Equipment Grant In Support Of A Series Of Studies Into The Biomechanical Properties Of Human
Tissue
Eric Goodyer, Bio-Informatics Research Group, De Montfort University
PART 1 - PREVIOUS RESEARCH TRACK RECORD - THE CENTRE FOR COMPUTATIONAL INTELLIGENCE
Eric Goodyer is an active member of the CCI research group, which is researching fuzzy logic, bio-health informatics, neural
networks, mobile robotics and creative computing. The CCI was rated 4 in RAE 2002 and is actively engaged in research with UK
and overseas partners. The Faculty has a specialist administrative structure to support all research activity.
ERIC GOODYER
Eric Goodyer has been engaged in research and development since the completion of his MSc by exam and dissertation in 1977 at
the Department of Electronics & Electrical Engineering, UMIST. He joined the Scientific Instruments Research Association
(Sira), as a Research Officer and was appointed head of his R&D group in 1982. Much of this early work was commercially
confidential restricting his ability to publish; however active involvement with various IEE and similar professional groups was
encouraged. Section 9 lists conference proceedings that relate to his early interest in instrumentation, particularly in the field of
novel sensors and low power techniques [1,2,3,4,5]
He joined DeMontfort University in 1994, whilst maintaining strong links with industry; much of his work from 1994 was also of
a confidential nature. Following an invitation to join a collaborative team at Harvard Medical School investigating tissue
engineering, he took the opportunity to undertake research in the true academic sense. Since 2003 he has secured grants totalling
over £407k, and has published joint papers with international collaborators from Germany, Stockholm and the USA. His last two
EPSRC grants were rated as ‘Tending to Outstanding’.
RECENT RELEVANT RESEARCH
Bio-Mechanical Properties of the Stratum Corneum
A specific area of the Group’s research is the visco-elastic measurement of human tissue. As a result of a long-standing research
partnership with Procter & Gamble (in the UK and the USA) a range of specialist and novel and methodologies have been
developed that have enabled a series of bio-mechanical studies to be undertaken in Europe and the USA [6,7,8,9]. Research
undertaken with Unilever has resulted in the launch of a new product range, and our methodologies were used to support a recent
patent application [10]. The inclusion of a chapter on the LSR in a recent publication by the CRC Press, ‘Bioengineering of the
Skin’ [11], as well as a study on bio-mechanical measurement methodologies [12] has resulted in further interest in the technique.
The Group remains in contact with Procter & Gamble, Unilever and other skin care companies, as well as collaborating with
colleagues in De Montfort's Health & Life Sciences Faculty, who are also researching the biomechanics of skin.
Bio-Mechanical Properties of The vocal fold
The Group’s research into the properties of the vocal fold has been undertaken through a series of international collaborations, as
summarised below.
 Collaboration with University Medical Centre Hamburg-Eppendorf (UKE)
Prof. Markus Hess, Head of the Department of Phoniatrics and Pediatric Audiology at UKE, is widely accepted as a leader in the
field of quantitative laryngology. His invitation to the Group to provide facilities for further research using human tissue
represented a superb opportunity to both further our research programme, and to gain valuable insight from a major figure in this
field. With EPSRC support (GR/S85849/01) a collaboration was entered into with Markus Hess and his team. Over the last two
years unique measurements of the elastic properties of excised human vocal folds have been obtained [13,14,15]. We have also
started to obtain in-vivo results from volunteer patients; and these results have been published [16]. The IGR relating to this
collaboration was deemed to be 'Tending to Outstanding'. The importance of this partnership is that it has brought together the
engineering skills of DMU with the life science skills at UKE. The result is a strong, and dedicated multi-disciplinary team that is
investigating the fundamental science and phenomena that is proving essential to the development of tissue engineering therapies
by DMU’s other international collaborators.
 Collaboration with Harvard Medical School at Massachusetts Eye & Ear Infirmary (MEEI)
The Harvard group, which includes laryngeal surgeons, voice scientists and engineers, is currently engaged in a major programme
to apply tissue engineering to repair/replace damaged vocal folds in patients who have voice disorders. A key part of the tissue
engineering process is to ensure that the implant matches the visco-elastic properties of the natural vocal fold. With EPSRC
support (GR/S23346/01) the joint research team successfully developed methodologies to map the bio-mechanical properties of
the vocal fold. Trials of the LSR using animal larynxes and a human larynx obtained from pathology were a success.
Measurements were obtained from the delicate vocal fold epithelium as well as surrounding tissue. From these results the first
iso-contour maps showing the variation of elasticity with respect to position were generated and published [13,14]. The team were
able to detect artificially stiffened tissue, which suggests that it could be used to quantify and map areas where vocal fold
properties have been changed by implants or by pathology. Changes in pliability after tissue augmentation implant were measured
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with respect to time, opening the possibility to 'tune' the pliability of the vocal fold during the implant procedure. These were the
first direct measurements ever made of the mechanical properties of the superficial layer of the human vocal fold in an intact
larynx. The IGR relating to this collaboration was deemed to be 'Tending to Outstanding'.
 Collaboration with Huddinge University Hospital, Stockholm
Prof. Stellan Hertegard of Huddinge University Hospital, Stockholm has been researching the effectiveness of different tissue
augmentation techniques. The most recent collaboration quantified the effectiveness of hyaluronic implants in a rabbit model. They
were split into 4 groups, one set were undamaged, one set had their vocal folds scarred, the 3 rd set had their vocal folds scarred and
then augmented using Hyaluronic Acid implants, and the 4 th group were similarly treated but with a control implant. The larynxes
were excised and measured using an Instron and a specially modified LSR. From this data it is possible to derive a range of elastic
properties. The results indicate that augmenting scarred tissue with hyaluronic acid implants can restore the elasticity of a damaged
larynx back to near normal. These results were announced in New York last year, and have been published [16,17]. A new research
programme, begun in 2005, is quantifying the effectiveness of stem-cell implants into damaged vocal folds. Early results indicate
that stem-cell implants into scarred vocal folds does restore the elasticity – further stem-cell studies are planned to further this
research programme.
 Collaboration with Wisconsin University Hospital, Madison USA
A large team, under the leadership of Dr Diane Bless, is planning to investigate new techniques to restore scarred vocal fold tissue
employing a range of tissue engineering techniques. Early this year the team successfully characterised 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.
A major NIH grant to further this work has been applied for, primarily to investigate tissue engineering techniques and therapies.
Our methodologies, alongside others, will be used to quantify the effectiveness of these new therapies.
 New Collaborations
We have recently been asked to join a team based at the Department of Ophthalmology, Guy's and St. Thomas' NHS Foundation
trust to assist in a programme to investigate the elastic properties of the human cornea. An initial study has demonstrated that it is
possible to map the variation of the stiffness of the cornea with respect to both position and angle. A more extensive study is
planned in 2007 to examine the use of tissue augmentation as a therapy for keratoconus. We are developing a new partnership with
Queen’s Medical Centre (QMC) Nottingham, to investigate the dynamics of the mucosal in-vivo using optical techniques. We are
starting a new study with EPSRC support to investigate reinervation treatment as a potential cure for vocal fold paresis with UCLA
Medical Centre.
REFERENCES
1. Goodyer EN. Overview of a range of novel automotive sensors. Proceedings of the Institution of Mechanical Engineers
(London), pages 79, October 1989.
2. Goodyer EN. Novel sensors for measuring fuel flow and level. Proceedings of the SPIE - The International Society for Optical
Engineering , pages 150-4, 1989.
3. Goodyer EN. Application of CMOS Technology To Process Instrumentation: Some Case Studies. Software & Microsystems
3(3):75-77, June 1984.
4. Goodyer EN. Application of low power microtechnology to process instrumentation: some case examples. IEE Colloquium on
Low Power Microprocessor Systems (Digest No.85), pages 1-5, 1983.
5. Goodyer EN. A microprocessor-based gas flow computer. Application of Microprocessors in Devices for Instrumentation and
Automatic Control. pages 57-66, 1980.
6. P Matts, E Goodyer. A New Instrument To Measure the Mechanical Properties of the Human Stratum Corneum. Journal of
Cosmetic Science, volume 49, pages 321-323, Sep/Oct 1988.
7. W Mok, B Bautista, K Hoyberg, P Kirnos, K Subramanayam. Mechanical Properties of Ageing Skin – Stratum Corneum vs.
Dermal Changes. Stratum Corneum III, 12-14th September 2001, Basel Switzerland.
8. P.J. Matts. Sensitive Measurement of Stratum Corneum Mechanical Properties Using the Linear Skin Rheometer. Stratum
Corneum IV, June 2004, Paris
9. K. P. Ananthapadmanabhan, David J. Moore, Kumar Subramanyan, Manoj Misra & Frank Meyer. Cleansing Without
Compromise: The Impact Of Cleansers On The Skin Barrier And The Technology Of Mild Cleansing Dermatologic Therapy,
Volume 17 Issue s1 Page 16 - February 2004
10. Liquid Cleansing Composition Having Simultaneous Exfoliating And Moisturizing Properties. United States Patent
Application 20040091446
11. 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
12. Rodrigues L. EEMCO Guidance to the in vivo Assessment of Tensile Functional Properties of the Skin. Skin Pharmacol Appl
Skin Physiol 2001;14:52-67 (DOI: 10.1159/000056334)
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PART 2: DESCRIPTION OF THE PROPOSED RESEARCH AND ITS CONTEXT
1. INTRODUCTION
With the support of the EPSRC (GR/S85849/01, GR/S23346/01, EP/D025591/1,EP/E04087X/1) the DMU research team has been
enabled to collaborate with 5 international partners, working in the field of vocal fold bio-mechanics and tissue engineering. The
end objective of this work is to assist and promote the development of new and novel tissue repair techniques. To date this has
focused on the use of implants, such as hyaluronic acid, to restore vocal fold pliability. Tissue Engineering is an exciting and
challenging alternative therapy that must be researched in more depth. Recent stem cell research [23] has indicated that it is
possible to stimulate the self-regeneration of scarred vocal fold tissue. DMU are actively assisting the investigations into stem-cell
therapy being carried out at the Karolinska Institute. The DMU team has also been invited to assist in a $1.8M 5-year NIH funded
programme at Wisconsin University Hospital. This programme will be examining the use of genetic transfection to control,
stimulate and suppress tissue regeneration, as well as the use of mesh scaffolds and growth factors to manage new tissue
regeneration. The DMU input has been essential to tissue repair researchers world-wide, resulting in the formulation of leading
edge precision instrumentation that has been used to measure the extremely low forces exerted by the delicate vocal fold tissues.
No such similar techniques or methodologies are available from other sources that directly quantify vocal fold mechanical
behaviour.
Our methodologies are used to characterise the biomechnical properties of healthy tissue, and to quantify the effectiveness of tissue
augmentation and repair procedures. We have now been approached by other research groups investigating other pathologies. One
example being a pilot study recently carried out at St Thomas and Bart’s Hospital, who are investigating the eye disease of corneal
keratoconus. This disease is the result of weakness in the cornea that results in a malformed structure that causes optical
aberrations. The researchers are investigating a possible cure using cross-linked collagen augmentation to the mid-cornea region.
DMU’s role is to deploy our point-specific elasticity measuring methodology to map the variation of tissue stiffness across the
surface of the cornea, before and after treatment with the collagen implant.
2. RESEARCH CONTEXT
 Characterisation of the Vocal Fold using Excised Tissue
This proposal is an essential extension of our preceding structured and coherent research ethos. The key objective is to evaluate and
promote the use of new and novel tissue repair techniques, such as hyaluronic acid augmentation, and tissue engineering including
stem cell stimulated self-regeneration. The original research at Harvard Medical School and UKE, using animal tissue and a
limited set of freshly excised human larynxes, enabled us to develop and refine the methodologies that we are now successfully
deploying to support more fundamental research and to support tissue engineering programmes. Our results represent the first
published material [13,14,15] that shows the variation of elasticity over the surface of the human vocal fold. Detailed mappings
have been obtained from both animal and human excised tissue. Figure 1 shows iso-contour maps obtained at Harvard, and the
experimental set-up used by Wisconsin University Hospital to verify our methodologies.
Figure 1 Iso-Contour Map from Harvard (left)
Supporting Experimental Set-up from
Wisconsin (right)
To capitalise on these achievements we are now part way through a large-scale study obtaining data from 50 excised larynxes and
volunteer patients. This affords the opportunity to generate the mathematical models that will ultimately enable objective
assessment of the effectiveness of tissue repair surgery. Our interim results will be presented to AQL 2006 in Groningen. Figure 2
shows a pair of graphs with our latest results, obtained by two different methods, one employing a direct shear stress/strain
analysis; the other employing an indentometer. These methods are referred to as ‘The Shear Model’ and ‘The Indentometer
Model’. Note the similarity of the data sets, yielding shear modulus ranges of typically between 500 and 2500 Pascal, with male
larynxes tending to be stiffer than female, and a trend to increased stiffness with age.
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Vocal Fold Shear Modulus (indentometer model)
Vocal Fold Shear Modulus (shear model)
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Figure 2 Variation of Shear Modulus with Respect to Age
These results are highly novel, as there is very little published literature that details work carried out with human tissue. Our
methodologies are unique in that they use intact larynxes and are capable of relating the data obtained to a specific anatomical
position and direction of stress. The results are similar to those obtained by other methods, such as parallel plate rheometry; as
detailed in our publications. Wisconsin University Hospital has independently verified our methods; and they have now submitted
supporting results for publication. Wisconsin successfully generated contour maps, showing the variation of elasticity across the
surface of the vocal fold of canine, human and rat samples. The rat was particularly challenging in view of the extremely small
structures that the collaborating team were trying to attach to; however it was achieved, and it was shown possible to distinguish
between healthy and scarred tissue.
 Characterisation of Vocal Fold using in-vivo Data
Towards the end of 2004 a new measurement apparatus, designed by DMU, was successfully deployed that allowed quantification
of the bio-mechanical properties of healthy and diseased vocal folds, in-vivo, with volunteer patients at UKE. The most recent
comparable published work was that by Tran QT, Berke GS, Gerratt BR, Kreiman J, in 1993 [22]. Their work was groundbreaking,
but relied on a cumbersome apparatus that would be difficult to reconstruct. In contrast the DMU apparatus has been designed to
clamp on to a standard laryngoscope, such that it can be used repeatably in the operating theatre. The laryngeal tensiometer is
clamped to a standard size C laryngoscope (figure 3). 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. A detailed description of the design and methodology can be found in our most recent publication [14]. The graph
(figure 3) shows a series of readings taken from a larynx. 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. The paper details results obtained from two volunteer female patients of similar age. Elasticity measurements
were obtained from healthy tissues from both, 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. Since then we have obtained data from 8 volunteer patients, yielding
values of shear modulus for healthy tissue from between 673 Pascal to 2143 Pascal. This range is similar to that obtained from the
excised tissue, and in recent work published by R Chan and presented by J McGlashan. Chan obtained his data from excised vocal
fold covers; McGlashan used an optical in-vivo technique to derive modulus from measurements of the mucosal wave velocity.
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Figure 3 The In-Vivo Laryngeal Tensiometer and Typical Data Trace
 Tissue Engineering Studies
The results of our research to date demonstrate that the key milestone of obtaining meaningful data from both excised larynxes and
from volunteer patients in-vivo has been achieved. These data, and our methodologies are now in demand by other teams
investigating tissue engineering therapies. To date we have assisted Harvard [13] and Karolinska [17,18] to quantify the
effectiveness of hyaluronic acid tissue augmentation, as well as completing a recent study with Karolinska that examined stem-cell
therapy. We have now been invited to participate in a major NIH funded 5-year research programme based at Wisconsin
University. The EPSRC have partially funded our involvement, and a study has begun to assess the viability of using ultrasonics to
measure tissue properties in-vivo. Data obtained by this promising methodology is being compared to that obtained using our
existing electro-mechanical apparatus and similar equipment. If successful then it will give us another tool that will be able to
obtain data in a non-invasive manner in-vivo.
UCLA has requested our assistance for a study to quantify the change in vocal fold tissue tension resultant from nerve stimulation.
This will be carried out in-vivo using a canine model. If successful then this would enhance our understanding of the interaction
between the level of stimulation of the recurrent laryngeal nerve (RLN) and the resultant tension in the vocal fold cover. We
propose to carry out a short pilot study, based on four trips to UCLA, to determine if it is feasible to use either the Laryngeal
Tensiometer (LT) or the Linear Skin Rheometer (LR) to obtain in-vivo data from an anaesthetised dog.
St Thomas & Barts Hospital have requested our assistance to evaluate a technique of corneal collagen cross-linkage with
Riboflavin and UVA to strengthen the cornea, as a therapy for keratoconus.
Karolinska are planning a major series of studies using stem-cell implants, following the success of a trial study in 2005. It is our
intention to assist them with apparatus that will quantify tissue changes in-vivo.
4 The Apparatus
Detailed here is the apparatus that we wish to purchase, associated with the tissue studies that the apparatus will assist to progress.
4.1 Refurbished LSR
The LSR was invented by Eric Goodyer, and costs £12,000 plus VAT. DMU does not own an LSR, and has to date relied on an
early model of the device that belongs to Eric Goodyer. We have also been loaned on a long term basis a similar old style LSR by
Procter & Gamble. Both devices have been superseded by new mechanical enhancements that dramatically improve the
repeatability and stability for the force readings. Using other resources one of the devices has been upgraded by the manufacturers
at a cost of only £4000. This modernised device is now in permanent use on a range of laryngeal tissue studies, and has resulted in
a dramatic improvement in the coefficients of variance of the measured data. Typical CofVs using the older style device were
typically 8.9%, the modernised device is producing typical CofVs of 2.8%. This is a substantial improvement, and greatly enhances
the value of our research. We therefore request a similar investment to upgrade our second LSR. It will then be used for ad-hoc
studies at various institutions as detailed in this proposal.
4.2 Suction
Attaching the measurement probe of our various devices to human tissue, both excised and in-vivo, has proven to be the most
challenging problem. For excised tissue the most repeatable method has been the use of calibrated needles. This method enables
the team to obtain site specific data which has been used to generate our published iso-contour maps. However it has two
disadvantages; firstly the depth of penetration must be carefully controlled to ensure repeatability, secondly developing the
constitutive equations to derive fundamental measurands in term of modulus has not been possible to date. Therefore we have been
restricted to published results in terms of relative change in elasticity.
For in-vivo work we have been using a non-toxic adhesive based on methyl cellulose. It works very well in a moist environment,
but is difficult to use and must be cleaned off after use. For excised tissue we have used cyanoacrylate, as we do not have the same
restrictions as we do when dealing with volunteers. The advantage of this method is that we can develop a shear modulus model to
derive fundamental elastic properties in terms of material modulus. We are also able to measure the surface of attachment thus
enabling a calculation of applied shear stress. The disadvantage of this technique is that the attachment area is difficult to control,
as represented by our coefficients of variance when we quantify repeatability. With great care we can achieve CofV of typically
8% with excised tissue, but we cannot use as much time for the in-vivo work as it would be unethical to delay the patient’s medical
procedure. Our in-vivo CofV have been as poor as 15%.
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Our most reliable method has come from using suction. Suction is always available in an operating theatre, and we have use of an
old vaccum pump for lab use. We have constructed a range of attachment probes from light weight aluminium cannulars. CofV
have dramatically improved. The two figures shown here show a probe attached using methylcellulose and the resultant strain data.
Note the slippage within the adhesive after each mechanical transition, which does not occur at all with suction.
Force grams
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We request funds to enable us to construct a small, portable, battery operated suction probe for use in the operating room
environment. This means that we will not have to disturb any apparatus that is connected to the suction supply for the medical
procedure, and can comply with medical safety regulations which forbid the use of a direct connection to mains power when
apparatus will come in contact with a patient. A medically approved portable vacuum controller will cost around £800.
4.3 Precision Tissue Mounting Apparatus
A key objective of many of our studies is to quantify bio-mechanical properties with respect to anatomical position and direction of
applied stress. As we are measuring intact tissue samples, we do not have the advantage of being able to dissect out a layer and pin
it to a flat surface. Position and direction is determined using a range of available apparatus from other equipment, such as an index
head , a linear screw with a micrometer, and an old x-y translation table. None of this apparatus is mechanically compatible with
any other piece, and a lot of time is used setting up the equipment for a study. Our resources in terms of FEC funded time would be
much better served if we could invest in a complete new set of bench apparatus that was designed for our purposes. This would
improve the efficiency, in terms of research output and staff costs, and enhance the quality of the results obtained.
The most appropriate apparatus is optical bench equipment. We require the following components :
a basic optical slide with two mounting plates onto which we can mount the tissue and the measuring apparatus
a pair of goniometers to adjust the angle of both the plates with respect to the main axis, this is to ensure that the probe is
normal to the tissue sample
an X-Y translation table to enable us to select a specific measurement point at a calibrated distance for an anatomical
datum
A height adjustment to align the tissue surface plane with the measuring probe.
Competitive quotes have been obtained for this apparatus, and they cost is around £2000 per set. We will require 2 sets.
4.4 Battery operated Data Acquisition Apparatus
The laptop used for the in-vivo studies employs a data acquisition card with an external power supply; which is inappropriate for
use in an operating theatre and requires us to deploy a medically approved isolating transformer. In the laboratory we are using a
PCI based data acquisition card, which is acceptable but means that our apparatus is not portable.
Our efficiency and ability to set up studies speedily at other institutions will be greatly enhanced if we had a small battery operated
data acquisition unit that can be connected to a battery operated laptop device. Commercially available equipment will provide the
features that we require, but are expensive. We would prefer to design our own system that directly meets our needs; which will be
based on the in vivo data acquisition unit that we designed in-house. This part of the project will also enable us to offer some
practically based projects for final year and MSc students. We request funds to cover the PCB manufacture costs and components;
which are £1200.
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4.4 Cornea Mount
We are in the process of developing a new study in partnership with St Thomas & Bart’s to map the elastic properties of the human
cornea. This is in support of a study that proposes to evaluate the treatment of kerataconus with collagen implants. We will mount
donor cornea in an artificial eye mount as is usual, so that we can apply a known and controlled back pressure. What is required for
our study is an additional gimlet arrangement that will allow us to present any part of the inflated cornea to the measuring probe
such that the attachment is normal, and in any direction. This will require a mechanical designer to produce suitable drawings for
our mechanical workshop to work to. We are therefore seeking FEC costs for 2 weeks for a mechanical designer, plus mechanical
workshop time and materials.
4.5 Miniature Optical Sensor for Detecting Biomechanical Force
In the 1980s Eric Goodyers research team was responsible for the investigation, and subsequent commercial exploitation, of a
range of optical sensors. Amongst these devices was a successful means to photonically auto-calibrate a force sensing mechanism.
The great advantage of using an optical technique is the substantially higher sensitivity that can be obtained over using electromechancial methods. This technique [ref SPIE] has since been used as the basis for a precision accelerometer, dynamic load
cycling measurement and within active suspension systems in cars.
We are proposing a small investigation to determine if modern optical components, which are substantially smaller than in the
1980s, are capable of being used to measure the extremely low forces produced by biomechanical actions. This is a challenging
problem, but one that offers great rewards if achievable as optical sensors can operate from very low current sources, which is a
statutory requirement for in-vivo devices. The underlying principle is based on the use of a pair of matched optical sensors, and a
single emitter. The device is set up such that the desired measurand causes a differential optical signal to be detected, which is
photonically auto-normalised by dynamically adjusting the emitter output power. We established the mathematics over 20 years
ago, and demonstrated that in this configuration the differential optical signal is linearly proportional to the applied primary
measurand, and not a square relationship as would expected for a wave-front.
We are seeking sufficient funds for an exploratory study to investigate if we apply this proven phenomena to achieve superior
research results for our bio-medical studies.
RA 3 months workshop 2 weeks electronics 2 weeks materials 3k PI time 1 week
5 The Studies
Given here are all the studies that are either underway or planned for the near future.
5.1 Anisotropic Nature of the Vocal Fold
A controversial finding of our work to date is that the vocal fold is highly anisotropic. What is not known is whether this is due to
the way that the lamina propria is attached to the underlying tissue, or whether it is inherent in the LP itself. This is very significant
as all other published literature does not discriminate between the elastic properties of the vocal fold with respect to direction.
Anisotropic behaviour has significant implications for our research partners investigating tissue engineering and the use of growth
factors on scaffolds. If vocal fold tissue is anisotropic then it will be necessary to construct rectangular scaffolds that will result in a
reproduction of the anisotropic behaviour when the growth factors take effect. We will use the refurbished LSR and the precision
tissue mount for this research.
The research programme will use freshly excised human donor larynxes, typically 2 days post-mortem. The larynxes will be
sectioned along the saggital plane, to expose the vocal fold. Using the apparatus the elastic properties of the vocal fold cover will
be examined with respect to angle of applied shear stress and anatomical position. The lamina propria will be removed and set
aside for further studies. The measurements will then be repeated on the vocalis ligament. Finally the ligament will be removed and
the final set of readings will be obtained from the vocalis muscle.
Our pilot study using 8 excised larynxes has assisted us to develop the methods, and our initial findings have confirmed our
opinion that the vocal fold is anisotropic. We express our results so far in terms of the ratio of the stiffness of the tissue in the
transverse direction and the longitudinal direction. For a complete larynx the mean ratio is 0.4, for the muscle and ligament only it
is 0.5 and for the muscle only it is 0.6. This indicates that as the laryngeal layers are built up the anisotropic nature increases. We
plan to continue this study using the improved apparatus that will be provided by this grant, in order to obtain sufficient data to
quantify anisotropic nature with a statistically significant sample.
The lamina propria will then be subjected to a further study, in isolation of the underlying structures. Our results to date indicate
that the lamina propria itself is anisotropic. What is not known is if this is due to internal structure of the LP or is it due to the way
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it is attached to the underlying ligament and muscle. We have already constructed an apparatus to investigate this question. The
device is a tensiometer attachment to the LSR that can be used to measure stress/strain relationship when a small piece of tissue is
extended. The LP will be tested in orthogonal directions to determine if the anisotropic nature is inherent.
5.2 Corneal Study
The proposed research with St Thomas & Bart’s Hospital Department of Ophthalmic Medicine is intended to develop a tissue
augmentation therapy to treat keratoconus. This disease affects a large number of people, and is the result of a weakness in the
mid-cornea that results in a buckling of the whole cornea, resulting in a cone shape extruding from the centre. Focusing the eye
now becomes extremely difficult. The commonest therapy is to model the defect and to mould a specially designed contact lens
over the distorted cornea. Our research partners are of the opinion that augmenting the weakened part of the cornea with a collagen
implant could strengthen the structure, enabling the patient to self correct the vision. The role of DMU is that our apparatus is
capable of obtaining point specific measurement data of tissue elasticity; and our research partners require our expertise to map the
variation of elasticity over the surface of a healthy set of donor corneas. This will provide a baseline set of normative data to be
used to assess the tissue augmentation therapy. Once that data has been obtained we will then quantify the effectiveness of tissue
augmentation using donor corneas which will be injected with the collagen implants.
We will use the precision tissue positioning apparatus, the corneal mount, the refurbished LSR and the suction apparatus for this
study.
5.3 In-vivo tissue engineering assessment
We have assisted Prof Stellan Hertegard at the Karolinksa institute with two studies that quantified the effectiveness of tissue
engineering in rabbit models. One was the application of Hyaluronic Acid augmentation, and the second was the first study the
deployed stem-cells to treat vocal fold scarring. Our apparatus was capable of obtaining data from excised larynxes, but our
attempt to use the laryngeal tensiometer in-vivo was not a success. This was due to the dimensions of the apparatus, in that is was
designed for use in humans, and was too large for use with rabbits. Since then we have also shown that suction is a superior means
of tissue attachment than the use of adhesives. However the ability to monitor changes in tissue elasticity whilst any tissue
augmentation therapy takes affect is of great value to us; and we will continue to investigate methods to achieve this.
Eric Goodyer is a named consultant to the 5-year NIH funded tissue engineering research programme that has secured $1.8 million
funding from the NIH. This team, headed by Prof Diane Bless will be investigating genetic transfection, and the use of growth
factors to repair vocal fold scarring. The final stages of the programme will involve in-vivo studies in a canine model; and it is our
intention to deploy the new apparatus to measure the change in tissue properties in-vivo. Access to a portable data acquisition unit
will greatly enhance our ability to achieve this objective, as it will enable us to take our apparatus to the study site.
We will use the suction probe and the battery operated data acquisition unit for these studies.
5.4 Iso-contour mapping of the vocal fold
The development of iso-contour maps remains a core activity for our group. We are aiming to develop a set of normative base-line
data that maps the variation of the elastic properties of the vocal fold with respect to anatomical position and direction. Freshly
excised human larynxes are obtained within 2 days post mortem, split along the sagittal plane and mounted. Using the LSR a series
of readings are obtained along the mid-membranous line of the vocal fold, typically spaced 2mm apart. Similar data is obtained
along parallel lines both superior and inferior. The purpose is to build up a set of data from which it will be possible to develop a
baseline.
This study will use the precision tissue mount, a refurbished LSR and the suction apparatus.
5.5 Reinervation therapy as a potential cure for vocal fold paresis
This study will be carried out in partnership with UCLA. An initial visit has been made to the research centre, and the outline
experimental apparatus has been successfully deployed. Using a canine model it is intended to investigate in-vivo the relationship
between recurrent Laryngeal Nerve (RLN) stimulation and the stiffness that results in the vocal fold. Both the LSR and Laryngeal
Tensiometer (LT) will be deployed as appropriate. The LT has been deployed successfully in the OR environment to obtain in-vivo
data from humans, and deployed in Stockholm in a rabbit model. The gape of canines is large enough to insert the LT probe, which
will then be attached to the mid-membranous part of the vocal fold. The LSR probe should also be capable of such an attachment;
but with more difficulty due to the bulk of instrument’s measuring head. We are sure of successful attachment with the LT, and
have a reasonable confidence of success with the LSR. Both devices operate in a similar manner, the LT applies a known
displacement and measures force, and the LSR applies a known force and measures displacement. The applied stress is in the
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transverse direction to the axis of the Vocal Process to the Anterior Commisure. Use of suction ensures that the direct stress is
applied only to the epithelium. Force can be resolved to 10 micrograms, and displacement to 1 micron.
From this data we can derive a measure of the stiffness of the vocal fold cover in terms of force applied per distance displacement.
It is possible to derive an absolute estimate of the value for shear modulus using established mathematical procedures, and
applying the correct geometric model. However our primary interest is to measure the change in stiffness due to RLN stimulation,
therefore quantifying the relative change that is adequate for this study. At least 5 readings will be taken from the same point, using
different levels of stimulation. From this data the relative change in stiffness with respect to stimulation will be determined. We
will also take the opportunity to determine whether or not the tension changes with time, due to relaxation of the bio-mechanical
process. This will be achieved by taking a series of readings at known time intervals after the application of the nerve stimulus. The
above procedures are identical to our published and validated methodologies for obtain shear modulus data for the vocal fold
cover. They all rely on the subject or specimen being still, and the stress being applied by the measuring instrument.
The In-vivo Canine Model
Mongrel dogs (approximately 25 kg each) will be used. Each dog will be anaesthetised with intramuscular acepromazine (0.1 – 0.5
mg/kg), then intravenous sodium pentobarbital (Nembutal) (30 mg/kg) to maintain a level of corneal anaesthesia. Throughout the
procedure, general anaesthesia will be achieved using halothane. Maintenance intravenous fluid will be given at 2 ml/kg/hr. Core
temperature will be monitored with a rectal probe, and a heating pad will be used to maintain a homeostatic temperature. The vocal
folds will be visualised prior to operation to verify normal anatomy. Intravenous dexamethasone will be given periodically to
decrease nerve and vocal cord swelling.
Each animal will be placed supine, the neck prepped, and a midline incision from the hyoid bone to the sternal notch made. The
sternocleidomastoid and strap muscles are exposed and retracted laterally to expose the larynx and trachea. Neck exploration is
then performed to locate both recurrent laryngeal nerves (RLN) and superior laryngeal nerves (SLN) at their entrance into the
larynx. Both RLN will be isolated 5 cm inferior to the larynx. Custom designed rubber electrodes (monopolar, flexible, conductive
neopreme with silicone, and silicone insulation KE45) will be applied to the isolated nerves at the most proximal point dissected.
Electrical isolation of the two nerves will be confirmed by direct visualization of the vocal folds during phonation. The RLN will
be stimulated by a constant current nerve stimulator (WR Medical Electronics Co. Model 2SLH, St. Paul, Minnesota). These
nerves will be stimulated at 80 Hz with 0-3.0 mA for 1.5 msec pulse duration to achieve adduction.
Subject to the outcome of these studies it is planned to extend the work to use freshly excised human larynxes (typically less than 4
hours post-mortem). The results of these studies will lead directly to the development of the reinervation therapy.
These studies will require the portable data acquisition apparatus, a refurbished LSR and the suction apparatus.
RELEVANCE TO BENEFICIARIES
Vocal fold scarring and vocal fold paresis are debilitating diseases, resulting in partial or total loss of voice. Tissue augmentation
and engineering therapies offer the potential for damaged tissue to be restored to a state where the voice can be used again.
Reinervation therapy has been demonstrated to be of value in many cases, and UCLA are leaders in developing this treatment.
Success and partial success results in an immediate enhancement of the quality of life of the patient.
These studies will enable us to gain a greater understanding of how the vocal fold works, and will provide a set of normative
baseline data against which the outlined new therapies can be scientifically assessed and quantified.
DISSEMINATION AND EXPLOITATION
There is potential for dissemination by various means. To date DMU have successfully disseminated our work in 3 European
Journals, Folia Phoniatrica, The European Archives of OtoRhinoLaryngology and Acta OtoLaryngologica. Our pilot studies in
partnership with Wisconsin University Hospital have recently been accepted by the American Journal of Voice. The work of the
UCLA team is well published in a range of US based journals including The Laryngoscope. The results of all of these studies will
be presented on both sides of the Atlantic as appropriate. The main European Conference opportunity will be the Pan European
Voice Conference, to be held in Groningen in 2007; at which we may be able to make an initial announcement. The key US
Conferences will be the American Academy of Oto-Rhino Laryngology Annual meetings for 2008.
Any IPR resulting from the proposed research will be protected through established mechanisms at DMU.
13. Goodyer EN,Gunter H,Masaki A, Kobler J. Mapping the visco-elastic Properties of the Vocal Fold, AQL 2003, Hamburg.
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14. 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.
15. 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. 264:45-50,Jan 2007.
16. M Hess, F Muller, J Kobler, S Zeitels, E Goodyer. Measurements of Vocal Fold Elasticity Using the Linear Skin Rheometer.
Folia Phoniatrica, March 2006, vol 58, issue 3.
17. S Hertegård, Å Dahlqvist, E Goodyer, E Maurer. Viscoelasticity In Scarred Rabbit Vocal Folds After Hyaluronan Injection short term results. AAO-HNSF/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.
18. Hertegård S, Dahlqvist Å, Goodyer E, Maurer. Viscoelastic Measurements After Vocal Fold Scarring In Rabbits– Short Term
Results After Hyaluronan Injection. Acta Oto-Laryngologica – July 2006
19. Chhetri, D.K., Mendelsohn, A.H., Blumin, J.H., Berke, G.S. Long-term follow-up results of selective laryngeal adductor
denervation-reinervation surgery for adductor spasmodic dysphonia.. The Laryngoscope. 116 (4), pp. 635-642.
20. Chhetri, D.K., Berke, G.S.. Treatment of adductor spasmodic dysphonia with selective laryngeal adductor denervation and
reinervation surgery. Otolaryngologic Clinics of North America 39 (1), pp. 101-109.
21. Chhetri, D.K., Blumin, J.H., Vinters, H.V., Berke, G.S. Histology of nerves and muscles in adductor spasmodic dysphonia.
Annals of Otology, Rhinology and Laryngology 112 (4), pp. 334-341.
22. Tran QT, Berke GS, Gerratt BR, Kreiman J. Measurement of Young's modulus in the in vivo human vocal folds. Ann Otol
Rhinol Laryngol. 1993 : 102, 584-91.
23. Regeneration Of The Vocal Fold Using Autologous Mesenchymal Stem Cells. Kanemaru S, Nakamura T, Omori K, Kojima H,
Magrufov A, Hiratsuka Y, Hirano S, Ito J, Shimizu Y. Ann Otol Rhino Laryng. 2003 Nov;112(11):915-20.
24. Goodyer EN, Müller F, Licht AK, Hess M. In Vivo Measurement of the Shear Modulus of the Human Vocal Fold - Interim
Results from 8 Patients. European Archives of Oto-Rhino-Laryngology and Head & Neck. In print.
25. Dailey SH, Tateya I, Montequin D, Welham N, Goodyer EN. Viscoelastic Measurements of Vocal Folds Using the Linear
Skin Rheometer (LSR). Journal of Voice (in print)2007.
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Justification of Resources
The summary of resources requested is as follows
1 Apparartus
Refurbished LSR
Vaccum Pump x 2
Tissue mount x 2
Portable Data Acquisition
Cornea mount
£4000
£1600
£4000
£1200
£ 300
2 Research Staff Costs
Design Engineer 2 weeks £3474 FEC
PI Time 2 weeks
£3952 FEC
3 Technician Costs
Mechanical workshop 2 weeks
Electronic technician 2 weeks
£1500 FEC
£1500 FEC
4 Travel
Two trips to Hamburg
£500
Total project costs
£22,026
This investment by the EPSRC represents excellent value for money, as the apparatus will be deployed on a range of studies as
opposed to a single research programme. DMU have developed an extensive and credible range of research partners, and the key
barrier to progressing all these projects in parallel is the lack of suitable apparatus. Our research outputs to date, in terms of
published papers, conference presentations and international collaborations are testimony to our ability to apply limited resources
to good effect.
The prices quoted are based on obtaining competitive quotes where possible, or using our industrial contacts to achieve the lowest
price. The total equipment cost requested is less than the list price of a Bohlin Parallel Plate Rheometer (£25,000) which is widely
used for biomechanical studies by other institutions. Our apparatus is in contrast more flexible, and adaptable to different research
requirements.
In addition to the research outcomes we will be able to generate final year and MSc projects from this programme; representing
further value for money for this EPSRC investment.
As our key research partners are UKE Hamburg two visits will be required to formulate the design specifications, and to carry out
trials of the new apparatus.
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