10.14639/0392-100X-N2389
Objective. The involvement of the inner ear in otosclerosis may lead to the development of cochlear otosclerosis. The aim of this study was to analyse changes in the chemical composition and microstructure of the stapes in the course of otosclerosis compared to healthy stapes. Materials and methods. This analysis included 31 patients with otosclerosis and 9 patients without otosclerosis. Microanalytical and diffraction techniques were used to assess the elemental distribution and orientation topography of the stapes. Results. The concentration of Ca2+ in the study group was significantly lower in the area of the anterior crus of the stapes than in the posterior crus. A reduction in the Ca2+/P3+ ratio in the anterior crus was associated with deteriorated bone conduction and tinnitus. Degradation of the stapes microstructure in the area of otosclerotic lesions was observed with scanning electron microscopy. Conclusions. Bone remodelling is most significant at the closest location to typical otosclerotic lesions with hydroxyapatite porosity and scale-like bone formation according to scanning electron microscopy. There is a relationship between the disturbance of calcium metabolism and the development of clinical symptoms of cochlear otosclerosis.
text/xml
1827-675X
2389
en
Agnieszka Wiatr
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
stapes microstructure
Effect of stapes demineralisation on the development of cochlear otosclerosis
Text.Serial.Journal
https://www.actaitalica.it/
44
Otology
2024-02-29
2022-11-24
2024-04-23
2024-04-23
120-127
1
Agnieszka Wiatr
2024/02/29
10.14639/0392-100X-N2389
120
Department of Otolaryngology, Jagiellonian University Medical College, Kraków, Poland
1827-675X
stapes microstructure
en
127
Effect of stapes demineralisation on the development of cochlear otosclerosis
https://www.actaitalica.it/article/download/2389/1081
44
ACTA OTORHINOLARYNGOLOGICA ITALICA 2024;44:120-127; doi: 10.14639/0392-100X-N2389




Otology


Effect of stapes demineralisation on the development
of cochlear otosclerosis
Maciej Wiatr1, Robert Bartoszewicz2, Kazimierz Niemczyk2, Agnieszka Wiatr1
1
 Department of Otolaryngology, Jagiellonian University Medical College, Kraków, Poland; 2 Department of Otolaryngology, Head and
Neck Surgery, Medical University, Warsaw, Poland



SUMMARY
Objective. The involvement of the inner ear in otosclerosis may lead to the development of
cochlear otosclerosis. The aim of this study was to analyse changes in the chemical composi-
tion and microstructure of the stapes in the course of otosclerosis compared to healthy stapes.
Materials and methods. This analysis included 31 patients with otosclerosis and 9 patients
without otosclerosis. Microanalytical and diffraction techniques were used to assess the
elemental distribution and orientation topography of the stapes.
Results. The concentration of Ca2+ in the study group was significantly lower in the area of the
anterior crus of the stapes than in the posterior crus. A reduction in the Ca2+/P3+ ratio in the anterior
crus was associated with deteriorated bone conduction and tinnitus. Degradation of the stapes mi-
crostructure in the area of otosclerotic lesions was observed with scanning electron microscopy.
Conclusions. Bone remodelling is most significant at the closest location to typical oto-
sclerotic lesions with hydroxyapatite porosity and scale-like bone formation according to
scanning electron microscopy. There is a relationship between the disturbance of calcium
metabolism and the development of clinical symptoms of cochlear otosclerosis.
KEY WORDS: otosclerotic lesions, cochlear otosclerosis, stapes demineralisation,
scanning electron microscope, stapes microstructure


Introduction
Hearing loss at any age can significantly reduce quality of life, thus leading to
a lack of adaptation and social isolation. A related cause of acquired hearing
loss is otosclerosis.                                                                                       Received: November 24, 2022
Otosclerosis is the primary disease affecting the bony labyrinth. The essence                               Accepted: October 16, 2023
                                                                                                            Published online: February 29, 2024
of the disease is pathological remodelling of the temporal bone, the basis of
which involves the abnormal resorption of bone tissue and its remodelling,                                  Correspondence
thus leading to dysplasia. This disease, which occurs only in humans in the                                 Maciej Wiatr
temporal bone, is characterised by progressive hearing impairment and tin-                                  E-mail: maciej.wiatr@uj.edu.pl
nitus. The aetiology of otosclerosis is complex, multifactorial and can involve
both environmental and genetic factors.                                                                       How to cite this article: Wiatr M, Bartoszewicz
                                                                                                              R, Niemczyk K, et al. Effect of stapes deminer-
According to the current state of knowledge, the activity of osteoblasts and                                  alisation on the development of cochlear otoscle-
osteoclasts is not observed in the bony labyrinth in healthy people. During the                               rosis. Acta Otorhinolaryngol Ital 2024;44:120-
course of otosclerosis, processes of abnormal resorption and recalcification of                               127. https://doi.org/10.14639/0392-100X-N2389
the endochondral layer of the temporal bone occur. The endochondral layer is
                                                                                                            © Società Italiana di Otorinolaringoiatria
an area that allows small regions of immature cartilage (known as interosseous                              e Chirurgia Cervico-Facciale
globules) to be populated by osteoblasts and osteoclasts 1-3.
The disease process may involve all of the bony structures of the ear; however,                                            OPEN ACCESS

the initiating lesion is often adjacent to the fissula ante fenestram and spreads                           This is an open access article distributed in accordance with
                                                                                                            the CC-BY-NC-ND (Creative Commons Attribution-Non-
throughout the vascular canals to the remaining areas of the temporal bone                                  Commercial-NoDerivatives 4.0 International) license. The
through calcification of the annular ligament and remodelling of the stapes foot-                           article can be used by giving appropriate credit and mentio-
                                                                                                            ning the license, but only for non-commercial purposes and
plate. Involvement of the inner ear in the disease leads to degenerative changes                            only in the original version. For further information: https://
in the spiral ligament, vascular striatum, organ of Corti and cochlear neurons.                             creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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                                                                                            Bone tissue demineralisation in otosclerosis




Current surgical treatments (through the use of a prosthe-        in the tests, with carbon dioxide as the gas. Electron beam
sis) restore the mobility of the ossicular chain, but neither     accelerating voltages of 15-20 kV, electron beam currents
eliminate the disease nor inhibit further bone remodelling        of 5.66, 11.3 and 23 nA and working distances (i.e., the
in temporal bone.                                                 distance from the sample surface to the objective lens pole
An understanding of the mechanisms affecting stapes bone          piece) ranging from 8-14.6 mm were used. A low-vacuum
tissue in otosclerosis will form the basis for further re-        secondary electron detector was used to assess bone sur-
search on conservative treatments, which may be of key            face topography via secondary electrons, and a low-volt-
importance for people at risk of developing this disease 4-6.     age, high-contrast detector was used to record the backscat-
The aim of this study was to analyse changes in the chemical      tered electron (BSE) signal.
composition and microstructure of the stapes in otosclerosis      Qualitative analysis of the chemical composition in areas
with respect to healthy stapes. The specific objective was to     of the stapes was performed by using an EDS Genesis X-
analyse the impact of changes in the chemical composition         ray detector; PhiRoZet software was used at the quantita-
of bone in otosclerosis on the functions of the inner ear.        tive analysis stage.
                                                                  Chemical composition analysis was performed by using the
Materials and methods                                             Raymond Gauvin correction procedure. In addition, we used
                                                                  the variable pressure method, which consisted of measur-
A prospective analysis of patients who underwent surgery          ing two spectra at two different CO2 pressures (i.e. the first
from 2020 to 2021 was conducted. The study included 31            spectrum at a pressure that was twice as high as the second
patients with otosclerosis (77.5%) and 9 patients without         spectrum), after which we extrapolated the maximum values
otosclerosis, who constituted the control group (22.5%).          of the individual peaks to zero gas pressure values.
Thirty-four women and 6 men were analysed to determine            Descriptive statistics were used to describe the character-
various influential factors. The oldest patient was 61 years      istics of the studied groups, including the mean, median,
old, and the youngest was 32 years old. Mean age was 42           standard deviation (SD), first and third quartiles [inter-
years. Moreover, the group of patients with otosclerosis          quartile range (IQR)] and range. The normality of the
consisted of 26 women and 5 men aged 22-55 years. The             distribution of individual parameters that were assessed
mean age of these patients was 38 years.                          in this study was checked with the Shapiro-Wilk test.
Stapes suprastructures that were obtained as medical waste        The Mann-Whitney or Kruskal-Wallis U test was used to
during surgery were analysed by scanning electron micros-         analyse data with a non-normal distribution. Moreover,
copy (SEM). Thirty-one stapes from patients with otoscle-         the Dunn pairwise test was used to compare individual
rosis and 9 healthy stapes were included in the study. The        groups. To determine whether there were differences be-
healthy stapes constituted the control group; their removal       tween the results of particular analysed variables in par-
resulted from the use of the infracochlear approach for ves-      ticular periods (the dependent variables) in the study
tibular schwannoma excision.                                      group, the non-parametric Wilcoxon test or Student t test
In all patients with otosclerosis, typical preoperative otolar-   with Bonferroni correction for multiple testing was used.
yngological diagnostic tests were performed, which con-           To investigate the existence of monotonic relationships
sisted of a physical examination, medical history and audio-      between the variables, Spearman’s correlation coefficient
metric diagnosis, including accumetric examinations, tuning       was used. A mixed-effects linear model was used to inves-
fork tests, pure-tone audiometry and impedance audiometry.        tigate the relationships between the variables. The level of
The threshold values for air and bone conduction were             significance was set at p < 0.05.
determined by using the Madsen® MIDIMATE® 622 au-
diometer equipped with TDK 39® headphones. The audi-
ometer met the requirements of the ISO standards for air
                                                                  Results
(ISO0389-1985) and bone conduction (ISO7566-1987).                In the first stage, the chemical composition of the stapes
Pure-tone audiometry was immediately performed before             was analysed by comparing the head, anterior crus and pos-
surgery and at 12 months after stapedotomy. The obtained          terior crus in the test group.
data were analysed according to the guidelines developed by       A significant difference in the Ca2+ distribution was ob-
the Committee on Hearing and Equilibrium of the American          served among the different areas of the stapes (Kruskal-
Academy of Otolaryngology - Head and Neck Surgery 7.              Wallis test, p < 0.05) in the study group. In addition, a sta-
SEM examination was performed using a QUANTA 3D                   tistically significant difference in the Ca2+/P3+ ratio among
FEG scanning electron microscope in low-vacuum mode.              the analysed areas of the stapes was found in the study
A variable vacuum ranging from 0.3 to 0.6 Torr was used           group (p = 0.0481; Kruskal-Wallis test).



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M. Wiatr et al.




There was no significant difference in the distribution of P3+                                 A significant relationship between the occurrence of tin-
among the areas of the stapes in patients with otosclerosis                                    nitus and the duration of disease was demonstrated. The
(p = 0.302; Kruskal-Wallis test). The results are presented                                    lower Ca2+/P3+ ratio observed with increased duration of
in Table I.                                                                                    otosclerosis was significantly more likely in the presence
The applied Dunn post hoc tests allowed for precise deter-                                     of tinnitus (p = 0.0027; Mann-Whitney U test).
mination of which groups exhibited statistically significant                                   A reduction in the Ca2+/P3+ ratio in the anterior crus was
differences. The results are presented in Table II. The con-                                   highly correlated with deteriorated bone conduction (Spear-
centration of Ca2+ was significantly lower in the anterior                                     man’s correlation coefficient 0.531, p = 0.03; Wilcoxon
crus of the stapes than in the posterior crus (p < 0.05).                                      test). In the posterior crus and head, the correlation was
In the next stage, the analysis was performed for the control                                  low or weak (Spearman’s correlation coefficients: 0.028,
group, which showed no significant differences in the distri-                                  p = 0.905; Wilcoxon test and 0.165, p = 0.499; Wilcoxon
bution of Ca2+ or P3+ among the head, anterior crus and pos-                                   test, respectively).
terior crus (Tab. III). In the next stage of the study, the results                            However, when considering the posterior crus, there was a
obtained for the test and control groups were compared. A                                      significant correlation between the Ca2+/P3+ ratio and dis-
significant difference was demonstrated in the distribution of                                 ease duration in years (Spearman’s correlation coefficient
the Ca2+/P3+ ratio in the anterior crus of the stapes between                                  -0.474, p = 0.026; Wilcoxon test). This correlation was
patients in the test and control groups (p < 0.05; Mann-                                       negative and of an average strength. This means that for a
Whitney U test) (Tab. IV). Patients in the study group had
                                                                                               longer disease duration, the Ca2+/P3+ ratio in the posterior
a significantly lower Ca2+/P3+ ratio in the anterior crus than
                                                                                               crus was lower.
patients from the control group. The analysis of the Ca2+/
                                                                                               In patients with otosclerosis, changes in the surface micro-
P3+ ratio distribution in the posterior crus did not reveal any
                                                                                               structure of the stapes in the area of the anterior crus were
significant relationships (p = 0.8018; Mann-Whitney U test).
                                                                                               noticeable; specifically, degradation of the stapes micro-
In the next stage of the study, the relationship between the
chemical composition of the stapes and the presence of
symptoms typical of otosclerosis, such as tinnitus or sen-                                     Table II. Comparison of Ca2+ concentrations among stapes areas in the
sorineural hearing loss – indicative of cochlear otosclero-                                    study group by post hoc testing.
sis – was evaluated.                                                                            Compared areas                                            p value
In the study group, a significant correlation was observed be-                                  Head - Anterior crus                                          0.068
tween the Ca2+ level in the anterior crus and the occurrence                                    Head - Posterior crus                                         0.923
of tinnitus (p < 0.05; Mann-Whitney U test) (Tab. V). Tinni-                                    Anterior crus - Posterior crus                                0.014
tus patients had significantly lower Ca2+ levels in the anterior                               p: significance level.
crus than patients without tinnitus in the study group.                                        Significance level (p) < 0.05 in bold.



Table I. Comparison of the chemical composition of different areas of the stapes in the study group.
 Variable                    Parameter          Head of the stapes          Anterior crus        Posterior crus of                          Test         p value
                                                       (N = 31)             of the stapes      the stapes (N = 31)
                                                                               (N = 31)
 Ca2+
                                    Mean (SD)                    16.5 (7.4)               11.4 (5.4)                 17.2 (6.9)         Kruskal-Wallis   0.0245
                                   Median (IQR)                16 (10.3-19.8)           14.2 (8.6-15.6)            16.9 (12-20.4)
                                     Range                       7.3-35.1                  0.2-17.5                  5.3-35.1
 P3+
                                    Mean (SD)                     6.8 (4.3)                5.3 (2.7)                  6.8 (3)           Kruskal-Wallis   0.302
                                   Median (IQR)                 6.6 (3.5-9.8)            5.4 (4.5-7.2)             6.7 (5.6-8.6)
                                     Range                        0.9-15.6                 0.1-9.5                   1.4-15.6
 Ca2+/P3+ ratio
                                    Mean (SD)                     3.4 (2.4)                2.1 (3.4)                 3.3 (2.9)          Kruskal-Wallis   0.0481
                                   Median (IQR)                 2.2 (1.7-4.4)             2 (1.8-2.5)              2.3 (1.6-3.2)
                                     Range                        1.3-8.9                  1.3-17.7                  1.1-13.5
Ca: Calcium; P: Phosphorus; N: number of patients; p: significance level. Significance level (p) < 0.05 in bold.




122
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Table III. Comparison of chemical composition in different areas of the stapes in the control group.
 Variable              Parameter         Head of the stapes Anterior crus of the Posterior crus of                         Test                 p value
                                                (N = 9)               stapes (N = 9)        the stapes (N = 9)
 Ca2+
                             Mean (SD)                  4.7 (2.2)              14.1 (2.6)            14.6 (2)         Kruskal-Wallis            0.2745
                            Median (IQR)              4.9 (3.7-5.7)         14.9 (12.1-16.4)     15.6 (13.6-15.9)
                              Range                      1-8.2                 9.9-17.1             11.6-17.2
 P3+
                             Mean (SD)                   3 (1.3)                1.6 (0.2)           6.7 (0.2)         Kruskal-Wallis            0.0726
                            Median (IQR)              2.8 (2.2-3.9)           1.7 (1.4-1.9)       6.7 (6.6-6.9)
                              Range                        1-5                   1.2-2              6.3-6.9
 Ca2+/P3+ ratio
                             Mean (SD)                  1.6 (0.7)               8.7 (2.5)           2.1 (0.3)         Kruskal-Wallis            0.0611
                            Median (IQR)              1.3 (1.2-1.9)           8.3 (7.6-8.5)        2.2 (2-2.3)
                              Range                     0.7-3.2                 5.5-13.5            1.7-2.5
Ca: Calcium; P: Phosphorus; N: number of patients; p: significance level.
Significance level (p) < 0.05 in bold.



Table IV. Comparison of the chemical composition of the anterior crus between the study and control groups.
 Variable                                   Parameter                Study group           Control group                   Test                 p value
                                                                       (N = 31)               (N = 9)
 Ca2+
                                                       Mean (SD)              11.4 (5.4)           14.1 (2.6)        Mann-Whitney U             0.3736
                                                      Median (IQR)          14.2 (8.6-15.6)     14.9 (12.1-16.4)
                                                        Range                  0.2-17.5            9.9-17.1
 P3+
                                                       Mean (SD)               5.3 (2.7)            1.6 (0.2)        Mann-Whitney U             0.0973
                                                      Median (IQR)           5.4 (4.5-7.2)        1.7 (1.4-1.9)
                                                        Range                  0.1-9.5               1.2-2
 Ca2+/P3+ ratio
                                                       Mean (SD)               2.8 (3.4)            8.7 (2.5)        Mann-Whitney U             0.0198
                                                      Median (IQR)            2 (1.8-2.5)         8.3 (7.6-8.5)
                                                        Range                  1.3-17.7             5.5-13.5
Ca: Calcium; P: Phosphorus; N: number of patients; p: significance level.
Significance level (p) < 0.05 in bold.



structure was visible, with scale-like structures visible on                            hydroxyapatite with a variable Ca2+/P3+ ratio. In otosclerosis,
the bone on SEM (Fig. 1).                                                               bone tissue demineralisation was observed. At the level of
In patients from the control group, SEM clearly showed the                              biochemical analysis, the microscopic changes were accom-
fibrous structure of hydroxyapatite (which is characteristic                            panied by changes in the chemical composition, with measur-
of healthy bone tissue in the stapes), with no signs of de-                             able bone tissue decalcification visible in the immediate vicin-
generation (Fig. 2). In otosclerosis, hydroxyapatite poros-                             ity of the otosclerotic foci and significant differences observed
ity was observed by using BSE imaging, which resulted                                   relative to healthy bone.
from stapes bone degeneration (Fig. 3).                                                 In this study, a significant difference in Ca2+ distribution was
                                                                                        observed among different areas of the otosclerotic stapes, with
                                                                                        the lowest level occurring in the anterior crus, followed by
Discussion                                                                              the head and posterior crus (p = 0.0245, Kruskal-Wallis test).
In the present research, the chemical composition analysis                              Comparative analysis of the Ca2+ distribution in the healthy
confirmed that the main component of the bone structure was                             stapes (control group) did not show a significant difference



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M. Wiatr et al.




Table V. Comparison of the chemical composition of the anterior crus in the presence or absence of tinnitus in the study group.
 Variable                                    Parameter            Tinnitus present         Tinnitus absent                Test                              p value
                                                                       (N = 20)               (N = 11)
 Ca2+
                                                        Mean (SD)                  11.3 (3.5)                  18.8 (7.6)        Mann-Whitney U             0.0283
                                                       Median (IQR)               11 (8.4-13.9)              19.1 (15-22.7)
                                                         Range                       7.3-16                    7.3-35.1
 P3+
                                                        Mean (SD)                   5.2 (3.1)                   7.6 (4.7)        Mann-Whitney U             0.3802
                                                       Median (IQR)               4.4 (3.4-6.6)                7 (3.8-12)
                                                         Range                      1.6-10.3                   0.9-15.6
 Ca2+/P3+ ratio
                                                        Mean (SD)                   2.7 (1.5)                   3.7 (2.7)        Mann-Whitney U             0.4827
                                                       Median (IQR)               1.9 (1.6-3.9)               2.2 (1.8-4.4)
                                                         Range                      1.5-4.9                     1.3-8.9
Ca: Calcium; P: Phosphorus; N: number of patients; p: significance level. Significance level (p) < 0.05 in bold.



                                                                                               of mineralisation occur only transiently in immature foetal
                                                                                               bone; therefore, only the mature form of calcification rep-
                                                                                               resented by hydroxyapatite is found in adults 8.
                                                                                               The bony labyrinth, unlike most cranial bones, which are
                                                                                               formed as a result of intramembranous ossification, devel-
                                                                                               ops like long bones as a result of the endochondral ossifi-
                                                                                               cation process. The most suitable chemical model for the
                                                                                               main bone mineral phase is Ca5(OH)(PO4)3 hydroxyapa-
                                                                                               tite, containing approximately 5% to 10% CO32- groups
                                                                                               and approximately 5% to 10% HPO42- groups, in the
                                                                                               CaHPO4•2H2O configuration. In the bony labyrinth, there




Figure 1. Otosclerosis. Microstructure of the anterior crus of the stapes,
showing bone with a scale-like structure of hydroxyapatite on scanning elec-
tron microscopy (SEM) using a backscattered electron (BSE) signal; magnifi-
cation, 1000x.


among the head, anterior crus and posterior crus. There was
a significant difference in the distribution of the Ca2+/P3+ ra-
tio in the stapes between the otosclerosis and control groups.
Patients with otosclerosis had a significantly lower Ca2+/P3+
ratio in the anterior crus than patients in the control group
(p = 0.0198; Mann-Whitney U test).
According to the literature, the Ca2+/P3+ ratio in normal
bone is not always constant over time. Skerry 8 believes
that the initial amorphous phase of calcium phosphate in
healthy bones is gradually and successively converted to
octa-calcium phosphate Ca8(HPO4)2(PO4)4•5H2O, tricalci-                                        Figure 2. Control group. Microstructure of the anterior crus of the stapes on
um phosphate Ca3(PO4)2 and hydroxyapatite. These forms                                         SEM using a BSE signal; magnification, 1200x.



124
                                                                                                         Bone tissue demineralisation in otosclerosis




                                                                              control group. In the latter cases, SEM showed the typical
                                                                              fibrous structure of hydroxyapatite.
                                                                              The structure of healthy bone may vary depending on the
                                                                              anatomical location. According to the literature, approxi-
                                                                              mately 8% of PO4 ions can be replaced in the structure
                                                                              of healthy bone by CO3 ions; this represents B-type hy-
                                                                              droxyapatite. In the case of A-type hydroxyapatite, the OH
                                                                              groups are replaced by CO3 anions 11.
                                                                              Inner ear involvement in the process of osteodystrophy ob-
                                                                              served in otosclerosis is described as degenerative changes
                                                                              in the spiral ligament, vascular striatum, organ of Corti and
                                                                              cochlear neurons. In our observations, the progressive de-
                                                                              cline in the Ca2+/P3+ ratio in the anterior crus coexisted with
                                                                              deterioration in bone conduction (Spearman’s correlation
                                                                              coefficient 0.531, p = 0.03; Wilcoxon test). A significant
Figure 3. Otosclerosis. Anterior crus of the stapes, showing hydroxyapatite   dependence of tinnitus on the duration of disease was also
porosity on SEM using a BSE signal; magnification, 1200x.                     demonstrated. The lower anterior crus Ca2+/P3+ ratio ob-
                                                                              served with increased duration of otosclerosis was signifi-
                                                                              cantly more likely in the presence of tinnitus (p = 0.0027;
was no significant remodelling of bone tissue typical of                      Mann-Whitney U test). The typical location of otosclerotic
long bones, which is related to the lack of alternating bone                  foci was the anterior crus; the biochemical manifestation of
resorption and remodelling.                                                   the ongoing processes was a significant change in the Ca2+/
The metabolic rate of the bony labyrinth is approximately                     P3+ ratio in the anterior crus compared to other parts of the
0.13% per year, while in long bones, it is up to 10% per                      stapes.
year. This low rate of bone remodelling in the bony laby-                     Changes in the elemental composition and levels of toxic
rinth is due to high levels of osteoprotegerin (OPG), which                   metabolites released during bone remodelling, penetrating
inhibits osteoclastogenesis and consequent bone resorp-                       the inner ear, seem to negatively affect the inner ear and
tion. In otosclerosis, the level of OPG in the bony labyrinth                 be responsible for the clinical manifestations of cochlear
is reduced, resulting in a decreased level of bone resorption                 otosclerosis. Tinnitus intensification has been observed
(otospongiosis phase) in favour of an increased level of re-                  with the progression of otosclerosis and bone remodelling,
modelling (otosclerosis phase). This causes a change in the                   which, overlapping with hearing loss, deepens the subjec-
chemical composition of the bone tissue and then partial                      tive sense of hearing impairment 12.
conversion to other related mineral species, such as dahl-                    In a study by Vallejo-Valdezate et al. 13, a decrease in the
lite or struvite. These transformations are illustrated by the                Ca2+/P3+ ratio of the stapes similar to that in otosclerosis
quantitative determination of phosphorus (or phosphates)                      was observed in Van der Hoeve syndrome (VDHS). De-
and calcium, as their relative percentages in each of the                     spite the aetiological differences, both diseases are related
quoted mineral species are different. In the assessment of                    to changes in the bony labyrinth and progressive sensori-
the described processes, it is useful to determine the ratio                  neural hearing loss. While VDHS is caused by a disorder
of calcium to phosphorus (Ca2+/P3+) 9,10.                                     of type I collagen synthesis, it has been postulated that col-
The change in metabolic activity during the course of oto-                    lagen II autoimmunity is present in otosclerosis 13.
sclerosis has an impact on the degeneration of bone tissue                    The available reports, similar to our observations, explain
(as shown on SEM by the formation of scale-like bone and                      the biochemical changes observed in otosclerosis with the
porous tissue). The significant differences in the micro-                     probable transformation of hydroxyapatite into tricalcium
structure and chemical composition shown in the analysis                      phosphate and insufficient mineralisation of the collagen
concerned individual parts of the stapes in otosclerosis and                  matrix 14.
were considerably different with respect to stapes free from                  The influence of bone tissue demineralisation on the
otosclerotic changes.                                                         function of sensory cells of the inner ear has been widely
The process of stapes bone remodelling in patients with                       discussed in the otologic literature. Sensorineural hear-
otosclerosis was most strongly expressed on the surface of                    ing loss accompanying demineralisation is explained by
the anterior crus with a typical scaly structure, in contrast to              the accumulation of Ca2+ in inner hair cells. Numerous
the normal surface of the healthy stapes in patients from the                 reports have indicated a relationship between the reduc-



                                                                                                                                               125
M. Wiatr et al.




tion in bone tissue density and hearing impairment. Ji           The current analysis complements the observations of other
Yoon et al. showed that postmenopausal osteoporosis and          authors, thus demonstrating a significant role of demin-
the related disturbance of calcium metabolism favour the         eralisation of the stapes and disturbances in calcium ion
occurrence of sensorineural hearing loss 15. Moreover,           metabolism in the development of hearing loss in cochlear
Groschel et al. observed the effects of changes in cal-          otosclerosis.
cium levels with age on the occurrence of sensorineural
hearing loss, which also occurred in the upper levels of         Conclusions
the auditory pathway 16.
According to research by Xingqi Li et al., the reduced Ca2+      Stapes bone remodelling is most significant in the closest
concentration in the perilymph mediated by reductions in         location to typical otosclerotic changes (i.e., in the fissula
the Ca2+ concentration in inner hair cells has a protective      ante fenestram). There is a relationship between the distur-
effect on hearing when exposed to noise 17. During the oto-      bance of calcium metabolism in otosclerosis and the devel-
sclerotic process, bone decalcification promotes the accu-       opment of clinical symptoms of cochlear otosclerosis. As
mulation of Ca2+ in inner hair cells.                            the otosclerotic process advances in the stapes, the porosity
Sensorineural hearing loss is also significantly more fre-       of hydroxyapatite resulting from bone tissue degeneration
quent in cases of hypoparathyroidism due to the distur-          and scale-like bone tissue formation increases, as observed
bance of calcium metabolism. Adverse effects on the inner        by SEM.
ear are explained by abnormal levels of calcium in the inner
ear fluid and/or a direct effect of vitamin D deficiency on      Acknowledgements
the inner ear 18,19.                                             Special thanks to the Scanning Electron Microscopy Labo-
Numerous studies have indicated that a reduced level of          ratory of the IMiM PAN in Krakow for help with the re-
vitamin D, in addition to disturbed Ca2+ metabolism and          search work.
abnormal microcirculation in the cochlea, are factors in-
fluencing the development of sensorineural hearing loss 20.      Conflict of interest statement
Decreased serum Ca2+ and vitamin D levels, which are of-         The authors declare no conflict of interest.
ten associated with osteoporosis via increases in bone me-
tabolism, are considered independent risk factors for hear-      Funding
ing impairment. Furthermore, decreased vitamin D levels          This study was created as a result of research project No.
appear to reduce auditory sensitivity by altering Ca2+ me-       2020/04/X/NZ5/00550 financed by the National Science
tabolism and microcirculation in the cochlea 21.                 Centre confirmed.
The accumulation of Ca2+ (with an increase in its con-
centration in the perilymph mediated by increases in the         Author contributions
concentration of glutamate) leads to inner hair cell dam-        MW, KN, AW: conception; MW, AW: design; MW, RB,
age 22. In inner hair cells, an increase in intracellular Ca2+   KN, AW: data collection; MW, RB, KN, AW: analysis;
causes the mobilisation of synaptic vesicles and the exo-        MW: funding; MW, RB, AW: literature; MW, KN: review;
cytotic release of glutamate at the base. The release of         MW, AW: writing.
glutamate modulates the activity of auditory nerve fibres
by activating specific receptors, including the ionotropic       Ethical consideration
glutamate receptors AMPA (α-amino-3-hydroxy-5-methyl-            The research was approved by the Institutional Bioethics
4-isoxazolepropionic acid receptor), NMDA (N-methyl-D-           Committee of Jagiellonian University, Kraków, Poland
aspartate receptor) and KARs (kainic acid receptors), as         (approval no. 1072.6120.311.2020). The research was con-
well as metabotropic receptors. Outer hair cells react com-      ducted ethically, with all study procedures being performed
pletely differently to changes in their membrane potential.      in accordance with the requirements of the World Medical
Their membranes contain a protein (prestin) that changes         Association’s Declaration of Helsinki. Written informed
conformation with the membrane potential and forces the          consent was obtained from each participant/patient for
cell length to change at acoustic frequencies 23.                study participation and data publication.
Increased Ca2+ activity via the CaV1.2L-type calcium
channel in the auditory ossicles and ear capsule through         References
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