Effect of stapes demineralisation on the development of cochlear otosclerosis| ACTA Otorhinolaryngologica Italica

Abstract

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.

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.

Otosclerosis is the primary disease affecting the bony labyrinth. The essence of the disease is pathological remodelling of the temporal bone, the basis of which involves the abnormal resorption of bone tissue and its remodelling, thus leading to dysplasia. This disease, which occurs only in humans in the temporal bone, is characterised by progressive hearing impairment and tinnitus. The aetiology of otosclerosis is complex, multifactorial and can involve both environmental and genetic factors.

According to the current state of knowledge, the activity of osteoblasts and osteoclasts is not observed in the bony labyrinth in healthy people. During the course of otosclerosis, processes of abnormal resorption and recalcification of the endochondral layer of the temporal bone occur. The endochondral layer is an area that allows small regions of immature cartilage (known as interosseous globules) to be populated by osteoblasts and osteoclasts ^1-3.

The disease process may involve all of the bony structures of the ear; however, the initiating lesion is often adjacent to the fissula ante fenestram and spreads throughout the vascular canals to the remaining areas of the temporal bone through calcification of the annular ligament and remodelling of the stapes footplate. Involvement of the inner ear in the disease leads to degenerative changes in the spiral ligament, vascular striatum, organ of Corti and cochlear neurons.

Current surgical treatments (through the use of a prosthesis) restore the mobility of the ossicular chain, but neither eliminate the disease nor inhibit further bone remodelling in temporal bone.

An understanding of the mechanisms affecting stapes bone tissue in otosclerosis will form the basis for further research on conservative treatments, which may be of key importance for people at risk of developing this disease ^4-6.

The aim of this study was to analyse changes in the chemical composition and microstructure of the stapes in otosclerosis with respect to healthy stapes. The specific objective was to analyse the impact of changes in the chemical composition of bone in otosclerosis on the functions of the inner ear.

Materials and methods

A prospective analysis of patients who underwent surgery from 2020 to 2021 was conducted. The study included 31 patients with otosclerosis (77.5%) and 9 patients without otosclerosis, who constituted the control group (22.5%). Thirty-four women and 6 men were analysed to determine various influential factors. The oldest patient was 61 years old, and the youngest was 32 years old. Mean age was 42 years. Moreover, the group of patients with otosclerosis consisted of 26 women and 5 men aged 22-55 years. The mean age of these patients was 38 years.

Stapes suprastructures that were obtained as medical waste during surgery were analysed by scanning electron microscopy (SEM). Thirty-one stapes from patients with otosclerosis and 9 healthy stapes were included in the study. The healthy stapes constituted the control group; their removal resulted from the use of the infracochlear approach for vestibular schwannoma excision.

In all patients with otosclerosis, typical preoperative otolaryngological diagnostic tests were performed, which consisted of a physical examination, medical history and audiometric diagnosis, including accumetric examinations, tuning fork tests, pure-tone audiometry and impedance audiometry.

The threshold values for air and bone conduction were determined by using the Madsen^® MIDIMATE^® 622 audiometer equipped with TDK 39^® headphones. The audiometer met the requirements of the ISO standards for air (ISO0389-1985) and bone conduction (ISO7566-1987).

Pure-tone audiometry was immediately performed before surgery and at 12 months after stapedotomy. The obtained data were analysed according to the guidelines developed by the Committee on Hearing and Equilibrium of the American Academy of Otolaryngology - Head and Neck Surgery ⁷.

SEM examination was performed using a QUANTA 3D FEG scanning electron microscope in low-vacuum mode. A variable vacuum ranging from 0.3 to 0.6 Torr was used in the tests, with carbon dioxide as the gas. Electron beam accelerating voltages of 15-20 kV, electron beam currents of 5.66, 11.3 and 23 nA and working distances (i.e., the distance from the sample surface to the objective lens pole piece) ranging from 8-14.6 mm were used. A low-vacuum secondary electron detector was used to assess bone surface topography via secondary electrons, and a low-voltage, high-contrast detector was used to record the backscattered electron (BSE) signal.

Qualitative analysis of the chemical composition in areas of the stapes was performed by using an EDS Genesis X-ray detector; PhiRoZet software was used at the quantitative analysis stage.

Chemical composition analysis was performed by using the Raymond Gauvin correction procedure. In addition, we used the variable pressure method, which consisted of measuring two spectra at two different CO₂ pressures (i.e. the first spectrum at a pressure that was twice as high as the second spectrum), after which we extrapolated the maximum values of the individual peaks to zero gas pressure values.

Descriptive statistics were used to describe the characteristics of the studied groups, including the mean, median, standard deviation (SD), first and third quartiles [interquartile range (IQR)] and range. The normality of the distribution of individual parameters that were assessed in this study was checked with the Shapiro-Wilk test. The Mann-Whitney or Kruskal-Wallis U test was used to analyse data with a non-normal distribution. Moreover, the Dunn pairwise test was used to compare individual groups. To determine whether there were differences between the results of particular analysed variables in particular periods (the dependent variables) in the study group, the non-parametric Wilcoxon test or Student t test with Bonferroni correction for multiple testing was used. To investigate the existence of monotonic relationships between the variables, Spearman’s correlation coefficient was used. A mixed-effects linear model was used to investigate the relationships between the variables. The level of significance was set at p < 0.05.

Results

In the first stage, the chemical composition of the stapes was analysed by comparing the head, anterior crus and posterior crus in the test group.

A significant difference in the Ca²⁺ distribution was observed among the different areas of the stapes (Kruskal-Wallis test, p < 0.05) in the study group. In addition, a statistically significant difference in the Ca²⁺/P³⁺ ratio among the analysed areas of the stapes was found in the study group (p = 0.0481; Kruskal-Wallis test).

There was no significant difference in the distribution of P³⁺ among the areas of the stapes in patients with otosclerosis (p = 0.302; Kruskal-Wallis test). The results are presented in Table I.

The applied Dunn post hoc tests allowed for precise determination of which groups exhibited statistically significant differences. The results are presented in Table II. The concentration of Ca²⁺ was significantly lower in the anterior crus of the stapes than in the posterior crus (p < 0.05).

In the next stage, the analysis was performed for the control group, which showed no significant differences in the distribution of Ca²⁺ or P³⁺ among the head, anterior crus and posterior crus (Tab. III). In the next stage of the study, the results obtained for the test and control groups were compared. A significant difference was demonstrated in the distribution of the Ca²⁺/P³⁺ ratio in the anterior crus of the stapes between patients in the test and control groups (p < 0.05; Mann-Whitney U test) (Tab. IV). Patients in the study group had a significantly lower Ca²⁺/P³⁺ ratio in the anterior crus than patients from the control group. The analysis of the Ca²⁺/P³⁺ ratio distribution in the posterior crus did not reveal any significant relationships (p = 0.8018; Mann-Whitney U test).

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 sensorineural hearing loss – indicative of cochlear otosclerosis – was evaluated.

In the study group, a significant correlation was observed between the Ca²⁺ level in the anterior crus and the occurrence of tinnitus (p < 0.05; Mann-Whitney U test) (Tab. V). Tinnitus patients had significantly lower Ca²⁺ levels in the anterior crus than patients without tinnitus in the study group.

A significant relationship between the occurrence of tinnitus and the duration of disease was demonstrated. The lower Ca²⁺/P³⁺ ratio observed with increased duration of otosclerosis was significantly more likely in the presence of tinnitus (p = 0.0027; Mann-Whitney U test).

A reduction in the Ca²⁺/P³⁺ ratio in the anterior crus was highly correlated with deteriorated bone conduction (Spearman’s correlation coefficient 0.531, p = 0.03; Wilcoxon test). In the posterior crus and head, the correlation was low or weak (Spearman’s correlation coefficients: 0.028, p = 0.905; Wilcoxon test and 0.165, p = 0.499; Wilcoxon test, respectively).

However, when considering the posterior crus, there was a significant correlation between the Ca²⁺/P³⁺ ratio and disease duration in years (Spearman’s correlation coefficient -0.474, p = 0.026; Wilcoxon test). This correlation was negative and of an average strength. This means that for a longer disease duration, the Ca²⁺/P³⁺ ratio in the posterior crus was lower.

In patients with otosclerosis, changes in the surface microstructure of the stapes in the area of the anterior crus were noticeable; specifically, degradation of the stapes microstructure was visible, with scale-like structures visible on the bone on SEM (Fig. 1).

In patients from the control group, SEM clearly showed the fibrous structure of hydroxyapatite (which is characteristic of healthy bone tissue in the stapes), with no signs of degeneration (Fig. 2). In otosclerosis, hydroxyapatite porosity was observed by using BSE imaging, which resulted from stapes bone degeneration (Fig. 3).

Discussion

In the present research, the chemical composition analysis confirmed that the main component of the bone structure was hydroxyapatite with a variable Ca²⁺/P³⁺ ratio. In otosclerosis, bone tissue demineralisation was observed. At the level of biochemical analysis, the microscopic changes were accompanied by changes in the chemical composition, with measurable bone tissue decalcification visible in the immediate vicinity of the otosclerotic foci and significant differences observed relative to healthy bone.

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 the head and posterior crus (p = 0.0245, Kruskal-Wallis test). Comparative analysis of the Ca²⁺ distribution in the healthy stapes (control group) did not show a significant difference among the head, anterior crus and posterior crus. There was a significant difference in the distribution of the Ca²⁺/P³⁺ ratio 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, tricalcium phosphate Ca₃(PO₄)₂ and hydroxyapatite. These forms of mineralisation occur only transiently in immature foetal bone; therefore, only the mature form of calcification represented by hydroxyapatite is found in adults ⁸.

The bony labyrinth, unlike most cranial bones, which are formed as a result of intramembranous ossification, develops like long bones as a result of the endochondral ossification process. The most suitable chemical model for the main bone mineral phase is Ca₅(OH)(PO₄)₃ hydroxyapatite, containing approximately 5% to 10% CO₃^2- groups and approximately 5% to 10% HPO₄^2- groups, in the CaHPO₄•2H₂O configuration. In the bony labyrinth, there was no significant remodelling of bone tissue typical of long bones, which is related to the lack of alternating bone resorption and remodelling.

The metabolic rate of the bony labyrinth is approximately 0.13% per year, while in long bones, it is up to 10% per year. This low rate of bone remodelling in the bony labyrinth is due to high levels of osteoprotegerin (OPG), which inhibits osteoclastogenesis and consequent bone resorption. In otosclerosis, the level of OPG in the bony labyrinth is reduced, resulting in a decreased level of bone resorption (otospongiosis phase) in favour of an increased level of remodelling (otosclerosis phase). This causes a change in the chemical composition of the bone tissue and then partial conversion to other related mineral species, such as dahllite or struvite. These transformations are illustrated by the quantitative determination of phosphorus (or phosphates) and calcium, as their relative percentages in each of the quoted mineral species are different. In the assessment of the described processes, it is useful to determine the ratio of calcium to phosphorus (Ca²⁺/P³⁺) ^9,10.

The change in metabolic activity during the course of otosclerosis has an impact on the degeneration of bone tissue (as shown on SEM by the formation of scale-like bone and porous tissue). The significant differences in the microstructure and chemical composition shown in the analysis concerned individual parts of the stapes in otosclerosis and were considerably different with respect to stapes free from otosclerotic changes.

The process of stapes bone remodelling in patients with otosclerosis was most strongly expressed on the surface of the anterior crus with a typical scaly structure, in contrast to the normal surface of the healthy stapes in patients from the 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, approximately 8% of PO₄ ions can be replaced in the structure of healthy bone by CO₃ ions; this represents B-type hydroxyapatite. In the case of A-type hydroxyapatite, the OH groups are replaced by CO₃ anions ¹¹.

Inner ear involvement in the process of osteodystrophy observed in otosclerosis is described as degenerative changes in the spiral ligament, vascular striatum, organ of Corti and cochlear neurons. In our observations, the progressive decline 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 dependence of tinnitus on the duration of disease was also demonstrated. The lower anterior crus Ca²⁺/P³⁺ ratio observed with increased duration of otosclerosis was significantly more likely in the presence of tinnitus (p = 0.0027; Mann-Whitney U test). The typical location of otosclerotic foci was the anterior crus; the biochemical manifestation of the ongoing processes was a significant change in the Ca²⁺/P³⁺ ratio in the anterior crus compared to other parts of the stapes.

Changes in the elemental composition and levels of toxic metabolites released during bone remodelling, penetrating the inner ear, seem to negatively affect the inner ear and be responsible for the clinical manifestations of cochlear otosclerosis. Tinnitus intensification has been observed with the progression of otosclerosis and bone remodelling, which, overlapping with hearing loss, deepens the subjective sense of hearing impairment ¹².

In a study by Vallejo-Valdezate et al. ¹³, a decrease in the Ca²⁺/P³⁺ ratio of the stapes similar to that in otosclerosis was observed in Van der Hoeve syndrome (VDHS). Despite the aetiological differences, both diseases are related to changes in the bony labyrinth and progressive sensorineural hearing loss. While VDHS is caused by a disorder of type I collagen synthesis, it has been postulated that collagen II autoimmunity is present in otosclerosis ¹³.

The available reports, similar to our observations, explain the biochemical changes observed in otosclerosis with the probable transformation of hydroxyapatite into tricalcium phosphate and insufficient mineralisation of the collagen matrix ¹⁴.

The influence of bone tissue demineralisation on the function of sensory cells of the inner ear has been widely discussed in the otologic literature. Sensorineural hearing loss accompanying demineralisation is explained by the accumulation of Ca²⁺ in inner hair cells. Numerous reports have indicated a relationship between the reduction in bone tissue density and hearing impairment. Ji Yoon et al. showed that postmenopausal osteoporosis and the related disturbance of calcium metabolism favour the occurrence of sensorineural hearing loss ¹⁵. Moreover, Groschel et al. observed the effects of changes in calcium levels with age on the occurrence of sensorineural hearing loss, which also occurred in the upper levels of the auditory pathway ¹⁶.

According to research by Xingqi Li et al., the reduced Ca²⁺ concentration in the perilymph mediated by reductions in the Ca²⁺ concentration in inner hair cells has a protective effect on hearing when exposed to noise ¹⁷. During the otosclerotic process, bone decalcification promotes the accumulation of Ca²⁺ in inner hair cells.

Sensorineural hearing loss is also significantly more frequent in cases of hypoparathyroidism due to the disturbance of calcium metabolism. Adverse effects on the inner ear are explained by abnormal levels of calcium in the inner ear fluid and/or a direct effect of vitamin D deficiency on the inner ear ^18,19.

Numerous studies have indicated that a reduced level of vitamin D, in addition to disturbed Ca²⁺ metabolism and abnormal microcirculation in the cochlea, are factors influencing the development of sensorineural hearing loss ²⁰. Decreased serum Ca²⁺ and vitamin D levels, which are often associated with osteoporosis via increases in bone metabolism, are considered independent risk factors for hearing impairment. Furthermore, decreased vitamin D levels appear to reduce auditory sensitivity by altering Ca²⁺ metabolism and microcirculation in the cochlea ²¹.

The accumulation of Ca²⁺ (with an increase in its concentration in the perilymph mediated by increases in the concentration of glutamate) leads to inner hair cell damage ²². In inner hair cells, an increase in intracellular Ca²⁺ causes the mobilisation of synaptic vesicles and the exocytotic release of glutamate at the base. The release of glutamate modulates the activity of auditory nerve fibres by activating specific receptors, including the ionotropic glutamate receptors AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor), NMDA (N-methyl-D-aspartate receptor) and KARs (kainic acid receptors), as well as metabotropic receptors. Outer hair cells react completely differently to changes in their membrane potential. Their membranes contain a protein (prestin) that changes conformation with the membrane potential and forces the cell length to change at acoustic frequencies ²³.

Increased Ca²⁺ activity via the CaV1.2L-type calcium channel in the auditory ossicles and ear capsule through bone remodelling affects the auditory and vestibular functions of the inner ear and underlies the pathophysiology of otosclerosis and other osseous disorders of the ear ²⁴.

The current analysis complements the observations of other authors, thus demonstrating a significant role of demineralisation of the stapes and disturbances in calcium ion metabolism in the development of hearing loss in cochlear otosclerosis.

Conclusions

Stapes bone remodelling is most significant in the closest location to typical otosclerotic changes (i.e., in the fissula ante fenestram). There is a relationship between the disturbance of calcium metabolism in otosclerosis and the development of clinical symptoms of cochlear otosclerosis. As the otosclerotic process advances in the stapes, the porosity of hydroxyapatite resulting from bone tissue degeneration and scale-like bone tissue formation increases, as observed by SEM.

Acknowledgements

Special thanks to the Scanning Electron Microscopy Laboratory of the IMiM PAN in Krakow for help with the research work.

Conflict of interest statement

The authors declare no conflict of interest.

Funding

This study was created as a result of research project No. 2020/04/X/NZ5/00550 financed by the National Science Centre confirmed.

Author contributions

MW, KN, AW: conception; MW, AW: design; MW, RB, KN, AW: data collection; MW, RB, KN, AW: analysis; MW: funding; MW, RB, AW: literature; MW, KN: review; MW, AW: writing.

Ethical consideration

The research was approved by the Institutional Bioethics Committee of Jagiellonian University, Kraków, Poland (approval no. 1072.6120.311.2020). The research was conducted ethically, with all study procedures being performed in accordance with the requirements of the World Medical Association’s Declaration of Helsinki. Written informed consent was obtained from each participant/patient for study participation and data publication.

Figures and tables

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

Figure 2.Control group. Microstructure of the anterior crus of the stapes on SEM using a BSE signal; magnification, 1200x.

Figure 3.Otosclerosis. Anterior crus of the stapes, showing hydroxyapatite porosity on SEM using a BSE signal; magnification, 1200x.

References

1. Quesnel AM, Ishai R, McKenna MJ. Otosclerosis: temporal bone pathology. Otolaryngol Clin North Am. 2018; 51:291-303. DOI
2. Palacios-Garcia J, Ropero-Romero F, Aguilar-Vera F. Short-term audiological outcomes of stapedotomy: microdrill at low revolutions versus manual perforator to perform a small footplate fenestra. A prospective observational study. Otolaryngol Pol. 2021; 75:45-50. DOI
3. Tavernier LJM, Fransen E, Valgaeren H. Genetics of otosclerosis: finally catching up with other complex traits?. Hum Genet. 2022; 141:939-950. DOI
4. Szyfter W, Gawęcki W, Bartochowska A. Conductive hearing loss after surgical treatment of otosclerosis – long-term observations. Otolaryngol Pol. 2021; 75:1-6. DOI
5. Viza Puiggrós I, Granell Moreno E, Calvo Navarro C. Diagnostic utility of labyrinth capsule bone density in the diagnosis of otosclerosis with high resolution tomography. Acta Otorrinolaringol Esp (Engl Ed). 2020; 71:242-248. DOI
6. Wiatr A, Składzień J, Świeży K. A biochemical analysis of the stapes. Med Sci Monit. 2019; 25:2679-2686. DOI
7. American Academy of Otolaryngology – Head and Neck Surgery. Committee on Hearing and Equilibrium guidelines for the evaluation of results of treatment of conductive hearing loss. Otolaryngol Head Neck Surg. 1995; 113:186-187.
8. Skerry TM. The response of bone to mechanical loading and disuse: fundamental principles and influences on osteoblast/osteocyte homeostasis. Arch Biochem Biophys. 2008; 473:117-123. DOI
9. Sørensen MS, Frisch T, Bretlau P. Dynamic bone studies of the labyrinthine capsule in relation to otosclerosis. Adv Otorhinolaryngol. 2007; 65:53-58. DOI
10. Bloch SL. On the biology of the bony otic capsule and the pathogenesis of otosclerosis. Dan Med J. 2012; 59:B4524.
11. Wang YC, Xu WL, Lu YP. Investigation of nature of starting materials on the construction of hydroxyapatite 1D/3D morphologies. Mater Sci Eng C Mater Biol Appl. 2020; 108:110408. DOI
12. Lima AF, Moreira FC, Costa IE. Tinnitus and otosclerosis: an exploratory study about the prevalence, features and impact in daily life. Int Arch Otorhinolaryngol. 2021; 26:e390-395. DOI
13. Vallejo Valdezate LA, Martín Gil J, José-Yacamán M. Otosclerosis and Van der Hoeven’s syndrome: a contribution. Acta Otorrinolaringol Esp. 2001; 52:85-93. DOI
14. Vallejo-Valdezate LA, Martín-Gil J, José-Yacamán M. Scanning electron microscopy images and energy-dispersive x-ray microanalysis of the stapes in otosclerosis and Van der Hoeve syndrome. Laryngoscope. 2000; 110:1505-1510. DOI
15. Yoon Kim J, Bin Lee S, Ho Lee C. Hearing loss in postmenopausal women with low bone mineral density. Auris Nasus Larynx. 2016; 43:155-160. DOI
16. Gröschel M, Hubert N, Müller S. Age-dependent changes of calcium related activity in the central auditory pathway. Exp Gerontol. 2014; 58:235-243. DOI
17. Li X, Yu N, Sun J. Prophylactic effect of Ca2+ - deficient artificial perilymph perfusion on noise-induced hering loss. Chin Med J. 2003; 116:440-443.
18. Ikeda K, Kobayashi T, Kusakari J. Sensorineural hearing loss associated with hypoparathyroidism. Laryngoscope. 1987; 97:1075-1079. DOI
19. Joseph ADD, Sirisena ND, Kumanan T. Hypoparathyroidism, sensorineural deafness and renal disease (Barakat syndrome) caused by a reduced gene dosage in GATA3: a case report and review of literature. BMC Endocr Disord. 2019; 19:111. DOI
20. Ikeda K, Kobayashi T, Itoh Z. Evaluation of vitamin D metabolism in patients with bilateral sensorineural hearing loss. Am J Otol. 1989; 10:11-13.
21. Bigman G. Deficiency in vitamin D is associated with bilateral hearing impairment and bilateral sensorineural hearing loss in older adults. Nutr Res. 2022; 105:1-10. DOI
22. Naples JG. Calcium-channel blockers as therapeutic agents for acquired sensorineural hearing loss. Med Hypotheses. 2017; 104:121-125. DOI
23. Sendowski I, Holy X, Raffin F. Magnesium in the central nervous system [Internet]. University of Adelaide Press: Adelaide (AU); 2011.
24. Cao C, Oswald AB, Fabella BA. The CaV1.2 L-type calcium channel regulates bone homeostasis in the middle and inner ear. Bone. 2019; 125:160-168. DOI