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

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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 non-

constant 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 3-

kinase 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 year-

long 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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Research Proposal

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 Finish Thesis Proposal Draft

Winter 2011-2012 Revise Thesis Proposal

Spring 2012 Finalize Thesis Proposal

Present Thesis Proposal (March)

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Research Proposal

Submit HHMI, NSF, and ACCIAC grant applications

Complete laboratory training

Create team website

Fall 2012

Lab work for Specific Aim 1

Data analysis for Specific Aim 1

Present at Junior Colloquia

Spring 2013

Write up for Specific Aim 1

Lab work for Specific Aim 2

Data analysis for Specific Aim 2

Outline Team Thesis

Present at Undergraduate Research Day

Submit IACUC application

Summer 2013Assemble device

Start writing team thesis

Fall 2013

Lab work for Specific Aim 3

Data analysis for Specific Aim 3

Finish analyzing data

Complete writing team thesis

Winter 2013-2014Revise team thesis

Prepare thesis presentation

Spring 2014Prepare for thesis conference

Present at thesis conference

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Research Proposal

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 2K2/2, 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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Research Proposal

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

Specific AimExperimental

Groups

Control

GroupsTime Points Replicates

Required Animal

Subjects

3 1 1 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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Research Proposal

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

Appendix A: Methods

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Research Proposal

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, short-

term 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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Research Proposal

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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Research Proposal

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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Research Proposal

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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Research Proposal

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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Research Proposal

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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Research Proposal

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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Research Proposal

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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Research Proposal

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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Research Proposal

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

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Research Proposal

Vascularization: the formation of new blood vessels in tissues

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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Research Proposal

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