Individual Grant Review – Final Report
Quantifying the Effectiveness of Stem-Cell Implants To
Promote Self-Healing Of The Vocal Fold
Prepared by Eric Goodyer Oct 2007
EPSRC Reference EP/D025591/1
1 Background, Context & Summary
The structure of the vocal fold can be very simply described as an outer layer, the epithelium, that encloses a complex
structure of soft inner layers, the lamina propera. Phonation is achieved by a combination of air flow control and
muscular action that results in a wave propogating across these tissue structures. Various researchers are currently
engaged in the development of tissue augmentation techniques to restore the bio-mechanical properties of damaged
vocal fold tissue, thus restoring the power of phonation to patients suffering from aphonia or dysphonia. This research
has identified two requirements. One is the need to quantify the biomechanical properties of healthy vocal folds, taking
account of the variation of these properties with respect to position and depth of tissue under test. The other is to
develop measurement techniques to ensure that tissue implants, and regeneration procedures, can recreate these
properties.
With EPSRC support, and additional funding from the Royal Society, the Royal Academy of Engineering and the
Regional Innovation Fund, DeMontfort University has been enabled to work with a number of international
collaborators in the field of vocal fold characterisation and tissue engineering. These include Harvard Medical School,
Wisconsin University Hospital, UCLA, Universitat Klinic Eppendorf (UKE) Hamburg Germany, and the Karolinska
Institute in Sweden.
The primary purpose of this grant was to enable DMU to work with Wisconsin University Hospital in order to carry out
the initial studies in support of their application for funding for a 5 year Tissue Engineering research programme. The
input requested from DeMontfort was the development and deployment of techniques to quantify the visco-elastic
properties of the vocal fold, and the effectiveness of tissue engineering therapies. I am very pleased to report that
Wisconsin was successful in this application, and have been awarded $1.8 million by the National Instate of Health to
fund a further 5 years research. Eric Goodyer is a named Consultant to this research programme, and the input from
DMU was included in the application to the NIH.
The opportunity was also taken to continue our working relationships with UKE and Karolinska. We were invited to
assist with Prof Stellan Hertegard’s ground breaking stem-cell study in 2005, which indicated that stem-cell implants
into scarred vocal fold in a rabbit model did result in a restoration of tissue pliability. A number of studies were
undertaken with UKE, which has resulted in a number of publications as listed below.
Finally new opportunities for collaboration were explored. Dr David O’Brart of Department of Ophthalmology, Guy's
and St. Thomas' NHS Foundation trust, has requested our assistance with a project to investigate the use of collagen
implants into the cornea as a treatment for keratoconus. Mr Julian McGlashan, a consultant surgeon at QMC
Nottingham has asked DMU to join them in an investigation of the use of optical techniques to quantify vocal fold
dynamic behaviour. Dr Dinesh Chettri of UCLA has asked Dmu to assist in a project to investigate reinervation therapy
as a potential cure for vocal fold paresis; EPSRC have recently awarded DMU a small grant of £15700 to assist with
the UCLA project.
The publications produced during the period of this grant are as follows.
1.1 Abtract for Wisconsin University Hospital’s Tissue Engineering research Programme
Grant Number:
2R01DC004428-06A1
PI Name:
BLESS, DIANE M.
PI Email:
PI Title:
Project Title:
bless@surgery.wisc.edu
PROFESSOR
Characterization and Treatment of the Scarred Vocal Fold
Abstract: DESCRIPTION (provided by applicant): Vocal fold scarring is the single greatest cause of poor voice after
vocal fold surgery (Hirano, 1995; Woo, Casper, Colton and Brewer, 1994). Such scarring results in replacement of
healthy tissue by fibrous tissue, which can irrevocably alter vocal fold function, leading to a decreased or absent vocal
fold mucosal wave (Benninger, Alessi, Archer et al., 1996). Even small foci of scar can impede vocal fold function
when separation of the body (muscle) and cover (epithelium and the superficial layer of the lamina propria) is
compromised. Because there is currently no consistently effective treatment for the scarred vocal fold, it is one of the
most challenging laryngeal disorders to treat (Thibeault and Ford, 2003) and is the focus of this competing renewal on
characterizing taryngeal scars. The long-range aim of this project no longer focuses exclusively on sulcus vocalis, but
now encompasses the characterization and treatment of the more universal phenomenon of vocal fold scarring. This
long-range objective will be accomplished by characterizing laryngeal scarring and its influence on phonation, both
pre- and post-treatment, using 1) controlled laboratory experiments, and 2) computer modeling experiments. The
laboratory experiments will investigate treatment effects at the cellular level in animal's tissues and excised larynx
models. Building on experiments completed over the last five years, we will investigate the role of growth factors and
vitamin A in preventing and treating scars, and we will attempt to better characterize scars in the vocal fold and their
vibratory consequences. We have found it necessary to combine modeling and systematic laboratory investigations to
resolve the complex interactions between tissue characteristics, geometry and surgical requisites necessary to create
a suitable clinical outcome. This proposal is timely because no consistently effective treatment modality is known for
scarring, and it remains one of the unsolved problems of phonosurgery. While our initial five years of study have
yielded several key hypotheses to guide treatment options, the current proposal will enable us to substantiate and/or
further clarify these hypotheses so that they will have a greater impact.
2 Key Advances and Supporting Methodology
The 5-year programme based at Wisconsin University has only just started, and no major research results have yet to
be produced. Two exploratory studies have been completed. The first study investigated the feasibility of mapping the
variation of elasticity over the surface of a range of animal larynxes, this included a dog, a rabbit, a rat and a human
larynx. The second study involved an investigation of the use of ultrasound as a means to visualise the underlying
structures of the vocal fold, before and after augmentation.
The research programme at UKE has progressed well, and resulted in two major advances. First we have succesfully
deployed a new technique to measure the biomechanical properties of the human vocal fodl in-vivo. Results from our
inititial study with two colunteer patients have been published [1]
2.1 Wisconsin University Hospital Madison USA
A final visit was made to Wisconsin University Hospital in Jan 2005. A large team, under the leadership of Dr Diane
Bless, is planning to investigate new techniques to restore scarred vocal fold tissue that do not employ the use of
tissue implants.
The purpose of the visit was to allow them to assess if the LSR and Tensiometer techniques could be of value in
assessing the effectiveness of innovative cell regeneration techniques. The opportunity was taken during the visit to
characterise a range of vocal folds, mainly canine, but also human and rat. The team was able to generate a series of
surface maps that show the variation of the elastic properties of the vocal fold, and were able to use these surface
maps to contrast the difference between healthy and scarred tissue. Some of which are shown below
It is now intended to apply for a major NIH grant to further this work, with myself as a named collaborator. Part of the
new research programme will be an investigation into the use of bone-marrow stem cells to promote self-generated
vocal fold reconstruction.
Figure 12 Contour Maps Showing Elasticity Variations over the Surface of a Canine Vocal Fold
2.1 Study of an Intact Male Donor Larynx
Abstract: The Linear Skin Rheometer, which measures skin visco-elasticity, was adapted for measurements of vocal
fold properties. In an excised male donor human larynx small patches of mucosa were driven sinusoidally at 0.3 Hz
over 1-2mm distances using a small probe. Forces of the order of 1 gram gave optimal measurements. Stiffness
values were derived from stress/strain data and using a simple shear model.
Measurements were initially taken from an intact larynx, along the axes of the vocal folds. The larynx was then split,
and the measurements repeated on the intact hemi-sections. We believe that these are the first readings taken of the
biomechanical properties of an intact excised human larynx; with the variation of elasticity quantised with respect to
position.
Figure 1 Experimental Set-up Using Intact Larynx
Figure 2 Typical Results Screen
Figure 3 is a graph that shows the variation of the Dynamic Spring rate (DSR) of both vocal folds, measured within an
intact excised larynx. Two sets of readings were taken on consecutive days in order to assess changes with time and
desiccation. Of importance is the way in which the elasticity of the vocal fold changes with respect to the point at
which it is measured. It is not a homogenous structure, and its’ bio-mechanical properties are dependant upon the
tissue’s situation and adjoining structures. This has important implications for those seeking to develop tissue
augmentation procedures. The change in results over 1 day was not significant, indicating that the mechanical
properties of the structure do not need to be taken immediately. This same larynx was re-tested after being frozen for
1 month, and still yielded similar data.
Figure 3 Measurements from an Intact Human
Larynx
The larynx was then split and two more sets of readings were taken from each half, with a greater degree of positional
accuracy. This demonstrates the main disadvantage of using an intact larynx, in that it is far more difficult to take
measurements from a repeatable set of positions. Similar results were obtained.
Figure 4 Experimental Set-up using a Split Larynx
Figure 5 Graph Showing the variation of DSR with Position
Our conclusions were that the LSR is capable of taking repeatable measurements of the DSR of the vocal fold, and
that proceeding to a large-scale study would be worthwhile.
2.2 Development of New In-vivo Laryngeal Tensiometer
Abstract: The research to date has been carried out using excised tissue only. The development and application of an
in-vivo measuring tool is considered to be an essential extension of this research programme. A further research grant
was secured during the lifetime of this project that enabled an engineering team at De Montfort University to develop a
new surgical tool capable of measuring biomechanical properties in-vivo. The design of this new surgical instrument
was strongly influenced by our relationship with Prof. Markus Hess and his team, which was facilitated by this EPSRC
grant.
The laryngeal tensiometer is clamped to a standard size C laryngoscope loaned to us by Storz. A load cell, similar to
that used by the LSR system, is rigidly clamped to the laryngoscope. A probe is inserted down the laryngoscope, and
is attached to the vocal fold. A sprung hand grip arrangement allows the surgeon to displace the probe by 1mm, which
on release provides a smooth displacement during which time the change in force is logged.
The design challenges were in two areas
1) How do we attach the probe to the vocal fold tissue
2) How do clamp the probe to the load cell, without damaging the sensor
Three methods of attachment were examined at MEEI and Eppendorf, the use of a needle, the use of suction and the
use of adhesives. All methods were found to work satisfactorily, and repeatably. The advantage of suction over a pin
is that the tissue is not punctured. However it is known that bio-mechanical properties of tissue vary with the depth of
penetration of the motion – this is far more controllable and repeatable if a calibrated pin used, as suction can result in
differing quantities of tissue being sucked up into the cannular probe. It was also felt that the pin would allow the user
to more precisely define the position from which the measurement is taken. However the final choice was to use
adhesives, as this represents the minimum invasion of the patient and was considered to be more appropriate for this
pilot study.
The clamping arrangement posed the greater challenge. In order to measure the extremely low forces that are exerted
during the measurement cycle, typically 1g, a 25g load cell was employed. Such a delicate sensor will be destroyed if
forces of more than 50g are applied to the sensing element. Providing a rigid mounting arrangement was not an
option, as it is essential that the surgeon has the freedom to move the pin to any position on either vocal fold. A range
of options were examined, including a pair of clamps, a ball and socket joint, adhesives, putty and a magnetic
attachment. Using a rapid prototyping technique different clamping arrangements were speedily ‘grown’ in a rigid
resin, and tested. The final arrangement that allowed the greatest flexibility was an ingenious use of a magnetised ball
bearing to replace the ball & socket arrangement most favoured by the medical practitioners.
This arrangement is shown below. A nylon attachment was manufactured that fitted securely onto the sensing element
of the load cell. This attachment is hollow, and a small magnet is inserted into the hollow, a steel ball bearing is
inserted at the end of the channel such that a small section of its’ surface protrudes. A steel rod or cannular can now
be inserted down the laryngoscope, and attached to the tissue under test. Once the surgeon is content with the
position it can be attached magnetically to the ball bearing. The holding force of the magnetic coupling was measured
to be 8g. The maximum force that we apply to the vocal fold is 1g, therefore we are able to take our measurements
and easily move the metal rod to a new position.
A series of test were carried out using excised tissue, which gave DSR results similar to those obtained by the LSR.
These tests also allowed us to experiment with a range of adhesives, essentially based on formulations used for
denture fixings. These types of adhesive are inherently water soluble, and non-toxic.
The graph below shows a series of readings taken using the arrangement shown in figure 6. Each step represents a
compression change in the handgrip, and release. The data is noisy, but the noise threshold is well below the force
difference that we are trying to measure. Similar data is obtained from the LSR, and a good signal can be recovered
using digital signal processing techniques; these will be added to future versions of this instrument.
0
1
307 613 919 1225 1531 1837 2143 2449 2755 3061 3367 3673 3979 4285 4591 4897 5203 5509 5815 6121 6427 6733 7039 7345 7651 7957 8263
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Figure 6 Experimental Set-up Showing Magnetic
‘ball joint’ Arrangement and Adhesion to a Split Larynx
Figure 7 Data Obtained from an Excised Larynx
Using a least squared fit the differential force for each compression was determined, giving a mean DSR result of 0.57
g/mm with a standard deviation of 8% (Standard Deviation/Mean expressed as a %).
These results are comparable with data obtained using the LSR in both Germany and the USA.
The instrument was then used on two volunteer female patients of similar age. Elasticity measurements were obtained
from healthy tissues from both volunteers, and were found to be similar and in line with findings from excised tissue. In
addition we measured the elasticity in the region of a large polyp, which resulted in a reading showing far lower
stiffness than that for healthy tissue; this is the result that is to be expected. It is now proposed to continue this in-vivo
work with more volunteers over the next 2 years, subject to further EPSRC funding.
Figure 8 Setting up the In-vivo Tensiometer in Surgery
Figure 9 Showing Measuring Probe Adhered to Vocal Fold
2.3 The Effectiveness of Hyaluronan Implants
abstract: Prof. Stellan Hertegard of Huddinge University Hospital, Stockholm has been researching the effectiveness
of different tissue augmentation techniques. Following the announcement of our results at the AQL conference in 2003
I was invited to assist him in the final stages of a 6-month study into the effectiveness of Hyaluronan implants. 15
rabbits were sacrificed for this study. They were split into 4 groups, one set were undamaged, one set had their vocal
folds scarred, the third set had their vocal folds scarred and then augmented using Hyaluronic implants, and the 4th
group were similarly treated but with a control implant.
The larynxes were excised and measured using an Instron and the LSR. As the vocal fold tissue was so small a
different technique was developed to measure the elasticity of the samples. Instead of pulling/pushing the tissue
cyclically the LSR was re-arranged to be an indentometer. A 1mm diameter rod was slowly pushed into the tissue,
whilst logging both force and displacement. From this data it is possible to derive a range of elastic properties. The
box plot shown below indicates that augmenting scarred tissue with Hyaluronan implants can restore the elasticity of a
damaged larynx. These results were announced last year in New York [3].
Box Plot
Split By: Group
Rabbit 11 mid 1
6
2
5.5
1.5
4.5
Norm
4
1
Units
movement
5
0.5
Sc
3.5
3
Sc+Hylaf
2.5
Sc+Rest
2
0
0
5
10
15
-0.5
1.5
1
.5
force
Figure 10 Typical Indentometer Data Showing The
Predicted Logarithmic Response
LSR Y
Figure 11 Box Plot Showing that Hyaluronan Implants
can Restore the Elasticity of Damaged Tissue
3 Future Direction
This grant has been very successful. Two major teaching hospitals in Germany and the USA are applying for grants
for major studies into vocal fold properties, and their repair. I am a named collaborator in both bids.
Regardless of the outcome of those bids, I will continue to research the visco-elastic properties of the vocal fold with
my German and Swedish partners. This work will use both excised tissue and volunteer patients. A secondary
outcome of this proposed research will be the commercialisation of the in-vivo tensiometer tool.
1.
Goodyer EN, Hemmerich S, Müller F, Kobler JB, Hess M. The shear modulus of the human vocal fold,
preliminary results from 20 larynxes. European Archives of Oto-Rhino-Laryngology. 2006. on line first edition.
2.
Goodyer EN, Hemmerich S, Mueller F, Licht AK, Hess M. Characterisation of the Elasticity of the Human
Vocal Fold using Electromechanical Measurement Techniques. 7th International Conference Advances in
Quantitative Laryngology, Voice and Speech Research, October 2006.
2.
Goodyer EN, Muller F, Bramer B, Chauhan D, Hess M. In Vivo Measurement of the Elastic Properties of the
Human Vocal Fold. European Archives of Oto-Rhino-Laryngology. 263(5):445-462, May 2006.
3.
Hess M, Muller F, Kobler JB, Zeitels S, Goodyer EN. Measurements Of Vocal Fold Elasticity Using The Linear
Skin Rheometer. Folia Phoniatrica. 58(3)2006.
5.
Hertegård S, Dahlqvist Å, Goodyer E, Maurer. Viscoelastic Measurements After Vocal Fold Scarring In
Rabbits. Acta Oto-Laryngologica. 126(7):758-763, July 2006.