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3970Z

Investigating the Ultrastructure of the Tympanic Membrane in Rats

April 10th 2013

Gord Locke

Supervisors: Dr. Hanif Ladak & Dr. Jian Liu

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Introduction

The ear is a highly complex organ used to detect sounds and detect the

internal balance of the body. The ear can be subdivide into three different parts: the

inner ear, the middle ear and the outer ear. The outer ear is responsible for

gathering the sound waves and focusing them on the middle ear. The middle ear is

responsible for transmitting and magnifying the sound waves and the inner ear for

converting them into neurological signals. A disruption in any of these areas can

lead to hearing loss but the most frequent area of disruption is in the middle ear. 3

out of 4 children will experience a middle ear infection by the time they are 3 years

old [1]. These infections can become chronic and damage the middle ear.

The tympanic membrane, which is commonly referred to as the eardrum, is a

key component in the middle ear. Things such as trauma, disease and loud noises

can affect the integrity of the tympanic membrane, which can lead to temporary or

permanent hearing loss. If the membrane becomes ruptured, the membrane will

heal the wound but the underlying structure of collagen fibers may remain damaged

[2]. Using scanning electron microscopy, the structure of the collagen fibers in the

tympanic membrane can be observed which will allow for better artificial eardrums

to be produced.

The purpose of this research was to examine the arrangement of fibers in rat

tympanic membrane, measure the diameter and subunit frequency of individual

fibers and look at the how the fibers differ in various areas of the tympanic

membrane.

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Theory

The Middle Ear

In both rat and human ears, the middle ear is comprised of the tympanic

membrane, three small bones called the ossicles and several muscles. The middle

ear primary function is to act as an impedance matcher for sound waves [3]. If the

middle ear did not act as an impedance matcher, then most of the energy carried in

sound wave would be reflected and lost. The middle ear by acting as an impedance

matcher prevents this energy from being reflected and increases the sensitivity of

the ear greatly.

The middle ear also takes advantage mechanical properties when

transmitting sound. The natural position of the ossicles is arranged in a lever

system. As shown in figure 1, as the tympanic membrane is shifted from the sound

waves, the stapes is moved with far greater magnitude, which allows for the

vibrations to be magnified. The surface area of the tympanic membrane is far larger

than that of the oval window as shown in figure 2, which the sound waves are

transmitted through. The energy of the sound wave is distributed across the large

area of the tympanic membrane. Through the principle of conservation of energy,

the same amount of energy is applied on the oval window, which has a far smaller

surface area. This causes the sound waves to be magnified and improves the

performance of the ear.

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Figure 1: This figure is of the ossicles showing how the size and orientation of the

bones make the ear an effective lever. The lever arm attached to the membrane, Lm

is longer than the lever arm attached to the inner ear, Li. As a result of Lm having a

larger arm than Li, Li will have a greater displacement relative to the displacement

of Lm.

Figure 2: figure is of the ossicles showing how the surface areas of the tympanic

membrane, Ad and the oval window, Af relate to each other. Pressure is defined as

Force per area and as Pressure is conserved the force on the oval window is higher

relative to the force on the tympanic membrane. This is a way that the middle ear

amplifies the sound waves.

Figure 1 Figure 2

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The Tympanic Membrane

The tympanic membrane is not a uniform and flat surface. There are several

distinct regions of the membrane that contribute to the properties of the eardrum.

The pars tensa is the main structural component of the membrane. The manubrium

is the attachment site of the malleus; one of the ossicles. The pars flaccida is a

smaller and more compliant part of the membrane. These areas are shown in figure

3.

The tympanic membrane is composed of four different layers as shown in

figure 4; an epidermal layer, a mucosal layer and two layers that contain collagen

fibers. One of these layers contains collagen distributed in a radial manner,

extending from the center of the eardrum to the outer diameter. The other layer

contains fibers that are organized in a circumferential manner [4]. The organization

of these fibers is shown in figure 5.

Figure 3: This figure is a diagram of a human tympanic membrane

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These layers of collagen fibers are the primary structures, which determine

the shape, and the structure. It is thought that the radial collagen fibers are

primarily responsible for the conduction of sound across the membrane and the

circumferential fibers responsible for providing structural strength to the

membrane.

Scanning Electron Microscopy

Scanning electron microscopy or SEM is an imaging technique that allows for

both biological and non-biological objects to be imaged with a very high

magnification. With this magnification SEM is able to resolve points that are less

than 1 nanometer apart. In order for biological materials to be imaged, they need to

be coated with a heavy metal. This is necessary because the surface of the object to

be imaged needs to be electrically conductive in order to interact with the electrons

being focused on them. Biological samples must also contain no liquids as SEM

requires a vacuum to function, if liquid remained in the specimen it would

evaporate and disturbed the imaging.

Figure 4: The layers of the tympanic membrane

Figure 5: The organization of collagen fibers in the tympanic membrane

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Methods

The tympanic membranes were obtained through surgically removal from

healthy deceased rats and then incubated in a 1 % tripsin in phosphate buffer

solution for 24 hours to remove the epidermal layer and the mucosal layer from the

membrane. The membranes were then rinsed with a phosphate buffer solution and

then fixed in 2.5% glutaraldehyde solution. The membranes were then fixed in

ascending concentration series of ethanol and critical point dried with liquid carbon

dioxide. The membranes were then coated with 3nm of osmium and imaged with a

scanning electron microscope.

The images were then exported into ImageJ, an open source image-

processing tool. Using imageJ, the contrast of the fibers was adjusted using

histogram equalization. The diameter and subunit frequency of the fibers were then

measured using imageJ.

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Results

Figure 6: This figure is an overhead view of a rat tympanic membrane. a. The manubrium of the eardrum b. a tear in the eardrum the occurred during the surgical removal from the rat. c. The pars tensa of the eardrum d. The pars flaccida of the eardrum.

Figure 6

a

b

c

d

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Figure 7: This figure is an image of collagen fibers located in the pars tensa. The

Figure 7

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lines on the figure indicate the trend of radial fibers having aligned orientation.

Figure 8

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Figure 8: This figure is an image of radial fibers in the pars tensa of the tympanic membrane. The arrow in the figure is showing

Figure 9

Figure 9: This figure shows collagen with axial periodicity exhibited in the bands. This image was taken at the upper manubrium of the ear. As these bands are not exhibited in the radial fibers of the pars tensa.

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Figure 10

Figure 10: This figure shows collagen fibers taken at the manubrium. There are distinct bundles of collagen fibers that share common orientation but overall the fibers are randomly oriented compared to the fibers located in the pars tensa.

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Figure 12: This figure is a histogram of the measured diameters of collagen fibers in the pars tensa. The mean diameter of the fibers was 36.1nm with a standard deviation of 3.6nm from a sample n=30

Figure 9

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Figure 13: This figure is a histogram of the measured subunit periodicity of collagen fibers in the manubrium. The mean subunit periodicity of the fibers was 52.2 nm with a standard deviation of 5.7 nm from a sample n=26

Figure 10

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Discussion

While the structure of the tympanic membrane has been looked at

previously, the advances made in the imaging field allow for higher resolutions

views of the tympanic membrane and allow for individual fibers to be measured.

The mean diameter of the collagen fibers was measured and the histogram of the

collected data exhibited a low standard deviation and a very clear peak Images of

collagen that were taken at manubrium exhibited an axial banding periodicity that

was not seen in radial collagen fibers in the pars tensa. The presence of this banding

pattern indicated that these fibers are a different type of collagen than the collagen

fibers in the pars tensa.

The orientations of the fibers provide some insight into their function in the

membrane. While the radial fibers are highly organized and exhibit uniform

direction, which is consistent with the results found in literature [5], other collagen

structures were found that had not been reported in the literature. Collagen fibers at

the manubrium were organized in comparatively small bundles and did not exhibit

the collective orientation unlike the radial fibers. As the manubrium is the

attachment site for the malleus, it is likely that these fibers primary purpose is to

attach and secure the tympanic membrane and the malleus and that the random

orientation provides better attachment.

Incorporating these orientations and sizes into computer models of the

tympanic membrane are one avenue in which this research hold potential. Finite

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element models of the eardrum have existed for several decades but the models are

constantly being refined to become more accurate. Due to the anisotropy of the

tympanic membrane, it responds unequally in different areas of the membrane to

different frequencies. Human speech has a spectrum from 20 to 20,000 Hz [6] so it

is important to have an accurate model that responds accurately to different

frequencies. Geometry is the primary factor that determines how a finite element

model responds and by incorporating the structure of the collagen fibers into the

model design, the model can provide improved accuracy. The degree of attachment

of the manubrium to the malleus is an aspect of finite element modeling that has a

significant impact of the results of the model [7,8].

This research also has applications in improving existing medical technology.

Currently, a ruptured eardrum is left to heal without medical intervention. However,

there are some ruptures that are too large for the body to heal on its own or chronic

infection prevents the healing of the eardrum. Currently, eardrum grafts primary

goal are to close the hole in the eardrum. They accomplish this by providing

scaffolding for the endothelial and mucosal layers of the tympanic membrane to

grow over. Eventually this graft will disintegrate and the rupture will be sealed but

the collagen fibers will not be replaced. This has been shown to affect the detection

of high frequency sound waves [9]. The loss of these high frequencies can affect an

individual’s ability to distinguish phonetics sounds both in person and over

telecommunications. By learning more about the structure and arrangement of the

collagen fibers, grafts which properly mimic the structure of the collagen fibers can

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be developed and prevent the hearing loss that would otherwise be an acceptable

loss in the healing process.

When measuring the diameter and subunit periodicity sample sizes were

chosen based on previously published literature. The numbers of samples could be

increased to reduce potential error, especially with respect to the axial periodicity,

which as shown with the histogram which exhibited a skew, which could be due to

the number of fibers measured.

Conclusion

Using scanning electron microscopy, collagen fibers in the tympanic

membrane were imaged. The diameters of the collagen fibers and the axial

periodicity of the collagen fibers along the manubrium were measured. Qualitative

data about the orientation of the fibers was acquired and analyzed. Radial fibers

exhibited common uniform orientation and fibers found on the manubrium were

disorganized and composed of a different type of collagen.

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References

1. National Institute on Deafness and Other Communication Disorders. Ear infections in children. http://www.nidcd.nih.gov/health/hearing/Pages/Default.aspx. Updated 2013. Accessed 04/01, 2013.

2. L. Feenstra FEK. The concept of an artificial tympanic membrane. Clinical Otolaryngol. 1984;9:215.

3. Hemilä S, Nummela S, Reuter T. What middle ear parameters tell about impedance matching and high frequency hearing. Hearing Research. 1995;85(1-2):33.

4. W. Funnell CL. A critical review of experimental observations on eardrum structure and function. ORL. 1982;44(4):181.

5. N. Bonzon, X. Carrat, C. Deminière, G. Daculsi, F. Lefebvre, M. Rabaud. New artificial connective matrix made of fibrin monomers, elastin peptides and type I + III collagens: Structural study, biocompatibility and use as tympanic membranes in rabbit. Biomaterials. 1995;16(11):881.

6. B.Masterton HH, and R.Ravizza. The evolution of human hearing. Journal of the Acoustical Society of America. 1969;45(4):966.

7. Ladak HM FW. Finite-element modeling of the normal and surgically repaired cat middle ear. J. Acoust. Soc. Am. 1996;100(2):933.

8. Funnell WRJ, Khanna SM, Decraemer WF. On the degree of rigidity of the manubrium in a finite-element model of the cat eardrum. J. Acoust. Soc. Am. 1992;91(4):2082.

9. G. Volandri, F. Di Puccio, P. Forte, C. Carmignani. Biomechanics of the tympanic membrane. Journal of Biomechanics. 2010;44(1219).