Abstract
Objective
The aim of our study was to investigate the relationship between the destruction of temporal bone structures, ossicular chain destruction, dissemination of cholesteatoma and the expressions of bone morphogenetic proteins (BMPs), BMP-2, BMP-4 and BMP-6 in patients with acquired cholesteatoma.
Material and Methods
This study was performed in a total of 80 patients with cholesteatoma and without cholesteatoma who had undergone surgery due to chronic otitis media. The patients were grouped as the study and the control groups. The study group comprised patients with primary acquired cholesteatoma, and the control group consisted of chronic otitis media patients without cholesteatoma. The samples were obtained from cholesteatoma tissue and the external acoustic meatus skin in study group patients and they were obtained from the external acoustic meatus skin only in control group patients. The Reverse Transcriptase Polymerase Chain Reaction method was used for the measurements of BMPs, BMP-2, BMP-4 and BMP-6 expressions. Polymerase Chain Reaction was studied by isolation of Ribonucleic Acid from the tissue samples.
Results
When the expressions of BMP in the external acoustic meatus skin were compared between the study and the control groups, the BMPs, BMP-2 and BMP-6 were determined to have a statistically significant relation in the study group (p < 0.05), but BMP-4 was not significant (p > 0.05). When the expression of BMP in cholesteatoma tissue was investigated in the study group patients, the BMPs, BMP-2 and BMP-6 were determined with statistically significant positivity (p < 0.05), but there was no significant positivity for BMP-4 (p > 0.05). In the study group, there was no statistical significance between the expressions of BMPs, BMP-2, BMP-4 and BMP-6 in cholesteatoma tissue, in the external acoustic meatus skin, and temporal and ossicular chain destruction, and dissemination of cholesteatoma (p > 0.05). A statistically significant positivity for BMPs expression in cholesteatoma tissue was determined in patients with destruction of the incus + malleus + stapes (p < 0.05).
Conclusion
The expressions of BMPs, BMP-2 and BMP-6, were elevated in cholesteatoma tissue. Furthermore, the positivity of BMPs expression was statistically significant in patients with destruction of all the ossicles, and we think that this marker can be used for evaluation of the aggressiveness of cholesteatoma.
1
Introduction
Despite the developing techniques and antibiotherapy, the complications of otitis media are still important at present because of its mortality. Since early diagnosis affects the prognosis significantly, it is important to be careful and remember the possibility of complications at all times .
Cholesteatoma may be defined as skin in the wrong place, which causes middle ear chronic inflammation, leading to ossicles and bone erosion . Cholesteatoma is a benign but destructive tumor of the middle ear characterized with progressive accumulation of hyperproliferative epithelia with keratin . It has been reported that intracranial and extracranial complications of chronic otitis media (COM) are more common in chronic otitis cases with cholesteatoma .
Bone morphogenetic proteins (BMP) were first described by Marshall Urist in 1960, who determined that these proteins increased the formation of ectopic bones, cartilage and connective tissue in rodents . Marianne et al. determined in a study that BMP-2 expression was increased in cholesteatomas.
In our study, we aimed to research the general expression of BMPs, BMP-2, BMP-4 and BMP-6 on cholesteatoma tissue in acquired cholesteatoma and their effect on aggressiveness, postoperative imaging of acquired cholesteatoma patients, the degree of their direct clinical dissemination perioperatively and the relationship between BMPs, BMP-2, BMP-4 and BMP-6 expressions and ossicles and bone destruction.
2
Material and methods
This study was conducted following the approval from the Local Ethic Committee of Fırat University School of Medicine, in the Ear, Nose and Throat Ward on 80 patients who had been operated for chronic otitis with or without cholesteatomas between November 2009 and September 2010.
Perioperatively, tissue samples from the cholesteatoma tissues and the external acoustic meatus (EAM) skin of chronic otitis patients with cholesteatoma for the study group and from the EAM skin for the control group were obtained and preserved at − 80 °C until the series were completed. As the series were completed, the samples were evaluated by the Medical Biology and Genetics Laboratory.
The patients were divided into two groups as the study and the control groups.
- 1.
Study group (n = 40): Primary acquired chronic otitis patients with cholesteatoma
- 2.
Control Group (n = 40): Chronic otitis patients without cholesteatoma
The staging of cholesteatoma dissemination was performed by Saleh and Millis classification on patients who had been operated for COM with cholesteatoma. The main aim of this classification was to enable the surgeon to correlate the type of operations performed and their outcome to disease stage, ossicular condition and the presence or absence of preoperative complications. Change in the level of transcript expression of target genes in order to assess whether the RT-PCR method was used. The measurements of BMP-2, BMP-4 and BMP-6 expression levels were also made using the Semi Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) method. RNA isolation was accomplished according to the manufacturer’s instructions (Invitrogen., Kibbutz Beit Haemek, Israel). RNA concentration was quantified by spectrophotometry at 260 nm. cDNA synthesis was performed using RevertAid Premium First Strand cDNA Synthesis Kit (Fermentas, USA). Six micrograms of RNA was used for reverse transcription in a final volume of 20 μL. PCR was performed with the stated primers. Semi-quantitative analysis was carried out employing GAPDH as the internal standard. Amplifications were carried out on an Eppendorf Master Gradient thermal cycler (Nethler-Hinz GmbH, Hamburg, Germany). The amplification conditions were 1 min at 94 °C for initial denaturation, followed by 40 s at 94 °C, 40 s of annealing at 56 °C, and 40 s of elongation at 72 °C. The amplification was achieved at 35 cycles for GAPDH and BMPs. The PCR mix was composed of a thermal concentration of 1.75 mm MgCl2, 0.1 mm deoxyribonucleoside triphosphates (each), and 100 pm primers (each) in final volume of 50 μL with 0.5 U of Sigma Taq DNA polymerase and 2 ng of cDNA. Amplification products were stored at 4 °C, and 15 μL reaction products were electrophoresed on 2% agarose gel ( Fig. 1 ). The gel image optical densities were analyzed using a computerized software program (LabWorks 4.0; UVP, Inc. Cambridge, UK).
The data collected from the patient follow-up forms were transferred to electronic media following the composition of the data basis and evaluated using the SPSS (Statistical Package for Social Sciences) for Windows 11.5 Program.
The significance level was accepted as 0.05 in the statistical analysis. In the presence of the two categories of dependent and independent variables according to the availability of distribution, the Pearson Chi-Square Test or the Fisher’s Exact Chi-Square Test was used, and in the presence of the two categories of dependent variables and five categories of independent variables, the Kolmogorov–Smirnov Z tests were used, since the distribution was not suitable for the chi-square test.
2
Material and methods
This study was conducted following the approval from the Local Ethic Committee of Fırat University School of Medicine, in the Ear, Nose and Throat Ward on 80 patients who had been operated for chronic otitis with or without cholesteatomas between November 2009 and September 2010.
Perioperatively, tissue samples from the cholesteatoma tissues and the external acoustic meatus (EAM) skin of chronic otitis patients with cholesteatoma for the study group and from the EAM skin for the control group were obtained and preserved at − 80 °C until the series were completed. As the series were completed, the samples were evaluated by the Medical Biology and Genetics Laboratory.
The patients were divided into two groups as the study and the control groups.
- 1.
Study group (n = 40): Primary acquired chronic otitis patients with cholesteatoma
- 2.
Control Group (n = 40): Chronic otitis patients without cholesteatoma
The staging of cholesteatoma dissemination was performed by Saleh and Millis classification on patients who had been operated for COM with cholesteatoma. The main aim of this classification was to enable the surgeon to correlate the type of operations performed and their outcome to disease stage, ossicular condition and the presence or absence of preoperative complications. Change in the level of transcript expression of target genes in order to assess whether the RT-PCR method was used. The measurements of BMP-2, BMP-4 and BMP-6 expression levels were also made using the Semi Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) method. RNA isolation was accomplished according to the manufacturer’s instructions (Invitrogen., Kibbutz Beit Haemek, Israel). RNA concentration was quantified by spectrophotometry at 260 nm. cDNA synthesis was performed using RevertAid Premium First Strand cDNA Synthesis Kit (Fermentas, USA). Six micrograms of RNA was used for reverse transcription in a final volume of 20 μL. PCR was performed with the stated primers. Semi-quantitative analysis was carried out employing GAPDH as the internal standard. Amplifications were carried out on an Eppendorf Master Gradient thermal cycler (Nethler-Hinz GmbH, Hamburg, Germany). The amplification conditions were 1 min at 94 °C for initial denaturation, followed by 40 s at 94 °C, 40 s of annealing at 56 °C, and 40 s of elongation at 72 °C. The amplification was achieved at 35 cycles for GAPDH and BMPs. The PCR mix was composed of a thermal concentration of 1.75 mm MgCl2, 0.1 mm deoxyribonucleoside triphosphates (each), and 100 pm primers (each) in final volume of 50 μL with 0.5 U of Sigma Taq DNA polymerase and 2 ng of cDNA. Amplification products were stored at 4 °C, and 15 μL reaction products were electrophoresed on 2% agarose gel ( Fig. 1 ). The gel image optical densities were analyzed using a computerized software program (LabWorks 4.0; UVP, Inc. Cambridge, UK).
The data collected from the patient follow-up forms were transferred to electronic media following the composition of the data basis and evaluated using the SPSS (Statistical Package for Social Sciences) for Windows 11.5 Program.
The significance level was accepted as 0.05 in the statistical analysis. In the presence of the two categories of dependent and independent variables according to the availability of distribution, the Pearson Chi-Square Test or the Fisher’s Exact Chi-Square Test was used, and in the presence of the two categories of dependent variables and five categories of independent variables, the Kolmogorov–Smirnov Z tests were used, since the distribution was not suitable for the chi-square test.
3
Results
Patients who had undergone previous operations, patients who had been diagnosed in a shorter time than two years, those with tympanic membrane ruptures, particularly those with secondary acquired cholesteatomas due to inversion of the epithelial tissue to the medial ear from marginal and attical perforations, and those with congenital cholesteatomas were excluded from the study.
42 patients were male (52.5%) and 38 (47.5%) were female. The ages of the patients ranged between 18 and 61 years (mean age: 33.46 ± 14.14 years).
For the types of operations performed by groups, in Group 1, two patients (5%) had undergone canal wall up mastoidectomy, 22 (55%) patients had undergone radical mastoidectomy, and fifteen (37.5%) patients had undergone modified radical mastoidectomy (Bondy procedure). In group 2, 38 (95%) patients had undergone canal wall up mastoidectomy and two patients (5%) had undergone Bondy procedure.
As the bone destructions were evaluated intraoperatively, in Group 1, four patients (10%) had destruction on the sigmoid sinus wall, five patients had destruction in the tegmen bone (tegmen antri and tegmen tympani), nine patients (22.5%) had destruction in the canalis facialis, and nine patients (22.5%) had destruction in the EAM wall. 13 (32.5%) patients had no destruction. In group 2, there was no destruction.
The ossicular chains of all the patients were assessed intraoperatively. In Group 1, destruction was observed in the ossicles of 30 patients (75%). Seven patients (17.5%) had destruction of the incus, five patients had destruction of the malleus and the incus, four patients (%10) had destruction of the incus and the stapes, and 14 patients (35%) had destruction of the malleus + incus + stapes. In Group 2, ossicle destruction was observed in 10 patients (25%). Two patients (10%) had incus, six patients (15%) had incus + stapes, and two patients (10%) had malleus + incus + stapes destructions.
On evaluation of the dissemination of the cholesteatoma, the patients were commonly in phase 2 (n = 12) (30%) when the disease was disseminated to another site and in phase 5 (n = 11) (27.5%) when the disease was disseminated to four and more sites beside the primary site.
When the expression of BMP in cholesteatoma tissue was investigated in the study group patients, BMPs, BMP-2 and BMP-6 were determined as statistically significantly positive (p < 0.05), but there was no significant positivity for BMP-4 (p > 0.05) ( Table 1 ).
