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Keywords:

  • Cholesteatoma;
  • bone resorption;
  • pH;
  • permeability;
  • filaggrin

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

Objectives/Hypothesis

The etiopathology of bone resorption in cholesteatoma is unclear. We studied pH in middle ear cholesteatoma tissue and the permeability of the cholesteatoma epithelium in an attempt to elucidate the mechanism of bone resorption in this disease.

Study Design

Laboratorial study.

Methods

Cholesteatoma tissue was collected from patients with primary acquired middle ear cholesteatoma. The pH of the keratin debris of cholesteatoma was measured using a pH meter. The cholesteatoma epithelium was examined under a confocal laser scanning microscope, and under a transmission electron microscope. Expression of filaggrin in the cholesteatoma tissue was explored by fluorescence immunohistochemistry and by quantitative reverse transcription-polymerase chain reaction.

Results

The pH of the keratin debris of cholesteatoma was acidic. The pH of the basal layer of the cholesteatoma epithelium was significantly lower than that of the antrum mucosa. Transmission electron microscope showed distinct penetration of lanthanum in the intercellular space of the basal, spinous, and granular layers of the cholesteatoma epithelium, but only a small amount of lanthanum in the granular layer in the normal skin. The expression of filaggrin mRNA was significantly lower in the cholesteatoma tissue than in the normal skin.

Conclusions

These results indicate that acid leakage through the cholesteatoma epithelium probably participates in the resorption of the underlying bone structure. The increased permeability of the cholesteatoma epithelium may be explained by a decrease in filaggrin expression.

Level of Evidence

N/A. Laryngoscope, 124:245–250, 2014


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

Cholesteatoma is an epidermal cyst that contains desquamated keratin of the epidermis. It usually arises from an invagination of a part of the tympanic membrane. A deep invagination is referred to as a retraction pocket, the wall of which is composed of a reversed keratinizing stratified squamous epithelium. As the retraction pocket extends medially into the middle ear cavity, desquamated keratin, the so-called “keratin debris,” accumulates inside the pocket, forming into a cholesteatoma. The lesion then erodes adjacent bone structures and further expands, leading to a vicious circle of the expansion of cholesteatoma.

Cholesteatoma in the middle ear cavity consequently causes not only hearing loss and otorrhea, but also vertigo, deafness, facial nerve palsy, sigmoid sinus thrombosis, and even intracranial complications such as meningitis and epidural/subdural/brain abscesses. Such a destructive nature is rarely seen in chronic otitis media without cholesteatoma. There is no effective medical treatment for middle ear cholesteatoma; therefore, most patients with this disease are forced to sustain surgical treatment.

Since the 1970s, a variety of cytokines, chemical mediators, and enzymes have been detected in cholesteatoma tissue, and these factors have been reported as possibly causative of bone resorption.[1-20] However, the etiopathology of this aggressiveness remains controversial. In the present study, we examined pH in middle ear cholesteatoma tissue and the permeability of the cholesteatoma epithelium in an attempt to elucidate the mechanism of bone resorption in this disease.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

Patients and Sample Collection

Samples were collected from patients who underwent tympanomastoidectomy or neck surgery in the Department of Otorhinolaryngology at the University of Occupational and Environmental Health. Cholesteatoma tissue was collected from patients with primary acquired middle-ear cholesteatoma. In addition, middle ear mucosa of the antrum was collected from patients with chronic otitis media without cholesteatoma. Normal skin of the neck was collected from patients with benign cervical tumors. Informed consent was obtained from all patients, and the study was approved by the institutional review board of the University of Occupational and Environmental Health.

Measurement of pH of Keratin Debris from Cholesteatoma

Samples of keratin debris from cholesteatoma were placed in polyethylene tubes and weighed. Fourfold weight of saline was added and homogenized, and the pH of the debris suspension was measured using a pH meter (ThermoOrion model 290A; Orion Research, Inc., Boston, MA).

Measurement of Cholesteatoma Epithelium pH

The pH of the basal layer of the cholesteatoma epithelium and that of the antrum mucosa were measured with a pH indicator-conjugated phospholipid, N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (fluorescein-DHPE; Invitrogen Corp., Carlsbad, CA), using an excitation ratio method. Samples were incubated with 25 μM fluorescein-DHPE in saline for 1 hour at room temperature, briefly washed with saline, and placed in a thin-bottomed Petri dish. Then, samples were examined under a confocal laser scanning microscope (LSM 5 PASCAL; Carl Zeiss Co., Heidelberg, Germany). The light source was an argon ion laser. The excitation wavelengths were 488 and 458 nm, and the emitted fluorescence was passed through a 505 to 530 nm band-pass filter. Images were digitally captured using the supplied software (LSM 5 software Version 4.0; Carl Zeiss Co., Heidelberg, Germany). The fluorescence intensity was displayed in a 4096-step arbitrary scale of 0 (no fluorescence) to 4095 (most intense fluorescence) in each pixel of the image. In order to determine an absolute pH value, the ratio of the fluorescence intensity at 488 nm excitation to that at 458 nm excitation (Ex ratio 488/458) was calculated. As a control, the antrum mucosa of patients with chronic otitis media without cholesteatoma was treated and examined in the same manner. A calibration curve was drawn by measuring the Ex ratios 488/458 of pH-controlled 100 mM phosphate buffers.

Transmission Electron Microscopy

In order to investigate the permeability of the cholesteatoma epithelium, transmission electron microscopy using lanthanum chloride as a tracer was performed, as described previously.[21] The collected cholesteatoma epithelium was immersed in a mixture of 2.5% glutaraldehyde and 2% lanthanum chloride in 0.1 M cacodylate buffer at 4°C for 2 hours. After being washed with 0.1 M cacodylate buffer at 4°C overnight, the sample was post-fixed with 1% osmium tetroxide in 0.1 M cacodylate buffer at 4°C for 2 hours, dehydrated through a graded series of acetone, and embedded in epoxy resin. Ultrathin sections were prepared using an ultramicrotome, stained with lead citrate, and then examined under a JEM 1200 EX electron microscope. As a control, normal skin of the neck was examined in the same way.

Fluorescence Immunohistochemistry

The cholesteatoma tissue and normal skin of the neck were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 (PB) at 4°C overnight. The fixed samples were transferred into 20% sucrose in 0.1 M phosphate buffered saline at pH 7.4 (PBS), and incubated at 4°C for two nights with three to four changes of the solution. The samples were then embedded while frozen in the Tissue-Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan) and stored at −80°C before sectioning. Seven-μm-thick sections were prepared using a cryostat, mounted on silane-coated glass slides (Superfrost; Matsunami Glass Industries, Osaka, Japan), and air-dried. The sections were hydrated in PBS with 0.3% Triton X-100 (PBST) for 20 minutes, and treated with 1.5% normal goat serum in PBST for 1 hour. They were then incubated with mouse anti-human filaggrin antibody (Abcam, Tokyo, Japan) diluted 1:200 in PBST containing 0.5% bovine serum albumin (BSA) at 4°C overnight. After a brief rinse with PBST, the sections were reacted with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen; Molecular Probes, Eugene, OR) diluted 1:1000 in PBST containing 0.5% BSA at room temperature for 2 hours. The sections were cover-slipped with Prolong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Invitrogen, Molecular Probes, Eugene, OR) and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The light source was an HBO 103 W/2 mercury vapor lamp. The light was passed through a 475 to 495 nm bandpass filter for the excitation of Alexa Fluor 488, or through a 340 to 380 nm bandpass filter for DAPI. The emitted fluorescence was allowed to pass through a 515 to 565 nm bandpass filter for Alexa Fluora 488, or through a 435 to 485 nm bandpass filter for DAPI. Images were captured using a Carl Zeiss AxioCam digital camera attached to the microscope. As a negative control, the primary antibody was omitted from the process.

Preparation of Total RNA

For quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the specimens were soaked in RNA stabilization reagent (Qiagen Inc., Valencia, CA) at 4°C overnight. Total RNA was extracted using an RNeasy Midi Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The purity of RNA was assessed by determining the ratio of light absorption at 260 to that at 280 nm (an A260/A280 ratio in the 1.9–2.1 range was considered acceptable). The RNA concentration was determined from A260.

Quantitative RT-PCR

The total RNA was reverse-transcribed to cDNA with a High-Capacity RNA-to-cDNA Kit (Applied Biosystems Inc., Foster City, CA), which uses random primers. The qRT-PCR analysis was performed with an Applied Biosystems StepOnePlus real-time PCR system using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems) for filaggrin mRNA and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as a housekeeping gene, according to the manufacturer's specifications. The TaqMan Gene Expression Assays for filaggrin (assay identification number: Hs00863478_g1) and GAPDH (assay identification number: Hs00951455_m1) were purchased from Applied Biosystems. One ng/μl of cDNA was mixed with TaqMan Universal PCR Master Mix with AmpErase (uracil N-glycosylase) and the primer/probe set of the TaqMan Gene Expression Assays, and the mixture was subjected to PCR amplification with real-time detection. The thermal cycler conditions were as follows: holding at 95°C for 2 minutes, followed by two-step PCR of 40 cycles of 95°C for 1 second, followed by 60°C for 20 seconds. Each sample was assayed in duplicate.

The measured threshold cycle (CT) was normalized by subtracting the CT for GAPDH of each sample from that for filaggrin. From the obtained ΔCT, the ratio of filaggrin mRNA to GAPDH mRNA was calculated as follows:

  • display math

Statistics

The data are expressed as means ± SEM. The statistical significance of differences was analyzed using the Mann-Whitney U test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

pH of Keratin Debris from Cholesteatoma

The pH of the keratin debris from cholesteatoma was measured in 55 patients and was determined to be 6.55 ± 0.07, ranging from 4.95 to 7.70. Of the 55 samples examined, 53 showed a pH of <7.40 and 46 showed a pH of <7.00.

pH of Cholesteatoma Epithelium and Antrum Mucosa

Figure 1 shows representative confocal laser scanning micrographs of the cholesteatoma epithelium and antrum mucosa. At 488 nm excitation, the basal layer of the cholesteatoma epithelium was uneven and moderate to weak fluorescence, whereas that of the antrum mucosa showed moderate and relatively uniform fluorescence. The pH of the basal layer of the cholesteatoma epithelium was significantly lower on average than that of the antrum mucosa (6.17 ± 0.14 vs. 6.67 ± 0.18, P = 0.027; Fig. 2).

image

Figure 1. Confocal laser scanning micrographs. Cholesteatoma epithelium (A) and antrum mucosa of chronic otitis media without cholesteatoma (B) were incubated with 25 μM fluorescein DHPE in saline for 1 hour at room temperature, and the samples were examined under a confocal laser scanning microscope. The images are optical sections vertical to the epithelial plane at 488 nm excitation. Note that the basal layer of the cholesteatoma epithelium exhibits uneven and moderate to weak fluorescence, whereas that of the antrum mucosa shows moderate and relatively uniform fluorescence. Scale bar = 50 μm.

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image

Figure 2. The pH of the basal layer of the cholesteatoma epithelium and antrum mucosa. The pH value was determined from the ratio of the fluorescence intensity at 488 nm excitation to that at 458 nm excitation (Ex ratio 488/458) on confocal laser scanning microscopic images.

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Permeability of Lanthanum Chloride Through Cholesteatoma Epithelium

Transmission electron microscopy using lanthanum chloride was performed to observe the cholesteatoma from six patients and normal skin from six patients. Figures 3A and B represent electron micrographs of the cholesteatoma epithelium and normal skin, respectively. Distinct penetration of the tracer was observed in the intercellular space of the basal, spinous, and granular layers of the cholesteatoma epithelium, whereas the normal skin showed only a small amount of the tracer in the granular layer.

image

Figure 3. Transmission electron micrographs. Cholesteatoma epithelium (A) and normal skin (B) were immersed in a mixture of 2.5% glutaraldehyde and 2% lanthanum chloride in 0.1 M cacodylate buffer at 4°C for 2 hours, post-fixed with 1% osmium tetroxide in 0.1 M cacodylate buffer, and embedded in epoxy resin. Ultrathin sections were stained with lead citrate, and then examined under a JEM 1200 EX electron microscope. The bottom of the micrographs is assigned to the basal side of the tissue. Arrows and Ns indicate lanthanum deposition and cell nuclei, respectively. Note that distinct deposition of the tracer is observed in the intercellular space of the basal, spinous, and granular layers of the cholesteatoma epithelium, whereas the normal skin shows only a small amount of the tracer in the granular layer. Scale bar = 1 μm.

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Expression of Filaggrin

Figure 4 shows photomicrographs of fluorescence immunohistochemical staining for filaggrin of the cholesteatoma tissue and normal skin. Immunoreactivity for filaggrin was observed in the horny layer in both tissues. The qRT-PCR results are presented in Figure 5. The expression of filaggrin mRNA was significantly lower in the cholesteatoma tissue than in the normal skin (P = 0.018).

image

Figure 4. Photomicrographs of fluorescence immunohistochemical staining for filaggrin. Figures show positive staining/negative control pairs of the cholesteatoma tissue (A/B) and normal skin (C/D). Green and blue colors express the fluorescence of Alexa Fluor 488 and DAPI, respectively. Note that immunoreactivity for filaggrin is observed in the horny layer in both tissues. Scale bar = 60 μm.

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image

Figure 5. Expression of filaggrin mRNA in the cholesteatoma tissue and normal skin. Extraction of total RNA and qRT-PCR were performed as described in Materials and Methods.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

In the present study, we demonstrated acidic pH of the keratin debris and cholesteatoma epithelium, and penetration of a low-molecular-weight tracer through the cholesteatoma epithelium. These observations suggest the possibility that acidic pH in the cholesteatoma content directly causes decalcification of the adjacent bone structures.

On the basis of clinical and experimental observations, bone resorption by cholesteatoma has conventionally been explained by pressure exerted on the bone.[22-26] Such a “pressure necrosis” theory is currently rejected by most researchers; various cytokines, chemical mediators, and enzymes have been presumed to be candidates that play pivotal roles in bone resorption in cholesteatoma. Interleukin-1α is among the cytokine candidates that has been most thoroughly investigated as a bone-resorbing factor in cholesteatoma.[5, 6, 10, 11, 14, 16] There are a number of other substances that have been detected in the cholesteatoma tissue in connection with bone-resorbing activity; namely, cytokines such as tumor necrosis factor−α,[14, 16] interleukin-6,[9] and macrophage-colony stimulating factor;[17] prostaglandin E2;[3, 5] parathyroid hormone-related protein;[4, 8] platelet-derived growth factor;[7] annexin;[13] glycosidases;[19, 20] and proteases such as collagenase,[1, 2] matrix metalloproteinase,[12] and cathepsin K.[15] More recently, the expression of receptor activator of nuclear factor κB and osteoprotegerin and their ligands have been demonstrated in the cholesteatoma tissue; possible roles for these factors in the stimulation and differentiation of osteoclasts have been suggested.[17, 18] Despite such extensive investigations, the pathogenesis of bone resorption in cholesteatoma is not yet fully understood.

The occurrence of chemical lysis of the bone in cholesteatoma was already suggested in the 1950s.[27] A later investigation by Kaneko et al.[28] revealed that bone destruction in cholesteatoma is seen at the site of the rupture of the cholesteatoma epithelium, together with the leakage of keratin debris, and they speculated about the possible relevance of acidic activity of the cholesteatoma content to bone resorption. Iino et al.[29] detected fatty acids and lactate in the keratin debris of cholesteatoma, and surmised that acidity generated by these organic acids in the cholesteatoma content may participate in bone resorption. Co-occurrence of bone destruction and epithelial rupture in cholesteatoma also has been described in a more recent report.[30]

The major inorganic component of the bone is hydroxyapatite, which is a hexagonal crystal form of calcium phosphate. Hydroxyapatite is highly insoluble in an aqueous solution at physiological pH with solubility of less than 10−5 mol/L (5 mg/L), but solubility drastically increases as the pH of the solution decreases to the acidic range.[31]

The horny layer of the epidermis is naturally kept under weakly acidic conditions by various exogenous and endogenous substances.[32] Such acidity plays an important role in maintaining an effective epidermal barrier that separates the external environment from the interior of the body. The present results demonstrated that the keratin debris of cholesteatoma was weakly acidic in most cases. Interestingly, the basal layer of the cholesteatoma epithelium exhibited uneven fluorescence of the pH-indicator (Fig. 1A). Such heterogeneity of pH distribution may be explained by spotty disruptions of the cholesteatoma epithelium, as has been suggested in previous reports.[28, 30] The pH of the basal layer of the cholesteatoma epithelium was significantly lower on average compared to that of the antrum mucosa, as shown in Figure 2. Furthermore, using lanthanum chloride as a tracer, we next revealed the breakdown of the barrier function of the cholesteatoma epithelium by an electron microscopic experiment. This finding is consistent with the macroscopic properties of this tissue; that is, it is often noticed during surgery that the cholesteatoma epithelium is much more fragile than the skin. These results indicate that acid leakage through the cholesteatoma epithelium probably participates in the resorption of the underlying bone structure.

Dental caries is another disease state caused by acid lysis. The outermost layer of the tooth, enamel, is the hardest mineralized tissue in the human body, but it softens at the pH <6.3.[33] The fluorine content of the tooth is one of the important factors responsible for acid resistance.[34] The bone contains less fluorine than enamel,[35, 36] indicating the former is more acid-sensitive than the latter. The present study demonstrated that the pH of the cholesteatoma epithelium was slightly above 6.0 (6.17 ± 0.14). These lines of evidence suggest again that acid lysis of the bone likely occurs on the boundary between the bone and cholesteatoma epithelium.

The present result also showed the pH of the basal layer of the antrum mucosa was weakly acidic (6.67 ± 0.18). Although relatively rare, mild bone erosion may occur in chronic otitis media without cholesteatoma, as reported previously.[37, 38] On the other hand, even more aggressive bone resorption is often seen in otitis media with cholesteatoma. Such a large difference in the degree of the destructive nature between the two diseases may be explained by the physicochemical property of hydroxyapatite; that is, critical pH value for the solubility of hydroxyapatite may be below 6.67.[39]

Filaggrin is one of the major constituent proteins in the horny layer of the epidermis. It binds to keratin, leading to keratin filament aggregation, which is essential for the barrier function of the skin.[40] Aberrant expression of filaggrin has been shown in skin diseases associated with impairment of the skin barrier, such as atopic dermatitis, eczema, and contact dermatitis.[41, 42] The present results clarified that filaggrin is expressed in the horny layer of the cholesteatoma epithelium, as well as in the normal skin, but that the mRNA expression of filaggrin is significantly lower in the cholesteatoma tissue than in the normal skin. This observation implies that a decrease in filaggrin may be responsible for the increased permeability of the cholesteatoma epithelium.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

We investigated pH in middle ear cholesteatoma tissue and the permeability of the cholesteatoma epithelium. The pH of the keratin debris and cholesteatoma epithelium was shown to be acidic. Breakdown of the epithelial barrier was also identified in the cholesteatoma tissue at the ultrastructural level. These results strongly suggest the role of acid lysis of the bone in cholesteatoma. The pathogenesis of the increased permeability of the cholesteatoma epithelium may be explained by a decrease in filaggrin expression. Further studies remain to be conducted to elucidate the roles of other epithelial barrier-related components such as tight junction proteins.

Acknowledgments

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY

We would like to thank Miss Satoe Nozoe for her technical assistance.

BIBLIOGRAPHY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgments
  9. BIBLIOGRAPHY
  • 1
    Abramson M, Huang CC. Localization of collagenase in human middle ear cholesteatoma. Laryngoscope 1977;87:771791.
  • 2
    Moriyama H, Honda Y, Huang CC, Abramson M. Bone resorption in cholesteatoma: epithelial-mesenchymal cell interaction and collagenase production. Laryngoscope 1987;97:854859.
  • 3
    Jung TT, Juhn SK. Prostaglandins in human cholesteatoma and granulation tissue. Am J Otol 1988;9:197200.
  • 4
    Cheshire IM, Blight A, Ratcliffe WA, Proofs DW, Heath DA. Production of parathyroid-hormone-related protein by cholesteatoma cells in culture. Lancet 1991;338:10411043.
  • 5
    Kurihara A, Toshima M, Yuasa R, Takasaka T. Bone destruction mechanisms in chronic otitis media with cholesteatoma: specific production by cholesteatoma tissue in culture of bone-resorbing activity attributable to interleukin-1α. Ann Otol Rhinol Laryngol 1991;100:989998.
  • 6
    Kakiuchi H, Kinoshita K, Katoh Y, Tabata T. Interleukin-1 of cholesteatomatous keratinocytes. Ann Otol Rhinol Laryngol Suppl 1992;157:3238.
  • 7
    Fujioka O, Huang CC. Platelet-derived growth factor in middle ear cholesteatoma. Eur Arch Otorhinolaryngol 1994;251:199204.
  • 8
    Cheshire IM, Blight A, Ratcliffe WA, Proops DW. In vitro production of parathyroid hormone-related protein by cholesteatoma and normal skin. Clin Otolaryngol Allied Sci 1995;20:448452.
  • 9
    Bujia J, Kim C, Ostos P, Kastenbauer E, Hultner L. Role of interleukin-6 in epithelial hyperproliferation and bone resorption in middle ear cholesteatomas. Eur Arch Otorhinolaryngol 1996;253:152157.
  • 10
    Bujia J, Kim C, Ostos P, Sudhoff H, Kastenbauer E, Hultner L. Interleukin 1 (IL-1) and IL-1-receptor antagonist (IL-1-RA) in middle ear cholesteatoma: an analysis of protein production and biological activity. Eur Arch Otorhinolaryngol 1996;253:252255.
  • 11
    Bujia J, Kim C, Boyle D, Hammer C, Firestein G, Kastenbauer E. Quantitative analysis of interleukin-1α gene expression in middle ear cholesteatoma. Laryngoscope 1996;106:217220.
  • 12
    Schonermark M, Mester B, Kempf HG, Blaser J, Tschesche H, Lenarz T. Expression of matrix-metalloproteinases and their inhibitors in human cholesteatomas. Acta Otolaryngol 1996;116:451456.
  • 13
    Kim TT, Chen CT, Huang CC. Expression of annexin II in human middle ear cholesteatoma. Otolaryngol Head Neck Surg 1998;118:324328.
  • 14
    Akimoto R, Pawankar R, Yagi T, Baba S. Acquired and congenital cholesteatoma: determination of tumor necrosis factor-a, intercellular adhesion molecule-1, interleukin-1α and lymphocyte functional antigen-1 in the inflammatory process. ORL J Otorhinolaryngol Relat Spec 2000;62:257265.
  • 15
    Hansen T, Unger RE, Gaumann A, et al. Expression of matrix-degrading cysteine proteinase cathepsin K in cholesteatoma. Mod Pathol 2001;14:12261231.
  • 16
    Yetiser S, Satar B, Aydin N. Expression of epidermal growth factor, tumor necrosis fastor-α, and interleukin-1α in chronic otitis media with or without cholesteatoma. Otol Neurotol 2002;23:647652.
  • 17
    Hamzei M, Ventriglia G, Hagnia M, et al. Osteoclast stimulating and differentiating factors in human cholesteatoma. Laryngoscope 2003;113:436442.
  • 18
    Jeong JH, Park CW, Tae K, et al. Expression of RANKL and OPG in middle ear cholesteatoma tissue. Laryngoscope 2006;116:11801184.
  • 19
    Olszewska E, Olszewski S, Borzym-Kluczyk M, Zwierz K. Role of N-acetyl-β-d-hexosaminidase in cholesteatoma tissue. Acta Biochim Pol 2007;54:365370.
  • 20
    Olszewska E, Borzym-Kluczyk M, Olszewski S, Zwierz K. Catabolism of glycoconjugates in chronic otitis media with cholesteatoma. J Investig Med 2007;55:248254.
  • 21
    Fujimura T, Suzuki H, Shimizu T, et al. Pathological alterations of strial capillaries in dominant white spotting W/Wv mice. Hear Res 2005;209:5359.
  • 22
    Ruedi L. Cholesteatosis of the attic. J Laryngol Otol 1958;72:593609.
  • 23
    Chole RA, McGinn, MD, Tinling SP. Pressure-induced bone resorption in the middle ear. Ann Otol Rhinol Laryngol 1985;94:165170.
  • 24
    Wolfman DE, Chole RA. Osteoclast stimulation by positive middle-ear air pressure. Arch Otolaryngol Head Neck Surg 1986;112:10371042.
  • 25
    Orisek BS, Chole RA. Pressure exerted by experimental cholesteatomas. Arch Otolaryngol Head Neck Surg 1987;113:386391.
  • 26
    Huang CC, Yi ZX, Yuan QG, Abramson M. A morphometric study of the effects of pressure on bone resorption in the middle ear of rats. Am J Otol 1990;11:3943.
  • 27
    Walsh TE, Covell WP, Ogura JH. The effect of cholesteatosis on bone. Ann Otol Rhinol Laryngol 1951;60:11001113.
  • 28
    Kaneko Y, Yuasa R, Ise I, et al. Bone destruction due to the rupture of a cholesteatoma sac: a pathogenesis of bone destruction in aural cholesteatoma. Laryngoscope 1980;90:18651870.
  • 29
    Iino Y, Hoshino E, Tomioka S, Takasaka T, Kaneko Y, Yuasa R. Organic acids and anaerobic microorganisms in the contents of the cholesteatoma sac. Ann Otol Rhinol Laryngol 1983;92:9196.
  • 30
    Suzuki C, Ohtani I. Bone destruction resulting from rupture of a cholesteatoma sac: temporal bone pathology. Otol Neurotol 2004;25:674677.
  • 31
    Chen ZF, Darvell BW, Leung VWH. Hydroxyapatite solubility in simple inorganic solutions. Arch Oral Biol 2004;49:359367.
  • 32
    Chan A, Mauro T. Acidification in the epidermis and the role of secretory phospholipases. Dermatoendocrinol 2011;3:8490.
  • 33
    Barbour ME, Parker DM, Allen GC, Jandt KD. Human enamel dissolution in citric acid as a function of pH in the range 2.30<pH <6.30: a nanoindentation study. Eur J Oral Sci 2003;111:258262.
  • 34
    Magalhaes AC, Wiegand A, Rios D, Buzalaf MA, Lussi A. Fluoride in dental erosion. Monogr Oral Sci 2011;22:158170.
  • 35
    Linck G, Petrovic A, Shambaugh GE Jr. Fluoride and calcium content of bone in otosclerotic patients. Arch Otolaryngol 1967;86:412418.
  • 36
    Torrisi L, Foti G, Campisano SU. Fluorine microanalysis in teeth. Clin Mater 1990;5:139145.
  • 37
    Wright CG, Meyerhoff WL. Pathology of otitis media. Ann Otol Rhinol Laryngol Suppl 1994;163:2426.
  • 38
    Gerami H, Naghavi E, Wahabi-Moghadam M, Forghanparast K, Akbar MH. Comparison of preoperative computerized tomography scan imaging of temporal bone with the intra-operative findings in patients undergoing mastoidectomy. Saudi Med J 2009;30:104108.
  • 39
    Barbour ME, Lussi A, Shellis RP. Screening and prediction of erosive potential. Caries Res 2011;45(suppl 1):2432.
  • 40
    O'Regan GM, Sandilands A, McLean WH, Irvine AD. Filaggrin in atopic dermatitis. J Allergy Clin Immunol 2009;124(suppl 2):R2R4.
  • 41
    Novak N, Baurecht H, Schafer T, et al. Loss-of-function mutations in the filaggrin gene and allergic contact sensitization to nickel. J Invest Dermatol 2008;128:14301435.
  • 42
    Kim BE, Leung DY. Epidermal barrier in atopic dermatitis. Allergy Asthma Immunol Res 2012;4:1216.