10.14639/0392-100X-N1413
Objective. The damaging effect of cholesteatomas presents mainly as bone resorption by osteoclasts located in the space between the bone and perimatrix. This process is initiated by the molecular cascade of osteoclast differentiation factors. The aim of the study is to ana- lyse cholesteatoma microstructures via scanning electron microscope (SEM), and associate them with risk and grade of bone erosion.Methods. Pathological middle ear tissue fragments with cholesteatoma visible under a mi- croscope were collected from 58 patients operated on for chronic otitis media with features of bone defects in the middle ear walls. These fragments were examined under a scanning electron microscope.Results. Analysis of the cholesteatomas’ surface under a SEM revealed both regular and irregular structure of the matrix, most being the latter. Irregular matrix structures were ob- served in cases with a short disease history and in patients for whom this was the first surgical procedure. In our analysis, a cholesteatoma matrix with regular structures was associated with less bone destruction of the middle ear space.Conclusions.The microstructure of cholesteatomas that showed regular layers under SEM coincides with reduced destruction of the middle ear bone walls. An irregular structure (pathognomonic for a process with a short medical history, and in patients operated on for the first time) is characterised by a tendency towards deeper destruction of bone tissue.
text/xml
1827-675X
1413
en
Maciej Wiatr
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
bone defects
Pattern of cholesteatomas under a scanning electron microscope - a risk factor for bone resorption
Text.Serial.Journal
https://www.actaitalica.it/
41
Otology
2021-09-14
2021-01-23
2021-09-15
2021-09-15
371-376
1
Maciej Wiatr
2021/09/14
10.14639/0392-100X-N1413
371
Department of Otolaryngology, Jagiellonian University Medical College in Kraków, Poland
1827-675X
bone defects
376
Pattern of cholesteatomas under a scanning electron microscope - a risk factor for bone resorption
https://www.actaitalica.it/article/download/1413/657
41
ACTA OTORHINOLARYNGOLOGICA ITALICA 2021;41:371-376; doi: 10.14639/0392-100X-N1413




Otology


Pattern of cholesteatomas under a scanning electron
microscope - a risk factor for bone resorption
Modello di colesteatomi al microscopio elettronico a scansione - un fattore di rischio
per il riassorbimento osseo
Agnieszka Wiatr, Katarzyna Job, Maciej Wiatr
Department of Otolaryngology, Jagiellonian University Medical College in Kraków, Poland


SUMMARY
Objective. The damaging effect of cholesteatomas presents mainly as bone resorption by
osteoclasts located in the space between the bone and perimatrix. This process is initiated
by the molecular cascade of osteoclast differentiation factors. The aim of the study is to ana-
lyse cholesteatoma microstructures via scanning electron microscope (SEM), and associate
them with risk and grade of bone erosion.
Methods. Pathological middle ear tissue fragments with cholesteatoma visible under a mi-
croscope were collected from 58 patients operated on for chronic otitis media with features
of bone defects in the middle ear walls. These fragments were examined under a scanning
electron microscope.
Results. Analysis of the cholesteatomas’ surface under a SEM revealed both regular and
irregular structure of the matrix, most being the latter. Irregular matrix structures were ob-       Received: January 23, 2021
served in cases with a short disease history and in patients for whom this was the first             Accepted: February 27, 2021
surgical procedure. In our analysis, a cholesteatoma matrix with regular structures was
associated with less bone destruction of the middle ear space.                                       Correspondence
Conclusions.The microstructure of cholesteatomas that showed regular layers under SEM                Maciej Wiatr
coincides with reduced destruction of the middle ear bone walls. An irregular structure              Department of Otolaryngology, Head and Neck Sur-
(pathognomonic for a process with a short medical history, and in patients operated on for           gery of the Jagiellonian University, Jakubowskiego
the first time) is characterised by a tendency towards deeper destruction of bone tissue.            2 street, Cracow, Poland
                                                                                                     Tel./Fax +48 12 400 27 50
KEY WORDS: chronic cholesteatoma otitis media, microstructure of cholesteatoma,                      E-mail: maciej.wiatr@uj.edu.pl
scanning electron microscope, bone defects
                                                                                                     Funding
RIASSUNTO                                                                                            This research was supported by statutory funds
Obiettivo. L’effetto dannoso dei colesteatomi si presenta principalmente come riassorbimen-          of the Department of Otolaryngology of the
                                                                                                     Jagiellonian University, Krakow, Poland.
to osseo da parte degli osteoclasti situati nello spazio tra l’osso e la peri-matrice. Questo
processo è avviato dalla cascata molecolare dei fattori di differenziazione degli osteoclasti.
                                                                                                     Conflict of interest
Lo scopo dello studio è analizzare le microstrutture del colesteatoma tramite microscopio
                                                                                                     The Authors declare no conflict of interest.
elettronico a scansione (SEM) e associarle al rischio e al grado di erosione ossea.
Metodi. Sono stati raccolti frammenti patologici di tessuto dell’orecchio medio con cole-
steatoma visibile al microscopio da 58 pazienti operati per otite media cronica con difetti            How to cite this article: Wiatr A, Job K, Wiatr M.
ossei delle pareti dell’orecchio medio. Questi frammenti sono stati esaminati al microsco-             Pattern of cholesteatomas under a scanning elec-
pio elettronico a scansione.                                                                           tron microscope - a risk factor for bone resorp-
Risultati. L’analisi della superficie dei colesteatomi al microscopio elettronico a scansio-           tion. Acta Otorhinolaryngol Ital 2021;41:371-376.
                                                                                                       https://doi.org/10.14639/0392-100X-N1413
ne ha rivelato una struttura irregolare della matrice nella maggioranza dei casi. Essa in
particolare è stata osservata nei pazienti con una breve storia di malattia e nei pazienti
                                                                                                     © Società Italiana di Otorinolaringoiatria
sottoposti ad una prima procedura chirurgica. Nella nostra analisi, una matrice di coleste-          e Chirurgia Cervico-Facciale
atoma con strutture regolari è stata associata a una minore distruzione ossea dello spazio
dell’orecchio medio.                                                                                                OPEN ACCESS
Conclusioni. 1. La microstruttura dei colesteatomi che mostravano strati regolari al SEM coinci-
de con una ridotta distruzione delle pareti ossee dell’orecchio medio. 2. Una struttura irregolare   This is an open access article distributed in accordance with
                                                                                                     the CC-BY-NC-ND (Creative Commons Attribution-Non-
(patognomonica per un processo con una storia medica breve, e nei pazienti operati per la prima      Commercial-NoDerivatives 4.0 International) license. The
volta) è caratterizzata da una tendenza alla distruzione più profonda del tessuto osseo.             article can be used by giving appropriate credit and mentio-
                                                                                                     ning the license, but only for non-commercial purposes and
PAROLE CHIAVE: colesteatoma cronico otite media, microstruttura del colesteatoma,                    only in the original version. For further information: https://
microscopio elettronico a scansione, difetti ossei                                                   creativecommons.org/licenses/by-nc-nd/4.0/deed.en




                                                                                                                                                             371
A. Wiatr et al.




Introduction                                                    damage. The group included 27 women and 31 men. The
                                                                youngest patient was 27 years old, and the oldest was 70.
A joint opinion on the definition and classification of cho-
                                                                The Local Bioethics Committee issued approval no.
lesteatoma was announced at the 10th International Confer-
                                                                112.6120.206.2016 for this study on 27 June 2016. The
ence on Cholesteatoma and Ear Surgery in June 2016 in
                                                                procedures were performed from 2016 to 2019. All meth-
Edinburgh 1. Histologically, a cholesteatoma is made up of
                                                                ods were performed in accordance with relevant guidelines
keratinised stratified squamous epithelium and keratin, re-
                                                                and regulations. Informed consent was obtained from all
sulting from the process of desquamation of keratinocytes
                                                                subjects or their parents and/or legal guardian if subjects
without nuclei. These elements are parts of the cholestea-
                                                                were under 18 years old. Afterwards, the histopathological
toma matrix and together with stromal cells form the chole-
                                                                characteristics were observed.
steatoma mass. About 3-6 in 100,000 people are diagnosed
                                                                The extracted material was prepared by fixing it in 4%
with cholesteatoma, and can be classified as either acquired
                                                                formalin, and then dehydrated in a series of ethyl alco-
or congenital.
                                                                hols at increasing concentrations: 35%, 50%, 75%, 96%
The cholesteatoma perimatrix is made of connective tissue
                                                                and 99.9%. Once prepared, the samples were subjected to
(collagen and reticulin fibres) and inflammatory cells (neu-
trophils, histiocytes and lymphocytes). Operated patients       air-drying and subsequently attached onto slides using glue
who were diagnosed with chronic cholesteatomatous otitis        (Electrodag 915 Silver Paint by TAAB). The samples were
media tend to suffer from bone tissue erosion. The dam-         then sprayed with a thin layer of gold using a JEOL JEE-4x
aging effect of cholesteatoma presents mainly in the de-        vacuum evaporator. An analysis of the prepared samples
struction of the ossicular chain, caused by bone resorption     was performed with the use of the JEOL JSM35CF scan-
by osteoclasts located in the space between the bone and        ning microscope at the Laboratory of Scanning Micros-
the cholesteatoma perimatrix. The process of bone resorp-       copy.
tion in cholesteatoma is initiated by a molecular cascade of    SEM analysis took into account the following parameters
osteoclast differentiation factors, which include osteopro-     and data:
tegerin (OPG), the receptor activator of nuclear factor κB      1. Analysis of images at 30x, 100x, 200x, 1000x, 2000x
(RANK), located on osteoclasts and the receptor activator          magnification.
of nuclear factor κB ligand (RANKL), located on the su-            a. The structure of the cholesteatoma: matrix and peri-
perficial layer of osteoblasts. This system is the most im-            matrix (m/p).
portant cascade regulating bone erosion. The long process          b. Matrix structure (regular and irregular).
of the incus is usually the fastest to erode. Bone defects         c. Blood vessels present in cholesteatoma (matrix and
may occur within the prominence of the lateral semicircu-              perimatrix).
lar canal and the facial canal. The inflammatory process           d. Surgery I – first operation, II – subsequent operation.
may spread to the labyrinth, sigmoid sinus and into the cra-    The statistical analysis was performed using Statistica soft-
nial cavities 2-4.                                              ware. A Chi-square test with cross tabulation was used to
The purpose of the study is to analyse cholesteatoma mi-        verify non-parametric hypotheses. This test was used for
crostructures via scanning electron microscope (SEM) and        qualitative variables. The test was then confirmed by the
associate them with risk and degree of bone erosion.            Chi-square goodness of fit test. Statistically significant re-
The aim of the analysis is to assess the relationship between   sults were considered for p < 0.05 5.
the microstructure of cholesteatoma and degree of damage
to adjacent bone structures.                                    Results
The specific goal is to identify variants of the cholesteato-
ma characterised by a tendency towards aggressive osteoly-      Between 2016 and 2019, 440 operations were performed
sis of bone tissue and consequently, causing damage to the      on patients due to chronic otitis media. Cholesteatoma was
elements of the auditory ossicle chain as well as the bones     found in 130 cases. Destruction of the bone walls of the
of the middle ear, which opens the way for the development      middle ear was present in 58 patients with cholesteatoma,
of ear-related complications                                    which constitutes about 10% of patients operated on for
                                                                chronic otitis media. These patients were further analysed.
                                                                Analysis of the cholesteatoma specimens under SEM in all
Materials and methods                                           discussed cases showed structures consisting of a matrix
Pathological middle ear tissue fragments with cholesteato-      and perimatrix.
ma visible under SEM were collected from 58 patients op-        In 35% of cases (20 of 58 patients), the matrix structure
erated on due to chronic otitis media with middle ear wall      was regular, with separate layers. In the remaining 65% of



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                                                                                               Microstructure of cholesteatomas as a risk factor of bone damage




Table I. The differences between regular and chaotic matrix structure of cholesteatoma.
                                                                                               Regular                                Irregular
                                                                                             (20 patients)                          (38 patients)
 Facial nerve canal defect                                                                      18 (90%)                               22 (70%)
 Lateral semicircular canal defect                                                               1 (5%)                                 4 (16%)
 Base of middle cranial fossa defect                                                             1 (5%)                                12 (16%)
 Vascularisation of cholesteatoma                                                                  No                                      No
 Perimatrix evaluation                                                                    Very high cell density                 Very high cell density
 First surgery                                                                                  4 patients                            29 patients
 Reoperation                                                                                   16 patients                             9 patients



cases (38 of 58 patients), the structure was chaotic and ir-
regular.
In cases with a regular matrix structure, bone destruction
was visible under SEM at 100x-200x magnification and
greater. In cases of irregular matrix structure, the damage in
some patients was already visualised at 30x magnification.
Most cases of regular, layered structure in cholesteatomas
were found in patients who were reoperated on, with a long
disease duration. An irregular structure of cholesteatoma
was observed in 65% of patients, mostly operated on for
the first time due to chronic otitis media. In these patients,
the features of chronic otitis media were observed no later
than 2 years prior to surgical treatment, indicating a short
medical history.
The differences between patients with regular and irregular
cholesteatoma matrix structures are presented in Table I.                       Figure 1. Matrix, x2000. (Focal corneocyte desquamation in the matrix.
In all patients, damage to the elements of the ossicular                        Squamous keratinocytes of the matrix, showing unordered stacking pattern
chain was observed, most often involving 2 or 3 ossicles.                       and irregular microplicae on the surface. Workshop of Scanning Microscopy).
Cholesteatoma with regular, layered structures were char-
acterised by a significantly lower comorbidity of bone wall
defects in the middle ear (p < 0.05). In this group, facial
nerve fistulas were often observed.
In cases of irregularly structured cholesteatoma, defects in
the bone walls with the potential for ear-related complica-
tions were statistically significant (p < 0.05) compared to
cholesteatomas with regular structures. These complica-
tions can be both intratemporal (defects of the facial nerve
canal, or defects in the horizontal semicircular canal) and
intracranial (defect of the skull in the medial fossa).
A defect in the base of the middle cranial fossa was ob-
served in 12 patients. In 4 patients, a fistula on the horizon-
tal semicircular canal was also present. In these patients,
the most common form of bone defect was damage to the
facial nerve canal, found in over 60% of cases with an ir-
regular matrix structure.
The following results were obtained from the SEM assess-
ment of cholesteatoma (Figs. 1-4).
No vessels were found in any of the preparations analysed,                      Figure 2. Matrix, x1000. (Squamous keratinocytes of the matrix, regular mi-
regardless of the structure of the cholesteatoma. In each                       croplicae on the surface. Workshop of Scanning Microscopy).



                                                                                                                                                          373
A. Wiatr et al.




                                                                              ment, and are the basis for further analyses aimed at devel-
                                                                              oping an effective method for combating this disease. The
                                                                              analysis carried out in the presented work complements
                                                                              numerous publications on this topic.
                                                                              We conducted a SEM analysis of the surface of the chole-
                                                                              steatomas, which revealed both regular, and in most cases,
                                                                              irregular matrix structures. An irregular matrix structure
                                                                              was observed in cases with a short disease history and
                                                                              in patients who underwent their first surgical procedure.
                                                                              Observations of this type of structure are in line with the
                                                                              research by Miodoński et al. 6. Similar to previous find-
                                                                              ings, this suggests that the proliferation of keratinocytes
                                                                              in cholesteatomas is much less coordinated than in normal
                                                                              epidermis, where the pattern is regular. Such surface fea-
                                                                              tures suggest abnormal keratinisation and a predominance
Figure 3. Perimatrix/Matrix, x6000. (Single settlements of bacteria on the    of cells in the formation of cholesteatoma.
border perimatrix/matrix, erythrocyte. Workshop of Scanning Microscopy,       The regular arrangement of layers in cholesteatoma was
magnification x 6000 to improve analysis in this case).
                                                                              observed using SEM during the investigation by Youngs
                                                                              and Rowels 7. Based on our observations, this type of ma-
                                                                              trix system was present in patients with many years of
                                                                              medical history, who underwent multiple surgeries due to
                                                                              recurrence of cholesteatoma. In our analysis, the regular
                                                                              structure of the cholesteatoma matrix was associated with
                                                                              less bone destruction of the middle ear space. This implies
                                                                              that a regular matrix structure and its associated tight in-
                                                                              tercellular connections between keratinocytes reduces mo-
                                                                              lecular permeability, while limiting the destructive activity
                                                                              of the cholesteatoma on the bone structures, compared to a
                                                                              chaotic and irregular matrix structure. It is worth consider-
                                                                              ing whether or not the regularity of the matrix structure of
                                                                              the cholesteatoma contributes to its overall permeability to
                                                                              agents involved in the complex mechanism of bone resorp-


Figure 4. Matrix x100. (Squamous keratinocytes of the matrix, irregular mi-
croplicae on the surface. Workshop of Scanning Microscopy).


case, the perimatrix was characterised by a very high cell
density. Parallel histopathological examination confirmed
the diagnosis of acquired cholesteatoma wherever relevant.
Microscopic analysis of defects and degree of damage to
the surface of the ossicular chain revealed changes in the
form of bone loss, fissured cracks and irregular surfaces
(Fig. 5).

Discussion
Advanced histological and immunohistochemical studies
have allowed for more detailed discoveries regarding the                      Figure 5. Incus, x30, (Defect of the ossicle’s surface). Workshop of Scan-
complex process of cholesteatoma expansion and develop-                       ning Microscopy).



374
                                                                            Microstructure of cholesteatomas as a risk factor of bone damage




tion in cholesteatoma. This could be similar to how healthy      which is the main bone resorption factor, often excluding
skin tissue acts as a barrier to disease, but irregular, less    their use as reconstruction materials 15. In our observation,
healthy skin is more permeable to foreign agents. Moreo-         the process of destruction of the ossicular chain elements
ver, it is worth considering whether or not the lower ex-        was at varying degrees of advancement, and in most cases
pression of transient receptor potential channels (TRPVs),       prevented the use of these elements for the reconstruction
observed in cholesteatomas in the studies by Ba Hung Do          stage.
et al. is important in the formation of a regular or irregular   The conducted study indicated the microstructure of cho-
structure 8. Studies conducted by Koizumi et al. 9 compared      lesteatoma as a prognostic factor of the destruction of bone
the increased permeability of the cholesteatomas’ epithe-        elements. On the one hand, this can lead to hearing im-
lium with skin tissue. The difference in the permeability of     pairment, but on the other it can also open the way for the
molecules between cholesteatoma epithelium and normal            development of complications of chronic otitis media. The
skin may be a clue to understanding the mechanism of bone        most aggressive course of cholesteatoma with the develop-
resorption in cholesteatoma by acid lysis. Answers to this       ment of ear-related complications was correlated with the
question require further research. However, it seems likely      presence of an irregular, chaotic structure combined with
that a better understanding of this mechanism may lead to
                                                                 a short medical history. The above observation is very im-
the development of a more effective treatment for chole-
                                                                 portant in clinical practice because it justifies the earliest
steatoma.
                                                                 possible initiation of surgical treatment after diagnosis of
Herein, particular attention was paid to the occurrence and
                                                                 chronic cholesteatoma otitis media.
degree of erosion of bone tissue due to the destructive effect
                                                                 The long-term course of cholesteatoma was reflected in the
of cholesteatoma. Based on the literature, destruction of the
                                                                 evolution of its microstructure observed under SEM to a
middle ear bone wall in the skull base was observed in ap-
proximately 5-10% of surgical operations, and intracranial       regular pattern in the majority of patients, with less aggres-
complications were found in 1% of patients operated on           siveness towards bone tissue. The main limitation is the ne-
as a result of diseases of the middle ear 10. The destruction    cessity to prepare the collected material in vitro according
of bone elements leads to damage to the ossicular chain,         to the SEM sample evaluation protocol, which limits the
and consequently to frequently worse outcomes in terms           simultaneous evaluation of biochemical pro-inflammatory
of hearing improvement. The occurrence of bone structure         factors responsible for the bone destruction process in the
defects within the temporal bone is an introduction to the       preparations analysed.
development of intratemporal and intracranial compli-
cations. According to the literature, the frequency of the       Conclusions
defect in the semicircular canal found during surgery for
chronic cholesteatoma in otitis media varies between 2.7%        The microstructure of cholesteatoma assessed using SEM
and 12.5%, and in most cases affects the lateral semicir-        in the form of regular layers coincides with reduced de-
cular canal. Grewal et al. showed this in 96% of cases and       struction of the middle ear bone walls. An irregular matrix
Faramarzi et al. in 95.8% of all fistulas described, which       structure (pathognomonic for a process with a short medi-
is consistent with our observations 11-13. It should be noted    cal history, patients operated on for the first time) is char-
that the analysis and statistical comparison of the results      acterised by a tendency for deeper destruction of the bone
of bone defects of the middle ear structures in the present      tissue.
work was difficult due to the small number of patients, and
is only an additional aspect analysed in the context of the      References
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