Eur Arch Otorhinolaryngol (2008) 265:1179–1188

DOI 10.1007/s00405-008-0602-3

OTOLOGY

Laser-assisted cholesteatoma surgery: technical aspects, in vitro implementation and challenge of selective cell destruction

Philipp P. CaYer · Ulrike Marzahn · Andrea Franke · Holger SudhoV · Sergije Jovanovic · Andreas Haisch · Benedikt Sedlmaier

Received: 16 November 2007 / Accepted: 24 January 2008 / Published online: 6 February 2008© Springer-Verlag 2008

Abstract Cholesteatoma is a destructive ear conditionrequiring complete surgical removal. One major problemlies in the frequent occurrence of residual cholesteatomacaused by squamous epithelium remaining in the middleear. Our aim is to develop a laser treatment that is selec-tively directed against residual cholesteatoma cells andcan be performed after cholesteatoma surgery in the samesession. In a Wrst trial, we studied the photodynamic eVectof argon (AL) and diode lasers (DL) on cholesteatoma tis-sue. Intraoperatively harvested monolayer-cultured cho-lesteatoma cells were stained in vivo with diVerentabsorption enhancers: neutral red (NR), Xuorescein diace-tate (FDA), and indocyanine green (ICG). In vitro, stain-ing tests on enhanced cellular dye absorption and lasertests were followed by cytotoxicity measurements todetermine the respective amount of damage. To achieveselective cell destruction, antibody-mediated staining of

cholesteatoma and middle ear mucosa cells was examinedin a second trial. Cell cultures (cytospin and coverglassgrowing) and paraYn-embedded cholesteatoma tissuesections were studied immunohistochemically to determinethe binding of monoclonal mouse antibodies againsthuman cytokeratins CK5, CK10, CK14 and the epidermalgrowth factor receptor EGFR. Intracellular staining withabsorption enhancers increased the optical density at thewavelength corresponding to the dye. Staining and subse-quent laser irradiation destroyed up to 92% of culturedcholesteatoma cells. Unstained irradiated tissue was notaVected. In cytospins, the antibody against CK5/6 showedstrong staining of cholesteatoma and weak staining ofmucosa cells. Reactivity for CK14 and EGFR was posi-tive in both tissues. In coverglass cultures, staining ofcholesteatoma cells was positive for CK5/6, CK14 andEGFR. Mucosa cells were positive for EGFR but negativefor cytokeratins. Both cell types were negative for CK10.In embedded cholesteatoma tissue, CK5/6 and CK14 werelocalized in the basal layers of the matrix, while CK10was situated in the suprabasal layers, and EGFR was pres-ent in all layers of the matrix and perimatrix. As for thetechnical aspects of laser-assisted cholesteatoma surgery,AL and DL have proved to be suitable devices; ICG andFDA are eVective nontoxic absorption enhancers. Theinvestigated antibodies against cytokeratins and EGFRshow nonselective staining and thus appear to be inappro-priate for avoiding unwanted cell damage. For safe andspeciWc intraoperative application to intact tissue, thechromophore should be coupled to a particular antibodythat binds solely to an easily accessible speciWc antigen atthe surface of cholesteatoma cells.

Keywords Cholesteatoma · Laser radiation · Cell culture · Photodynamic eVect · Antibody-mediated targeting

The authors conWrm the absence of Wnancial involvement in any organization with a direct commercial interest and the absence of any oV-label or investigational use.

P. P. CaYer (&) · B. SedlmaierDepartment of Otorhinolaryngology, Head and Neck Surgery, Charité, University Medicine Berlin, Campus Charité Mitte, Charitéplatz 1, 10117 Berlin, Germanye-mail: philipp.caYer@charite.de

U. Marzahn · A. Franke · S. Jovanovic · A. HaischDepartment of Otorhinolaryngology, Head and Neck Surgery, Charité, University Medicine Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany

H. SudhoVDepartment of Otorhinolaryngology, Head and Neck Surgery, Städtische Kliniken Bielefeld, Klinikum Mitte, Teutoburger Str. 50, 33604 Bielefeld, Germany

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Introduction

The development of light ampliWcation by stimulated emis-sion of radiation (LASER) for medical use in the early1970s opened up a new era of treatment for patients withotorhinolaryngologic (ORL) diseases. In many cases, thecorrect laser in the correct clinical situation oVers patientssigniWcant advantages over standard non-laser methods(e.g., rapid and bloodless operations, reduced anesthesiatime, less postoperative edema, less pain, and good sponta-neous epithelialization of defects). Owing to the develop-ment of many diVerent surgical laser systems, ideal lasersare now available for most applications in ORL surgery[13–15, 23]. However, cholesteatoma is a destructive earcondition requiring complete surgical removal. The patho-logic correlative is growth of squamous epithelium in anabnormal location such as the middle ear or petrous apex.Irrespective of the various traditional techniques in choles-teatoma surgery, one major problem lies in the frequentoccurrence of residual cholesteatoma caused by squamousepithelium remaining in the middle ear. Recurrence rates of30–60% have been described with more frequent relapsesin children than in adults [6, 17, 29]. Our aim was todevelop an adjuvant laser treatment that is selectivelydirected against cholesteatoma cells and can be performedafter cholesteatoma surgery in the same session to eliminateany residual squamous epithelium and thus reduce relapserate.

For safe and speciWc laser-assisted cholesteatoma sur-gery, the residual cells would have to be selectively stainedwith a special dye and irradiated with a suitable laser wave-length. For this purpose, the dye must be nontoxic andavailable for application in clinical practice; it should alsocorrespond to the laser wavelength in its main absorptionband. The argon laser (AL) and diode laser (DL) are com-mon and suitable medical laser devices owing to their radi-ation properties in biological tissues. They have anappropriate penetration depth (AL: 0.3–2 mm; DL: 1–3 mm) and couple to particular endogenous (e.g., hemoglo-bin) and synthetic chromophores. In our study, intravitalstaining was done with nontoxic dyes, and tests onenhanced cellular dye absorption and laser tests were thenfollowed by cytotoxicity measurements to evaluate theincreased damaging eVect of suitable wavelengths on thecultured cells. Results were optimal when only the stainedsquamous epithelia are devitalized by the potentiated lasereVect, and the unstained tissue is not aVected.

Thus, selective destruction is achieved by speciWcallystaining cholesteatoma cells with the aid of antibody-medi-ated targeting. Cytokeratins are intracellular intermediaryWlaments, which determine the cell type and diVerentiationstatus. Known to be speciWc for squamous epithelium, thecytokeratins CK5, CK10 and CK14 were investigated as

potentially useful markers [4, 19, 20, 24]. The epidermalgrowth factor receptor (EGFR) is important for prolifera-tion, diVerentiation, adhesion, migration und apoptosis ofepithelial cells. EGFR was chosen as an additional extracel-lular marker because of its (over-)expression in many epi-thelial tumors [5, 11, 27]. The selectivity of these cellulartargets was checked by comparison of cholesteatoma andmiddle ear mucosa cells. Immunohistochemistry (IHC) wasused to study the binding of antibodies against the chosenmarkers in cell cultures (cytospin and coverglass growing)and paraYn-embedded cholesteatoma tissue sections.While cytospins were done to detect general antibody-anti-gen binding, the coverglass cultures served to demonstratethe impact and binding of antibodies in living cells. Thecholesteatoma sections were examined to determine whichtissue layers contain the respective cytokeratins.

Materials and methods

Cell culture and tissue processing

In the Wrst trial, immortalized keratinocytes from theHaCaT cell line (Department of Dermatology, CharitéCBF, Berlin, Germany) were cultured in Dulbecco’s Modi-Wed Eagle Medium/ Ham’s solution F-12 medium (DMEM/Ham’s; 1:1; 1£) and served as the experimental model foroptimizing the staining methods and laser settings. Theoptimized conditions were tested in cholesteatoma cells ofprimary cultures from fresh material obtained during cho-lesteatoma surgery.

The respective tissue was minced and distributed on cellculture plates. After adding 1 ml of 0.25% trypsin solution,the plates were refrigerated overnight at 4°C. The matrixwas removed after the exposure period. The material wasleft to dry for 10 min at 37°C so that tissue componentsadhered to the plate bottom and cells grew out of the tissue.To prevent germ contamination, the cells were cultured inkeratinocyte serum-free medium (SFM; 1£·) with the addi-tion of 1% penicillin/streptomycin and 2.5 �g/ml of ampho-tericin B. The material was completely covered by Wllingthe plates with 10 ml of the medium. Cell growth tookplace under standardized incubation conditions (37°C; 5%CO2). Seven passages were possible in the initially pro-cessed cell line.

To perform staining and laser tests and record theabsorption spectra of the unstained cells, the plates weretrypsinized, and the cells were absorbed and counted in aNeubauer hemocytometer (microscope: CK40-SLP, Olym-pus, Japan). The cells were diluted with the medium toachieve the desired cell density of 2 £ 105 cells/ml forstaining tests and 4 £ 105 cells/ml for laser treatments. Ameasure of 100 �l of the cell suspension was pipetted into

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the test-speciWc wells of 96-well cell culture plates. After24 h incubation the cell density was microscopically moni-tored. The cells formed an adherent monolayer to the baseof the wells.

In the second trial, intraoperatively harvested cholestea-toma and mucosa tissue was used for speciWc antibody-mediated staining. IHC examinations of cytospins and cov-erglass cultures were carried out with cells from the sameculture in order to obtain comparable results. For cytospins,cells were passaged, counted, and diluted to a concentrationof 3 £ 105 cells/ml. Microscope slides, a Wlter and a funnelWlled with 100 �l cell suspension were mounted in a Cyto-spin 2 Centrifuge (Shandon Southern, USA). Cells werecentrifuged onto the slides for 5 min at 500 rpm. Until IHCstaining, culture cells were Wxed with cold acetone for10 min and stored at ¡20°C. This Wxation was assumed toincrease cell membrane permeability, which enabled bind-ing of extracellular antibodies to intracellular intermediaryWlaments. To avoid this Wxation eVect, cells of the cover-glass cultures were not Wxed. A coverslip with 0.5 ml oftype I collagen was placed in each well of a 24-well cellculture plate. After passaging and counting, cells weretransferred onto the collagen-coated coverslides and incu-bated with the corresponding antibody. Each coverglasswas then mounted on the microscope slide with Kaiser’sglycerol gelatin. For histological investigations, the biopticcholesteatoma tissue was Wxed in Somogyi’s solution at8°C for at least 24 h. After processing and paraYn-embed-ding, Harris hematoxylin/eosin-stained slides and Masson’strichrome-stained slides were prepared from the paraYn-embedded block.

Staining tests and dye absorption measurements

For (unspeciWc) staining, the neutral red (NR) and indocya-nine green (ICG) stains and the 3-(4.5-dimethylthiazole-2-yl)-2.5-diphenyltetrazolium bromide (MTT) used for thetoxicity test were dissolved in phosphate-buVered saline(PBS) solution without calcium or magnesium. The volumeof starting solutions was Wxed at 10 ml. The starting con-centration was 4 g/l for the NR dye, while ICG was used ina solution of 2, 4, or 5 g/l. The toxicity test was performedwith MTT in a concentration of 5 g/l. The Xuorescein diac-etate (FDA) dye was dissolved in 1 ml of acetone; the start-ing solution had a concentration of 10 g/l. To stain the cells,the medium was aspirated and the cells were washed with100 �l of PBS. One hundred Wfty microliters of the dyewere added to the cells in the respective concentrations.After a predeWned exposure period (90 s to 2 h), the dyewas aspirated, and the cells were washed three times with100 �l of PBS and resuspended in fresh medium. To deter-mine dye toxicity, at least six wells of the cell culture plateremained unstained and served as a negative control for

calculating the damage. Triple assessments were done toverify the results (Table 1). Before and after staining andlaser irradiation tests, the cells were photographed with adigital camera (Camedia C3030 zoom, Olympus, Japan)attached to an inverted microscope (CK 40-SLP).

After staining, the light absorption was photometricallymeasured, and the Xuorescence intensity of the absorbeddye was determined (Spectra Max 340 PC/ Spectra MaxGemini, Molecular Devices, USA). Absorption spectrawere detected over a wavelength range of 350–750 nm forNR-stained cells and 550–850 nm for ICG-stained cells.The Xuorescence intensity of Xuorescein, the Wssion prod-uct of FDA, was measured at an excitation wavelength of485 nm and an emission wavelength of 516 nm. Absorptionmeasurements of the dyes ICG and Xuorescein were per-formed in the medium. NR measurements were Wrst takenin the medium and later in PBS with calcium and magne-sium, since there was a strong intrinsic absorption of themedium of HaCaT cells at 550 nm. Control measurementsof the spectra of dyes had been carried out previously incell-free medium.

SpeciWc antibody-mediated staining was done using theavidin-biotin complex (ABC) method as the standard tech-nique for IHC staining of cells in cytospins, coverglass cul-tures and paraYn-embedded sections. Specimens wereincubated overnight at room temperature with unlabeledprimary monoclonal mouse antibodies against human CK5/6 (clone D5/16 B4; an isolated antibody against CK5 wasnot available), CK10 (clone DE-K10), CK14 (clone CBL197) and EGFR (clone 2–18C9). After washing with TrisbuVer (pH 7.2), biotinylated secondary antibody and subse-quently a complex of avidin-biotin peroxidase were appliedfor 10 min each. In the case of a positive result, the peroxi-dase was developed by the alkaline phosphatase substrateFast Red to obtain red staining. All cells without a reactionwere counterstained by Mayer’s hemalaun. DiVerent incu-bation periods were examined, since applying the antibodies

Table 1 Summary of concentrations and exposure times of dyes

NR neutral red, ICG indocyanine green, FDA Xuorescein diacetate

Dye Type of test Concentration (g/l) Exposure time

NR Staining test 0.0016–0.4 3 h

0.1/0.4 0.5, 1.5, 3 h

0.5–4 3 h

Laser test 0.0625–0.25 1.5 h

ICG Staining test 0.0625–2 0.5, 2 h

1.25/2.5/5 15, 30, 120 min

Laser test 0.125–0.5 2 h

FDA Staining test 0.01/0.02/0.04 1.5, 2 h

0.01/0.02/0.04 15, 30, 45, 60, 75, 90, 105, 120 min

Laser test 0.01/0.02/0.04 30 min, 2 h

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during surgery requires knowledge of their incubation timein the ear in situ. Furthermore, two diVerent media wereused as antibody diluents. ChemMate antibody dilutionbuVer (DAKO, Germany) contains the cytotoxic compo-nent azide, which would be unsuitable for local applicationin the middle ear. RPMI-1640 medium with sodium bicar-bonate and L-glutamine (Biochrom KG, Germany) wastherefore used as an alternative antibody diluent.

Laser tests and cytotoxicity measurements

Light is emitted by the AL (� = 499 nm) in the blue-greenrange of the visible electromagnetic spectrum and by theDL (� = 810 nm) in the near infrared area. The absorptionspectra of the applied stains were identical or very close tothe emission spectra of the lasers. Cells stained with NR(475–500 nm) and FDA (488 nm) were treated with the AL(DLS 5, Aesculap Meditec, Germany). A defocussingdevice on the handpiece was used to increase the diameterof the AL beam, which measured about 3 mm at a distanceof 1.1 cm from the irradiated object. The distance of 1.1 cmcorresponded to the rim height of the wells on the 96-wellcell culture plates. The DL (6020, ESC-Sharplan, Israel)was applied to cells stained with ICG (790–810 nm) viaWber (diameter 800 �m). The Wber was positioned in themiddle of the well at rim level so that the target beam Wlledthe entire Xoor of the well.

Preliminary experiments were done to optimize the dyeconcentration and laser parameters in stained HaCaT cells.Four rows of the culture test plate were stained with a con-centration of the dye, while the four remaining rows wereleft unstained. Stained and unstained cells were irradiatedwith rising laser powers and diVerent exposure times. ALtests were conducted in NR-stained cholesteatoma cellsafter incubation for 1.5 h at concentrations of 0.0625,0.125, and 0.25 g/l; they were performed in FDA-stainedcells after incubation for 30 min at concentrations of 0.01,0.02, and 0.04 g/l (AL power: 0.3–1.3 W; pulse time: 5 s;applied energy: 1.5–6.5 J). In DL trials, cultured cholestea-toma cells were stained for 2 h with 0.125, 0.25, and 0.5 g/l of ICG and lasered with increasing powers (DL power:2–20 W; pulse time: 2 s; applied energy: 4–40 J). Prior tothe tests, a power measurement was carried out for bothlasers (Type 200 Power Meter, Coherent, USA). A possi-ble temperature rise in the solutions was measured afterthe single laser irradiations with a NiCr–Ni thermal ele-ment (2ABAC 025 TM, Philips, Germany) on the base ofthe wells.

The staining tests on enhanced cellular dye absorptionand the laser tests were followed by a toxicity test to deter-mine the amount of damage caused by the dye and by thelaser treatment. The applied MTT test measures the mito-chondrial dehydrogenase activity, which transforms the

MTT into dark blue formazan. Absorption of formazan at awavelength of 595 nm shows a linear correlation to cellvitality. In damaged cells, there is no or less formation offormazan dye [18]. The degree of damage was determinedby comparing the treated cells with the untreated controlgroup. In the laser experiments with diVerent dye concen-trations, part of the plate was tested on the next day, sincethe eVects of laser treatment often became visible only afterthis time. For this purpose, another 100 �l of medium wasadded to that already present on the cells and incubatedwith 20 �l of MTT solution.

Results

Staining tests

HaCaT and cholesteatoma keratinocytes were stainedweakly by NR at low concentrations; the absorptionsrecorded were 0.163 at 0.25 g/l and 0.375 at 2 g/l after 3 h.The dye-induced cell damage ranged from 7 to 50% afterexposure to low concentrations (0.0125–0.5 g/l for 3 h).Higher NR concentrations increased the rate of damage,75% of cells being devitalized at 4 g/l. Cells incubated withNR for only 1 h showed lower cytotoxicity (Fig. 1, left).

FDA absorption measured by emission recordingrevealed strong Xuorescence intensity. High dye absorptionand Xuorescein formation already took place after 15–30 min, while the Xuorescence intensity dropped again withlonger exposure times. The cell damage in the MTT testreached a maximum value of 23% for FDA concentrationsbetween 0.01 and 0.04 g/l and did not increase with longerexposure.

ICG at concentrations of 2 g/l resulted in strong stainingwith absorptions of 0.25 after 30 min and 0.38 after 2 h.Increasing the dye concentration to 5 g/l caused onlyslightly stronger staining with absorption values of 0.35after 30 min and 0.42 after 2 h. Regarding cytotoxicity,ICG concentrations of up to 2 g/l induced a maximum celldamage rate of 28%. ICG staining at concentrations of0.25–1 g/l caused less cell damage after 2 h than after30 min of exposure, which was interpreted as a recoveryphenomenon of culture cells (Fig. 1, right).

Laser tests

AL irradiation of NR-stained cholesteatoma cells resultedin extinctions nearly identical to those associated withequally stained HaCaT cells. Staining alone had alreadydamaged 49–62% of the cells (Fig. 2, top). The post-ALMTT test showed an additive destruction of 15–28% incells stained with 0.25 and 0.125 g/l of NR. Cells stainedwith 0.0625 g/l of NR showed less pronounced primary

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damage by the dye but had similar laser-induced damagerates ranging up to 92%, which corresponded to an additivedamage of 43%.

Regarding AL treatment of FDA-stained cholesteatomakeratinocytes, the emissions recorded after staining werehigher than those of unstained HaCaT cells. The MTT testconducted immediately after the AL treatment showeddamage to 8% of stained cells at concentrations of 0.01 and0.04 g/l, while 20% were damaged by a concentration of0.02 g/l. Subsequent laser treatment caused no relevantadditive cell destruction. A second MTT test performed onthe next day (22 h after AL) showed stronger damage(Fig. 3, left). Hence, while the primary damage ranged from2 to 7%, laser irradiation caused 56% additive destructionto cells stained with 0.02 and 0.04 g/l of FDA. AL irradia-tion damaged 6–21% of cells stained with 0.01 g/l of FDA(Fig. 2, middle). Unstained cells were not aVected by ALtreatment (Fig. 3, right). The mean temperature rise in thesolutions after the single laser irradiations was 0.9°C(power 1.3 W; pulse duration 5 s).

As regards DL irradiation of ICG-stained cells, theMTT test revealed that staining alone had damaged 11–21% of cholesteatoma keratinocytes. Lasering causedadditive cell destruction of 13–25%. The unstained cellswere not damaged by DL treatment. The second MTT testconducted the next day (22 h after DL) showed lower dam-age rates with less than 5% primary ICG-induced celldestruction. In contrast to HaCaT cells, which were notfurther damaged by DL power exceeding 6 W (appliedenergy 12 J), cholesteatoma cells required a power of13 W (applied energy 26 J) to reach the highest damagerates. DL-induced additive cell destruction was 65% at0.5 g/l, 55% at 0.25 g/l, and 32% at 0.125 g/l of ICG(Fig. 2, bottom). Lasering damaged up to 8.2% of theunstained cells. The mean temperature rise in the solutionsafter the single laser irradiations on the base of the wellswas 3.6°C (power 13 W; pulse duration 5 s).

IHC examinations

The kind of antibody diluent used (antibody dilution buVervs. RPMI) did not play an essential role in the mode ofreactivity. Within one hour, staining of the cells progres-sively increased during incubation. A longer incubation ofantibodies did not lead to further alterations in stainingintensity.

For cytospins, Fig. 4 illustrates the IHC results of Wxedcholesteatoma and mucosa culture cells from one passage.The antibody against CK5/6 showed weak pink staining inmucosa cells, which was assessed as negative reactivity. Incontrast, strong pink staining was visible in cholesteatomacells. Both cell types showed negative reactivity for CK10but positive CK14 and EGFR staining. Altogether, the par-ticular antibodies used revealed no diVerences between thecell types except for the strength of staining.

IHC staining of unWxed cholesteatoma and mucosa cellsfrom the coverglass cultures yielded diVerent results for thebinding of antibodies in both tissues (Fig. 5). Mucosa cellsresponded positively with EGFR, while the reactivity withcytokeratins was so weak that it was evaluated as negative.In contrast, staining of cholesteatoma cells was positive forCK5/6, CK14 and EGFR. Again, both cell types were nega-tive for CK10. Existing cells were visualized by the phasecontrast microscope.

The sections of embedded cholesteatoma tissue revealedthe antibodies in diVerent layers. CK5/6 and CK14 werelocalized in the basal layers of the matrix, while CK10 wasfound in the suprabasal layers and EGFR in all layers of thematrix and perimatrix (Fig. 6).

Discussion

Various laser systems such as the AL (499 nm), KTP(532 nm), Nd:YAG (1,064 nm), Er:YAG (2,940 nm), and

Fig. 1 Time- and dye-concen-tration-dependent cytotoxicity measurements after staining cholesteatoma cells. Left MTT test after staining with NR. Right MTT test after staining with ICG

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CO2 laser (10,600 nm) have been used for diVerent indica-tions in middle-ear surgery [9, 12, 16, 21, 30]. Schindlerand Lanser [28] reported on their idea to apply the CO2 andKTP laser in selected patients with small cholesteatomasand to ensure the postoperative follow-up with a secondoperation and with the aid of high-resolution CT scanningof the petrous bone. Raslan et al. [26] tested the AL forvaporization of cholesteatoma fragments. They also stainedsquamous epithelium with several dyes and found that invitro staining with Janus green signiWcantly increased the

ablation of cholesteatoma tissue. In a comparative prospec-tive clinical investigation, Hamilton [10] showed that therelapse rate after cholesteatoma surgery was lower withthan without additional application of the KTP laser. Thiswas attributed to a more complete removal of microscopicfragments of residual squamous epithelium originating, forexample, from the auditory ossicles.

The hitherto published laser applications in cholestea-toma treatment are nonspeciWc. The laser irradiation mustbe directed at the target tissue and acts equally on choleste-atoma cells and adjacent structures. This type of applicationfails to eVectively cover nonexposable areas and unrecog-nizable very small fragments of residual epithelium. Anovel approach would be selective staining to enhanceabsorption of in situ squamous epithelial cells, which aredevitalized by extensive low-dose laser irradiation. Theapplication system should provide circular emission oflaser radiation in order to cover the entire operating areawith a single pass. Lasers suitable for this indication rangein wavelength from 400 to 1,000 nm. The laser radiationshould have a certain tissue penetration depth and couple toa particular chromophore. AL radiation (488 nm) has anabsorption peak within the range of the hemoglobin mole-cule. Thus, small vascular structures can be treated withthis wavelength. The DL emits a radiation of 810 and940 nm. Depending on the mode of application, thesewavelengths show a tissue penetration depth in the submil-limeter range.

Chromophores whose absorption spectra lie in the ALand DL wavelength range can be used as absorptionenhancers and enable selective laser therapy by accumulat-ing in particular target tissues. Suitable nontoxic dyes havealready been clinically tested and are used for various indi-cations. Experimental assays have shown enhanced absorp-tion of NR by proliferating cells. VanderWerf et al. [33]demonstrated in vitro a photodynamic eVect of this dye in asquamous-cell-carcinoma line after irradiation with theKTP laser. According to our measurements, the absorptionspectrum of NR in PBS lies between 475 and 500 nm witha broad peak. The present study used the AL (488 nm),since its wavelength is precisely within this range. Fluores-cein has long been intrathecally applied in vivo for safelocalization of CSF Wstulae. The excitation and emissionspectra are 488 and 516 nm; hence the AL is also suitablehere. ICG has been intravascularly applied for about30 years to determine liver function, plasma volume, andcardiac output [22] and is now also used for the photody-namic treatment of malignancies [1].

DiVerent chromophores have been used to speciWcallyenhance the laser eVect in the photodynamic therapy ofmalignant tumors [7, 25]. Particularly suitable are pro-cesses characterized by superWcial growth with a low depthof invasion on the skin and mucosae. Photosensitizers

Fig. 2 Dye-concentration- and laser-power-dependent cytotoxicitymeasurements after laser irradiation of stained cholesteatoma cells.Top MTT test directly after NR staining for 1.5 h and AL treatmentwith 0.3–1.3 W for 5 s (applied energy: 1.5–6.5 J). Middle MTT test22 h after FDA staining for 30 min and AL treatment with 0.3–1.3 Wfor 5 s (applied energy: 1.5–6.5 J). Bottom MTT test 22 h after ICGstaining for 2 h and DL treatment with 1–13.25 W for 2 s (applied en-ergy: 2–26.5 J)

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introduced into the cells act cytotoxically by forming freeoxygen radicals when irradiated with a laser wavelengthcorresponding to the absorption of the molecule. Celldestruction results after a certain latency. Teatini et al. [32]studied hematoporphyrin absorption in experimentallyinduced cholesteatomas and suggested that photodynamictreatment would also be feasible in this benign tumor.Another mechanism of intracellular absorption enhance-ment, i.e., the direct thermal eVect of laser irradiationthrough the presence of a suitable dye, was investigated byFlock and Jacques [8] with intravascular ICG and alexan-drite laser irradiation for vascular occlusion.

The present study used cholesteatoma keratinocytes as amodel for the laser dosimetric examinations. In the irradia-tion tests, unstained cells were never aVected regardless ofthe laser wavelength or laser parameters applied. NR

staining already caused primary damage to a large part ofthe cells (up to 75%). High concentrations were chosen,since preliminary experiments showed that a markedincrease in extinction was not measurable below a concen-tration of 0.25 g/l. The initial intention was to achievedirect photothermal cell damage. VanderWerf et al. [33]described a photodynamic eVect without dye-induced celldestruction below a concentration of 0.05 g/l. The additivedamage caused solely by the AL irradiation ranged from 30to 50%.

FDA resulted in negligible primary cell damage at theconcentrations applied. With this dye, the eVect of AL irra-diation was evident in the second vitality test after 24 h butnot in the Wrst one. The AL destroyed about 60% of thestained cells. A photodynamic eVect may be assumed here,whereas temperature measurements make a photothermal

Fig. 3 AL-induced cholesteatoma cell destruction (magniWed 1:400).Left Damage after staining with 0.04 g/l of FDA for 30 min and ALirradiation (power: 0.65 W; pulse time: 5 s; applied energy: 3.25 J).The cells had lost their oblong shape and had become contracted andstrongly rounded. Some cells were ruptured, but most still had an intact

cell membrane. Right No damage to unstained cells after AL treatment(power: 1.3 W; pulse time: 5 s; applied energy: 6.5 J). The cells had notbecome contracted or rounded but were conXuent and adhered to thesurface

Fig. 4 IHC staining of Wxed mucosa culture cells (a; upper row) and cholesteatoma culture cells (b; lower row) in cytospins by the ABC method and using the substrate Fast Red. Antibod-ies against (a1/b1): CK5/6; (a2/b2): CK10; (a3/b3): CK14; (a4/b4): EGFR; (dilution 1:200 in antibody dilution buVer, incuba-tion 1 h, magniWcation £400)

Fig. 5 IHC staining of unWxed mucosa culture cells (a; upper row) and cholesteatoma culture cells (b; lower row) in cover-glass cultures by the ABC meth-od and using the substrate Fast Red. Antibodies against (a1/b1): CK5/6; (a2/b2): CK10; (a3/b3): CK14; (a4/b4): EGFR; (dilution 1:200 in RPMI, incubation 1 h, magniWcation £400)

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eVect unlikely. ICG also showed only low dye-induced tox-icity, even at higher concentrations. Likewise, DL-inducedcell damage was recognizable after a latency of 24 h.Baumler et al. [3] identiWed free oxygen radicals as thecytotoxin developing after irradiation of ICG-stained cellsby inhibiting the eVect with radical quenchers. The MTTtest indicated that up to 65% of the exposed cholesteatomakeratinocytes were devitalized and that particularly periph-eral cells could escape the DL treatment. This was due tothe more inhomogeneous radiation proWle for lasering indefocus and to the irradiation Weld being somewhat smallerthan the area of cultured cells.

Regarding the antibody-mediated selective staining ofcholesteatoma cells, our IHC results conWrm the detectionof CK5/6 and CK14 as proliferation markers. In cytospins,the antibody against CK5/6 showed strong staining of cho-lesteatoma and weak staining of mucosa cells. Fixationmakes the cell membrane permeable for antibodies againstintracellular fragments. However, the weak staining inmucosa cells was unexpected and might be due to nonspec-iWcity of the applied antibody clone D5/16 B4. In cover-glass cultures without Wxation, the binding was weak incholesteatoma and negative in mucosa cells. CK14 stainingwas positive in Wxed cholesteatoma and mucosa cells ofcytospins. Again, the evidence in mucosa tissue is unclearbut also be found in other studies [4, 19]. Besides, theseWndings could be explained by a history of frequently

recurring middle ear infections, which might have alteredthe protein expression of mucosa cells. In contrast, CK14staining was positive for cholesteatoma and negative formucosa cells in unWxed coverglass cultures. The embeddedcholesteatoma tissue sections revealed CK5/6 and CK14 inthe basal layers of the matrix.

CK10 is a squamous epithelial marker that stains thesuprabasal layers of cholesteatoma tissue. However, theapplied clone DE-K10 did not detect the antigen in ourWxed or unWxed cholesteatoma cell cultures. Arriaga et al.[2] demonstrated growth inhibition of keratinocyte culturesby the addition of a monoclonal antibody against CK10 andpostulated a possible therapeutic relevance in the treatmentof residual squamous epithelium after cholesteatoma sur-gery. Our IHC results did not verify CK10 as a diVerentialmarker for epithelial cells in the dediVerentiated culturedcholesteatoma cells. As reported in literature, mucosa cellswere negative for CK10.

Another speciWc antigen may be the membrane-basedreceptor for the epidermal growth factor EGF. IHC investi-gations by SudhoV et al. [31] showed that this receptorstructure is detectable in cholesteatoma cells but not inadjacent middle-ear mucosa cells. In contrast, our resultsdemonstrated positive EGFR staining in both tissues. Whilethis evidence was expected in proliferating cholesteatomacells, a (weaker) detection was also possible in mucosacells. No conWrmation of EGFR evidence in growing and

Fig. 6 IHC staining of diVerent layers in paraYn-embedded choleste-atoma tissue by the ABC method and using the substrate Fast Red.Localization of antibodies against a CK5/6; b CK10; c CK14; d EGFR

(dilution 1:200 in antibody dilution buVer, incubation 24 h, magniWca-tion £400, Masson’s trichrome stain)

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proliferating mucosa cells was found in the literature. Inour cholesteatoma tissue sections, EGFR was localized inall layers of the matrix and perimatrix.

In summary, the investigated antibodies against theapplied cytokeratins and EGFR appear to be inappropriatefor selective staining. Furthermore, cytospins should not beused in future investigations. There are no intercellular con-nections between the Wxed single round cells. Presumably,the manipulation of cell characteristics makes them accessi-ble for antibodies against intracellular components andleads to false positive results. In coverglass cultures, how-ever, the cells are unWxed and intact, and they have contactto each other, which results in weaker staining. Moreover,we assume that the chromophore has to be coupled to a par-ticular antibody that binds exclusively to an easily accessi-ble speciWc antigen at the surface of cholesteatoma cells.

Finally, a critical assessment of our study leads to theimplication that future trials should investigate potentialproblems of clinical application to produce more reliableestimates of treatment eVects in vivo. Before this newmethod can be considered for intraoperative application inintact tissue, it must be evaluated by animal experiments toclarify important questions such as possible ototoxicity,details of intraoperative dye application (e.g., dose, stainingduration), eVective laser parameters and consequences ofpotential temperature rises, the healing process, and thelong-term outcome. Concerning safety, experience withdiVerent lasers in middle ear surgery of up to 25 years dura-tion in humans has not indicated a generally increased riskfor intraoperative inner ear trauma or threshold shifts. Sincelaser-assisted cholesteatoma surgery aims to irradiatesuperWcial tissue without contact, drill vibration, or bonecutting, it is promising to increase surgical accuracy withsimultaneous prevention of mechanic and acoustic trauma,as well as bleeding. However, as with all middle ear surgi-cal treatments, long-term vigilance in this regard is recom-mended. Finding a speciWcally binding dye providingcholesteatoma-selective treatment will be the priority sub-ject of upcoming work.

Conclusions

Our results suggest that laser-assisted cholesteatoma sur-gery is technically possible and promising to achieve alower rate of residual cholesteatoma than with the conven-tional methods. Applying absorption enhancers enablesstained cells to be damaged by laser energies that do notaVect unstained cells. However, staining is still nonspeciWc.The investigated antibodies against cytokeratins and EGFRappear to be inappropriate for avoiding unwanted celldamage. Problems include accessibility of the antigensand cross-reactions with mucosa cells. As intracellular

intermediate Wlaments, cytokeratins are reached only aftercell membrane passage. Thus a parietal macromoleculeseems to be more promising for speciWc antibody-mediatedtargeting of cholesteatoma cells.

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