Middle Ear Cholesteatoma Tissue Removal

Middle Ear Cholesteatoma Tissue Removal
Kristen Fetchko, Paige Hartsoe, Otto Juhl, Pranita Muralidhar
Department of Biomedical Engineering, University of Delaware, Newark, DE
Introduction: Cholesteatoma is a proliferating mass of keratinized squamous epithelial cells abnormally located
in the middle ear. Cholesteatoma creates pressure in the middle ear and erodes important structures. Dizziness,
facial muscle paralysis, and permanent hearing loss due to ossicle damage can occur. It is commonly resolved
with a mastoidectomy and tympanoplasty. Aesthetically, the cholesteatoma tissue is almost identical to healthy
tissue of the middle ear; this poses a severe limitation towards successful treatment since multiple invasive
surgeries are required per patient in order to remove the tumor in its entirety. Cholesteatoma removal is
performed in about 1 in every 10,000 children, necessitating better surgical planning and intra-operative guidance
techniques. Therefore, a combinatorial approach that both selectively fluoresces cholesteatoma tissue during
surgery and induces cell death by photoablation will significantly advance the otorhinolaryngology field.
Materials and Methods: Fixed, deidentified cholesteatoma samples from the Nemours Alfred I. DuPont Hospital
for Children were used for this study. To test for selective targeting, immunohistochemistry (IHC) was
performed. Primary antibodies used were anti-cytokeratin 10 (Abcam ab9026) and anti-EGFR (Abcam ab2430).
MDCK, MC3T3, and Neuro-2a cell lines to represent the middle ear mucosa, the ossicles and the mastoid bone,
and the facial nerve, respectively, were subjected to the same treatment and used as controls to ensure integrity of
healthy middle ear tissue. 150nm silica core gold shell nanoparticles were
functionalized with anti-EGFR using a 5000kDa PEG-SH linker. Modified
nanoparticles were characterized using dynamic light scattering (DLS) and
zeta potential. Cholesteatoma tissue was treated with the modified
nanoparticles for 1 hour, and washed. Tissue sections were subjected to 3
minutes of near IR laser exposure, and an infrared thermal imaging sensor
was used to measure the temperature of the tissue in order to test if induction
of cell death by photoablation is possible. As a control, a cholesteatoma
sample was treated with PEG-only coated nanoparticles, and subjected to the
same treatment otherwise. Two-photon microscopy was used to show
selective binding of the modified nanoshells to the tissue.
Figure 1: IHC results for selective
targeting of three tissue sections.
Results and Discussion: IHC results of n=3 tissue sections stained
Table 1: DLS and zeta potential results showing
for each antibody at ideal concentrations are shown in Figure 1.
an increase in diameter and positive charge.
Corresponding light images are shown for clarity of the tissue
outline. All cell control lines showed no fluorescence, as expected.
DLS and zeta potential results are shown in Table 1. A Leica 780
Confocal was used for two-photon microscopy, which can be seen in
Figure 2. Images were taken with transmitted light for clarity of
tissue edges. Cholesteatoma tissue is fluoresced red, while
nanoshells are fluoresced green. Temperature readings during near
IR laser exposure on modified-nanoparticle treated tissue yielded about a
PEG-only
Anti-EGFR test
15°F increase, while the control PEG-only group did not experience an
control
increase. Future studies will involve testing on more control cell lines
and live tissue, and suspending the nanoparticle treatment in a gel to
prevent passage into the inner ear.
Conclusions: We have shown that we can successfully conjugate gold
nanoparticles with antibodies and can selectively target the cholesteatoma
tissue. Also shown is the ability to ablate the tissue using an IR laser.
Figure 2: Two-photon microscopy results.
This research clearly demonstrates the ability to target cholesteatoma
Tissue is in red, nanoparticles are in green.
tissue and ablate it setting the groundwork for a new surgical procedure
that could more effectively eliminate the unwanted tissues in patients afflicted with this disease.