Introduction to Revised Application
The project investigators have carefully considered the study section reviewers’
critiques. Reviews by the 5 experts were thoughtful and comprehensive, and we have
incorporated their ideas into this resubmission. We are grateful for the many positive
comments noted in the reviews. The strengths of the original proposal, as cited in the
reviews, have been maintained in our revised application. These strengths include: 1)
the highly innovative work proposed in Aims #1 and #2, 2) the highly-qualified, excellent
collaborative team with complementary skills, 3) a research environment that is wellsuited to the proposed work, and 4) proposed studies that capitalize on the foundation
laid by the productive record of the investigators.
The most substantive changes in this grant application are a reduction in scope,
clarification of productivity and use of animals/experiment. Most importantly all 5
reviewers suggested that the original proposal was overly ambitious and the clinical arm
(Aim 3) was not sufficiently well developed. The clinical arm of the original proposal has
been eliminated to enable the project investigators to more carefully focus on the other
aims. It is our impression that this reduction in proposed work takes care of most of
questions raised in the critiques. Based on the initial 5 years of funding we are confident
we can accomplish the remaining aims. Responses to critiques not concerned with the
clinical study or ambitiousness are addressed below:
Critique #1
No timeline provided & needed for clinical studies. No clinical studies but timeline
now included.
Critique #3
The physical model described in section C.1.2 and D.2.2.1 was borrowed by the
subcontractor. In the former Section C.1.2, we tried to explain that because of
limitations of the original physical model proposed, we chose to focus our efforts on
other aspects of scarring not included in the original proposal, including the
determination of appropriate scaffolds, cells and growth factors to regenerate the
scarred vocal fold, as well as an examination of their therapeutic effects.” In particular,
we explained that “in the previous physical model of the vocal fold mucosa (Titze et al,
1995; Chan et al., 1997), the epithelium was made of silicone, and the superficial layer
of the lamina propria was made of water.” Accordingly, “in that model, it was not
possible for the scar to extend further into the vocal fold tissues than the epithelium.
Thus, it was not possible to duplicate conditions commonly seen in the clinic, or the
conditions previously studied in the finite element model. However, in the new physical
model (Thomson, 2004), the scar can extend much further into the vocal fold mucosa,
allowing us to simulate scarring conditions observed clinically, and conditions previously
tested in the finite element model.” [Quotes are from previously reviewed grant
submission].
Also, in the former section C.1.2., our intent was to propose the revival of the
physical model component of the grant using a new physical model supplied to Dr. Berry
by the author (Thomson, 2004). However, in his most recent peer-reviewed journal article
[Thomson, S.L., Mongeau, L., Frankel, H.F. (2005). Aerodynamic transfer of energy to the
vocal folds, J. Acoust. Soc. Am. 118, 1689-1700] on page 1695, Dr. Thomson publicly
acknowledges the work of Dr. Berry’s laboratory at UCLA in adapting his physical model
for other investigations: “
In the above-referenced paper the physical model was constructed
independently in Dr. Berry’s laboratory. While the reviewer is correct that the “creation”
of such a physical model is not a “trivial” task, Dr. Berry has been perfecting these
procedures for over a year. Several intricate aspects regarding the successful
construction of the physical model have included (1) complete mixing of the various
elements using a specialized shaker, (2) a low-pressure vacuum chamber to degas and
prevent the formation of bubbles, and (3) post-curing of the “folds” in a heated oven.
While Dr. Thomson is actively pursuing advanced body/cover adaptations of his
physical model, he is well aware of Berry’s adaptations of the model for vocal fold
scarring. Dr. Thomson and Dr. Berry are in frequent contact with each other, have a
collegial relationship, and frequently exchange ideas on laryngeal modeling. To include
both of them as consultants on the same research proposal would be unnecessary. The
proposed procedural adaptations of the model for vocal fold scarring, while difficult if not
impossible for the Titze/Chan physical model (because the superficial layer of the lamina
propria is a liquid), are relatively straight forward for the Thomson physical model.
Proposal states that a gap size of 1mm will be used. The unit is wrong and should
read 0.1 -300 rad/s (corresponding to 0.016 - 48 Hz). The gap size was 1mm for our
earlier work involving normal scarred tissue over this same frequency range ( Rousseau
et al,2003) . Data was analyzed by Roger Chan. For the proposed work the gap will be
varied to account for the desired force
Not clear that cellular work presented in D.1.6.1 and C.2.3 covers significant new
territory compared to work by Verdolini in Pittsburgh or Thibeault in Utah Our
systematic approach to using growth factors, gene therapy ,functional outcomes
,development of a rat model are novel approaches and make unique contributions on an
important topic. Dr. Susan Thibeault has joined our group as a consultant, read the
grant proposal and concurs with our assessment. A letter of support is included with
document.
The linear skin Rheometer does not yield absolute numbers. The correlation of
the relative numbers to absolute numbers should be clarified. The LSR is a
precision electro-mechanical device designed to specifically measure the visco-elastic
properties of human and animal tissue. The design is such that it can be very flexibly
applied, and configured in different ways to obtain data by five different methods. Two of
these techniques have been applied to excised human larynxes by Hess and Goodyer,
and yield similar values and will be used in our studies. The figure and conversion factor
from dynamic spring rate to viscosity (antipoise) is detailed in our latest manuscript
included in the Appendix (Dailey, Welham et al. Submitted 2005).
Shear properties are measured by attaching a 'probe' to the chuck which is
attached to the tissue under test. The micro-positioner can then stress the tissue in the
shear mode in a methodical manner (typically by applying a sinusoidal force cycle). The
force and displacement sensors capture the absolute stress & strain data from which
shear modulus is derived. The raw data is expressed in terms of Dynamic Spring Rate,
which is the complex term made up of the Elastic Spring Rate and the viscous Loss
Rate. Using the usual mathematical transformations, based on the geometry of the test
set-up (i.e. tissue thickness and probe attachment area) the complex shear modulus
term can be derived. It is also possible to resolve out the elastic and viscous modulii, as
the instrument calculates the phase angle of the between the sinusoidal force and
resultant position cycles.
The indentometer approach is to derive a stress/strain characteristic by logging
the change in force as the tissue is compressed. Shear modulus can be derived from
this data by applying the mathematical transformation derived by Hayes in the 1970's.
This methodology was developed specifically
to determine the shear modulus from indentometer data obtained from soft tissue, and is
widely used by many researchers today. The raw data takes the form of a classic
indentometer stress/strain curve, which shows
hysteresis when there is a viscous element to the tissue characteristics. The Hayes
solution requires knowledge of the Poisson's ratio and the thickness of the tissue. As soft
human tissue is highly incompressible its' Poisson's ratio is usually estimated to lie
between 0.45 and 0.49, tissue thickness can be measured empirically. Therefore it is
possible to derive absolute values for shear modulus.
It should be clarified why growth factors are used instead of a mechanical
environment like a bioreactor.
Cells are affected by various factors such as transcription factors, growth factors, and
microenvironment surrounding cells including mechanical stress. Titze proposed a
bioreactor for engineering vocal fold tissue and indicated that vibratory strain alters the
expression levels of extracellular matrix-related genes. Influence of mechanical stress
on cell character is already reported in other organs, such as bladder wall and tendon, in
which cells are exposed to mechanical stress and it is reasonable that fibroblasts in
vocal folds are affected by mechanical stress as well. Nevertheless, we believe growth
factors are the strongest tool for engineering vocal fold tissues. Various kinds of growth
factors have been identified and the functions of each growth factor have been
documented. The great advantage of growth factors is each growth factor has different
function which enables one to control cell characteristics more precisely and abundant
knowledge has been established in growth factors although there is a lot still unknown.
The knowledge makes it easier to tune cells. For example, numerous growth factors
have been used to differentiate stem cells to desired cells. Thus, we believe growth
factor is an ideal tool to tune vocal fold fibroblasts.
D.1.8.2 discusses software but there appears no reference or source of software
and its availability.
References are provided for the source of analysis software. At this time neither the Yan
GAW or Jiang software are commercially available.
The budget for year 5 lists “supplies” and “other expenses” as continuing from
previous years. One should be less since year 5 should not contain much in
terms of laboratory work but more analysis and publication preparation We do
not anticipate stopping data collection or increased analysis during the 5th year of the
project. We presume we will continue with a steady program of data collection, analysis
and publication throughout the period of funding. This is absolutely critical in an
integrated multidisciplinary project that uses the results of studies to formulate
hypotheses in the other related areas such as we do in relating one type of model to
another.
Critique #5
It is not clear if excised rat larynges can be phonated. Given the small size of the
structure, some feasibility data on this should be provided. Feasibility data is
provided in D.1.7. Movement of rat vocal folds are visualized with creative optics using a
microscope and high speed camera. Movement is best visualized in movie pictures (i.e.
AVI format). AVI movies of normal and scarred rat larynges can be provided if
requested.
No figures or tables were used in this or any of the reports. No figures are
provided to demonstrate the LSR map Figures and tables have been added to
illustrate work completed and feasibility of proposed studies.
The application implies that the investigators tried a variety of animal models
during the initial project period but now are mainly using rats. This is likely for
cost reasons. It is true that cost is an important issue, but it is not the main reason why
we’ve shifted toward rats from bigger animals. Rats appear to have
advantages compared to dogs and rabbits. The vocal fold lamina propria of rats
displays characteristics similar to that of humans. Like the human, the rat LP has 3
layers with the deep layer containing more collagen fibers than the superficial layer. In
contrast the fibrous components are denser in the superficial layer than in the deep layer
in dogs LP. Also, rats can be expected to show quicker wound healing because of their
short life span. In using dog and rabbit models, 6-month postoperative time frame was
necessary for a vocal fold scar to mature whereas rats need only 2 months. This is a
great advantage in conducting our research because we can address more questions
and have longer term follow-up in a shorter time frame. Another rationale for focusing
on rats is that there is an abundance of genetic information available. Moreover
numerous basic scientific techniques, especially in molecular biology, are already
established in rats. The availability of genetic information and developed techniques
provide a short cut allowing us to focus on specific molecules in a timely fashion. Thus,
rats appear to be an ideal model for investigation of wound healing in the vocal folds as
further described under justified use of vertebrate animals.
With regard to Aim 2, the relation of the aging rat vocal fold model studies from
the preliminary studies to the overall goal of scar prevention and treatment and
the proposed experiments is not explained.
Three factors have motivated us to include life-span studies including the aging rat : 1)
Fetal wound healing is known to do so scarlessly; 2) Cellular structure of the vocal fold is
known to change across the life span but characteristics and regulatory agents are not
well understood, and 3) Histological characteristics of aged and scarred vocal folds are
similar. The ultimate goal of treating vocal fold scarring is to reconstruct healthy
tissue. In this regard, there is a great deal of knowledge to be gained from studying the
processes by which fetal and neonatal vocal folds develop into the exquisite adult vocal
fold. Fetal wound healing is known to heal without scar and fetal tissue should contain
the keys to develop therapies for scarless healing. Our previous study in neonatal and
young rat vocal folds suggested that vitamin A storing cells are present in rat vocal folds
as well as in human vocal folds, the vitamin A-containing lipid droplets in MF of rat vocal
folds increased with age, and the cell shapes in MF change with age. These results
imply the relationship of vitamin A to vocal fold maturity (Tateya T, in press 2005). (In
addition, our preliminary data (unpublished) showed distinct keratinization of vocal fold
epithelial cells in vitamin A deficient rats. Vitamin A may be involved in the development
and maintenance of normal vocal fold structure.) Moreover, histological characteristics
of aged and scarred vocal folds share some similar characteristics in terms of excessive
collagen and reduced HA. (Hirano, et al unpublished data). This suggests that aged
vocal folds can provide with an experimental model similar to the scarred vocal fold
model and that the same therapeutic strategy can be applied to aged and scarred vocal
folds. Our previous studies about the feasibility of growth factors support this. Growth
factor is a potent regulatory element that affects cells and their functions. We have
revealed that hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF)
significantly increases the production of hyaluronic acid (HA) and decreases collagen
production from vocal fold fibroblasts in vitro studies. In vivo studies, we have found that
local injection of bFGF improved aged vocal folds of rats in terms of histology. Our
recent data also shows that local injection of bFGF improved the chronic vocal fold scar
in a rat model in-vivo and in-vitro.
There are a large number of investigators and consultants that will require
organization to manage the many projects. Of the key personnel listed as at UWMadison, one currently resides in Japan and two others will be leaving the U.S. for
Japan this year. They are key investigators in Aim 2. Plans for replacement of
these positions are not spelled out in the application. During the first five years of
funding we have demonstrated the organization and ability to manage many projects.
Conference calls, electronic messaging and face-to-face meetings have facilitated
coordination, interaction and operations with all key personnel and consultants.
Additionally, for the past 20 years there have been Research Fellows from Japan
working with Dr. Bless. This arrangement will continue and provide the in-site
replacement. Drs. I Tateya, T Tateya and Hirano will continue to be part of the research
team as consultants.
A. SPECIFIC AIMS
A.1. Statement of the Problem
Vocal fold scarring is the single greatest cause of voice impairment following
vocal fold surgery. 1,2 Such scarring results in the replacement of healthy tissue by
fibrous tissue, a process that can irrevocably alter vocal fold function and lead to a
decreased or absent vocal fold mucosal wave .3 Even small foci of scar can impede
vocal fold function when separation of the body of the vocal fold (muscle) and its cover
(epithelium and the superficial layer of the lamina propria) are compromised. Because
there is currently no consistently effective treatment for scarred vocal folds, vocal fold
scarring is one of the most challenging laryngeal disorders to treat . 4The long-range aim
of this project no longer focuses exclusively on sulcus vocalis; it now encompasses the
characterization and treatment of the more universal phenomenon of vocal fold scarring.
We will accomplish this long-range objective by characterizing laryngeal scarring and its
influence on phonation, both pre- and post-treatment, using (1) clinical and surgical
trials, (2) controlled laboratory experiments, and (3) computer-modeling
experiments. We will use laboratory experiments to investigate treatment effects at the
cellular level in animal tissues and excised larynx models. Building on experiments
completed over the last 5 years, we will also extend our initial laboratory investigations to
the clinical population. It is necessary to combine clinical surgical treatment with
systematic laboratory investigations to resolve the complex interactions between tissue
characteristics and geometry and the surgical procedures required to create a suitable
clinical outcome.
A.2. Hypotheses
A.2.1. Hypothesis 1. Vocal fold scarring results in predictable changes to vocal fold
vibration and phonatory output. We will test specific subhypotheses and attempt varying
the properties of the laryngeal scar in several models of vocal fold vibration, including a
computational model, a physical model, and an excised larynx model.
Subhypothesis 1. The location of the scar along the anterior-posterior and
inferior-superior axes determines the severity of the impairment of oscillation and voice
much more than the depth of the scar. For example, an anterior scar induces
significantly greater impairment than a posterior scar. In addition, there is a critical area
within ± 2mm of the superior-medial edge in which scarring is most detrimental to
phonation.
Subhypothesis 2. A linear increase in the viscoelasticity of the scarred tissue
results in an exponential increase in phonation-threshold pressure, making phonation
impossible beyond a certain critical threshold of viscoelasticity in scarred tissue.
Subhypothesis 3. For mild to moderate scarring, a unilateral scar impedes
phonation significantly more than an equivalent bilateral scar. Only for severe scarring
does a bilateral scar impede phonation more than an equivalent unilateral scar.
A.2.2. Hypothesis 2. The extracellular matrix (ECM) response associated with laryngeal
scarring is a major cause of refractory dysphonia that can be prevented and/or
minimized with treatment. We will test this hypothesis in the following ways: (1) we will
measure the histologic, rheologic, and biomechanical properties of the treated vocal fold
during acute and chronic stages of wound repair in a rat model and compare these
properties with those of untreated, wounded vocal folds, (2) using cell-culture models,
we will culture normal and diseased laryngeal fibroblasts from humans and animals and
examine the effects of treatment on the in vitro deposition of the extracellular matrix.
Subhypothesis 1. The prevention and/or reversal of changes produced in the
vocal fold by scarring will be manifest in treated vocal folds compared with control vocal
folds that have been scarred but not treated.
Subhypothesis 2. Treated larynges will demonstrate improved aerodynamic
properties for phonation characterized by increased vocal economy and reduced
phonation-threshold pressure when compared with untreated larynges.
Subhypothesis 3. Growth factors will induce an antifibrotic wound-healing
response characterized by increased hyaluronan and decreased collagen when
compared with untreated, wounded vocal folds.
Subhypothesis 4. Upregulation of hyaluronic acid synthase genes will result in
an antifibrotic wound-healing response characterized by increased hyaluronan and
decreased collagen when treated vocal folds are compared with untreated, wounded
vocal folds.
Subhypothesis 5. Treatment of a vocal fold scar in rats with a 585nm pulseddye laser (PDL) will induce increased collagen and elastin and decrease the amount of
disorganized collagen when treated vocal folds are compared with untreated, scarred rat
vocal folds. Treatment with the PDL will improve the pliability of the vocal fold and
reduce its aerodynamic inefficiency when it is compared with untreated vocal folds.
A.2.3. Hypothesis 3. The location of the laryngeal scar combined with measures of the
severity of impaired tissue pliability, geometrical tissue deformity, and glottal
insufficiency provide a useful framework for treatment decisions. The impact of glottic
insufficiency as a consequence of scarring is an independent variable that can
significantly impede entrainment, oscillation, and glottic function.
A.3. Aims
A.3.1. Aim 1. To measure and contrast the histologic, viscoelastic, and vibratory
properties of the pre- and post-treatment tissues associated with vocal fold scarring and
to establish functional relationships between these biomechanical variables and
phonatory output. This aim will be addressed using systematic data obtained from in
vitro models, in vivo models, physical models, and computational models of the vocal
folds.
A.3.2. Aim 2. To determine the appropriate scaffolds, cells, and growth factors needed
to regenerate a scarred vocal fold and examine their therapeutic effects using the same
pre- and postoperative measures discussed in Aim 1. Using animal models, we will
address this aim with both in vitro and in vivo studies.
A.3.2. Aim 3. To determine the role of inflammatory mediators on the development of
vocal fold scarring
A.4. Significance
In the 1995 Chevalier Jackson Lecture entitled "Phonosurgery: past, present, and
future," Minoru Hirano suggested that the 2 major laryngeal problems awaiting
improvement in the future are scarring and sulcus.1 However, little has changed since
this lecture was given because there are no effective treatments for either problem. Both
cause deformity of the vocal fold edge, an increase in stiffness of the vibratory structure,
and glottic incompetence. Our current proposal is designed to meet this challenge with
successful therapies for vocal fold scarring. Our work is important because scarred vocal
folds are a significant source of disability. Predicting the clinical course of laryngeal
wound healing and identifying optimal therapeutic options can save time, morbidity, and
health-care dollars. Thus, this proposal is significant not only because it promises to
develop effective treatments for laryngeal scarring but also because it could significantly
reduce the amount of health-care dollars spent on addressing this problem.
B. BACKGROUND
Clinically, the scarred vocal fold represents one of the most challenging laryngeal
disorders to treat, and it is regarded as the single greatest cause of voice impairment
following phonosurgery.2,3 Currently, no consistently effective forms of treatment for
vocal fold scarring exist. Our studies conducted over the past 5 years have helped to
quantify the pathophysiologic characteristics of the scarred vocal fold. 5-30 Studies have
revealed differences between the acute and chronic stages of vocal fold wound healing,
suggesting that the acute and chronic stages of tissue repair have sufficiently different
histologic characteristics and may require different treatment approaches.
This proposal aims to build on knowledge acquired during the last 5 years to
further the understanding of wound-healing events leading to scar formation and to
develop better treatment approaches for different presentations of vocal fold scarring
based on its underlying pathophysiology. Outcomes of this research have the potential
to lead to a greater understanding of the acute and chronic stages of vocal fold wound
healing.
B.1. Current Treatment Approaches
Most treatments for vocal fold scarring are aimed at excising and/or breaking up
the scarred tissue or manipulating the ECM to create a tissue environment with optimal
biomechanical properties. Current methods of treatment include surgical implantation
and/or injection of a desirable substance into the vocal fold lamina propria.3 One of the
drawbacks to injectables is that repeat injections are often necessary to maximize
efficacy. A downside of implantable materials is that they are subject to the impact forces
of vocal fold vibration and may be extruded from the site of implantation. Despite the
limitations of these treatment approaches, ECM modification appears to hold the most
promise for both the treatment and prevention of scarred vocal folds. 31
Because of limitations inherent in all of the currently available treatments, there is
clear need for an improved understanding of what works, what fails, and why. The
method that may prove to be most useful in ECM modification is one that stimulates
vocal fold fibroblasts to synthesize desired ECM substances and minimize the
production of molecules that promote fibrosis. In other words, the goal is to optimize the
molecular environment to facilitate an ideal environment for tissue regeneration. The
ideal substance would not extrude or migrate when implanted, would have long
residence time in tissue, and would recreate the natural biomechanical properties of the
vocal fold lamina propria. Alternately, modification of cellular function would provide an
optimal situation because cells could produce a more natural extracellular matrix.
B.2. Vocal Fold Models
One of the problems in studying vocal fold scarring in a clinical population is that
presentations of scar are often quite different among patients. Despite the lack of
phonation in animals, animal models nevertheless provide a unique solution to this
problem. Vocal fold injury can be systematically manipulated in an animal model so that
the depth and extent of injury can be controlled, and periodic follow up can be
undertaken to assess the different stages of wound healing. Among the different animal
models used in laryngeal research are dogs, pigs, rats, and rabbits. Ideally, an animal
model appropriate for investigations of scarring should have a larynx that can be
phonated in excised-larynx studies for in vitro studies of vocal fold vibratory
characteristics post-treatment, a larynx that provides enough tissue for completion of
rheologic experiments for in vitro measurements of viscoelastic shear tissue properties,
and a larynx that displays similar tissue and biomechanical properties with humans. One
of the most frequently used animal models in laryngeal research is the canine model.32
While it is not a perfect animal model for all voice research because of the difference in
differentiation of laryngeal layers between dogs and humans16 the length and thickness
of the lamina propria in the canine larynx make it an ideal model for the investigation of
the surgical management of scarred vocal folds. In addition, the canine larynx can be
easily phonated experimentally, the lamina propria provides enough tissue to carry out
both histologic and rheologic experiments using the same vocal fold, and the ECM
components are similar to those in human larynges. Most important for our work, sulcus
scar formation occurs naturally in dogs secondary to kennel cough. 14,35
Of all the animal models used, the rat is particularly good for the study of the
prevention and treatment of laryngeal scarring in humans because the vocal fold has a
3-layered structure with ECM components similar to those in humans. It is also small
and easy to handle, it regularly produces sound, and it has a short lifespan facilitating
long-term follow up of treatments. The application of the rat model to our proposed
studies is well justified because we have found that rats demonstrate developmental
changes in ECM similar to those reported in humans .20,23 Most important, we have
found that rats demonstrate altered tissue characteristics following scarring similar to
those observed in humans. Furthermore, wound healing in rats demonstrates
morphologic and functional changes in vocal fold fibroblasts and hyaluronic acid, and
these changes parallel those observed in human vocal folds.20-23
B.3. The Histology of Vocal Fold Scarring
The histologic ultrastructure of vocal folds plays a crucial function in the
biomechanical properties of the lamina propria. The ECM of the vocal fold lamina propria
consists of a number of important molecules that contribute to tissue characteristics
during oscillation. 27,36 These molecules display important biomechanical functions and
play critical roles in providing the ECM with structural support that influences tissue
viscoelastic properties, regulates the thickness of the lamina propria, and affects fluid
content. The vocal fold lamina propria consists of fibrous proteins and interstitial
constituents. Collagen and elastin are 2 of the fibrous proteins found in the vocal fold
lamina propria. Collagen and elastin contribute to tissue strength and flexibility. The
interstitial constituents of the vocal fold lamina propria consist of proteoglycans and
glycosaminoglycans. Proteoglycans and glycosaminoglycans, such as hyaluronic acid,
occupy the spaces between fibrous proteins. The fibrous and interstitial components of
the vocal fold lamina propria have important biomechanical functions that contribute to
tissue characteristics during oscillation. These molecules play an important role in
determining tissue viscoelasticity, and they may affect clinical voice measures, such as
phonatory threshold pressure and vocal fundamental frequency. Thus, an increased
understanding of the role of these molecules during wound remodeling should provide
an important component in understanding how vocal fold scarring inhibits oscillation and
might ideally be repaired to produce normal vibration patterns.
Histologically, the scarred vocal fold is characterized by increased or
disorganized collagen, decreased elastin, occasionally decreased hyaluronic acid,
increased fibronectin, and decreased decorin, which destroys the viscoelastic property of
the vocal fold.16,17,20,24,28,37-39 In this sense, the scarred vocal fold loses properties that
allow it to be the primary source of vocal vibration. To regenerate normal tissue
properties and associated vibratory function, histologic changes must be understood and
addressed. Recently, tissue engineering has been used to regenerate tissues and/or
their function. The underlying concept of tissue engineering is that tissue consists of 3
elements: scaffolding, cells, and growth factors. Because ECM(ECM) is primarily
produced by fibroblasts in the lamina propria of the vocal fold, one strategy to restore
normal organization of ECM to the scarred vocal fold would be to control fibroblasts
using growth factors. Other reasonable strategies would be to administer cells, such as
normal fibroblasts or stem cells, and to provide an appropriate scaffold in which cells can
grow and function properly. Our proposed studies aim to determine the appropriate
scaffolds, cells, and growth factors needed to regenerate the scarred vocal fold and then
examine their therapeutic effects. The studies consist of in vitro studies, in vivo studies
using animal models, and clinical studies in human subjects.
B.5. Different Stages of Wound Repair
The acute and chronic stages of wound healing appear to have sufficiently
different histologic characteristics. Thus, it is necessary to investigate both stages to
completely understand wound repair, determine treatment effectiveness for different
presentations of scar, and improve clinical decision making. For example, Rousseau et
al (2003) observed increased procollagen and decreased elastin in a vocal fold scar 2
months postoperatively. Elastin fibers were characteristically tangled and disorganized.
However, after 6 months, the vocal fold scar revealed decreased elastin and increased
collagen. Collagen fibers also formed thick, disorganized bundles, while the elastin fibers
were disorganized throughout the entire scarred lamina propria. Biomechanical
measurements revealed increased stiffness and dynamic viscosity for scarred tissue
samples in both the acute and chronic scar.
Further analysis of the scarred tissue samples for the adhesion molecules
fibronectin, cadherin, syndecan-1, and syndecan–4 was also done.11,40,41 Results
revealed a significant increase in fibronectin in the superficial layer of the scarred vocal
fold lamina propria at both 2 and 6 months and a significant overexpression of
syndecan-4 along the basal layer cells of the epithelium during both time periods. These
studies have shed some light on the histologic ultrastructure of the scarred vocal fold
lamina propria, as well as on factors that might contribute to increased stiffness and
tissue viscosity. The studies demonstrated that changes in tissue viscoelastic properties
might take place prior to scar maturation (as early as 2 months prior) and that while
abundant collagen deposition (observed at 6 months) might influence viscoelastic shear
properties, disorganization of collagen and/or elastin fibers (observed during both time
periods), thick bundle collagen formation, or the interplay of several of these factors
might also play a role. Moreover, results from Hirano et al (2003) suggested that the
binding characteristics of fibronectin to collagen, proteoglycans, and other structural
proteins might contribute to increased stiffness and dynamic viscosity of the scarred
vocal fold.
Thibeault et al (2002) studied the histologic and rheologic changes of a vocal fold
scar 2 months postoperatively in a rabbit animal model. One of the most important
findings from this study was that collagen was decreased during an acute phase of
injury, yet stiffness and viscosity were increased. This was important because,
traditionally, collagen has been regarded as the single most important cause of
biomechanical tissue changes post-scarring. Thibeault and colleagues demonstrated
that this notion was not the case during an acute phase of vocal fold injury, and they
raised speculation that interstitial proteins may play a more important role in contributing
to biomechanical tissue changes than previously thought.
B.6. Hyaluronan
Hyaluronan is an important glycosaminoglycan found in the vocal fold lamina
propria that influences tissue viscosity, tissue flow, tissue osmosis, and wound healing.
38
Hyaluronan is also well suited for the binding of water molecules. This latter
characteristic makes hyaluronan an excellent molecule for filling up space. Accordingly,
Hertegard et al (2002) and Hallen et al (2001) have had success using cross-linked
hyaluronan derivatives (hylan B gel) and the dextranomers in hyaluronan for the
treatment of glottal insufficiency due to vocal fold bowing, paresis, paralysis, atrophy,
and scarring. Although modified forms of hyaluronan provide increased residence time in
tissue, they do not always address the biomechanical properties of the lamina propria
sufficiently. Natural hyaluronan likely provides the most ideal substance for optimal
tissue biomechanics. Unfortunately, little is known about ways to increase natural
cellular production of hyaluronan.
Interestingly, hyaluronan is the most prominent ECM molecule in injured fetal
tissue, which heals without scarring.44 In fact, this property happens to be one of the
major differences between fetal wounds and adult wounds. There are other factors that
differentiate fetal wounds from adult wounds, such as an abundance of macrophages
and lack of neutrophils during the first 4 days post-injury; a unique cytokine profile that
may serve to promote tissue regeneration; and the earlier presence of fibronectin, which
may serve as an important scaffold for cell-cell adhesion, cell-ECM adhesion, and
epithelial cell migration. 44,45 Each of these factors has the potential to influence the
biomechanical properties of the scarred vocal fold. For example, it has been found that
the enzymatic removal of hyaluronan from the human vocal fold lamina propria leads to
decreased stiffness of the vocal fold cover on the order of 35%, while dynamic viscosity
is increased by 70% at frequencies above 1Hz .46
Chan and his colleagues have suggested that hyaluronan likely contributes to the
maintenance of the optimal tissue viscosity necessary to facilitate phonation. Hyaluronan
may also contribute to the optimal tissue stiffness that is important for vocal fundamental
frequency control. Currently, data regarding hyaluronan deposition in the scarred vocal
fold are limited to 2- and 6-month time frames post-injury.15-17,27 Thibeault, Rousseau,
and associated collaborators found hyaluronan levels in the scarred vocal fold at 2
months post-injury to be similar to control levels in a rabbit animal model. Similarly,
Rousseau found hyaluronan levels to be similar to control levels at both 2 and 6 months
post-injury in a canine animal model, although in some cases, hyaluronan was mainly
observed in the deepest layer of the scarred vocal fold. These findings suggest that
although hyaluronan levels are stabilized (return to preoperative levels) by 2 months
post-injury, there may be existing differences in the distribution of hyaluronan in the
remodeling scar that in turn may affect biomechanical tissue properties. Thus, much can
be learned from studying hyaluronan alterations in vocal fold wound healing.
An equally important area of study lies in manipulating hyaluronan levels during
various stages of wound healing because of hyaluronan’s impact on the ECM, which in
turn impacts viscosity and vibratory characteristics. ECMmanipulation has been
suggested as a possible treatment technique for vocal fold scarring. 10,46 The goal of
ECMmanipulation is to arrive at an optimal balance of ECMconstituents by manipulating
1 or more tissue components. Because hyaluronan is an important molecule contributing
to tissue viscosity and scarless fetal wound repair, techniques aimed at maximizing
hyaluronan levels during acute wound healing may provide a useful method for restoring
optimal tissue viscoelasticity and preventing postoperative scar formation.
Our preliminary data with a small sample also suggest that autocross-linked HA
gel has an effect on the functional outcomes of vocal folds, such as phonatory threshold
pressure and voice production efficacy (Hirano et al. unpublished). HA is synthesized by
3 subtypes of hyaluronic acid synthase (Has) and digested by hyaluronidase (Hyal) in
mammals. Using a rat model and real-time polymerase chain-reaction analysis, we’ve
observed increased expression of Has1 and Has2 mRNA in the very early phase of
vocal fold injury that returned to normal within 2 weeks. We’ve interpreted this finding to
suggest that maintaining the upregulation of Has genes may be necessary to achieve
scarless wound healing of vocal folds similar to that of a fetus. Consequently, we
propose to transfect Has1 and Has2 genes into the injured vocal folds of rats to examine
the effect of Has genes on the prevention of vocal fold scarring.
B.8. Cell Transplantation
Analyses of cellular location and concentration in the vocal fold lamina propria
imply that fibroblasts distributed in the lamina propria of the vocal fold are primarily
responsible for the production of ECM components. 51,52 Thus, to regenerate vocal fold
tissue, it is important to control fibroblasts. It would appear that one means to restore
normal organization of ECM to the scarred vocal folds would be to administer drugs,
such as growth factors or genes. Other strategies would be to administer cells, such as
normal fibroblasts or stem cells, or to provide an appropriate scaffold in which cells could
grow and function properly. The studies we propose are significant because they aim to
determine appropriate cells and scaffolds to regenerate the scarred vocal fold and then
examine their therapeutic effects. The studies consist of in vitro studies, in vivo studies
using animal models.
B.9. Tissue Engineering
Advanced tissue-engineering techniques enable us to regenerate new tissue in
cell culture. Appropriate scaffolding, cells, and growth factors are needed to complete
this task. Scaffolds should be biodegradable and provide a cell-seeded substrate that
can be implanted into the vocal fold lamina propria. There are many possible
approaches to developing an appropriate scaffold. For example, cell sheet
engineering53,54 is one possible strategy. Cultured cells can be collected as a sheet by
coating a culture plate with a temperature-responsive polymer (PIPAAm) that is
detached from the plate by reducing the temperature of the polymer.
The cultured cell sheet can be easily moved and piled onto another cultured cell
sheet to create a 3-dimensional (3-D) cell culture. This technique enables one to
construct organs by laying different types of cell sheets. The 3-Dl cell culture was carried
out with rat's cardiac cells.54 Lamina propria consists of 3 layers with different
concentrations of collagen, HA, and elastin. This engineered cell sheet is suitable as a
scaffold to regenerate vocal fold tissue in 3 layers. Our proposed studies aim to find the
appropriate scaffolds, cells, and growth factors needed to create a tissue-engineered
vocal fold.
B.10. In Vitro Inflammation Model of Vocal Fold Fibroblasts
Healing of scarless fetal wounds is known to occur without a significant
inflammatory processes.57 Since a key feature of scarless fetal-wound healing is an
elevated HA level,38 there may be a link between inflammation, specifically through the
cyclooxygenase-2 (COX-2) pathway,59 and the production of ECM components (HA and
collagen) by proliferating fibroblasts during the wound-healing process. However, this
link has not been demonstrated. We plan to establish an in vitro inflammation model of
vocal fold fibroblasts and confirm whether any such link exists. This research is
significant because it would provide a critical step in explaining the basis for current
treatment techniques used to increase HA levels under conditions of inflammation. It
would also provide the knowledge necessary to seek novel, more specific therapeutic
agents.
B.11. Growth-Factor Therapy
Growth factors are potent regulatory elements that affect cells and their
functions. We have discovered that hepatocyte growth factor (HGF) significantly
increases the production of hyaluronic acid and decreases collagen production in vocal
fold fibroblasts in vitro.6,7,58 We also have found that local injection of HGF prevented
scar formation in injured vocal folds in a rabbit model and even improved the scarred
vocal fold in a canine model.55,56 Yet, there are many problems as yet unanswered, such
as what dosage of HGF is ideal, how frequently HGF should be injected, and what the
best route of the administration of HGF might be.
B.12. Cell Therapy
Fibroblasts are distributed in the lamina propria of the vocal fold and are primarily
responsible for the production of all extracellular matrix.11,12,38,40 Fibroblasts in the
scarred tissue have been reported to be altered in terms of their ability to synthesize
ECM.11 Injection of fibroblasts into a scarred vocal fold has been suggested as an
alternative method of regenerating the vocal fold.41,72 In order to test this assumption, it
is necessary to determine which fibroblast should be used and how to control the chosen
fibroblast. This step is necessary because the functions of vocal fold fibroblasts, skin
fibroblasts, and scar fibroblasts may be quite different. Regenerative medicine uses
stem cells. Stem cells can be induced and differentiated into any type of mature cells.
Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to become
other mature cells as well as bone-marrow cells. MSCs can be easily obtained from
humans to avoid problems of allergic response or ethical issues that arise in association
with the use of embryonic stem cells. MSC implants into injured vocal folds in the canine
model have improved wound healing with less scar formation. Our studies aim to show
how to control MSCs and the role they play in the treatment of vocal fold scarring.
B.13. Functional Outcome Measures
Phonatory threshold pressure (PTP) is a commonly used measure, defined as
the minimum amount of lung pressure required to produce vocal fold oscillation.61,62 It is
a quantitative measure of the relative ease of phonation. Onset PTP has been described
as the minimum lung pressure needed to initiate vocal fold oscillation from rest, while
offset PTP is defined as the minimum lung pressure needed to sustain vocal fold
oscillation once it has already started.62 In the laboratory setting, onset and offset PTP
can be obtained by directly manipulating subglottal air pressure. Vocal economy is
typically used to investigate the efficiency with which the vocal folds are able to convert
aerodynamic power into acoustic power, and it can be measured from simultaneous
recordings of subglottal pressure, airflow, and sound intensity. Vocal economy can be
defined as the ratio of acoustic power to aerodynamic power. Thus, the vocal economy
measure employed in studies IV and V involved a dimensionless output-cost ratio (OCR)
between the acoustic intensity (output) and subglottal pressure (cost). The combination
of PTP, OCR, and histologic measures is important in assessing the impact of vocal fold
scarring on phonation. Because functional measures of phonation are likely influenced
by the underlying tissue structure of the vocal fold, all of these measures should be
taken into account when reporting the effects of vocal fold scarring on vocal fold
vibration. More studies are needed to investigate the effects of vocal fold scarring on
these measures.
B.14. Viscoelastic Measurements of Vocal Folds
Vocal fold scarring continues to be a challenging clinical entity in the field of
laryngology. Multiple treatment options, including medialization thyroplasty, scar/sulcus
excision, and vocal fold augmentation with collagen, fascia, and fat, have all been
attempted with less-than-optimal results. Failure to achieve an optimal approach is likely
based on a lack of understanding of the fundamental nature of vocal fold scarring and
how to characterize it.
Historically, vocal fold scarring has been characterized by histologic,
biochemical, aerodynamic, acoustic, stroboscopic, and rheologic parameters. Rheology
of the tissues being tested has been limited by technological barriers. Parallel plate
techniques require investigators to excise the entire vocal fold soft tissue away from its
cartilaginous attachments and record measurements based on the tissue as a whole.
The ability of this technique to record spatially important changes is poor. Parallel plate
technology cannot, for example, map the depth at which vocal fold stiffness resides (for
example, the epithelium, superficial lamina propria, vocal ligament, or thyroarytenoid
muscle). In addition, it cannot localize stiffness along the long and vertical axes of the
vocal fold. Importantly, scarring is localized to the epithelium and superficial lamina
propria and occupies particular sites along the long and vertical axes. This shortfall in
parallel plate technology prevents precise characterization of a vocal fold scar in both
site and severity, limiting ability to track focal interventions.
As the variety of interventions grows to accommodate the spectrum of problems
that are manifest in vocal fold scarring, a more refined characterization of the scar itself
will be required to tailor the treatment to the problem. Use of a linear skin rheometer
(LSR) in the characterization of vocal fold scarring addresses the shortfalls that parallel
plate technology cannot. We intend to characterize the local and regional rheometric
changes to animal vocal folds using an LSR in both acute and chronic scar models. We
will record rheometric changes induced by the application of growth factors, the 585nm
pulsed dye laser, and genetic transfer techniques. We will then integrate the rheometric
vocal fold maps into mathematical models of vocal fold oscillation.
C.PRELIMINARY STUDIES AND PROGRESS REPORT
Following is a list of work accomplished during the first granting period. Not included in
the list but noteworthy is that between 2001-2005 seven of the presentations made at
scientific society meetings were given peer review awards for scientific merit including
ALA Young Faculty Research Award, ABEA Broyles Malony Award, ABEA Scientific
Poster Award and the Voice Foundation David W. Brewer for Best Scientific Poster,
1) Hirano S, Kojima H, Tateya I, Ito J. Fiberoptic Laryngeal Surgery for Vocal Process
Granuloma. Ann Otol
Rhinol Laryngol 111:789-793, 2002.
2) Hirano S, Thibeault S, Bless DM, Ford CN, Kanemaru SI. Hepatocyte Growth Factor
and its Receptor CMet in Rat and Rabbit Vocal Folds. Ann Otol Rhinol Laryngol 111:661-666, 2002.
3) Thibeault S, Gray S, Bless DM, Chan RW, Ford CN. Histologic and Rheologic
Characterization of Vocal
Fold Scarring. Journal of Voice 16:96-104, 2002.
4) Thibeault S, Gray S, Li W, Ford CN, Smith M, Davis RK. Genotypic and Phenotypic
Expressions of Vocal
Fold Polyps and Reinke’s Edema: A Preliminary Study. Ann Otol Rhinol Laryngol
111:302-309, 2002.
5) Thibeault S, Ford C. The Scarred Vocal Fold. In: Ossoff R, Shapshat S, Woodson G,
Netterville J, Eds.
The Larynx. Philadelphia, Pa: Lippincott Williams and Wilkins, 2002: 431-440.
6) Kriesel K, Thibeault S, Chan RW, Suzuki T, VanGroll PJ, Bless DM, Ford CN. Treatment
of Vocal Fold
Scarring: Rheological and Histological Measures of Homologous Collagen Matrix.
Ann Otol Rhinol
Laryngol 111:884-889, 2002.
7) Suzuki T, Connor NP, Lee K, Bless DM, Ford CN, Inagi K. Age-Related Alterations in
Myosin Heavy Chain
Isoforms in Rat Intrinsic Laryngeal Muscles. Ann Otol Rhinol Laryngol 111:962-967,
2002.
8) Hirano S, Bless DM, Heisey D, Ford CN. Effect of Growth Factors on Hyaluronan
Production by Canine
Vocal Fold Fibroblasts. Ann Otol Rhinol Laryngol 112:617-624, 2003
9) Hirano S, Bless DM, Rousseau B, Welham N, Scheidt T, Ford CN. Fibronectin and
Adhesion Molecules
on Canine Scarred Vocal Folds. The Laryngoscope 113:966-972, 2003.
10) Hirano S, Bless DM, Heisey D, Ford CN. Roles of Hepatocyte Growth Factor and
Transforming Growth
Factor β1 in Production of Extracellular Matrix by Canine Vocal Fold Fibroblasts. The
Laryngoscope
Both acute phase of vocal fold injury and chronic vocal fold scar will be examined
in terms of the alteration of cells and ECM components in vocal fold lamina propria using
animal models.
There are two approaches of therapeutic research for vocal fold scarring; to
prevent scar formation in acute vocal fold injury and to restore healthy tissue in chronic
vocal fold scar. Preliminary data: In normal rat vocal folds, vimentin-positive cells
existed in the deep layer of lamina propria and the basement membrane zone, whereas
very few myofibroblasts existed in the lamina propria. Following injury, fibroblasts
proliferated in the injured site of lamina propria with a peak at day 3 and produce ECM
components. Myofibroblasts increased from days 5 and 7 returning to near normal at
day 14. The inflammatory granuation tissue was thickest at days 3 and 5 gradually
decreasing between 1and 2 weeks. The period of tissue contraction was almost in
complete synchrony with the period of myofibroblasts. (Figure 7)
HA
appeared in
injured vocal
folds by day
3, peaked at
day 5 and
thereafter
decreased
showing
lower level
than control
(Figure 8).
Collagen type
I also
appeared in
injured vocal
folds by day
3, peaked at
day 5 and
thereafter
gradually
decreased
though it kept
higher level
Figure 8: The alteration of ECM components in
Figure 7: Cell component
than control.
vocal fold lamina propria after rat vocal fold
changes in rat vocal folds after
Collagen type
stripping. The scale bar indicates 50m.
vocal fold stripping. Cells are
III appeared
detected by
at day 1 and continued to be intense at all time points after
immunohistochemical staining
for vimentin, a general marker
day 3. Fibronectin also appeared at day 1 and continued to
of fibroblasts, and alfa smooth
be intense for 4 weeks and then decreased close to the
muscle actin (alfa-SMA), a
control level at 8 and 12 weeks.19,20,21
marker of myofibroblast. The
Significance of the preliminary data: These results
scale bar indicates 200m.
suggested that: 1) What serve as a cell source of vocal folds
in response to injury are the fibroblasts in the injured site of the lamina propria. The
macula flava do not act as a cell sourse if they are not injured. 2) Myofibroblasts play an
important role in producing contraction of the vocal fold scarring and that around 1week
following injury is a critical period for wound contraction of the vocal fold scarring. 3) The
expression of these ECM components peaks in the period of day 3-5.
4) The tissue remodeling process in scarred vocal folds slows down around 2 months
after wounding.
5) The characteristics of vocal fold wound healing are similar to that of skin in the early
phases but differ during the subsequent remodeling phase.
C2.1.3. Vocal fold development and aging: a rat model
Because fetal wounds heal scarlessly
characterization of vocal folds through
Figure 9: A) Expression of
vimentin (purple) and
prenatal, postnatal and aging process are
collagen type III (green) in
important. Vitamin A has been identified in
3-day-old, 3-week-old and
the macula flava (MF) of human vocal folds
8-month-old rat vocal folds
and rats. It is generally accepted to be a
that are sectioned into axial
morphogen that controls the differentiation
slices. B) Vimentin-positive
cells (purple) with nuclei
and morphogenesis of cells and therefore it is
staining (green) in mid
expected to be related to the development
membranous portion (MM)
and maintenance of vocal folds and may play
and posterior macula flavae
a significant role in wound healing. We will
(MF) of 3-day-old, 3-weekold and 8-month-old rat
attempt to identify the key molecules that
vocal folds lamina propria.
cause age-related changes. To study the
role of vitamin A we will use a vitamin a
deficient rat model. Preliminary data: MF
was observed as a mass of cells that expressed vimentin (general marker of fibroblasts)
intensively in the cytoplasm. MF showed denser hyaluronic acid and collagen type I
than the mid membranous portion of the vocal folds lamina propria. Clear
developmental changes were evident in macula flava and other regions. The vimentinpositive cells of MF in 3-day-old vocal folds were mainly oval-shaped and had less
cytoplasm, whereas those in 8-month-old vocal folds were spindle and stellate-shaped
and had more cytoplasm, similar to that reported in humans. Vitamin A-containing lipid
droplets, were limited to the MF of 3-week-old and 8-month-old rat vocal folds and were
not present in 3-day-old rat vocal folds. 23 Significance of the preliminary data: These
results suggest that a rat model is useful in studying of vocal folds development and that
the presence of vitamin A changes with age.
C.2.1.4. In-vitro inflammation model of vocal fold fibroblasts
Various treatments have been tested in-vivo and in-vitro. One of these
treatments involves Hepatocyte Growth Factor (HGF). HGF has mitogenic, proliferative
and anti-fibrotic effects and is produced by vocal fold fibroblasts.55 Increased HA
production was observed upon application of HGF to in-vitro vocal fold fibroblast
cultures.11 HGF is also known to affect certain cell types and their production of
cytokines, and may play a role in inflammation.73 We suggest here that HGF may affect
HA production through inflammation.
Scarless fetal wound healing is known to occur without significant inflammatory
processes. Since a key feature of scarless fetal wound healing is also elevated HA
levels57, there may be a link between inflammation, specifically the cyclooxygenase-2
(COX-2) pathway59, and the production of extracellular matrix components (HA and
collagen) by proliferating fibroblasts during the wound healing process. However, this
link has not been demonstrated in existing literature. This study aims to confirm if any
such link exists, to explain current treatment methods aimed at increasing HA levels
from an inflammation point of view, and to seek novel, more specific therapeutic agents
based on this knowledge.
The study will first establish the in-vivo expression of pro-inflammatory factors TNFalpha, Interferon-gamma and NFkappa-B in vocal folds following injury by real-time
PCR. These factors will then be administered to in-vitro vocal fold fibroblast cultures, and
COX-2 expression will be monitored as a
NFkB
marker of inflammation. HA and collagen
synthase expression monitored as well by
Realtime PCR. Introducing pro-inflammatory
factors and then HA-level boosting agents will
also be studied with relation to COX-2. The
adjacent figure shows our preliminary data of
mRNA expression level of inflammatory factors
in the lamina propria following rat vocal fold
4hrs
8hrs
16hrs 24hrs 72hrs
4hrs
injury examined by real time PCR (N=5 in each
time point). Y axis shows expression levels of
COX-2
a target gene in injured vocal folds compared
to those in normal vocal folds. Nuclear factor
1.00E+01
kappa beta (NFkB) showed earliest onset
8.00E+00
bursts following injury with peaks at 8hrs.
6.00E+00
Tumor necrosis factor-alpha (TNF-alpha)
4.00E+00
peaked at 8hrs after injury. Cyclooxygenase-2
2.00E+00
(Cox-2) showed relatively slow response with a
0.00E+00
smaller peak at 4hrs and a larger peak at
4hrs
4hrs
8hrs
16hrs 24hrs 72hrs
16hrs after injury. Transforming growth factorbeta (TGF-B) showed slowest response with
relatively weaker expression at 4, 8, and 16hrs than other factors and gradually
increased at 24hrs and 72hrs. These expression patterns of inflammatory factors
following injury are consistent with those reported in other regions such as skin.
C.2.2. Development of growth factor therapy
Growth factor is a potent regulatory element that affects cells and their functions.
We have revealed that hepatocyte growth factor (HGF) and basic fibroblast growth factor
(bFGF) significantly increases the production of hyaluronic acid (HA) and decreases
collagen production from vocal fold fibroblasts in vitro studies. 40,41,56 We have revealed
that HGF significantly increases the production of hyaluronic acid (HA) and decreases
collagen production from vocal fold fibroblasts in in-vitro studies .40,41 We also have
found that local injection of HGF prevented scar formation of injured vocal folds in a
rabbit model, and even improved the scarred vocal fold in a canine model 15 In vivo
studies, we have found that local injection of HGF prevented scar formation of
injured vocal folds in a rabbit model 16,26, and even improved the scarred vocal
fold in a canine acute phase model to some extent .50 We have also found that
local injection of bFGF improved aged vocal folds of rats in terms of histology,
which raises the notion that bFGF may also be effective
for the treatment of vocal fold with chronic scar which
has similar histological characteristics to aged vocal
folds. Indeed, our recent data shows that local injection
of bFGF improved the chronic vocal fold scar in a rat
model in-vitro (Figure 10) and in-vivo (Figure 11).22
We are now attempting to determine the ideal
combination of growth factors, dosages, the best
frequency of injection and the best route of
Figure 10. Effect of bFGF on
administration to maximize the effect on the vocal fold
cultured fibroblasts obtained
1.20E+01
1.20E+05
1.00E+01
1.00E+05
8.00E+00
8.00E+04
6.00E+00
6.00E+04
4.00E+00
4.00E+04
2.00E+00
2.00E+04
0.00E+00
0.00E+00
TNF-alpha
8hrs
16hrs
24hrs
72hrs
24hrs
72hrs
TGF-B
1.40E+02
1.20E+02
1.00E+02
8.00E+01
6.00E+01
4.00E+01
2.00E+01
0.00E+00
8hrs
16hrs
120
100
80
Normal-Has1
Scar-Has1
60
Normal-Has2
Scar-Has2
40
20
0
0hr
3hrs
6hrs
12hrs
24hrs
from chronic rat vocal fold
scar examined by real-timeFigure 11. Effect of growth
RT-PCR. Has 2 mRNA factors on chronic rat vocal
fold scar in vivo(Alcian Blue
expression level in
fibroblasts from scarred staining for HA). HA in bFGF
vocal fold increases higherinjected rats have
significantly higher HA than
than that in fibroblasts from
sham and HGF injected rats.
normal vocal fold.
scarring, and ultimately to provide the bases to initiate clinical studies.
The problems left are how much dosage of HGF and bFGF is ideal, and how
frequently HGF and bFGF should be injected, and which of them has a better effect on
acute and/or chronic vocal fold scar. The best route of the administration should be
warranted.
C.2.3. Prevention of scar and regeneration of vocal fold: for a future treatment
These studies aim to develop new treatment methods of acute/chronic vocal fold
scar for a future clinical treatment. Animal models are used for this study. Gene therapy
and tissue engineering techniques will be used for the regeneration of the vocal fold
tissue.
C.2.3.1. Gene therapy
Human vocal fold lamina propria consists of ECM components and cell components, and
hyaluronic acid (HA), collagen, and elastin are main components of
ECM that play vital roles in mechanical and biological functions. 46,48.74
Our preliminary studies have supported the notion that HA has
important biological functions other than acting as a space filling
material and that it serves both as a pathway for cell migration and
influences cell proliferation and differentiation. Using a rat model and
real-time PCR we observed an increased expression of Has1 and
Has2 mRNA in the very early phase of vocal fold injury which returned
to normal within 2 weeks. This suggested that maintaining
upregulation of Has genes may be necessary to achieve scarless
wound healing of vocal folds similar to that seen in a fetus. In
Figure 12. mRNA expression of Has
proposed studies we will transfect Has1 and Has2 genes into injured
1, 2, and 3 genes following rat vocal
folds of rats to examine the effect of Has genes on prevention of vocal
fold injury revealed by real-time PCR
fold scarring. Our preliminary studies (Figure 12, 13) have also shown
the effect of bFGF and HGF on vocal fold scarring. Administration of
bFGF for the chronic vocal fold scar returned HA to normal level and
HGF injection for acute phase scar returned collagen level to normal.
However,. bFGF wasn’t effective in terms of collagen and HGF
treatment didn’t improve the aerodynamics. One of the reasons for
the insufficient effect is thought to be a short half-life of growth factors,
only a few days. Because transfection techniques maintain
Figure 13. Expression of Has2 mRNA
following rat vocal fold injury revealed
expression of a target gene for a longer period Tateya sought training
by in-situ hybridization.
and gathered pilot data. In pilot studies bFGF and HGF were
overexpressed by transfection to prevent/restore vocal fold scarring using a
rat model. Of transfection strategies we opted for non-viral methods
because they are non invasive and considered to be suitable for the
treatment of benign conditions, such as vocal fold scarring. In-vivo
electroporation technique was used as a first step in order to maximize the
transfection efficacy . The adjacent figure shows a coronal section of
bilateral rat vocal folds 2 weeks after electroporation of enhanced green
fluorescence protein (EGFP) gene. Transfected cells in the injected side of
the vocal folds shows strong green fluorescence whereas little signal is
observed on the contralateral control side.
C2.3.2. Cell transplantation
Lamina propria consists of ECM components and cell components, mainly
fibroblasts. Our preliminary work has shown the distribution of fibroblasts in the lamina
propria of the vocal fold of developing rats. It has also demonstrated that the fibroblasts
may be primarily responsible for the production of every ECM components.51,52 We’ve
also determined to regenerate vocal fold tissue, it is important to control fibroblasts by
administrating drugs, such as growth factors or genes, to restore normal organization of
ECM to the scarred vocal fold. Our continued studies aim to find out appropriate cells
and scaffolds to regenerate the scarred vocal fold, and then examine their therapeutic
effects. The studies consist of in vitro, in vivo using animal models, and human subjects.
C2.3.3. Establishment of in-vitro inflammation model of vocal fold fibroblasts
As previously stated in preliminary work various treatments have been tested invivo and in-vitro. One of these treatments involves Hepatocyte Growth Factor (HGF).
HGF has mitogenic, proliferative and anti-fibrotic effects and is produced by vocal fold
fibroblasts .55 Increased HA production was observed upon application of HGF to in-vitro
vocal fold fibroblast cultures.11 HGF is also known to affect certain cell types and their
production of cytokines, and may play a role in inflammation.73 We suggest here that
HGF may affect HA production through inflammation.
Scarless fetal wound healing is known to occur without significant inflammatory
processes.. Since a key feature of scarless fetal wound healing is also elevated HA
levels .57 there may be a link between inflammation, specifically the cyclooxygenase-2
(COX-2) pathway59, and the production of extracellular matrix components (HA and
collagen) by proliferating fibroblasts during the wound healing process. However, this
link has not been demonstrated in existing literature. Our continued work aims to confirm
if any such link exists, to explain current treatment methods aimed at increasing HA
levels from an inflammation point of view, and to seek novel, more specific therapeutic
agents based on this knowledge.
D. RESEARCH DESIGN AND METHODS
This section is divided into two sections; methods and research design. A table
summarizing the relationship of the methods to each specific aim is provided in the
Appendix. The methods that will be commonly used in our series of experiments are
summarized here and referred to by letter. Method A describes animal models of vocal
fold scarring; Method B tissue preparation; Method C histology; Method D protein
analysis; Method E gene analysis; Method F rheologic examination; Method G excisedlarynx acoustic, aerodynamic, and movement analysis; and Method H clinical
assessment. These methods are based on knowledge and experience gained during the
last 5 years of work on laryngeal scarring. Methods that are unique to an individual
experiment are described separately. The number of rats determined for each
experiment were based on needs to meet adequate statistical power and numbers that
could be easily handled each year without posing any hardship on staff or facilities.
D.1. METHODS
D.1. Method A: Animal Models of Vocal Fold Scarring
D.1.1. Rat videolaryngoscopic surgery. Rat videolaryngoscopic surgery will be
performed as described by Tateya.20 Rats will be anesthetized with an intraperitoneal
injection of ketamine (90mg/kg) and xylazine (9mg/kg). The animals will be placed on an
operating platform in a near-vertical position. A suspension microlaryngoscope
fabricated from a
1mm diameter
Figure 14 (A) Rat videolaryngoscopic
surgery. A rat is placed on an
steel wire20 will be
operating platform in a near-vertical
inserted though the
position. A suspension
animal’s mouth to
microlaryngoscope fabricated from
help visualization
1mm diameter steel wire is inserted
of vocal folds.
though the mouth to help visualize
the vocal folds. (B) A rat larynx
Vocal folds will be
visualized by monitoring with a
visualized by
1.9mm diameter telescope with an
angle of 25. (C) Left vocal fold
stripping is performed using a 25G
needle. The right side is kept intact
and used as a control.
monitoring with a 1.9mm diameter telescope with an angle of 25 (Richard Wolf, Vernon
Hills, IL, USA). Using a 25G needle and microforceps, unilateral vocal fold stripping will
be performed for histologic study and in situ hybridization, and the thyroarytenoid muscle
will be exposed. The contralateral side will be kept intact and used as a control. For realtime polymerase chain-reaction (PCR) study, bilateral vocal fold stripping will be done to
get enough sample volume to perform real-time PCR, and control samples will be
collected from untreated rats.
The rats will be anesthetized with a mixture of ketamine (90mg/kg) and xylazine
(9mg/kg) and humanely euthanized by injecting a euthanasic agent (0.22mL/kg)
intracardially.
D.1.2. Canines with kennel cough exhibiting general edema and unilateral or
bilateral sulcus. The larynges of these canines will be obtained from the kennel cough
cases that have been sacrificed for other purposes and identified by veterinarians in the
School of Veterinary Medicine, University of Wisconsin-Madison. These excised canine
larynges with naturally occurring sulci will first be run on the excised-larynx system
(Method G) to determine the impact of the sulcus on vibration. The same specimens will
subsequently, be histologically studied (Method C) to determine how the location and
depth of the sulcus impact vibration. No surgery will be done on these specimens.
D.1.2. Method B: Tissue Preparation
D.1.2.1. Human and canine larynges. Human larynges will be obtained from autopsy
cases and canine larynges will be obtained from the cases used and euthanized in the
School of Veterinary Medicine, University of Wisconsin-Madison.
For histology specimens (Method C), vocal fold tissue will be cut into pieces of
approximately 0.5mm thickness coronally or horizontally, quickly frozen with a
combination of acetone and dry ice, and preserved in a –80oC freezer. Ten to 30µm
cryostat sections of vocal folds will be prepared, air-dried, and stored at -20oC until
they’re used. For electron microscopy, larynges will be sectioned into pieces of 0.5-1.0 x
5 x 10mm.
For rheologic examination (Method F) and excised-larynx studies (Method G),
whole larynges will be removed, quickly frozen with liquid nitrogen, and then preserved
in a –80oC freezer until they are used.
D.1.2.2. Rat tissue preparation. Rat tissue will be obtained using rat
videolaryngoscopic surgery under the procedure described in Method A. For histologic
study (Method C), whole excised rat larynges will be harvested soaked in embedding
medium (optical coherence tomography compound, Tissue-Tek, Kyoto, Japan), quickly
frozen with a combination of acetone and dry ice, and kept in a freezer at –80oC. For
conventional stains, 10µm cryostat coronal or axial sections of vocal folds will be
prepared, air-dried, and stored at -20oC until used. For immunohistochemistry study, 10
to 30µm cryostat coronal or axial sections of vocal folds will be prepared, air-dried, and
stored at -20oC until used. For protein analysis (Method D) and gene analysis (Method
E), whole excised rat larynges will be harvested soaked in embedding medium, quickly
frozen with a combination of acetone and dry ice, and kept in a freezer at –80oC.
Additionally, for protein analysis (Method D) and real-time reverse-transcription PCR
(RT-PCR), a microdissection technique will be used to collect the lamina propria from
larynges accurately. Sixty µm cryostat axial sections of the vocal folds will be prepared
and the entire lamina propria dissected by visualizing it with a microscope and using
30G needles. The tissue will be collected in tubes and kept in a freezer at –80oC. For
rheology (Method F) and excised-larynx study (Method G), the whole larynx will be
removed, quickly frozen with liquid nitrogen, and then preserved in a –80oC freezer until
it is used.
D.1.3. Method C: Histology
D.1.3.1. Conventional stains. Elastica-van-Gieson (EVG) staining will be performed to
identify collagen and elastin separately. Masson’s trichrome staining will be used to
detect collagen fibers more clearly than with EVG staining. Alcian blue staining (pH 2.5)
will be used to identify HA. The hyaluronidase digestion technique will be used to detect
HA. For the hyaluronidase digestion procedure, 50mg of bovine testes hyaluronidase
(Sigma, St. Louis, MO) will be diluted in a 100mL phosphate-buffered saline (PBS)
solution. Each section will be incubated in this solution for 1 hour at 37oC. Next, the
sections will be stained with Alcian blue (pH 2.5). HA will be detected by comparing the
sections without digestion to those with digestion. Five slides will be used for each stain
per animal.
D.1.3.2. Immunohistochemistry. Tissue sections will be unfixed or fixed in 4%
paraformaldehyde and washed 3 times in a PBS solution. For immunofluolescence
study, sections will be blocked with 5% normal goat serum and 0.1% Triton-X in PBS,
incubated with adequately diluted first antibodies with 1% normal goat serum and 0.1%
Triton-X, washed in PBS, and incubated with adequately diluted second antibodies and
TOTO-3 (200nm). Finally, samples will be washed 3 times in PBS and mounted on
coverslips in Vectashield (Vector Labs, Burlingame, CA) for observation under a laserscanning confocal microscope (Bio-Rad H600, Hercules, CA). A DakoCytomationLabelled Streptavidin-Biotin2 System (Dako, Carpinteria, CA) will also be used for lightmicroscopy observation using the same first antibodies and counterstaining them with
Mayer’s Hematoxylin.
D.1.3.3. Electron microscopy (Immuno-SEM). Immunohistochemstry is done the same
method as stated above in Method C except for using the thick sliced tissue (see Method
B) and gold- conjugated secondary antibody. After immunolabeling, the tissue is fixed in
1% glutaraldehyde for 1 hour, dehydrated in a graded series of concentrations of ethanol
and dried using a dryer for 1 hour, and coated with an iron beam coater.
Cd \
Md Observation is conducted with a Hitachi S900 High Resolution, Low Voltage, FieldEmission Scanning Electron Microscope (Hitachi High Technologies America, Inc.
Pleasanton, CA).
D.1.3.4. Optical coherence tomography (OCT).. All of the laryngeal scars studied in
our excised-larynx experiments will be imaged with an OCT system obtained from the
Ophthalmology Department at the University of Wisconsin-Madison (see letter of
support). This system is the same one reported in our preliminary studies. Because this
technology is currently being adapted for clinic use, the laboratory techniques used to
characterize laryngeal scarring should eventually be available in clinics. Indeed, OCT
used in operating rooms has already helped guide the surgical treatment of laryngeal
carcinoma 79 although the additional restrictions of endoscopy introduce new challenges
as surgeons and engineers attempt to achieve clarity and precision of in vitro images.
80,81
Attempting to use OCT to image the laryngeal mucosa in an outpatient office setting
would present additional challenges .82
D.1.4. Method D: Protein Analysis
D.1.4.1. Enzyme-linked immunosorbent assay (ELISA). Hyaluronic acid production
will be examined using a sandwich ELISA test. Ninety-six-well microtiter plates (Fischer
Scientific, PA) will be coated with 25μL of 0.5μg/mL hyaluronic acid binding protein
(HABP; Seikagaku America, MA) and incubated overnight at 4oC. The plates will be
blocked with 2% bovine serum albumin (BSA) in a sodium phosphate buffered solution
(pH 7.1), and incubated for 2 hours. The plates will be washed 3 times with PBS
containing 0.05% Tween 20, and then twice with PBS. Twenty-five microliter aliquots of
standard HA (Seikagaku America, MA) or samples will be dispensed into the wells and
the plates incubated for 1 hour. Samples will be examined in duplicate. After the plates
are washed, 25 microliter aliquots of 1μg/mL biotin-labeled hyaluronic acid binding
protein (B-HABP; Seikagaku America, MA) will be added into the wells. The plates will
be incubated for 1 hour and then washed. After the addition of 25 microliter aliquots of
1μg/mL peroxidase-conjugated streptavidin (Pierce Endogen, IL), the plates will be
incubated for 30 minutes and then washed again. An aliquot (40μL) of 3,3',5,5'tetramethylbenzidine (TMB) (KPL Inc., MD) in an acidic buffer will be added. The
reaction will be stopped with 40 microliter TMB stop solution. Absorbance will be read at
450nm by a microplate reader (ELX800, Bio-Tek Instrument Inc., VT). The
concentrations of HA in samples will be extrapolated from the standard curve.
Soluble collagen type I in supernatant media will be detected by capture ELISA
kit (MD Biosciences Inc., Montreal, Canada) and fibronectin production will be examined
using an inhibition ELISA kit (Chemicon International Inc., CA).
D.1.4.2. SDS-PAGE. Samples will be immediately placed in a skinning solution (100mM
KCl, 10mM imidazole, 1mM MgCl2, 2mM EGTA, 4mM ATP, 1% Triton X-100, and 1%
protease inhibitor cocktail; pH 7.0) at 4°C for 60 minutes and then washed in an identical
solution without Triton at 4°C for 2 minutes. Next, the samples will be placed in 50µL
boiling buffer solution (5% SDS, 10% glycerol, 60mM Tris; pH 6.8), heated, and vortexed
for up to 30 minutes to facilitate dissolution. The samples will then be frozen and stored
at -80°C in preparation for electrophoresis.
Two-dimensional SDS-PAGE will be performed using the method described by
O’Farrell et al. 69 Isoelectric focusing will be performed in glass tubes (2.0mm inner
diameter) using 2.0% pH 3.5-10 ampholines (Amersham Pharmacia Biotech,
Piscataway, NJ) for 9600 V-hours. Fifty ng of an isoelectric focusing internal standard
(either carbonic anhydrase or tropomyosin) will be added to each sample. Carbonic
anhydrase migrates with a molecular weight (MW) of 29kDa and an isoelectric point (pI)
of 7.3. Tropomyosin migrates with a MW of 33kDa and pI of 5.2. A pH gradient plot for
the set of ampholines will be generated using a
surface pH electrode. The gels will be silver-stained
using the protocol described by Oakley et al .70 The
gels will be scanned using an Agfa Arcus II flatbed
scanner (Agfa, Mortsel, Belgium) in transparent mode
at 200 dpi resolution and 24-bit image depth. Image
analysis will be performed using Melanie 4.02
software (GeneBio, Geneva, Switzerland) and
Melanie II-a software (Swiss Institute of
Bioinformatics, 2003). This software package utilizes
Figure 15. Illustration of quantifiable protein-spot
a protein-spot detection strategy incorporating a
features, visualized using (A) an intensity variation
profile and (B) a 3-D representation of the
diffusion-smoothing algorithm, a second derivative
highlighted area. (MW = molecular weight, pI =
feature-detection algorithm, and a minimum pixel-area
isoelectric point, Int. = pixel intensity.)
parameter. Quantifiable spot features (used as indices
of relative protein quantity) include optical density (OD), area, and volume (Figure 15).
The OD will be calculated as the difference between the maximum pixel intensity in a
given spot and the minimum pixel intensity in the background region immediately
adjacent to the spot. The area (inside the spot border) will be calculated at 0.75 of OD
(as measured from the point of maximum pixel intensity). The volume will be calculated
as the sum of the intensity of each pixel within the spot border. Estimated MW and pI
values for identified spots will be determined from interpolation and extrapolation
(logarithmic for MW; linear for pI) of values from known internal standards.
D.1.5. Method E: Gene Analysis
D.1.5.1.Real-time RT-PCR. Tissue from Method B will be treated with proteinase K.
mRNA will be extracted using an RNeasy Micro kit (Qiagen, Valencia, CA, USA) and
treated with RNase free DNase I (Qiagen, Valencia, CA, USA) to digest potentially
contaminated genomic DNA. Reverse transcription will be performed using Superscript
III (Invitrogen, Carlsbad, CA, USA) to synthesize first-strand cDNA. Real-time PCR will
be performed in a 20µL volume using the manufacturer's protocols and the reaction mix
will be examined. The mix is composed of 2µL of template cDNA, 2µL of LightCycler
DNA Master SYBR Green I (Roche Applied Science, Indianapolis, IN), 4mM MgCl2,
0.5µM final concentration of each primer, and RNase-free H20 to 20µL and expression of
target genes, such as Has1, Has2, Has3, Hyal1, Hyal2, Hyal3, Hyal4, matrix
metalloproteinase (MMP)-1, MMP-8, MMP-9, MMP-13, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) genes.
Amplification will be performed under the following conditions: 1 cycle of 95°C for 110
seconds, followed by 40 cycles of 95°C for 10 seconds, 55°C for 10 seconds, and 72°C
for 20 seconds, and 1 cycle of 60°C to 95°C to make a melting curve. Fluorescence will
be detected with a Smart Cycler II system (Cepheid, Sunnyvale, CA) and accompanying
software. RT(-) samples, for which reverse transcriptionase is not added during RT, will
also be treated with PCR reaction as a negative control sample to clarify that an
amplified DNA band is not made from contaminated genomic DNA. Primer dimmer
production will be checked using melting curves and gel electrophoresis after PCR.
Quantitative analysis will be performed based on a delta-CT method using standard
curves, and the ratio of target gene concentration to GAPDH will be calculated.
Statistical analysis will be performed by non-repeated ANOVA with a significant p value
of 0.05.
D.1.5.2. In situ hybridization. In situ hybridization experiments will be performed as
described by Takebayashi, Akazawa, and Kageyama (1995) with some modifications84.
In brief, cDNA templates of a target gene will be generated by PCR. cDNA, including the
coding region, will be subcloned into pGEM-T Easy (Promega, Madison, WI) and used
as the template for generating digoxigenin (DIG)-labeled RNA probes by in vitro
transcription. The templates will be generated by linearization with restriction enzymes
for each antisense and sense probes. Antisense probes will be synthesized by runoff
transcription from a T7 promoter with DIG-uridine triphosphate using an RNA labeling kit
(Roche Applied Science, Indianapolis, IN). Meanwhile, DIG-labeled sense probes,
synthesized by runoff transcription from SP6 promoter will be used as negative control
probes. The tissue sections described in Method B will be fixed in 4% paraformaldehyde
(PFA) for 30 minutes, rinsed with 0.1M phosphate-buffered saline (pH 7.5) containing
0.1% Tween-20 for 5 minutes twice. Hybridization will be performed at 65°C overnight in
a hybridization buffer (50% formamide, 1% dextran sulfate, 5 x SSC, 50ug/mL heparin,
50ug/mL yeast tRNA) containing DIG-labeled RNA probes. The sections will be washed
in 5 x SSC at 65°C for 30 minutes, 2 x SSC at 65°C for 30 minutes 3 times, and washed
with TBST 3 times. The probes will be revealed by alkaline-phosphatase-coupled antiDIG antibodies (Roche Applied Science, Indianapolis, IN), which react with nitrobluetetrazolium-chloride and 5-bromo-4-chlor-indolyl-phosphate substrates for color reaction.
D.1.6. Method F: Rheologic Examination
D.1.6.1. Linear viscoelastic shear properties. Linear viscoelastic shear properties of
the scarred and normal lamina propria will be measured. Elastic shear modulus (G’) and
dynamic viscosity (Eta’) will be determined as a function of oscillation frequency (0.1-300
Hz). Larynges will be excised immediately postmortem and quick frozen using liquid
nitrogen and then stored in a freezer at –70oC. Prior to analysis, the larynges will be
thawed overnight at 4oC and warmed gradually to 37oC, at which time the lamina propria
will be dissected. The tissue samples will be maintained at 37oC +0.1oC in a water bath
until mounted in a rheometer for viscoelastic measurements. This temperature will also
be maintained in the rheometer via a temperature-control unit. Small amplitude
sinusoidal oscillatory shear deformation of the sample will be performed with a controlled
strain rheometer (RFS-3 Rheometric Scientific, Piscataway, NJ). A parallel plate (plate
on plate) testing geometry will be used, consisting of a stationary lower plate and a
rotating upper plate (diameter = 7.9mm) and a gap size of 1mm. The lamina propria
sample will be placed in the gap between the two plates and subjected to precisely
controlled sinusoidal torque. A sensitive transducer will monitor the resulting angular
displacement and angular velocity of the upper plate as a function of time.
Shear stress, shear strain, and the strain rate associated with oscillatory shear
deformation will be computed from the prescribed torque and the measured angular
velocity by a computer, and linear viscoelastic functions (elastic shear modulus G’ and
dynamic viscosity Eta’) will be quantified based on the theory of linear viscoelasticity. A
shear-strain amplitude of 1.0% will be used to ensure oscillation linearity, and
measurements will be obtained at a frequency of 0.1-316 rad/s (corresponding to 0.01650Hz) at 37oC. The viscoelastic data will be compared using Student’s paired t test for
the normal and treated side in each group, and Mann Whitney nonparametric testing will
be used for comparison between growth factor-treated and sham-treated vocal folds. A p
value < 0.05 is considered statistically significant.
D.1.6.2. LSR basic equipment..
Laryngeal specimens from animal sources will be prepared by sagittal
hemisection, taking care to leave the vocal fold attachment to the thyroid cartilage at the
anterior commissure region intact. The specimens will be affixed to a firm table-top. The
specimens will be kept moist with physiologic saline and measurements will be made at
room temperature (roughly 20oC). Measurements will be made using needle-tipped
probes that have a fine ridge carved into them 1mm from the distal tip so that consistent
depth penetration can be monitored. We will use the most effective of several designs
tested; the needle is made from a spring steel rod 1mm in diameter and 10cm in length
and bent to a right angle 5mm from one end. A fine insect pin
will be soldered to the short bent section so that it protrudes
1.5mm beyond the end of the rod. This needle will be inserted
into the tissue up to the rod, which controls the insertion depth
to 1.0mm.
Basic materials will include human, dog, and rat
larynges. Dog and rat vocal folds will be subjected to
manipulation with growth factors, a 585nm pulsed dye laser,
and genetic transfection. The vocal folds will be tested along
with controls using the materials described above. Dog vocal
folds will be tested several times at 24 to 30 points along the
Figure 16. Schematic drawing of a linear
folds for maximal mapping of rheologic changes. Rat vocal
skin rheomatic sensing head.
folds are too small to allow for mapping, so 1 central point will
be tested on the control side and on the test side, using multiple trials at this central
point to maximize statistical power.
Using these noninvasive measurements of histologic and viscoelastic properties
of the laryngeal mucosa, we can correlate the properties of the scarred folds with the
resultant acoustic (phonatory) signals. For our in vitro studies, we will be using an OCT
system provided to us by the Department of Ophthalmology at the University of
Wisconsin (see signed statement). For the LSR studies, we will
be using a system designed by Eric Goodyer (see Appendix for
additional details).
D.1.7. Method G: Excised Larynx Experiments
We will examine vocal fold vibration in larynges using an
excised larynx setup that maintains in vivo conditions of
Figure 17. Scheme of excised larynx setup
temperature and humidity while recording high-speed images of the vibratory pattern,
airflow, pressure, and acoustic signal. In addition, normal untreated larynges will be
examined in the identical manner and used as controls.
To better visualize the vocal folds of excised whole larynges, supraglottic
structures, such as the epiglottis, the false vocal folds, and the aryepiglottic folds, will be
removed following resection of the superior portion of the thyroid cartilage. Both right
and left arytenoid cartilages will be sutured to each
other by 5-0 or 6-0 nylon strings to obtain glottal
closure.
Figure 17 shows the general experimental setup
we will use to monitor vocal fold vibration. The larynx
will be mounted with the upper section of the trachea
mounted on top of a pipe. The trachea is tightly
clamped to the pipe with a hose clamp to avoid any air
leakage. The larynx will be stabilized with a circular
multipronged device. An air compressor will be used to
generate airflow. The flow quantity will be controlled
Figure 18. Measurement of mucosal
wave amplitude using a rabbit model.
throughout the experiment with a valve and measured
(A) closed phase, (B) maximum open
with a flow meter. The air will be humidified to 100%
phase. Mucosal wave amplitude = (d2humidity at 36 to 38oC by passing it through a heaterd1) / L. (d1, d2 = the distance from the
humidifier (3M, Bird Co., Palm Springs, CA). Continual
midline of the glottis to the free edge of
the vocal fold, L = the length from the
dripping of saline onto the vocal folds will also help
anterior commissure to the vocal
prevent dehydration of the vocal folds.
process.)
A high-speed digital imaging system (Kodak
Ektapro [Photron] 4540) will be used to record glottal
vibrations from a superior view using a Nikon lens
(200mm micro-Nikkor). The camera will be mounted 50cm above the larynx and the
image viewed on a monitor. The images will be recorded at a frame rate of 4500 frames
per second with a CCD array of 256 x 256 pixels and an 8-bit monochrome intensity
level per pixel. A pressure sensor (Micro switch PK8772-4, Honeywell Inc., IL) will be
mounted on the pipe directly beneath the vocal folds to monitor subglottal pressure.
Subglottal pressure will be increased by 2cmH2O increments from the phonatory
threshold pressure, and glottal images will be taken at each increase. The phonatory
threshold pressure, vocal efficiency, amplitude of mucosal wave, and glottal gap will be
compared across normal, treated, and sham groups. Vocal efficiency is defined by the
formula: sound pressure level [dB] over subglottal pressure [cmH2O]. The amplitude of
mucosal wave and glottal
gap will be examined
using image analysis
software (Scion Image
beta3b, Frederick,
Maryland). The distance
(d1) from midline of the
glottis to the free edge of
the vocal fold will be
measured at the
anteroposterior middle
portion of the vocal fold at
the closed phase, and then
the same distance (d2) will
be measured at the
maximum open phase
(Figure 18). We will define
the mucosal wave
amplitude by subtracting d1
from d2 and then
Figure 19. Measurement of
normalize it by dividing the
result by the length (L)
glottal gap using a rabbit
from the anterior
commissure to the vocal
model. Glottal gap = a / L2. (a =
glottal area, L = the length from
process: mucosal wave
amplitude = (d2-d1) / L.
the anterior commissure to the
The glottal gap will be
examined on the images
vocal process.)
obtained at the closed
phase. The glottal area (a)
will be measured and then normalized by dividing it by L2 (Figure 19): glottal gap = a / L2.
Perturbation measures will be obtained from the high-speed images using the Yan GAW
(2004) analysis program that converts the glottal area waveform into perturbation plots.77
Measures of the mucosal wave will be obtained from software developed by Jiang
(2005). These values will be calculated in each larynx at PTP and 18cmH2O of subglottal
pressure.85
D.1.9. Method I: Surgical Treatment: Animals and Humans
D.1.9.1. Pulsed dye laser in animal models. A 585nm PDL (Cynosure, Chelmsford,
MA) will be used in acute and chronic scar models in the rat. With the rat under
anesthesia and the vocal folds exposed (Method A), the PDL will be applied through a
600-micron flexible fiber. The fiber will be supported from the superstructure of the
device holding the rat. A non-test vocal fold will be covered by dark impermeable
material to prevent exposure to the PDL.
The total exposure of the vocal folds to the energy of the PDL will vary. The fiber
tip will be at variable distances from the test vocal fold to alter the fluence (laser energy)
applied. The energy per pulse can also be adjusted (approximately 500 millijoules), as
can the number of total pulses. The fiber tip will be introduced perpendicular to the
medial surface of the test vocal cord. Surgilube will be lightly applied to the vocal fold
prior to PDL application to prevent drying of the fold from the laser.
After application of the PDL, the rat will be awakened and maintained until serial
PDL applications are completed or euthanasia (Method A). Each experimental rat will
undergo at least 1 PDL application. One month after the last PDL application, each rat
will be euthanized and the larynx subjected to analysis using excised larynx vibration,
LSR, histology, and ELISA testing (see Methods G, F. A, C, and D, respectively).
D.2. RESEARCH DESIGN
D.2.1 Computational Model of the Vocal Fold
D.2.1.1. Different anterior-posterior lengths of a mucosal scar in the finite-element
model. In a finite-element study from our previous proposal (Berry et al, in press), we
have already investigated most of the variations enumerated in the physical model
experiment described in D.2.2 below. The only exception is that we have not yet studied
variations in the anterior-posterior length of the scar. There is some indication in the
literature that such variations in the viscoelastic tissue properties along the anteriorposterior length of vocal folds may be responsible for generating different anteriorposterior vibration modes in vocal folds (Neubauer, 2001). Furthermore, prior clinical
experience suggests that an anterior scar is more detrimental to phonation than a
posterior scar, although this observation or hypothesis has never been systematically
evaluated using laboratory or computational models of phonation. Thus, we propose to
investigate this hypothesis and implement anterior-posterior variations in scar length and
report the resultant tissue vibrations and acoustic output. We will use descriptive
statistics to summarize our findings from the finite element modeling.
D.2.1.2. Left-right asymmetry in the finite-element model. To achieve left-right
asymmetry, we will repeat experiment D.2.2 from the physical model experiment
described below on the finite-element model. The only exception is that a near-complete
set of permutations between left and right will be performed on the model, because
repeating an experiment thousands of times (72 x 72 = 5184) is feasible on a computer
model but not on a physical model. The results of the computer modeling will be
summarized with descriptive statistics.
D.2.2Physical Model of the Vocal Fold
D.2.2.1. Investigating the influence of the size and position of a vocal fold scar
using a physical model. A physical model enables precise positioning and sizing of the
sulcus in the vocal fold mucosa, much more so than in human subjects or excised-larynx
models. In addition, because a physical model’s oscillations are flow-induced in an
actual physical setup and are not subject to numerical instabilities, the results from these
experiments provide a completely independent assessment of the predictions generated
from computer studies. As described in our preliminary studies, a physical model of a
normal vocal fold will be made by curing a polyurethane compound into a cast or model
of the desired vocal fold shape. The scarred vocal fold will be generated as follows:
(1) Use the same cast as that for a normal vocal fold. (2) Using the appropriate
ratios of the polyurethane materials, generate a polyurethane block that is 3 to 30 times
as stiff as a normal vocal fold (the normal fold has a stiffness of approximately 14kPa).
Let the block cure. This block will serve as the material from which to extract the vocal
fold scar. (3) Out of this block, cut a cylindrical rod of desired diameter. Cut the rod in
half by placing a scalpel along the circular diameter of 1 of the ends of the rod and then
cutting the rod along its entire length. (4) Place the half-cylinder rod in the desired
location of the cast, with the flat end (e.g., the cut end) facing the bottom of the cast
(e.g., the medial surface of the fold). (5) Pour a polyurethane liquid into the cast over the
scar. (6) Wait until the polyurethane substance cures. (7) After creation of two such
vocal folds, the folds will be mounted into acrylic plates and then pressed together, with
the plates mounted over a supply of compressed air, as described in our preliminary
studies. (8) Subglottal pressure will be increased, starting from zero, until phonation
occurs.
The following variables will be measured: (a) phonatory threshold pressure, (b)
amplitude of vibration, (c) degree of glottal closure, (d) mucosal wave velocity, (e)
mucosal wave shape, (e) glottal airflow, and (f) acoustic output. We will measure the
vibration variables under the following conditions:
(1) The lateral depth of the scar will be changed at the following increments: 0.5,
1.0, 1.5, and 2.0mm (4 conditions). (2) The vertical thickness of the scar will be
implemented at 0.5 and 1.0mm (2 conditions).
(3) The top of the scar will be positioned below the top of the medial surface of the folds
at the following increments: 0.5, 1.5, and 2.5mm (3 conditions). (4) The anterior-posterior
length of the scar will be 2 mm, half the vocal fold length, and the full vocal fold length (3
conditions), always positioned mid-way between the anterior-posterior ends of the folds.
Thus, we propose to simulate the size and position of the scar under a total of 72
conditions, contrasting all the conditions according to the vibration variables. Phonatory
threshold pressure, acoustic intensity, and fundamental frequency will also be
measured, along with all the vibratory variables enumerated in Aim 1 (A.3.1). Finally,
the properties of the laryngeal scar will be correlated with the measured phonatory
variables. The relationship of the six "vibration" variables to the four scar variables will
be characterized with multiple regression. The same technique will be used to examine
how scar characteristics affect the phonatory variables.
D.2.2.2. Left-right asymmetry using a physical model. Vocal fold scarring can occur
unilaterally or bilaterally. However, we have not yet systematically investigated the
influence of a unilateral scar in any of our models because it requires the use of a full
larynx model and asymmetry between the left and right vocal fold. Significantly, our new
physical model allows us to phonate a full larynx just as easily as a hemilarynx . 43,44
Thus, for this experiment, we propose to repeat the scarring conditions explained in
D.2.2.3 for the left vocal fold only. The right vocal fold will not be scarred. Thus, we will
systematically investigate the influence of a unilateral scar on vocal fold vibration. The
statistical analysis will be similar to that for Experiment D.1.2.1. This will allow us to
estimate and compare the effect of bilateral versus unilateral scarring.
D2.2.3. Optimization of a common phonosurgical treatment of scarring using a
physical model. One condition we have not considered up to this point in our models is
the underlying idea of mucosal slicing, in which a series of vertical scars interrupts the
anterior-posterior fibrotic tension while at the same time allowing mucosal wave
propagation up the medial surface of the folds (at least in the non-scarred regions).
Indeed, a systematic laboratory investigation has never been conducted to contrast this
type of vertical scarring with 1 long longitudinal scar near the vocal fold edge. By
applying an adhesive to the medial surface of the physical model for the 2 different
conditions, we will compare these 2 techniques.
We will use ANOVA to examine differences between vertical and longitudinal scarring.
D.2.3 Excised-Larynx Model of Vocal Fold Vibration
The general methods for excised-larynx experiments have already been
described (Method G). Although scars cannot be simulated as precisely and
systematically on an excised larynx as on a physical model or computational model of
vocal fold vibration, one advantage of the excised larynx is that it is a closer
representation of the intact human larynx than the other models. For this reason, the
excised larynx can help bridge the gap between laboratory and clinical observations.
Thus, we propose to phonate a variety of excised larynges, using specimens from
humans, dogs, pigs, and rats. Scars will be induced on human, porcine, and rat larynges
using trichloracetic (TCA) acid, as described in earlier work .13 The naturally occurring
sulci or scars found on canines will also be studied . 14,13,35
The main focus of our new in vitro and in vivo studies will be to repeat our
previous studies using new non-invasive measures of tissue histology and
viscoelasticity. Tissue histology will be measured using OCT (Method C) and tissue
viscoelasticity will be measured using LSR (Method F). On the excised larynx, we will
attempt to simulate the size and position of the sulcus for the same 72 conditions
mentioned for the physical model using TCA acid as described in earlier work 13 and our
preliminary studies, using human larynges for all conditions, but porcine and rat larynges
for a subset of the conditions. We will also examine naturally occurring sulci on 12
different canine specimens. In all of the experiments, the viscoelasticity and the extent of
the induced (or naturally occurring) scar will be measured using LSR and OCT,
respectively. Phonatory threshold pressure, acoustic intensity, and fundamental
frequency will also be measured, along with all the vibratory variables enumerated in
Aim 1 (A.3.1). Finally, the properties of the laryngeal scar will be correlated with the
measured phonatory variables. The relationship between scar characteristics and the
viscoelastic and phonatory responses will be examined with multiple regression models.
D.2.4. Homeostasis of Extracellular Matrix Components
D.2.4.1. In vitro studies. In our series of studies designed to characterize the ECM
structure of the lamina propria, we will use these specimens:
(1) human vocal folds from autopsy cases and canine vocal folds from dogs with
kennel cough,
(2) rats at different ages and genders (previous work completed on young male
rats by Tateya et al 23 will be duplicated on old male rats and both young and old female
rats),
(3) rats having vocal fold wounds from videolaryngoscopic surgery (Method A).
The methods used will be those developed during the previous 5 years.
Immunohistochemistry and electron microscopy (Method B) will be used to reveal the
distribution and the fine structure of collagen subtypes, elastin, and SLRPs in humans,
canines, and rats. Using rat specimens, we will perform real-time RT-PCR to examine
the homeostasis of collagen, HA, and SLRPs in detail, and in situ hybridization will be
performed to locate the cells that produce ECM components in normal and scarred vocal
folds (Method E). Analysis of Variance models (ANOVA) will be used to examine
differences in homeostasis between e.g. sex or age.
D.2.4.2. Establishing an in vitro inflammation model of vocal fold fibroblasts. To
examine the effect of various factors on reducing inflammation, we will establish an in
vitro inflammation model of vocal fold fibroblasts. At first, we will determine the effect of
introducing pro-inflammatory or pro-apoptotic factors on the cells of the vocal fold. Rats
will be humanely euthanized (Method A), vocal fold lamina propria will be collected from
vocal folds under a microscope, and the specimens will be placed in a Petri dish and
chopped into pieces. The tissues will then be incubated in Dulbecco’s modified Eagle’s
medium (DMEM, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS,
Invitrogen, Carlsbad, CA), penicillin (10,000 IU, Invitrogen, Carlsbad, CA), streptomycin
(10,000μg/mL, Invitrogen, Carlsbad, CA), and amphotericin B (25μg/mL, Invitrogen,
Carlsbad, CA) in an incubator containing 5% carbon dioxide and 95% air at 37oC.
Fibroblasts at first passage will be subcultured in 6cm dishes and incubated with
DMEM and 10% FBS in an incubator containing 5% carbon dioxide and 95% air at 37oC.
After 90 to 100% confluence, the medium will be replaced by a non-serum medium
(AIM-V Media, Invitrogen, Carlsbad, CA). After incubating for 2 days, tumor necrosis
factor (TNF)-alpha, interferon-gamma, and nuclear factor (NF) kappa-B will be added
into each dish. The cell sample will be collected after culturing for 0, 3, 6, 12, and 24
hours. The culture medium will also be collected from each sample and used for ELISA
study (Method D) to analyze HA concentration.
Total RNAs will be extracted from the cells and reverse transcription will be performed to
obtain first strand cDNA. COX-2 mRNA expression will be examined by real-time PCR
(Method E). The pro-inflammatory factor that stimulates COX-2 expression most strongly
will be used as an initiation factor of the in vitro inflammation model. Collagen type
I/III/HA synthase expression and apotosis will also be monitored by real-time PCR.
Subsequently, the effect of introducing selected factors in the presence of
elevated levels of HA will be determined in order to examine the effect of high HA levels
on the inflammation process. Again, collagen type I/III/HA synthase expression and
apotosis will be monitored.
As the last step, growth factors, such as HGF, will be applied after initiating the in
vitro inflammation model, and the effect will be examined in terms of COX-2 expression
and HA production. The relationship between COX-2 expression and outcome of the
inflammation model in terms of HA concentration will be clarified. After establishing an in
vitro inflammation model, different factors will be applied to examine their effect on
reducing inflammation. In order to account for nesting in the experimental structure,
linear mixed effect models will be used to examine the inflammation reduction of the
different growth factors.
D.2.4.3. Optimizing the dosage, timing, and type of growth factor used in treating
acute or chronic vocal fold scars. To determine the ideal dosage and timing of HGF
and bFGF, drugs will be injected into acute (3 days postoperatively) or chronic (2 months
postoperatively) scarred rat vocal folds at different concentrations (10ng, 100ng, and
200ng). The injection will be repeated at different times (once/week x 1 and 5 times).
The effects in preventing or reducing a scar will be examined by histology, rheology, and
excised-larynx study. The results will be used to determine the most appropriate way of
administering these treatments and serve as the bases for further studies.
Three hundred and fifty-five Sprague-Dawley male rats (400-450g in weight) will
be involved in the proposed studies. Rat vocal fold stripping will be performed (Method
A). The other side will be kept intact and used as a control. Five percent of the rats will
be randomly selected and sacrificed 1 day following surgery to histologically confirm the
injury. The remaining rats will be randomly divided into 28 groups, each group consisting
of 12 rats.
The variables of interest defining the groups include: type of scar, acute (3 days
postoperatively) or chronic (2 months postoperatively); material injected (PBS, bFGF, or
HGF), volume of drug injected (10ng, 100ng, or 200ng); and number of injections
(ranging from 1 to 5 times). Ten microliters of PBS, HGF (10ng, 100ng, or 200ng in 10μL
of PBS, Sigma-Aldrich, St. Louis, MO), or bFGF (10ng, 100ng, or 200ng in 10μL of PBS,
Sigma-Aldrich, St. Louis, MO) will be injected into the scarred vocal fold of each group.
Injections will be given weekly for 1 or 4 weeks using a 27G Hamilton’s syringe
(Hamilton, Reno, NV) under videolaryngoscopy.
The rats will be humanely sacrificed 2 months after the last injection. Whole
larynges will be harvested and used for histologic, rheologic, and aerodynamic
examination. We will use Alcian blue stain, EVG stain, and immunohistochemistry to
evaluate hyaluronic acid, collagen, and elastin. For rheologic examination, larynges will
be examined by LSR. PTP values and vocal fold vibration amplitude will be analyzed by
excised-larynx study (see Methods B, C, F, and G). The histological, rheology, and
aerodynamic data will be analyzed with ANOVA models. With twelve rats per group, we
will be able to find a standardized treatment difference of 1.20 with 80% power using a
two-sided two-sample t-test at a 5% significance level. Power increases to 90% for a
standardized treatment difference of 1.39.
D.2.4.4. Achieving a long-lasting effect from growth factors. Methodology to improve
the longevity of locally administered growth factors will be developed after the best
combination of growth factors has been determined. Recently, gelatin-embedded HGF
has demonstrated a long-lasting effect.54,90 This compound is gradually digested in the
tissue and remains there for 1 to 2 months. Using an animal model (see Method A), we
will inject the scarred vocal folds with gelatin-embedded growth factor, and the effects
will be examined by histology, rheology, and excised-larynx study (Methods B, C, F, and
G) The longevity of the growth factor effect will be examined via ANOVA, by comparing
gelatin-embedded growth factor delivery against the standard injections.
D.2.5. Strategies to Prevent Scarring and Promote Regeneration
D.2.5.1. Gene therapy: in vivo electroporation. Rats will undergo unilateral vocal fold
stripping and be divided into 7 groups: PBS, enhanced green fluorescence protein
(EGFP), EGFP-Has1, EGFP-Has2, EGFP-bFGF, EGFP-HGF, and EGFP-EGF groups.
For these 7 groups, phosphate-buffered saline, and 6 kinds of recombinant plasmid
DNA-encoding-enhanced green fluorescence protein — EGFP, EGFP-Has1, EGFPHas2, EGFP-bFGF, EGFP-HGF, and EGFP-EGF — will be injected into the scarred
vocal folds and electroporation will be performed to transfect these genes.
Electroporation will be performed using the PulseAgile electroporation system (Model
PA-4000) with a programmable pulse switch (Model PA-201) (Cyto Pulse Sciences, Inc.,
Columbia, MD) and a bipolar electrode placed on the superior and inferior surfaces of
the vocal fold. Bidirectional transfection will be performed to maximize the efficacy of
gene delivery.
These procedures will be performed in the acute and chronic phase of injury. Rats will
be sacrificed at several time points (2 weeks, 1 month, 2 months, and 3 months). The
effects of these treatments will be examined by Alcian blue staining (HA detection),
trichrome staining (collagen), and immunohistochemistry for collagen subtypes (Method
B, C). Excised-larynx (Methods F, G) acoustic and aerodynamic parameters will also be
used to analyze the treatment effects. The efficacy of electroporation will be examined to
validate the results obtained. Transfected cells will be detected histologically by means
of immunohistochemistry using an anti-EGFP antibody. mRNA expression of EGFP and
target genes will also be examined by real-time RT-PCR (Method E). The gene that
shows the best results will be used for a naked DNA injection study.
For this study, we will need 340 rats to ensure adequate statistical power for the
treatment and control groups. The 340 rats will include 25 normal control rats that will
receive neither injury nor treatment. The remaining 315 rats will undergo unilateral vocal
fold stripping and be randomly divided into the 7 groups listed above (PBS, EGFP,
EGFP-Has1, EGFP-Has2, EGFP-bFGF, EGFP-HGF, and EGFP-EGF). For these 7
groups, PBS and 6 kinds of recombinant plasmid DNA-encoding-enhanced green
fluorescence protein — EGFP, EGFP-Has1, EGFP-Has2, EGFP-bFGF, EGFP-HGF,
and EGFP-EGF — will be injected into scarred vocal folds respectively and
electroporation will be performed to transfect these genes. These procedures will be
performed 5 days after vocal fold stripping. Rats will be sacrificed at 3 time points: 10
days, 4 weeks, and 8 weeks after vocal fold stripping. The effects of treatment will be
examined by the same method described above. This study has n=15 for each time
point of each of 7 treatment methods. ANOVA models will be used to examine the
effects of the different gene therapy groups. With n=15 rats per group, we will be able to
find a standardized treatment difference of 1.06 with 80% power using a two-sided twosample t-test at a 5% significance level. Power increases to 90% for a standardized
treatment difference of 1.23.
D.2.5.2. Naked DNA injection. Work in several laboratories has shown that naked
plasmid DNA (pDNA) can be delivered efficiently to cells in vivo and has great prospects
for basic research and gene therapy .87,88 Naked DNA injection is less invasive than
electroporation and other gene-delivery methods, and it appears to be the most ideal
way to deliver gene therapy for scarring.
The rats in our experiment will undergo unilateral vocal fold stripping (Method A),
and they will be injected with naked DNA in the acute and chronic phase of injury. The
rats will be euthanized at several time points (2 weeks, 1 month, 2 months, and 3
months after injection), and the effects of treatment will be examined by histology,
rheology, and excised-larynx study (Methods B, C, F, and G). The efficacy of
electroporation will also be examined to validate the results obtained, as described
above. The efficacy of naked DNA injection in the eight gene therapy groups will be
examined with ANOVA models. With n=12 rats per group, we will be able to find a
standardized treatment difference of 1.20 with 80% power using a 2-sided 2-sample ttest at .05 significance. Power increases to 90% for a standardized treatment difference
of 1.39.
D.2.5.3. Cell transplantation: fibroblasts. Fibroblasts in the scarred tissue are altered
in terms of their ability to synthesize ECM.49 Fibroblast injection into the scarred vocal
fold has been suggested as an alternative therapy to regenerate the vocal fold.72 The
most important issues associated with this therapy are related to which fibroblast should
be used and how to control the fibroblasts because the functions of various types of
fibroblasts (vocal fold fibroblasts, skin fibroblasts, and scar fibroblasts) may be different.
Therefore, we will characterize the functions of vocal fold, skin, oral mucosa, and scar
fibroblasts in vitro in terms of ECM production and the properties of proteins.
Two months after unilateral vocal fold stripping, the vocal fold lamina propria will
be collected from normal and scarred vocal folds under microscope dissection, and the
specimens will be placed in a Petri dish and chopped into pieces. Samples from skin and
oral mucosa will also be collected as controls. The tissues will then be incubated in
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) containing 10%
fetal bovine serum (FBS, Invitrogen, Carlsbad, CA), penicillin (10,000 IU, Invitrogen,
Carlsbad, CA), streptomycin (10,000μg/mL, Invitrogen, Carlsbad, CA), and amphotericin
B (25μg/mL, Invitrogen, Carlsbad, CA) in an incubator containing 5% carbon dioxide and
95% air at 37oC.
Fibroblasts at first passage will be subcultured in 6cm dishes and incubated with
DMEM and 10% FBS in an incubator containing 5% carbon dioxide and 95% air at 37oC.
After 90 to 100% confluence, the medium will be replaced by a non-serum medium
(AIM-V Media, Invitrogen, Carlsbad, CA). After incubating the fibroblasts for 2 days, the
cell samples will be collected after culturing for 0, 3, 6, 12, and 24 hours. The culture
medium will also be collected in each sample and is used for ELISA study. Total RNAs
will be extracted from the cells and reverse transcription will be performed to obtain first
strand cDNA. The production of HA, collagen, elastin, fibronectin, and decorin will be
examined using ELISA and real-time PCR (Methods D and E).
For protein analysis, protein will be extracted from fibroblasts from oral mucosa,
skin tissues, and normal and scarred vocal folds. Two-dimensional sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE) will be performed to
characterize the fibroblasts in terms of protein properties. The raw data will be analyzed
by Melanie software (Swiss Institute of Bioinformatics, Geneva, Switzerland), and the
spots that show a significant difference will be identified by amino-acid sequencing.
As a second step, we will examine the effects of growth factors and cytokines on
each fibroblast in cell culture in terms of alteration of ECM production. Growth factors will
be applied to cultured fibroblasts from oral mucosa, skin tissue, and normal and scarred
vocal folds. ELISA and real-time PCR will be performed to examine the effect of growth
factors on each cell line (Methods D and E). We will then determine the cell line that
shows similar characteristics to those of normal vocal folds and that produces
appropriate ECM components for transplantation.
As a third step, we will obtain fibroblasts from an animal model, culture them, and
implant them into the scarred vocal fold of the animal. The effects will be clarified using
histology, rheology, and excised-larynx studies of vocal fold vibration (Methods B, C, F,
and G). As the last step, we will implant fibroblasts into the scarred vocal fold in an
animal model and administer regulatory factors (growth factors, naked DNA, etc.) to
control the exogenous fibroblasts. The effects will be clarified using histology, rheology,
and vibratory examinations (Methods B, C, F, and G). Differences between the twelve
groups in the data from ELISA and PCR (Methods D, E) will be examined via linear
mixed effect models, to account for nesting due to the different culture periods. With
twelve rats in each of twelve groups, we will be able to find a standardized treatment
difference of 1.20 with 80% power using a two-sided two-sample t-test at a 5%
significance level. Power increases to 90% for a standardized treatment difference of
1.39.
D.2.5.4. Cell transplantation: stem cells. Mesenchymal stem cells will be cultured and
various growth factors and cytokines will be added. We will examine how best to induce
MSCs and differentiate them into other cells. The functions of the MSCs in terms of ECM
production will be demonstrated by ELISA and real-time PCR. The effects of MSC
implantation into the scarred vocal fold will be examined in an animal model. A
combination of growth factors will be administered. The effects will be assessed with
histologic, rheologic, and excised-larynx studies (Methods C, F, and G). Differences
between growth factors will be modeled with ANOVA. With twelve rats per group, we will
be able to find a standardized treatment difference of 1.20 with 80% power using a twosided two-sample t-test at a 5% significance level. Power increases to 90% for a
standardized treatment difference of 1.39.
D.2.5.5. Tissue engineering. Fibroblasts will be seeded on the polymer in cell-culture
dishes, and growth factors will be added to induce the production of appropriate ECM
components for each layer. Growth factors will be determined based on the results of the
experiment described in D.5.3. After an adequate incubation period, the sheets will be
laid and tissue-engineered lamina propria will be made after further incubation. The
engineered lamina propria will be examined by histology (Method C) focusing on the
organization of ECM. Rheologic examination will clarify the viscoelasticity of the vocal
fold (Method F). With twelve rats in each of three groups, power is the same as in
D.2.5.4.
D.2.5.6. Pulsed dye laser trials. Two introductory trials of the PDL will be performed to
understand the effects of PDL energy levels and the number of PDL applications on
tissue changes in rat vocal folds in the chronic scar model (Method A, I). Rats that have
undergone surgical damage to 1 vocal fold and have survived for 2 months from injury
will be selected for these tests. First, we will use the PDL to check that the energy being
applied is less than the energy necessary to produce immediate tissue destruction.
Then, using a fixed number of pulses and a fixed level of energy per pulse being
delivered from the unit in a given session, we will use different rat groups to test for
tissue changes as the distance of the fiber tip from the surface of the test vocal fold
increases. Distances from the vocal folds will be 3, 6, 9, 15, and 20mm. Thus, 5 groups
will be used, each group having 2 rats. Two scarred but untreated rats will be used as
controls. Each test rat will undergo only 1 PDL application session. One month after the
last PDL application, each rat will be euthanized and the larynx subjected to analysis
using excised-larynx vibration, LSR, histology, and ELISA testing (see Methods G, F, A,
C, and D). Second, the effect of serial PDL applications to chronically scarred rat vocal
folds will be tested. Four test groups, each with 3 scarred rats, will be subjected to PDL
applications in the manner described above. Two scarred, untreated rats will serve as
controls. The number of pulses, amount of energy per pulse, and distance of the fiber tip
from the vocal fold surface will be determined from experience in the first PDL
experiment. These variables will be kept constant. The 4 test groups will undergo 1, 2, 3,
and 4 total PDL applications respectively, with 3 weeks between each application. One
month after the last PDL application, each rat will be euthanized and the larynx
subjected to analysis using the excised-larynx platform, LSR, histology, and ELISA
testing (Methods B, G, F, A, C, and D). In the tests to ensure tissue destruction does
not occur, tissue changes due to energy, pulses, and distance from the vocal folds will
be modeled with regression analysis. With three rats per group in the non-control
groups, we will be able to find a standardized treatment effect of 3.07 with 80% power
using a two-sided two-sample t-test at a 5% significance level. Power increases to 90%
for a standardized treatment difference of 1.39.
TIMELINE
YEARS
Aim 1: Measure and contrast the histologic, viscoelastic and vibrational
properties of the pre- and post-operative tissues associated with vocal fold
scarring. Document any correlations which exist between these properties
and the resultant acoustical signal. Efforts will be directed toward completion
of Aim 1 throughout the five years of funding.
Aim 2: Determine appropriate scaffolds, cells and growth factors to regenerate
the scarred vocal fold, and examine their therapeutic effects.
2.1. Homeostasis of ECM components
2.1.1. Human vocal folds
1, 2 year
2.1.2. Animal
1, 2 year
2.1.3. Development and aging
1, 2, 3 year
2.1.4. In-vitro inflammation model of vocal fold fibroblasts 1, 2, 3 year
2.2. Development of growth factor therapy
2.2.1. Optimization of dosage, timing and a type of a growth factor for the
treatment of acute/chronic vocal fold scar
1, 2, 3 year
2.2.2 Achieving a long-lasting effect of the growth factor 3, 4 year
(2.2.3. Clinical trial)
2.3. Prevention of scar and regeneration
2.3.1. Gene therapy
2.3.1.1. In-vivo electroporation
1, 2 year
2.3.1.2. Naked DNA injection.
2, 3, 4, 5 year
2.3.2. Cell transplanation
2.3.2.1. Use of fibroblasts
1, 2, 3, 4 year
2.3.2.2. Use of stem cells
3, 4, 5 year
E. HUMAN SUBJECTS
Non Applicable.
F. VERTEBRATE ANIMALS
2. Justify use of animals selected in the proposed study: The use of experimental
animals is required in the present research, as our other models of investigation, such as
computer modeling would not allow us to look at the effects of treatment approaches or
stages of wound repair. Also clinical studies would not allow us to make systematic
changes in timing, magnitude and combinations of treatments necessitating the use of
animals.
Our particular strain of rat was chosen for this research because it is sufficiently
large that the larynx can be easily visualized for vocal fold stripping, vibration patterns
can be documented with acoustic, aerodynamic and high speed imaging, and OCT
measures of the viscoelasticity can also be obtained. A rat model was chosen for
analysis due to the investigators technical expertise with rats, and a number of scientific
considerations. First, the rat has a short life span, with median lifespan of 36 months. As
such, physiological and morphological changes associated with scarring can be realized
in a relatively short period of time. Second, the ease of handling rats allows rigorous
experimental control and measurement of multiple parameters, and examination of the
relationships among variables. Third, the rat is ubiquitous in biomedical research. When
developing interpretations of our findings, we can draw upon the work of others in a
large knowledge base. Rat models for studies of vocal fold tissue allow for a relatively
straightforward comparison to data derived via study of human vocal folds because of
similar characteristics in the lamina propria ECM, found abundantly in the literature.
Accordingly, knowledge gained by our research can add to the available data on wound
healing. Fifth, the relative magnitude of fibroblasts in these rodents corresponds to that
typically reported for humans. As such, the rat model is relevant to changes observed in
both the acute and chronic scar..
Statistical methods and power calculation have ensured that our sample size is
adequate for the full cadre of animals and for a conservative estimate of the animal
sample size incorporating potential loss (i.e., 80% of the sample used). The number of
animals for each experiment was determined via statistical power calculations performed
by Dr. Glen Leverson, the biostatistician consulting on this project. Based on 12 rats in
each group, if a treatment causes the population mean to shift by at least 1.2 standard
deviation units, we should have an 80% chance of declaring statistical significance (If the
relative treatment effect is 1.2, 12 rats per group will yield a power of 0.8 if testing is
done at the usual 5% critical level. The power was based on a pairwise contrast of two
means in the ANOVA). Under most circumstances a shift of 1.2 standard deviation units
is quite small so our design should be effective at detecting scientifically relevant effects.
Based on these calculations we have determined that minimal number of animals
necessary to address the questions posed. A summary table of how the animals will be
allocated to the studies follows.
Estimated number of
animals for 5 year
period (2005-10)
1 (human and
canine)
2 (rat; age and
gender)
3 (scarred
rats)
4 (In-vitro
inflammation)
D2-2 (GF therapy)
1
(Optimization
of GF)
2 (Long lasting
effect)
D2-3
1 (Gene
(Prevension&regenerat
therapy)
ion)
Number of
groups
Number
rats/group
Number of ra
4
12
48
2
12
24
2
12
24
6
12
72
4
12
48
1 (electroporation)
14
12
168
2 (naked DNA)
1 (fibroblasts)
8
12
12
12
96
144
2 (stem cells)
8
3
12
12
96
36
D2-1 (Homeostasis of
ECM)
2 (Cell
transplantation
)
3 (Tissue
engineering)
756
.The animals will be humanly treated. They will be anesthetized with ketamine
35mg/kg and Xylazine 5mg/kg in an intramuscular injection. Animals will be placed on a
operating table and the larynx exposed using a specially constructed laryngoscope.
Once the larynx is visualized with a Zeiss operating scope. One vocal fold will be
stripped and the contralateral fold left untreated for comparison. Animals will recover in
their cages in the animal facility until a scar is formed. All post-op animals will be
inspected daily for 7 days by the researcher for signs of distress. Animal caretakers at
the facility also inspect animals twice daily. Any animals experiencing severe distress or
pain will be sacrificed immediately (we do not expect this to be a common occurrence as
the animals have tolerated the procedure well in our previous studies).
Sacrifice will be carried out with the ketamine/Xylazine anesthesia previously
described, followed by pentobarbital overdose. Once the animal has ceased respiration
and heart beats, the larynx will be removed and prepared for histological , acoustical,
aerodynamic, vibratory motion analysis and rheological analysis.
6.B. As previously stated we have chosen rats because of their relative low cost,
wide availability, size, ability to visualize the larynx and ease of anesthesia and surgery.
Although many laryngeal experiments have been performed in dogs, we feel that rats
will provide us with sufficient information to study the laryngeal musculature without
excessive cost. The numbers of animals proposed for the experiment is necessary to
give statistically significant data and to allow a buffer for certain inevitable animal deaths
with this laryngeal experiment.
6.C. Veterinary care will be provided by the UW Research Animal Resources
Care (RARC) facility. All animals will be housed in the animal facility under the care of
two full-time veterinarians and several trained animal care workers. All persons working
with animals at the University of Wisconsin have passed a rigorous test on animal care
guidelines and are certified by the facility before being allowed to handle animals.
6.D. All procedures have been reviewed and approved by the RARC as not
causing undue pain or suffering to these animals. We are using standard surgical and
euthanasia procedures recognized by the Animal Care Committee. The surgery is
similar to a vocal fold biopsy procedures, which is commonly performed by the surgical
supervisor, Dr. Charles Ford , in a clinical setting under local anesthesia with little patient
discomfort.
6.E. Euthanasia will be performed on the animals using an overdose (.22mg/kg
I.V.) of prentobarbital. This method has been selected because of the ease of
administration and its proven usefulness for this purpose. It is consistent with
recommendations of the Panel on Euthanasia of the American Veterinary Medical
Association.
G. LITERATURE CITED
1)
Hirano M. Chevalier Jackson Lecture: Phonosurgery-Past, Present, Future. American
bronchoesophageal Association, 1995.
2)
Woo P, Casper J, Colton R, Brewer D. Diagnosis and treatment of persistent
dysphonia after laryngeal surgery: a retrospective analysis of 62 patients.
Laryngoscope 1994; 104(9):1084-1091.
3)
4)
Benninger MS, Alessi D, Archer S, Bastian R, Ford C, Koufman J et al. Vocal fold scarring:
current concepts and management. Otolaryngol Head Neck Surg 1996; 115(5):474-482.
Thibeault S, Ford C. The scarred vocal fold. In: Ossoff R, Shapshay S, Woodson G,
Netterville J, Eds. The Larynx. Philadelphia. Pa: Lippincott Williams and Wilkins.
2002:431-440.
5) Hirano S, Bless D, Heisey D, Ford C. Roles of hepatocyte growth factor and
transforming growth factor beta1 in production of extracellular matrix by canine vocal
fold fibroblasts. Laryngoscope 2003; 113(1):144-8.
6) Hirano S, Bless DM, Heisey D, Ford CN. Effect of growth factors on hyaluronan
production by canine vocal fold fibroblasts. Ann Otol Rhinol Laryngol 2003;
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H. CONSORTIUM/CONTRACTUAL ARRANGEMENTS
None
I. CONSULTANTS
See the attached letters from David Berry, Shigeru Hirano, Eric Goodyer, Bernard
Rousseau, Susan Thibeault, Ichiro Tateya, and Tomoko Tateya.
J. RESOURCE SHARING
Non-applicable