Quantifying the effectiveness of stem-cell therapies to stimulate self-healing of
the vocal fold
Prepared for the Royal Academy of Engineering
By Eric Goodyer
October 2005
1 Background
Phonation is achieved by the controlled exhalation of air through the vocal
folds. The resultant oscillation propagates through the vocal fold, producing
the mucosal wave. By changing the tension of this structure, using the
underlying muscle, the resonant frequency can be controlled, thereby allowing
us to phonate, or speak.
Vocal fold scarring changes the oscillatory nature of this complex structure,
resulting in disordered voice (dysphonia) or total loss of voice (aphonia).
I have been assisting a number of research teams who are investigating the
visco-elastic properties of the vocal fold, and tissue engineering therapies to
repair damaged vocal folds. The input from myself and my colleagues at
DeMontfort University is to engineer the precision measuring equipment that
is used to quantify changes in the bio-mechanical properties of vocal fold
tissue [1,2,3]. This particular study was carried out in collaboration with
Assistant Professor Stellan Hertegard at the Karolinska Institute, Stockholm;
in which we completed a study into the effectiveness of embryonic stem-cells
implants into damaged vocal folds.
The clinical outcome was inconclusive, however the engineering methodology
was proven. The data shows that damaged tissue that is injected with
embryonic stem-cells does regenerate, but the overall results were not
statistically significant enough to justify any claim more than ‘indicative’. A
further study will take place in 2006, employing a different methodology based
upon our findings to date.
The Ethics Committee governing animal research in Sweden approved the
study. DeMontfort University’s involvement was approved by the University’s
ethics committee.
2 The Study
10 rabbits were divided into 3 groups. One group had vocal fold scarring, a
second group had scarring that was treated with embryonic stem-cell
implants, and the third was a control group. The study lasted a period of one
month, after which the rabbits were humanely killed and their vocal folds
excised for analysis.
The engineering team applied two different techniques to measure the
biomechanical properties of the tissue. One was an in-vivo method; the other
was used to measure the excised tissue post-mortem. Both techniques were
designed to take measurements from intact larynxes, which were split to
reveal the vocal fold structure for the excised analysis.
After testing using DMU’s equipment the vocal fold tissue was dissected out
from the underlying layers, to be retested using a parallel plate rheometer,
and samples were sent for histology.
The objective is to obtain two sets of data from the excised vocal folds using
different mechanical techniques; and to use the in-vivo technique to quantify
the change in bio-mechanical properties with respect to time. The excised
tests were successful; the in-vivo tests were not.
3 In-Vivo Tests
We have been working in collaboration with Professor Markus Hess and his
team at Eppendorf University Hospital, Hamburg, for a number of years. Over
the last year we have successfully deployed a novel apparatus, the Laryngeal
Tensiometer, that is able to measure the tensile strength of the human vocal
fold. [4].
The instrument consists of a rigid clamp that is fixed to an adult laryngoscope,
after it has been inserted into the mouth and larynx by the surgeon. A load cell
is coupled to this arrangement such that the sensing element is located close
to the view-port of the laryngoscope. A simple slide arrangement allows a
user to move the load cell back and forth by a calibrated distance, which is set
to 1mm. A magnetic coupling is fixed to the sensing element of the load cell. A
1mm diameter rod is inserted along the axis of the laryngoscope; the end of
which is flattened such that it offers a platen (1mm x 2mm) to the vocal fold
tissue. A water-based adhesive, methylcellulose, is used to attach the platen
to the tissue. The other end of the rod is then fixed to the load cell using the
magnetic attachment.
Measurements are then taken by repeatably displacing the vocal fold tissue
by 1mm and logging the change in resultant force. From this data it is possible
to derive the Spring Rate of the vocal fold, and to convert that to a value for
the shear modulus of the tissue using the known geometry of the
experimental set up.
It was this equipment that we attempted to use for this study. It was not
successful due to the substantially smaller and different structures that are
found in a rabbit as opposed to a human. In brief it was virtually impossible to
create a clear line of site along the modified paediatric laryngoscope that we
used from the load cell to the vocal fold. To obtain meaningful data the probe
must not touch any other structures between the load cell and the vocal fold.
We decided that it was unethical to continue to use this method.
However, by chance, one specimen did offer an opportunity to take a
measurement, as a clear visual path became available. The data was very
noisy, and was perturbed by normal physiological actions, most notably
breathing which was clearly visible as a slow rhythmic oscillation overlaying
the data. We were able to estimate the shear modulus of one vocal fold at
around 7792 Pa.
4 Excised Tests
The equipment deployed for these tests was a modified Linear Skin
Rheometer [5,6,7,8]. This apparatus consists of a load-cell that is mounted in
a slide arrangement. Using a maxon miniature motor and a ball screw, the
complete assembly can be accurately positioned. The position is measured
using an LVDT. A chuck is fixed to the load cell sensor, such that it will
measure forces that are in the same axis as the direction of movement of the
slide arrangement.
A range of different attachments have been developed that fit to the chuck,
which are capable of obtaining data from the tissue under test in a variety of
ways. For these tests a 1mm rod was fitted to the chuck, the other end of
which is bent through 90 degrees and sharpened to give a needlepoint. The
needlepoint was inserted into the vocal fold tissue, such that the direction of
movement was 90 degrees to a line drawn between the anterior commisure
and the vocal process. In effect this electro-mechanical arrangement enabled
us to stretch the vocal fold in a similar direction to the motion of a mucosal
wave.
The tissue was cycled back and forth, by applying a sinusoidal force of +-0.5g
at a rate of 1/3 Hz. The resultant displacement was logged at a rate of 1 kHz.
A regression formulae solved for the best-fit sinusoidal wave to derive the
peak force, the peak displacement and the phase shift between the two
traces. From this data it is possible to calculate the Dynamic Spring Rate
(DSR) of the test tissue, and to resolve the DSR into a purely elastic and a
purely viscous term.
For the purpose of this study our only interest is in the DSR. The purpose is to
determine the mean DSRs for healthy tissue, damaged tissue and stem-cell
treated tissue.
Rabbit
0221 left
0247 right
0226 right
0221 right
0224 right
0212 right
0222 left
0227 left
0212 left
0222 right
0220 left
0247 left
0217 left
0222 right
DSR
g/mm
1.07
1.161429
1.32
1.348462
1.390909
1.392
1.403636
1.403636
1.47
1.518333
1.519
1.584
1.607143
1.796
CofV
0.026476
0.156514
0.070459
0.131648
0.087314
0.121953
0.039574
0.039574
0.097953
0.191315
0.079513
0.128426
0.021767
0.257763
modulus max
Pa
5937.875
6445.252
7325.229
7483.176
7718.732
7724.787
7789.36
7797149
8157.641
8425.861
8429.562
8790.275
8918.705
9966.751
Scar type/treatment
Small scar
Big scar – treated
Control
Scar – treated
Scar – treated
Scar - treated
Scar – treated
Big scar – treated
Scar
Scar – treated
Small scar
Small scar – treated
Big scar – treated
Small scar – treated
0227 right
0224 left
0222 left
0220 right
1.796
1.811667
1.863333
1.867692
0.257763
0.135183
0.071905
0.120957
9966.751
10053.69
10340.41
10364.6
Big scar – treated
Scar – treated
Big scar – treated
Control
The table above gives the full set of results. Column 1 identifies the tissue
sample, column 2 is the DSR is units of g/mm, column 3 is the coefficient of
variance, column 4 is an estimate of the shear modulus of the tissue, and
column 5 identifies what group the tissue belongs to.
The results are ranked in terms of most pliable to most stiff. It can be seen
that there are no statistically significant groups. One of the few points of
interest is that tissue that has severe scarring, which would be expected to
cluster near the bottom of table (very stiff) is represented throughout the table.
This indicates that some form of healing did take place in some of these
samples.
Further tests are awaited, which will not be available in time for inclusion in
this report. Samples of tissue will be subjected to histological analysis, to
determine if there has been any measurable change in collagen and elastin
constituents. The dissected out vocal fold tissue will then be tested using a
parallel plate rheometer. This will give provide an alternative measurement of
the tissue’s elastic properties.
5 Further Work
Whilst inconclusive the results do indicate that some samples have
demonstrated healing. This indication merits further analysis with a further
study, which is constructed to provide an alternative method of obtaining
statistically significant results.
The main problem is that examples of the control group can be found
throughout the table. This indicates that the natural variation in tissue pliability
mitigates against finding significant clusters classified as healthy, scarred and
treated. To overcome this problem a new study will be carried out in which
each animal acts as its’ own control, by scarring & treating one side of the
larynx.
Whilst use of the pin has been well proven in previous studies, it is known that
the variations in depth of penetration results in small discrepancies, and that
the angle of tension with respect to the vocal fold axis is critical. Therefore in
addition to carrying out direct measurements of shear we will in addition
reconfigure the apparatus to operate as an indentometer. There exists a
proven analytical method of deriving shear modulus direct from indentometer
data [9,10]; and this method avoids the problems of both angle and depth.
A further RAE travel grant will be requested to allow us to participate in this
further study, which is planned for next year.
6 Dissemination
We still await the histology and rheology results. However the data to date is
indicative that severely damaged tissue injected with embryonic stem-cells did
regenerate. We will therefore be disseminating these results in a peerreviewed journal.
1
2
3
4
5
6
7
8
9
10
Hertegård S, Dahlqvist Å, Goodyer E, Maurer. Viscoelasticity in scarred
rabbit vocal folds after hyaluronan injection - short term results AAOHNSF/ARO Research Forum during the 2004 Annual Meeting of the
American Academy of Otolaryngology-Head and Neck Surgery Foundation,
New York, New York, September 19-22, 2004.
Goodyer EN, Gunter H, Masaki A, Kobler J, AQL 2003, Hamburg, Mapping
the visco-elastic Properties of the Vocal Fold
Hess M, Mueller F, Kobler J, Zeitels S, Goodyer S. Measurements of Vocal Fold
Elasticity Using The Linear Skin Rheometer, Folia Phoniatrica, in print.
Goodyer EN, Hess M, Mueller F. In-vivo Measurements of the Human Vocal
Fold. European Archives of Oto-Rhino-Laryngology in print.
Matts PJ, Goodyer EN. Journal of Cosmetic Science, volume 49, pages 321323, Sep/Oct 1988, A New Instrument to measure the mechanical properties
of the human stratum corneum.
Matts PJ. The Linear Skin Rheometer. Stratum Corneum Podium
Presentation Abstracts, item 23, – 10-12 Sep 1998, Cardiff.
Mok W, Bautista B, Hoyberg K, Kirnos P, Subramanayam K.. Mechanical
properties of ageing skin – Stratum Corneum vs. dermal changes. Stratum
Corneum III, 12-14th September 2001, Basel Switzerland.
Bioengineering of the Skin, Skin Biomechanics. Chapter 8 the Gas Bearing
Electrodynamometer and the Linear Skin Rheometer. Published by CRC
Press. ISBN 0-8493-7521-5
Hayes WC, Keer LM, Herrmann G, Mockros LF. A Mathermaticval Analysis
for Indentation Tests of Articular Cartilage. J Biomechanics, 1972, Vol 5, 541555
Fung YC, Biomechnics: Mechanical Properties of Living Tissue. New York,
Spriger Verlag 1981