Using Growth Factors to Treat Chronic Tympanic Membrane Perforation in a
Mouse Model
Kendall Weierich
May 5th, 2016
USING GROWTH FACTORS TO TREAT CHRONIC TYMPANIC MEMBRANE
PERFORATION IN A MOUSE MODEL
An Honors Thesis Submitted to
the Department of Biology
in partial fulfillment of the Honors Program
STANFORD UNIVERSITY
by
KENDALL WEIERICH
MAY 5th, 2016
Preface
First and foremost, I would like to extend my deepest gratitude to Dr. Peter
Santa Maria for his guidance. It is hard to find words to describe my appreciation for
everything he has done to help me throughout this process. His enthusiasm, humor,
and investment made working with him an enjoyable and fantastic experience.
Not only did he care about me producing quality work, but he also cared about
ensuring my excitement for the project and gave me a valuable glimpse into the field of
otolaryngology.
I would also like to thank Dr. Yunzhi Peter Yang. I met Dr. Yang as my premajor advisor coming into Stanford. He was the first to expose me to the field of
research and so graciously invited me to join his lab. I have learned more than I could
have ever imagined under his mentorship, and have gained an entirely new
appreciation for science. His advice throughout my Stanford career has proved
indispensable.
Two other individuals have contributed to the success of this project and to the
formation of my academic interests at Stanford. The first is Dr. Sungwoo Kim of the
Yang Lab, who was very helpful in running the ELISA and providing additional
support as I executed the studies. The second is Dr. Kang Shen. As my major advisor
and second reader, Dr. Shen sparked my interest in neurobiology at Stanford. He was a
professor of mine for two quarters in the Biology core and in Bio 154: Molecular and
Cellular Neurobiology, one of the most challenging yet rewarding courses I have had
the privilege to take. His passion for neurobiology strengthened my own interest in the
track and is one of the reasons I am here today.
Two courses of mine have especially prepared me for my thesis-writing process:
PWR2: The Rhetoric of Science with Dr. Jennifer Stonaker, and Bio199W: Senior
Honors Thesis with Dr. Russ Carpenter and Sally Kim. These classes have
demonstrated to me the importance of effective scientific communication, and have
challenged me to improve my own writing, speaking, and presentation skills. Within
Bio 199W, I would like to thank my group members Bhaven Patel and Nick Flores for
critically engaging with my work, posing thoughtful questions, and providing fantastic
feedback.
This project would not have been possible without support from the Garnett
Passe and Rodney Williams Memorial Foundation, Stanford’s SPARK Stanford Child
Health Research Institute NIH R01AR057837 (NIAMS, Y.Y.), NIH R01DE021468
(NIDCR, Y.Y.), DOD W81XWH-10-1-0966 (PRORP, Y.Y.), and Wallace H. Coulter
Foundation (Y.Y.).
Finally, I would like to thank my family and friends for their encouragement
and support throughout this process. From their input on my work to their patience
with me as I practiced the same presentation over and over again, my family and
friends have been invaluable to me as both a scientist and scholar.
Table of Contents
List of Diagrams, Tables, and Figures ..............................................................................1
Abstract ...............................................................................................................................2
Introduction ........................................................................................................................3
Relative anatomy .....................................................................................................4
Current standard of care ........................................................................................4
Growth factors: A potential solution ......................................................................5
Reproduction of EGF, FGF-2 studies .....................................................................6
Exploring Efficacy of HB-EGF in TM perforation regeneration ...........................7
Materials and Methods ......................................................................................................9
Results ..............................................................................................................................13
Healing of chronic TM perforations by treatment group..................................................13
Macroscopic analysis of unhealed perforations................................................................13
Histological analysis of healed perforations.....................................................................14
Discussion ........................................................................................................................15
Bibliography .....................................................................................................................18
Diagrams...........................................................................................................................21
Tables ................................................................................................................................23
Figures ..............................................................................................................................25
List of Diagrams, Tables, and Figures
Diagrams
Diagram 1
Diagram of the anatomy of the ear
Diagram 2
Diagram of a healthy and a perforated tympanic membrane
Tables
Table 1
Table 1 outlines the treatment groups used in the study
Table 2
Table 2 shows the efficacy of HB-EGF, EGF and FGF-2 compared to control
Figures
Figure 1
Cytokeratin staining of chronic TM perforations after treatment with either HB-EGF,
FGF-2 or EGF
Figure 2
Hematoxylin and Eosin staining of perforation edges of chronic TM perforations after
treatment with HB-EGF, FGF-2, or EGF
1
Abstract
Hearing loss can occur at any age, and is caused by a multitude of factors. One
common source of conductive hearing loss is due to the rupture or perforation of the
eardrum. Such perforations of the eardrum [or tympanic membrane (TM)] are caused by
trauma or disease and can be classified as either acute or chronic. Acute perforations are
temporary and can heal spontaneously, whereas chronic perforations may require surgical
intervention (usually a grafting procedure called a tympanoplasty) to repair. A nonsurgical treatment for chronic TM perforation would benefit those unable to access
surgery and would reduce the cost. Previous work using nonsurgical growth factor
treatments has involved the use of epidermal growth factor (EGF) or fibroblast growth
factor 2 (FGF-2) to reinitiate wound healing in the TM. However, results from these
studies are difficult to interpret since the experiments used acute TM perforations, which
tend to heal spontaneously. Here, we explore whether heparin binding epidermal growth
factor like growth factor (HB-EGF) heals chronic TM perforations with greater efficacy
than FGF-2 and EGF in a validated mouse model of chronic tympanic membrane
perforation. To test this, we surgically created chronic perforations in the mouse
tympanic membrane and administered the above growth factors via an injectable
hydrogel polymer. Relative TM closure was observed macroscopically and histologically
over a three-month period. HB-EGF showed to be significantly more effective in closing
chronic TM perforations over EGF and FGF-2. The healing rates of the TMs after
treatment compared to control were 83.3% with HB-EGF, 15.8% with EGF, 31.6% with
FGF-2 and 27.8% with control (no growth factor). An effective nonsurgical treatment,
2
such as use of applied growth factors for chronic TM perforations would provide a safer
and cost-effective alternative for those unable to have surgery.
Introduction
Tympanic membrane perforation is a common problem in otolaryngology. Caused
by trauma or disease—particularly infection—TM perforations are often accompanied by
mild to severe hearing loss in 50% of cases that can be both temporary or permanent,
vertigo, and liquid discharge.1–3 These perforations can be classified as either acute or
chronic. Although most acute perforations heal spontaneously, chronic perforations—
those that persist for more than 3 months— fail to heal spontaneously and often require
surgical intervention. With over one hundred thousand surgeries being performed in the
United States every year, the current standard of care for chronic perforations consists of
a surgical autograft of the TM, a type 1 tympanoplasty (myringoplasty), to improve
hearing and reduce further infection4,5. However, initial graft take rates can be as low as
65%.5,6 The benefits of non-surgical treatment in areas where healthcare is readily
available would include a reduced need for general anesthetic, lower cost, and
convenience, and provide a treatment option to those who cannot have surgery. In areas
where surgical treatment is not as accessible, the benefits would extend treatment to these
populations and would reduce the chance of infection by eliminating the need for surgery.
To this end, a novel non-surgical solution for chronic TM perforation would provide an
superior alternative in an area of unmet need.
3
Relevant anatomy
The tympanic membrane (TM) is an integral component of the ear that separates
the external ear from the middle ear (Diagram 1).7 It is semitransparent and made of
three layers: an outer layer of stratified squamous epithelium, a middle fibrous layer that
anchors the TM in the ear canal, and an inner mucosal layer of squamous epithelium. The
TM is approximately 0.1 mm thick and 8-10 mm in diameter, and is responsible for
transmitting sound from the air to the auditory ossicles, which are small bones in the
tympanic cavity (middle ear).8 The membrane is held in place by a thick, flexible ring of
fibrous tissue (Diagram 2).9 The transduction of sound occurs when soundwaves strike
the tympanic membrane, causing it to vibrate with the force of the strike. The vibrations
are transmitted to the tympanic cavity where further sound transmittance and processing
occurs. Patients who experience a traumatic rupture of the TM can have difficulty hearing
and possibly complete hearing loss if the perforation remains open for a considerable
amount of time.
Current standard of care
Today, the most common method of repairing chronic tympanic membrane
perforation consists of a surgical repair with autologous tissue. Myringoplasty, which is
the surgical correction of a perforation in the eardrum, is often employed to address such
an issue and is currently regarded as the most reliable and effective treatment for TM
perforation.10,11 The procedure requires general anesthesia and utilizes the patient’s own
tissue to minimize rejection. Many autologous tissue types used for grafting include
muscle fascia, perichondrium, and cartilage.12 However, there is little evidence to support
4
any of the above tissue types as optimal substitutes in treating various types of tympanic
membrane perforations.5 In addition, as mentioned earlier initial autograft take rates have
been as low as 65%.5,6 A continued search for better materials and methods to achieve
optimal healing and hearing improvement is required in order to improve patient
outcomes.
Growth factors: A potential solution
Non-surgical strategies have explored the utilization of various scaffolds, cells, and
regenerative biomolecules in treating chronic TM perforations, but these have yet to
demonstrate superiority to surgery.13,14 One such strategy involves growth factors as
bioagents, which can be especially useful in this settling.
Growth factors have been commonly studied as regenerative biomolecules that can
aid in TM perforation healing. Known to be heavily involved in epithelial cell migration
and wound repair, growth factors (particularly those of the EGF family) play a key role in
cell growth and regulation, and have been researched in their ability to aid in both a
general wound healing process as well as the healing of the TM. Growth factors used in
the regeneration of the TM are they hope to result in a reconstitution of all three layers of
the TM.15
Application of various growth factors to the TM have been used to attempt
regrowth and migration of keratinocytes to TM wounds. Prior studies have claimed
success in using nonsurgical growth factor treatments which usually consist of
incorporating various growth factors with scaffolds such as gelfoam, paper patches, and
hydrogel polymers to reinitiate wound healing in the TM. 17–23 Two growth factor
5
treatments in particular, fibroblast growth factor (FGF-2) and epidermal growth factor
(EGF) have claimed efficacy in literature in healing chronic TMs.18–23 Our lab calls into
question the validity of these studies; they are difficult to interpret as many of the
treatments are executed in acute, not chronic, animal models. Acute perforations heal
spontaneously and because of this it is difficult to tell the extent to which the growth
factor treatments truly aid in the healing process. To this end, it is important to re-execute
these studies in a validated animal model of chronic (not acute) TM perforation.
Reproduction of EGF, FGF-2 treatment studies
First, we reproduced the TM perforation studies utilizing the same growth factors
that claimed success in former studies, which employed animal models such as rats, mice,
guinea pigs, and chinchillas. A suitable animal model is one that mimics the human
condition. Mice are often used as their TM is anatomically more similar to that of the
human when compared to an animal like the guinea pig, which had also been studied
previously.24 Due to this, we chose to use a mouse model of chronic TM perforation to
most closely represent that of a human.
Here, we used a hydrogel polymer delivery system with EGF at 250 µg/ml or
FGF-2 at 100 µg/ml. The hydrogel polymer delivery system used to deliver growth
factors has been previously described.29 This polymer was developed in and provided by
the Department of Orthopedic Surgery at Stanford University and contains a combination
of polyactide, chitosan, and fibrinogen using sodium metabisulfide as a crosslinking
agent.30,31 By utilizing the optimum dosages suggested by these studies (250 µg/ml EGF
and 100 µg/ml FGF-2) and closely replicating the methods in a true chronic TM mouse
6
model, we predicted that the results in these studies are in fact not due to the success of
growth factors in chronic TM wound healing, but because of the spontaneous nature of
acute perforation healing.
Exploring efficacy of HB-EGF in TM perforation regeneration
We then explored the efficacy of a third growth factor of interest, HB-EGF, in
chronic TM perforation healing in a validated mouse model as it was recently shown to
have potential efficacy.30,32 HB-EGF is a heparin-binding member of the EGF family that
was initially identified in the conditioned medium of human macrophages that binds to
the EGF receptor (EGFR) and ERBB4.33,34 EGF promotes cellular proliferation, survival,
and differentiation.35 In the inflammatory stage of cutaneous wound healing, growth
factors such as EGF are released from platelets. Neutrophils and then macrophages enter
the wound, and the macrophages then synthesize additional growth factors including HBEGF.36 HB-EGF and similar growth factors then aid in the migration of epithelial cells
and fibroblasts during the later proliferative and repair stages of cutaneous wound
healing. Both HB-EGF and EGF act by binding with high affinity to cell surface EGFR,
which initiates ligand-induced dimerization and activating tyrosine kinase activity.37 A
signal transduction cascade immediately afterward allows biochemical changes within
the cell that ultimately lead to synthesis of DNA and cell proliferation.38
HB-EGF has a wide array of pathological and physiological functions, and has
been shown to play a role in wound healing, cardiac development and vasculature.39 It is
the predominant growth factor involved in cutaneous wound healing and is a major
component of wound fluids.33,40 The efficacy of HB-EGF as a treatment for chronic TM
7
perforations has been previously shown in animal models; HB-EGF is also the only
growth factor to demonstrate efficacy in chronic TM perforations with chronic
suppurative otitis media or Eustachian tube dysfunction.41 The decision to use this growth
factor with a concentration of 5µg/ml was again determined by previous success in
literature.30
For the experimental growth factor treatments, chronic perforations in the mouse
tympanic membrane were surgically created and kept open with KB-R7785 (a
metallomatrix proteinase inhibitor to inhibit wound healing). Growth factors were then
administered via an injectable hydrogel polymer, and relative TM closure was observed
after a three-month period.
Here, it was hypothesized that HB-EGF would be more effective in inducing
healing of chronic TM perforations with 100% closure than will EGF or FGF-2 in an
animal model.
A non-surgical treatment for chronic TM perforation has the potential to be “the
greatest advance in otology since the cochlear implant”.42 Ideally, a therapeutic,
bioresorbable implant would be administered locally and deliver the effective bioagents
such as growth factors (GF) or stem cells to accelerate or enable the healing of a
perforated TM. Such a therapeutic implant allowing the TM to heal without surgery is the
“holy grail” for this unmet clinical need.
8
Materials and Methods
Ethical considerations
All animal work was approved by Stanford’s Administrative Panel on Laboratory
Animal Care. All mice used for all experiments were 6- to 10- week old male CBA/CAJ
(15-25g) mice purchased from Jackson Laboratories (Florida, USA). All otoscopy and
surgical interventions were performed using inhaled isoflurane at 3-4% for induction and
1-2% for maintenance. Determination of animal numbers was performed using STATA
version 13 aiming for an α of 0.05, a β of 0.8.
Creating chronic perforations
A previously validated mouse model was used to compare the treatment groups.30
Briefly, this animal model was created bilaterally using KB-R7785 (10mM), a
metallomatrix proteinase inhibitor, to inhibit wound healing over a week. The perforation
was left untouched for three months and allowed to become chronic. The KB-R7785
used in this experiment was synthesized by the Department of Chemistry at Stanford
University.
Treatment groups
After creation of the chronic perforations, the 37 mice whose perforations
remained after three months were included in the study and subsequently divided into
four treatment groups (Table 1). Each group received either recombinant mouse
Proheparin-Binding EGF-like Growth Factor (HB-EGF) purchased from Prospec (New
9
Jersey, USA, catalogue number CYT-068) at 5 µg/ml (n=18), recombinant mouse
fibroblast growth factor-basic (FGF-2) purchased from Prospec (New Jersey, USA,
catalogue number CYT-386) at 100 µg/ml (n=19), recombinant mouse epidermal growth
factor (EGF) purchased from Prospec (New Jersey, USA, catalogue number CYT-326) at
250 µg/ml (n=19) or no growth factor (control) (n=18).
Choice of growth factor concentrations
In order to determine the optimum concentrations of each growth factor, we
analyzed current literature surrounding the treatment of both acute and chronic tympanic
membrane (TM) perforations, comparing the dosage concentrations used in each study to
the presumed success rates in the healing of the TM. These prior studies made use of
various scaffolds such as hydrogel polymer, paper patches, and gelfoam to deliver either
EGF or FGF-2. Studies that focused on the healing of chronic TM perforations
(perforations maintained over a period greater than 3 months) were favored.
To determine the dosage concentration of EGF, we considered four studies
utilizing EGF to treat chronic TM perforation. One of these studies was discarded as the
authors did not use a chronic tympanic membrane model.43 The other three in vivo
studies used chinchillas with chronic TM perforations. One study applied 100 µg/mL
EGF via a paper patch and gelfoam.25 81% of the experimental TMs healed completely,
whereas only 25% of untreated TMs healed. Long-term success was not mentioned. The
second study applied 50 µL EGF of a concentration of 250 µg/mL via gelfoam.44 100%
of EGF-treated TMs healed compared to 80% of control TMs. Reperforations and
cholesteatomas lowered long term success rate of EGF treated TMs to 71%. The third in
10
vivo study we considered used 250 µg/mL EGF.27 This achieved partial healing on 100%
of EGF-treated TMs. Therefore, we decided to also use a dosage of 250 µg/mL EGF. By
utilizing the optimum dosages suggested by these studies and closely replicating the
methods in a true chronic model, we show that the results in these former studies are in
fact not results of chronic TM wound healing, but of acute, which heal spontaneously.
The same data collection was performed with FGF-2. Former studies using FGF-2
to treat chronic TM perforations were considered when deciding upon an appropriate
dosage concentration. One such study involved a human TM perforation model in which
FGF-2 was administered with atelocollagen at 100 µg/mL.20 Complete closure was
achieved in 92% of patients. Additionally, another study by the same author used a
guinea pig model to which the authors applied 100 µg/mL FGF-2 via a gelatin
hydrogel.28 100% of treated TMs healed. A dosage of 100 µg/mL FGF-2 was determined
appropriate given the reported success of these former studies.
Hydrogel polymer delivery
Each growth factor was delivered by a previously described hydrogel polymer.31
The treatment was injected via the external auditory canal, using a syringe and 27-gauge
needle, through and onto the TM to fill the middle ear and into the external ear. The total
volume delivered was approximately 0.4ml in each case. In control ears, only the
hydrogel polymer was injected. TMs were evaluated at four weeks, when the hydrogel
was no longer visible in the ear canal.
11
Histology
Histology was performed using Eosin and Hematoxylin staining according to a
previously published technique.45
Immunohistochemistry
To observe the keratin and keratinocyte layers of the TMs. Cytokeratin staining
was performed using Monoclonal Mouse Anti-Human Cytokeratin Clone MNF116
(Dako Cat #M 0821) and a previously published technique.43
Photographic recording
Perforations were assessed under the microscope, and all trans canal photographs
were taken with a Digital MacroView Otoscope (Welch Allyn). The images were then
used to calculate the size of the residual perforations in those that did not show complete
healing. ImageJ software was used to trace around the remaining perforation and present
it as a percentage of the pars tensa.
Statistical analysis
STATA version 13.0 was used for analysis. For statistical analysis on the rate of
healing, Pearson’s χ2 test for goodness of fit was performed with Bonferroni correction.
For statistical analysis of the size of the perforations in those samples that did not show
complete healing, a one-way ANOVA with Bonferroni correction was performed. A
significance level of 0.05 was used for the null hypothesis.
12
Results
Healing of chronic TM perforations by treatment group
The healing of the chronic TM perforations by treatment group, as defined by
macroscopic total closure of perforation observed under the microscope immediately post
mortem, is shown in Table 2. The healing rates of the TMs after treatment compared to
control were 83.3% (n =15/18) with HB-EGF, 15.8% (n = 3/19) with EGF, 31.6% (n =
6/19) with FGF-2 and 27.8% (n = 6/18) with control. HB-EGF is the only growth factor
tested with significant efficacy over control, EGF, and FGF-2. Control vs HB-EGF had a
χ2 value of 11.25, p=.003. In contrast, control vs EGF and FGF-2 were χ2=.78 (p=1.00)
and χ2=.06 (p=1.00), respectively. HB-EGF vs EGF had a χ2 value of 16.88 (p<.001),
HB-EGF vs. FGF-2 was χ2=10.09 (p=.003), and EGF vs FGF-2 was χ2=1.31 (p=0.75).
Macroscopic analysis of unhealed perforations
Among the samples that did not demonstrate complete healing, the sizes of the
residual perforations as percentage of the total pars tensa area were 28.8% for HB-EGF
(n=3, SD=12.9), 38% for EGF (n=13, SD=20.4), 25.1% for FGF-2 (n=13, SD=20.4), and
41.7% for control (n=13, SD=18.0). According to a one-way ANOVA test, there was no
significant difference between the residual perforation size in the treatment groups
(F(3,40)=1.55, p=.0216).
13
Histological analysis of healed perforations
Figure 1 demonstrates representative images of the cytokeratin staining of the
TMs after treatment. Figure 2 demonstrates representative images of the hematoxylin and
eosin staining of the TMs after treatment.
The HB-EGF treated groups show epithelial layers with greater thickness than in
comparable areas of the healed groups in EGF, FGF-2, or control groups. Some areas of
epithelialization appear with differing degrees of thickness (range of thickness was 0.0090.033mm). A few of these areas were comparable to the normal TM keratinocyte layers
where others were increased. Compared to the HB-EGF treated groups (Fig. 1), the FGF2 and EGF tympanic membranes showed a lack of keratin staining even when
macroscopically healed.
The chronic perforation edges (Fig. 2) in the FGF-2, EGF and control groups
demonstrate thickening of the connective tissue layer and in some cases show the
mucosal layer joining directly adjacent to the keratinocyte layer. There was no
discernable observed histological difference between EGF, FGF-2, and control groups.
14
Discussion
This project shows that there is an effective non-surgical approach to induce
healing of chronic TM perforations through the use of endogenous growth factors applied
on site.
Gaps in current research revolving around alternative methods for chronic TM
perforation regeneration are prevalent—notably, there is a lack of a suitable animal
model of chronic TM perforation. Prior studies utilizing growth factors (EGF or FGF-2)
injected with a hydrogel polymer to heal chronic TM perforations in an animal model
have suggested success.20,25,27,28,44 However, the animal models these studies rely on are
of acute perforations, and are thus likely to heal spontaneously. As a result, the extent to
which growth factors aid in the healing process cannot be definitively confirmed based
upon this potential shortcoming.
By successfully creating and establishing a validated animal model of a true
chronic TM perforation in our previous study,30 it is possible to test true effectiveness and
compare the relative efficacies of different growth factors under the same conditions. In
this project, we aimed to replicate this studies by testing the relative efficacies of EGF
and FGF-2, as well to introduce an alternative growth factor treatment using HB-EGF; a
growth factor that has shown promise in former studies.41
Perforations in the TM were surgically created in mice and maintained for three
months by applying a proteinase inhibitor to prevent acute perforation closure, and the
waiting of three months to ensure the perforations were still present then allowed us to
define these TM perforations as chronic. The mice were divided into four treatment
15
groups (Table 1) and corresponding growth factors incorporated in a previously described
hydrogel polymer29 were administered onto the TM via injection. Relative TM closure
was observed macroscopically and histologically over a three-month period.
Using a validated animal model of chronic tympanic membrane perforation, we
demonstrate that growth factor HB-EGF heals chronic TM perforations more effectively
than does EGF, FGF-2, or control (Table 2).
Among TM perforations that did not heal, there was no difference in the healing
area between groups. Additionally, among the perforations that did heal within the HBEGF, EGF, FGF-2, and control groups, only the HB-EGF experimental group tympanic
membranes showed significant histological and macroscopic improvements. HB-EGF
showed keratinocyte staining (Fig. 1) as well as a lack of abnormally thickened
connective tissue when observing the H&E stain (Fig. 2). FGF-2 and EGF- treated
tympanic membranes demonstrated a lack of keratin staining even when macroscopically
healed. If the lack of keratinization and thickening of connective tissue in the animal
model of EGF and FGF-2 translates with similar result in human, risk of re-perforation
will be increased. Here, we show that HB-EGF heals chronic TM perforations in an
animal model more effectively than does EGF or FGF-2.
A limitation of this research is the possibility of bias as the results were not
blinded from the treatment arms. In this case, blinding would have been difficult
considering the treatments and animal model. Additionally, histological assessment
requires experience and training. This study must also be interpreted with consideration
of the limitations of animal models used in mimicking the human condition.
16
Before reaching a clinical setting, additional research must be done with HB-EGF
to demonstrate safety and non-ototoxicity of HB-EGF. Although HB-EGF has been
determined to be non-ototoxic in a mouse model, ototoxicity must be demonstrated in a
second non-rodent animal model.41 These results, however, are promising and provide
foundation for further preclinical studies.
This study confirms the advantage of HB-EGF over two other commonly used
growth factors and is a promising non-surgical treatment of chronic TM perforations. The
introduction of a novel non-surgical solution to areas where surgery would not be feasible
would increase access to treatment by providing a cheaper, safer, and more effective
alternative to surgery.
17
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Diagrams
Diagram 1: Anatomy of the ear
The tympanic membrane as shown separates the outer ear (pinna, ear canal) from the
middle ear (tympanic cavity) and inner ear, composed of the auditory ossicles and the
semicircular canal, cochlea, and cochlear nerves, respectively.
21
Healthy TM
Perforated TM
Diagram 2: The tympanic membrane
Representative diagrams of a healthy tympanic membrane (left) and a tympanic
membrane that has been perforated (right). Line directs to the site of perforation. Chronic
perforations occur when the perforation attained through trauma or degeneration persists
for greater than three months.
22
Tables
Table 1 – Overview of Treatment Groups
Treatment
Group
Numberof
perforations
Dosage
Concentration
No growth factor
n=18
Control
FGF-2
n=19
100 µg/ml
EGF
n=19
250 µg/ml
HB-EGF
n=18
5 µg/ml
Table 1 – Overview of the treatment groups and the specific dosages of corresponding
growth factors used in the study.
Dosage concentrations were determined through reported successes from a literature
review.
23
Table 2 – Efficacy of Growth Factor Treatments in the Mouse Model of Chronic TM
Perforation
Treatment
Outcome
(Complete closure of
perforation)
Difference
HB-EGF
(5 µg/ml)
15/18 (83.3%)
vs. control χ2=11.25
p=.003
vs. EGF χ2=16.88
p<.001
vs. FGF-2 χ2=10.09
p=.003
EGF
(250 µg/ml)
3/19 (15.8%)
vs. control χ2=.78
p=1
vs. FGF-2 χ2=1.31
p=.75
FGF-2
(100 µg/ml)
6/19 (31.6%)
vs. control χ2=.06
p=1
Control
(polymer only)
5/18 (27.8%)
p=1
Table 2- The efficacy of HB-EGF, EGF and FGF-2 compared to control.
Only HB-EGF, here shown in bold, demonstrated significant benefit over control and
other growth factor treatments.
24
Figures
Figure 1 - HB-EGF, FGF-2 and EGF treatment of chronic TM perforations
Figure 1 demonstrates the tympanic membrane after treatment with (a) HB-EGF (b) FGF2 (c) EGF and (d) control. The cytokeratin (brown) staining is used to identify the
keratinocytes and keratin layers in the TM. The TM in the HB-EGF groups show healed
TMs with thick keratin layers compared to FGF-2, EGF and control which demonstrate a
thickened connective tissue layer and lack of keratin staining. Scale bar =10µm
(Magnification 20x)
25
Figure 2 – The perforation edge of chronic TM perforations compared after treatment
Figure 2 is an H&E stain that demonstrates the tympanic membrane after treatment with
(a) HB-EGF (b) FGF-2 (c) EGF and (d) control. The HB-EGF group shows a healed TM
compared to the thickened connective tissue layer seen at the chronic perforation edges
(arrows) with the FGF-2, EGF and control groups. Scale bar =10µm (Magnification 20x)
26