title: Laser-assisted cholesteatoma surgery: technical aspects, in vitro implementation and challenge of selective cell destruction.
abstract: Cholesteatoma is a destructive ear condition requiring complete surgical removal. One major problem lies in the frequent occurrence of residual cholesteatoma caused by squamous epithelium remaining in the middle ear. Our aim is to develop a laser treatment that is selectively directed against residual cholesteatoma cells and can be performed after cholesteatoma surgery in the same session. In a first trial, we studied the photodynamic effect of argon (AL) and diode lasers (DL) on cholesteatoma tissue. Intraoperatively harvested monolayer-cultured cholesteatoma cells were stained in vivo with different absorption enhancers: neutral red (NR), fluorescein diacetate (FDA), and indocyanine green (ICG). In vitro, staining tests on enhanced cellular dye absorption and laser tests were followed by cytotoxicity measurements to determine the respective amount of damage. To achieve selective cell destruction, antibody-mediated staining of cholesteatoma and middle ear mucosa cells was examined in a second trial. Cell cultures (cytospin and coverglass growing) and paraffin-embedded cholesteatoma tissue sections were studied immunohistochemically to determine the binding of monoclonal mouse antibodies against human cytokeratins CK5, CK10, CK14 and the epidermal growth factor receptor EGFR. Intracellular staining with absorption enhancers increased the optical density at the wavelength corresponding to the dye. Staining and subsequent laser irradiation destroyed up to 92% of cultured cholesteatoma cells. Unstained irradiated tissue was not affected. In cytospins, the antibody against CK5/6 showed strong staining of cholesteatoma and weak staining of mucosa cells. Reactivity for CK14 and EGFR was positive in both tissues. In coverglass cultures, staining of cholesteatoma cells was positive for CK5/6, CK14 and EGFR. Mucosa cells were positive for EGFR but negative for cytokeratins. Both cell types were negative for CK10. In embedded cholesteatoma tissue, CK5/6 and CK14 were localized in the basal layers of the matrix, while CK10 was situated in the suprabasal layers, and EGFR was present in all layers of the matrix and perimatrix. As for the technical aspects of laser-assisted cholesteatoma surgery, AL and DL have proved to be suitable devices; ICG and FDA are effective nontoxic absorption enhancers. The investigated antibodies against cytokeratins and EGFR show nonselective staining and thus appear to be inappropriate for avoiding unwanted cell damage. For safe and specific intraoperative application to intact tissue, the chromophore should be coupled to a particular antibody that binds solely to an easily accessible specific antigen at the surface of cholesteatoma cells.
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Charité, University Medicine Berlin, Campus Charité Mitte, Charitéplatz 1, 10117 Berlin, Germany. philipp.caffier@charite.de
authors.forename: Philipp P
authors.initials: PP
authors.lastname: Caffier
authors.forename: Ulrike
authors.initials: U
authors.lastname: Marzahn
authors.forename: Andrea
authors.initials: A
authors.lastname: Franke
authors.forename: Holger
authors.initials: H
authors.lastname: Sudhoff
authors.forename: Sergije
authors.initials: S
authors.lastname: Jovanovic
authors.forename: Andreas
authors.initials: A
authors.lastname: Haisch
authors.forename: Benedikt
authors.initials: B
authors.lastname: Sedlmaier
mesh_terms: D002478:Cells, Cultured; D018424:Cholesteatoma, Middle Ear / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D004396:Coloring Agents / Q000493:pharmacokinetics; D066246:ErbB Receptors / Q000378:metabolism; D005452:Fluoresceins / Q000493:pharmacokinetics; D006801:Humans; D007208:Indocyanine Green / Q000493:pharmacokinetics; D053547:Keratin-14 / Q000378:metabolism; D053555:Keratin-5 / Q000378:metabolism; D053553:Keratin-6 / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism / Q000473:pathology*; D053685:Laser Therapy / Q000379:methods*; D054018:Lasers, Excimer / Q000627:therapeutic use; D054023:Lasers, Semiconductor / Q000627:therapeutic use
chemical_list: D004396:Coloring Agents; D005452:Fluoresceins; D053547:Keratin-14; D053555:Keratin-5; D053553:Keratin-6; D066246:ErbB Receptors; D007208:Indocyanine Green; C018506:diacetylfluorescein