
Self-Mending Net. The Body Electric. New York, NY: William Morrow and Company
Inc.; 1985. p 55-86, 204-205.
Research Proposal
20.
43
Chapman-Jones D, Young S, Tadej M. Assessment of wound healing following
electrical stimulation with Accel-Heal. Product Review 2010;6(3):4.
21.
Zhao M, Bai H, Wang E, Forrester JV, McCaig CD. Electrical stimulation directly
induces pre-angiogenic responses in vascular endothelial cells by signaling through
VEGF receptors. J Cell Sci 2004;117(Pt 3):397-405.
22.
Gourcerol G, Chaput U, LeBlanc I, Gallas S, Michot F, Leroi AM, Ducrotte P. Gastric
electrical stimulation in intractable nausea and vomiting: assessment of predictive
factors of favorable outcomes. J Am Coll Surg 2009;209(2):215-21.
23.
Petrofsky JS, Lawson D, Suh HJ, Rossi C, Zapata K, Broadwell E, Littleton L. The
influence of local versus global heat on the healing of chronic wounds in patients
with diabetes. Diabetes Technol Ther 2007;9(6):535-44.
24.
Burdge JJ, Hartman JF, Wright ML. A study of HVPC as an adjunctive therapy in limb
salvage for chronic diabetic wounds of the lower extremity. Ostomy Wound Manage
2009;55(8):30-8.
25.
Callaghan MJ, Chang EI, Seiser N, Aarabi S, Ghali S, Kinnucan ER, Simon BJ, Gurtner
GC. Pulsed electromagnetic fields accelerate normal and diabetic wound healing by
increasing endogenous FGF-2 release. Plast Reconstr Surg 2008;121(1):130-41.
26.
Zhao M, Bai H, Wang E, Forrester J, McCaig C. Electrical Stimulation Directly Induces
Pre-Angiogenic Responses in Vascular Endothelial Cells by Signaling through VEGF
Receptors. The Journal of Cell Science 2004;117:397-405.
27.
Cinar K, Comlekci S, Senol N. Effects of a specially pulsed electric field on an animal
model of wound healing. Lasers Med Sci 2009;24(5):735-40.
Research Proposal
28.
44
Athanasiou A, Karkambounas S, Batistatou A, Lykoudis E, Katsaraki A, Kartsiouni T,
Papalois A, Evangelou A. The effect of pulsed electromagnetic fields on secondary
skin wound healing: an experimental study. Bioelectromagnetics 2007;28(5):362-8.
29.
Tonnesen MG, Feng X, Clark RAF. Angiogenesis in Wound Healing. Journal of
Investigative Dermatology Symposium Proceedings 2000;5:40-46.
30.
Sebastian A, Syed F, Perry D, Balamurugan V, Colthurst J, Chaudhry IH, Bayat A.
Acceleration of cutaneous healing by electrical stimulation: Degenerate electrical
waveform down-regulates inflammation, up-regulates angiogenesis and advances
remodeling in temporal punch biopsies in a human volunteer study. Wound Repair
Regen 2011;19(6):693-708.
31.
Caiado F, Carvalho T, Silva F, Castro C, Clode N, Dye JF, Dias S. The role of fibrin E on
the modulation of endothelial progenitors adhesion, differentiation and angiogenic
growth factor production and the promotion of wound healing. Biomaterials
2011;32(29):7096-105.
32.
Roy S, Driggs J, Elgharably H, Biswas S, Findley M, Khanna S, Gnyawali U, Bergdall
VK, Sen CK. Platelet-rich fibrin matrix improves wound angiogenesis via inducing
endothelial cell proliferation. Wound Repair Regen 2011;19(6):753-66.
33.
Nicosia RF, Nicosia SV, Smith M. Vascular endothelial growth factor, platelet-derived
growth factor, and insulin-like growth factor-1 promote rat aortic angiogenesis in
vitro. Am J Pathol 1994;145(5):1023-9.
34.
Graiani G, Emanueli C, Desortes E, Van Linthout S, Pinna A, Figueroa CD, Manni L,
Madeddu P. Nerve growth factor promotes reparative angiogenesis and inhibits
Research Proposal
45
endothelial apoptosis in cutaneous wounds of Type 1 diabetic mice. Diabetologia
2004;47(6):1047-54.
35.
Machida A, Hasegawa M, Koga I. Arbitrary waveform generator. 1991 14-16 May
1991. p 251-257.
36.
Nagasaka M, Kohzuki M, Fujii T, Kanno S, Kawamura T, Onodera H, Itoyama Y, Ichie
M, Sato Y. Effect of low-voltage electrical stimulation on angiogenic growth factors in
ischaemic rat skeletal muscle. Clin Exp Pharmacol Physiol 2006;33(7):623-7.
37.
Binhi VN, Goldman RJ. Ion-protein dissociation predicts 'windows' in electric fieldinduced wound-cell proliferation. Biochim Biophys Acta 2000;1474(2):147-56.
38.
Lau TW, Lam FF, Lau KM, Chan YW, Lee KM, Sahota DS, Ho YY, Fung KP, Leung PC,
Lau CB. Pharmacological investigation on the wound healing effects of Radix
Rehmanniae in an animal model of diabetic foot ulcer. J Ethnopharmacol
2009;123(1):155-62.
39.
Xie L, Zhang M, Dong B, Guan M, Lu M, Huang Z, Gao H, Li X. Improved refractory
wound healing with administration of acidic fibroblast growth factor in diabetic
rats. Diabetes Res Clin Pract 2011;93(3):396-403.
40.
Choi JS, Leong KW, Yoo HS. In vivo wound healing of diabetic ulcers using
electrospun nanofibers immobilized with human epidermal growth factor (EGF).
Biomaterials 2008;29(5):587-96.
41.
Feedar JA, Kloth LC, Gentzkow GD. Chronic Dermal Ulcer Healing Enhanced with
Monophasic Pulsed Electrical Stimulation. Physical Therapy 1991;71(9):10.
Research Proposal
42.
46
Weber SA, Vonhoff PA, Owens FJ, Byrne JA, McAdams ET. Development of a Multi –
Electrode Electrical Stimulation Device to Improve Chronic Wound Healing. 2009;
Minneapolis, Minnesota. IEEE. p 2145-2148.
43.
Stieger S, Bloch S, Foreman O, Wisner E, Ferrara K, Dayton P. Ultrasound assessment
of angiogenesis in a matrigel model in rats. Ultrasound in Medicine & Biology
2006;32(5):673-681.
44.
Li YH, Zhu C. A modified Boyden chamber assay for tumor cell transendothelial
migration in vitro. Clin Exp Metastasis 1999;17(5):423-9.
45.
Fisher JP, Jo S, Mikos AG, Reddi AH. Thermoreversible hydrogel scaffolds for
articular cartilage engineering. J Biomed Mater Res 2003:Submitted.
46.
Fisher JP, Lalani Z, Bossano CM, Brey EM, Demian N, Johnston CM, Dean D, Jansen JA,
Wong ME, Mikos AG. Effect of biomaterial properties on bone healing in a rabbit
tooth extraction socket model. J Biomed Mater Res 2004;68(3):428-38.