2.1

Patients and samples

The study was conducted on 2 patient groups with chronic otitis media. One group consisted of patients with cholesteatoma (n = 28) and the other group were patients without cholesteatoma (n = 24). The noncholesteatoma group consisted of patients with chronic otitis media with granulation (n = 8) and without granulation (n = 16). There were 15 men and 13 women in the cholesteatoma group. The noncholesteatoma group consisted of 4 men and 4 women (granulation), and 6 women and 10 men (without granulation). Patient ages ranged between 21 and 81 years (median age, 42 years). All patients clinically presented a certain level of conductive hearing loss. The specimens were obtained during middle ear surgery (canal wall down- or up procedures) performed on patients under general anesthesia in the Department of Otolaryngology, Medical University of Gdańsk. This study was approved by the Independent Bioethical Committee of the said university (Reg. No. NKEBN/42/2008). All patients had been informed about the study and the procedure before approving the obtaining of specimens. The information was verbal and written.

Immediately after resection (15–30 minutes), one part of the tissue was frozen and maintained at −80°C until Western blot analyses, whereas the other part was fixed in 4% formaldehyde solution and embedded in paraffin until histologic and immunohistochemical analyses.

2.2

Immunohistochemistry

For the immunohistochemical assay, 4- μ m-thick paraffin-embedded tissue sections were prepared and placed on the adhesive slides, covered with 10% solution of poly- l -lysin (Sigma P8920, Sigma‐Aldrich Sp z oo, Poznan, Poland). After deparaffinization and rehydration, the slides were placed in a 10 mmol/L citric acid buffer (pH 6.0) and incubated in a water bath at 99°C for 40 minutes to antigen retrieval. Endogenous peroxidase of each section was inhibited with 3% solution of hydrogen peroxidase for 10 minutes in room temperature. The incubation of the slides with primary antibodies was performed for 12 hours at 4°C and with a visualizing system for 30 minutes at room temperature. A secondary antibody and a streptavidin-biotin horseradish peroxidase kit (LSAB2/ HRP, Kit 0675, Dako, Carpinteria, CA, USA) were used for immunohistochemical staining (detection of bounded antibodies). Each section was immunoreacted with 5% solution of calf-fetal serum for 1 hour. The sections between sequential procedures were washed with phosphate buffered saline. The negative controls were specimens treated with nonimmunological serum. Sections were stained with an antibody to OPG (human OPG, goat polyclonal 1:25; AF805; R&D Systems, Inc) and an antibody to RANKL (human Trance, mouse monoclonal 1:50; clone 70525; R&D Systems, Inc, Minneapolis, USA) for 2 hours at room temperature. The staining reactions were then developed in 3,3′-diaminobenzidine tetrahydrochloride for 5 minutes. Specimens were dehydrated in alcohol and xylol, stained with Meyer’s hematoxylin, and embedded in Canadian balsam . The slides were examined under an Olympus BX51 light microscope. The positivity of the cells stained for OPG and RANKL was evaluated in the cholesteatoma epithelium and in subepithelial stromal tissue. The immunoreactivity was assessed semiquantitatively, and the expression rate, based on the quantity of immunopositive cells together with the intensity of the reaction, was counted for each case. The expression rate was graded as 0 for absent (<10% of cells positive), 1 for low (low positivity in >10%–25% positive cells with strong expression), 2 for intermediate (25%–50% positive cells with strong expression), and 3 for high (>50% strongly immunopositive cells). Two pathologists independently examined the stained tissues.

The intensity of the inflammatory process was examined histologically on hematoxylin and eosin–stained slides by counting the number of lymphocytes and macrophages per high-power field (HPF). The intensity of the inflammation was graded as 1 for low (scattered lymphocytes and macrophages, up to 10 per HPF), 2 for intermediate (>10 lymphocytes and macrophages per HPF and slight granulation tissue), and 3 for high (extensive active inflammation with exuberant granulation tissue).

2.3

Western blot analysis

The tissue of interest was homogenized in 3 volumes of lysis buffer (50 mmol/L Tris/HCl, pH 7.2; 5 mmol/L EDTA; 100 mmol/L NaCl; 1% Triton X-100; 10% glycerol; 10 mmol/L KH ₂ PO ₄ ; 1 mmol/L Pefabloc SC; 5 μ g/mL leupeptin) in a homogenizer with a power-driven Teflon pestle. An equivalent amount of protein from tissue extracts (50 μ g) was mixed with a loading buffer, boiled for 5 minutes, and subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis . The separated proteins were electrophoretically transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore Sp zoo, Warsaw, Poland). The membranes were blocked with 3% bovine serum albumin in Tris-buffered saline for 2 hours. Then the membranes were incubated overnight at 4°C with mouse anti-RANKL (2 μ g/mL) and goat anti-OPG (0.2 μ g/mL) (R&D Systems) antibodies. After being washed with Tris-buffered saline, membranes were incubated with appropriate alkaline phosphatase–conjugated secondary antibodies. Membrane-bound antibodies were visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. The developed bands were quantified by the use of the Gel Doc 2000 system (Bio-Rad Sp z oo, Warsaw, Poland), and relative amounts were compared using the Quantity One program (Bio-Rad). The p14-3-3 protein (a constitutively expressed protein) was used as an internal reference protein. The program calculated the area of the analyzed bands by summing the intensities of the pixels within the band boundary and multiplying by pixel area. The obtained value was adjusted for background intensity and expressed in arbitrary intensity units×square millimeters. The level of investigated protein was expressed as the intensity ratio of the OPG or RANKL band to the p14-3-3 protein band. To evaluate whether the values measured were in the linear portion of the detection curve, each protein extract was analyzed based on data obtained from at least 3 different dilutions. Protein concentrations were determined by the Lowry method with bovine serum albumin as standard .

2.4

Assessment of degree of bone destruction

A clinical analysis was performed to establish the 3 degrees of bone destruction as follows: (1) mild erosion of the scutum and ossicle; (2) moderate destruction of the tegmen and all ossicles; and (3) severe destruction of all ossicles, external ear canal, bony labyrinth, and Fallopian canal.

2.5

Statistical analysis

The statistical analysis was performed using the Statistica 8.0 PL statistical package (StatSoft Sp z oo, Krakow, Poland). Statistical significance between the 3 groups was determined using the one-way analysis of variance test. When the overall analysis of variance showed statistical significance, Tukey post hoc test (for unequal number of samples) was performed to explore the differences between any 2 groups. The Spearman’s rank correlation test was used to compare the RANKL and OPG expression level and to analyze the relation between the ratio of OPG/RANKL and clinical variables. P values less than .05 were considered as statistically significant.

2.1

Patients and samples

The study was conducted on 2 patient groups with chronic otitis media. One group consisted of patients with cholesteatoma (n = 28) and the other group were patients without cholesteatoma (n = 24). The noncholesteatoma group consisted of patients with chronic otitis media with granulation (n = 8) and without granulation (n = 16). There were 15 men and 13 women in the cholesteatoma group. The noncholesteatoma group consisted of 4 men and 4 women (granulation), and 6 women and 10 men (without granulation). Patient ages ranged between 21 and 81 years (median age, 42 years). All patients clinically presented a certain level of conductive hearing loss. The specimens were obtained during middle ear surgery (canal wall down- or up procedures) performed on patients under general anesthesia in the Department of Otolaryngology, Medical University of Gdańsk. This study was approved by the Independent Bioethical Committee of the said university (Reg. No. NKEBN/42/2008). All patients had been informed about the study and the procedure before approving the obtaining of specimens. The information was verbal and written.

Immediately after resection (15–30 minutes), one part of the tissue was frozen and maintained at −80°C until Western blot analyses, whereas the other part was fixed in 4% formaldehyde solution and embedded in paraffin until histologic and immunohistochemical analyses.

2.2

Immunohistochemistry

For the immunohistochemical assay, 4- μ m-thick paraffin-embedded tissue sections were prepared and placed on the adhesive slides, covered with 10% solution of poly- l -lysin (Sigma P8920, Sigma‐Aldrich Sp z oo, Poznan, Poland). After deparaffinization and rehydration, the slides were placed in a 10 mmol/L citric acid buffer (pH 6.0) and incubated in a water bath at 99°C for 40 minutes to antigen retrieval. Endogenous peroxidase of each section was inhibited with 3% solution of hydrogen peroxidase for 10 minutes in room temperature. The incubation of the slides with primary antibodies was performed for 12 hours at 4°C and with a visualizing system for 30 minutes at room temperature. A secondary antibody and a streptavidin-biotin horseradish peroxidase kit (LSAB2/ HRP, Kit 0675, Dako, Carpinteria, CA, USA) were used for immunohistochemical staining (detection of bounded antibodies). Each section was immunoreacted with 5% solution of calf-fetal serum for 1 hour. The sections between sequential procedures were washed with phosphate buffered saline. The negative controls were specimens treated with nonimmunological serum. Sections were stained with an antibody to OPG (human OPG, goat polyclonal 1:25; AF805; R&D Systems, Inc) and an antibody to RANKL (human Trance, mouse monoclonal 1:50; clone 70525; R&D Systems, Inc, Minneapolis, USA) for 2 hours at room temperature. The staining reactions were then developed in 3,3′-diaminobenzidine tetrahydrochloride for 5 minutes. Specimens were dehydrated in alcohol and xylol, stained with Meyer’s hematoxylin, and embedded in Canadian balsam . The slides were examined under an Olympus BX51 light microscope. The positivity of the cells stained for OPG and RANKL was evaluated in the cholesteatoma epithelium and in subepithelial stromal tissue. The immunoreactivity was assessed semiquantitatively, and the expression rate, based on the quantity of immunopositive cells together with the intensity of the reaction, was counted for each case. The expression rate was graded as 0 for absent (<10% of cells positive), 1 for low (low positivity in >10%–25% positive cells with strong expression), 2 for intermediate (25%–50% positive cells with strong expression), and 3 for high (>50% strongly immunopositive cells). Two pathologists independently examined the stained tissues.

The intensity of the inflammatory process was examined histologically on hematoxylin and eosin–stained slides by counting the number of lymphocytes and macrophages per high-power field (HPF). The intensity of the inflammation was graded as 1 for low (scattered lymphocytes and macrophages, up to 10 per HPF), 2 for intermediate (>10 lymphocytes and macrophages per HPF and slight granulation tissue), and 3 for high (extensive active inflammation with exuberant granulation tissue).

2.3

Western blot analysis

The tissue of interest was homogenized in 3 volumes of lysis buffer (50 mmol/L Tris/HCl, pH 7.2; 5 mmol/L EDTA; 100 mmol/L NaCl; 1% Triton X-100; 10% glycerol; 10 mmol/L KH ₂ PO ₄ ; 1 mmol/L Pefabloc SC; 5 μ g/mL leupeptin) in a homogenizer with a power-driven Teflon pestle. An equivalent amount of protein from tissue extracts (50 μ g) was mixed with a loading buffer, boiled for 5 minutes, and subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis . The separated proteins were electrophoretically transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore Sp zoo, Warsaw, Poland). The membranes were blocked with 3% bovine serum albumin in Tris-buffered saline for 2 hours. Then the membranes were incubated overnight at 4°C with mouse anti-RANKL (2 μ g/mL) and goat anti-OPG (0.2 μ g/mL) (R&D Systems) antibodies. After being washed with Tris-buffered saline, membranes were incubated with appropriate alkaline phosphatase–conjugated secondary antibodies. Membrane-bound antibodies were visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. The developed bands were quantified by the use of the Gel Doc 2000 system (Bio-Rad Sp z oo, Warsaw, Poland), and relative amounts were compared using the Quantity One program (Bio-Rad). The p14-3-3 protein (a constitutively expressed protein) was used as an internal reference protein. The program calculated the area of the analyzed bands by summing the intensities of the pixels within the band boundary and multiplying by pixel area. The obtained value was adjusted for background intensity and expressed in arbitrary intensity units×square millimeters. The level of investigated protein was expressed as the intensity ratio of the OPG or RANKL band to the p14-3-3 protein band. To evaluate whether the values measured were in the linear portion of the detection curve, each protein extract was analyzed based on data obtained from at least 3 different dilutions. Protein concentrations were determined by the Lowry method with bovine serum albumin as standard .

2.4

Assessment of degree of bone destruction

A clinical analysis was performed to establish the 3 degrees of bone destruction as follows: (1) mild erosion of the scutum and ossicle; (2) moderate destruction of the tegmen and all ossicles; and (3) severe destruction of all ossicles, external ear canal, bony labyrinth, and Fallopian canal.

2.5

Statistical analysis

The statistical analysis was performed using the Statistica 8.0 PL statistical package (StatSoft Sp z oo, Krakow, Poland). Statistical significance between the 3 groups was determined using the one-way analysis of variance test. When the overall analysis of variance showed statistical significance, Tukey post hoc test (for unequal number of samples) was performed to explore the differences between any 2 groups. The Spearman’s rank correlation test was used to compare the RANKL and OPG expression level and to analyze the relation between the ratio of OPG/RANKL and clinical variables. P values less than .05 were considered as statistically significant.