PART 2: DESCRIPTION OF THE PROPOSED RESEARCH AND ITS CONTEXT
2.1 Introduction
With the support of the EPSRC (GR/S85849/01, GR/S23346/01, EP/D025591/1) the DMU research team has been enabled to
collaborate with 4 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 [18] has indicated that it is possible to
stimulate the self-regeneration of scarred vocal fold tissue; and DMU are actively assisting the investigations into stem-cell therapy
being carried out at the Karolinska Institute. The DMU team have also been invited to assist in a 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.
Another more recent enquiry, and the cause of this application, is a request from Dr Dinesh Chhetri of the UCLA School of
Medicine, Division of Head and Neck Surgery. Dr Dhetri is part of an internationally recognised team in the field of vocal fold
pathology, which includes Professor Gerald Berke and Associate Professor Dave Berry. Dr Chhetri is investigating treatment for
vocal fold paralysis, involving the reinnervation of the vocalis muscle in cases of severe dysphonia [19,20,21]. Dr Chhetri has
requested our assistance for a study to quantify the change is vocal fold tissue tension resultant from nerve stimulation; to 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. Eric Goodyer of DMU
and Dave Berry of UCLA are both named consultants to the Wisconsin Tissue Engineering research programme. We propose to
carry out a short pilot study to determine if it is feasible to use either the Laryngeal Tensiometer (LT) of the Linear Skin Rheometer
(LR) to obtain in-vivo data from an anaesthetised dog. A further study will be carried out at a later date following analysis of the
data, and adjustments to the methods and equipment as required.
2.2 Characterisation of the Vocal Fold using Excised Tissue
This proposal should be viewed as 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 ever-published material [8,9] 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 2 shows iso-contour maps
obtained at Harvard, and the experimental set-up used by Wisconsin University Hospital to verify our methodologies.
Figure 2 Iso-Contour Map from Harvard (left)
Supporting Experimental Set-up from Wisconsin (right)
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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 3
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.
Vocal Fold Shear Modulus (shear model)
Vocal Fold Shear Modulus (indentometer model)
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Figure 3 Variation of Shear Modulus with Respect to Age
These results are virtually unique, 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.
2.3 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 [16]. 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 4). 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 [12].
The graph (figure 4) 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 measuremetns of the mucosal
wave velocity.
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Figure 4 The In-Vivo Laryngeal Tensiometer and Typical Data Trace
2.4 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 [8] and Karolinska [10] to quantify the effectiveness
of hyaluronic acid tissue augmentation, as well as completing a recent study with Karolinska that examined stem-cell therapy.
DeMontfort 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.
2.5 Aims and Objectives
Key Aim
To quantify the relationship between stimulation of the recurrent laryngeal nerve (RLN) and the resultant change is tension of the
vocal fold cover.
Secondary Aim
To quantify the change in vocal fold tension with respect to time
To achieve this our key objective is to adapt the vocal fold tissue measuring apparatus to enable it to be used for in-vivo studies in
a canine model.
2.6 Methodology
UCLA already have extensive experience of vocal fold research in a canine model. All experimental methods will be governed by
the ethical procedures required by UCLA, which will also be submitted for approval to DeMontofort University’s Ethical Approval
body.
Both the LT and the LSR devices could be of value for this project. 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 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 guaranteed of success of attachment
with the LT, and have a reasonable confidence of success with the LSR.
Both instruments can be attached using either a needle, or the methyl-cellulose based adhesive successfully used with humans.
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 transverse direction to the axis of the Vocal Process to the Anterior
Commisure. Use of an adhesive ensures that the direct stress is applied only the to epithelium, and careful control of the needle
penetration depth ensure that the direct stress is only applied to lamina propria and epithelium. Our previous published work
demonstrates that this is achievable, and that the stress mainly occurs in the vocal fold cover.
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
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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 is adequate for this study.
The recurrent laryngeal nerves on both sides of the neck will be exposed. The vocal fold muscle (thyroarytenoid muscle) will be
stimulated at various levels. The maximum adduction level will be measured using the tensionometer, and then current will be
increased by fifths, taking readings of tension at each setting. This procedure will be repeated for both vocal chords.
At least 5 reading 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 proven 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. On completion
of these tests, we will deploy a new technique that will measure the change in
2.6.1 Work Package 1 Apparatus Construction
The laryngeal measuring apparatus will be adapted based on our initial understanding of the experimental requirements, and
transported to UCLA.
2.6.2 Work Package 2 Data Acquisition (feasibility study)
An intitial visit and study will be carried out to determine the feasibility of the exiting experimental. Despite our confidence of
success there may be improvement that can be identified. This will be a two-way process, as this will be the first opportunity that
the UCLA team will have to deploy the laryngeal apparatus. This will allow the to modify the study protocls if required, and to
identify other studies that will be enabled due to access to DMUs expertise.
2.6.3 Work Package 3 Analysis
The outcome of work package will be a set of data that relates vocal fold cover tension to applied stimulation current. These
results will be analysed in the light of existing knowledge, and mathematical models either in the form of equations of graphs will
be prepared to describe our new findings.
The experimental setup will be reviewed, and recommendations for changes will be noted and acted upon.
Subject to the quality of the initial results a journal paper will be prepared for peer review. The feedback from the peer review
process will become part of the design review.
2.6.4 Work Package 4 Review and redesign of Experimental Procedures
The DMU team will revise the apparatus and methodologies as a result of a study of the results and feedback. The UCLA team will
review the experimental setup based on the initial results.
2.6.5 Work Package 5 Data Acquisition (main study)
Over a period of 1 year more data will be acquired using the revised methodologies derived from the design review.
As a minimum we will seek to obtain sufficient data to correlate vocal fold cover tension with stimulation current. As a target
each group of 5 readings will offer a Correlation Coefficient of better than 80%, which is considered to be a reasonable target for
an in-vivo study.
As well as modelling the relationship between stimulation and tension, we will also derive estimates for the change in tissue
modulus. It is known that that there is a relationship between the velocity of the mucosal wave (produced during phonation) and
tissue modulus and density. It should therefore be possible to relate the wave velocity back to the nerve stimulation current.
2.6.6 Work Package 4 Dissemination and Further Work
There is potential for dissemination by various means. To date DMU have successfully disseminated our work in 3 European
Journal, Folia Phoniatrica, The European Annals of OtoRhinoLaryngology and Acta OtoLaryngologica. The UCLA team are well
published in a range of US based journals ranging including The Laryngoscope. The results of this work will be presented on both
sides of the Atlantic as appropriate.
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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. UCLA will subsequently be able to present the full findings at one the major
Laryngological Conferences to be held in the USA in 2008.
It is our intention to continue with this partnership in the future. UCLA are currently preparing a grant application to support this
initial study and further work into tissue repair techniques. Liaison will be enhanced as both Dave Berry and Eric Goodyer and
named consultant to the Wisconsin University Hospital based 5 year programme into tissue engineering.
2.7 Relevance to Beneficiaries
Vocal fold paresis is a debilitating disease, resulting in partial or total loss of voice. Reinnervation 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. This study will enable us to gain a greater understanding of how the
therapy works, and will therefore result in an improved procedure in the operating theatre.
2.8 Justification of Resources
This proposal is a high cost effective research opportunity for the EPSRC,in view of the fact that much of the apparatus already
exists in the UK. An OTGS is considered most appropriate as UCLA are also well prepared for such studies, and all that is required
is the funding to enable us to collaborate. The study must take place in the US as UCLA will be responsible for securing the Ethical
approval for the research programme.
Similar OTGS awards have successfully resulted in quality research, IGRs and publications. We are confident of maintaining our
standard with this study.
Three return flights to Los Angeles are required. The first is for the initial evaluation study. The second is for a longer visit, to
carry out a more intensive study, and to train UCLA staff in the use of the apparatus. The final visit is close down the project and to
review the results. Costs given are the currently published rates for economy return flights from Heathrow to Los Angeles, plus the
costs of transfers at both ends and the cost of transporting the apparatus.
Total transport costs £4350
Overnight accommodation will be required for a total of 15 days. The first visit will be 3 nights and the subsequent 2 visits will be
for 6 nights each. Nearby basic chain hotels within walking distance have a typical rate of £75 per night, allowing for subsistence
and local taxes an allowance of £100 per night is realistic.
Total subsistence and hotel costs £1500
FEC costs. A total of 20 funded is requested for Eric Goodyer. This is to cover the time required for travel and work at UCLA, and
the time required to devise, prepare, test the apparatus and to analyse the results.
Total FEC costs £8640
Total funding requested £14490
2.9 References
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2.
3.
Chhetri, D.K., Mendelsohn, A.H., Blumin, J.H., Berke, G.S. Long-term follow-up results of selective laryngeal adductor
denervation-reinnervation surgery for adductor spasmodic dysphonia.. The Laryngoscope. 116 (4), pp. 635-642.
Chhetri, D.K., Berke, G.S.. Treatment of adductor spasmodic dysphonia with selective laryngeal adductor denervation and
reinnervation surgery. Otolaryngologic Clinics of North America 39 (1), pp. 101-109.
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.
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.
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.
Hirano S, Bless DM, Rousseau B, Welham N, Montequin D, Chan RW, Ford CN. Prevention of vocal fold scarring by topical
injection of hepatocyte growth factor in a rabbit model. Laryngoscope. 2004 Mar; 114(3): 548-56.
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4.
Rousseau B, Hirano S, Chan RW, Welham NV, Thibeault SL, Ford CN, Bless DM. Characterization of chronic vocal fold
scarring in a rabbit model. J Voice. 2004 Mar; 18(1): 116-24.
5. Hirano S, Bless DM, Massey RJ, Hartig GK, Ford CN. Morphological and functional changes of human vocal fold fibroblasts
with hepatocyte growth factor. Ann Otol Rhinol Laryngol. 2003 Dec; 112(12): 1026-33.
6. Perlmann AL, Titze IR, Cooper DS. (1984) Elasticity of Canine Vocal Fold Tissue. Journal of Speech & Hearing
Research. Vol 17 pp 212-219
7. Berke SB. (1992) Intraoperative Measurement of the Elastic Modulus of the Vocal Fold part 1 Device
Development. Laryngoscope 102, July 1992
8. Ishizaka K, Kaneko T. (1968) On Equivalent Mechanical Constants of the Vocal Fold. J Acoustical Society of
America, Vol 24, part 5 pp 312-313
9. Perlmann AL, Durham PL. (1987) In-Vitro Studies of Vocal Fold Mucosa During Isometric Conditions, in
Laryngeal Function in Phonation & Respiration. Thomas Bear (ed). College-Hill Publications
10. Chan RW, Titze IR. Viscoelastic Shear properties of Human Vocal Fold Mucosa. J. Acoustic Society of America.
1999;106, 2008-2021
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