Differences in otosclerotic and normal human stapedial osteoblast properties are normalized by alendronate in vitro.

OBJECTIVE: Identify and compare phenotypic properties of osteoblasts from patients with otosclerosis (OSO), normal bones (HOB), and normal stapes (NSO) to determine a possible cause for OSO hypermineralization and assess any effects of the bisphosphonate, alendronate. STUDY DESIGN: OSO (n = 11), NSO (n = 4), and HOB (n = 13) cultures were assayed for proliferation, adhesion, mineralization, and gene expression with and without 10(-10)M-10(-8)M alendronate. SETTING: Academic hospital. METHODS: Cultures were matched for age, sex, and passage number. Cell attachment and proliferation + alendronate were determined by Coulter counting cells and assaying tritiated thymidine uptake, respectively. At 7, 14, and 21 days of culture + alendronate, calcium content and gene expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were determined. RESULTS: OSO had significantly more cells adhere but less proliferation than NSO or HOB. Calcification was significantly increased in OSO compared to HOB and NSO. NSO and HOB had similar cell adhesion and proliferation rates. A dose-dependent effect of alendronate on OSO adhesion, proliferation, and mineralization was found, resulting in levels equal to NSO and HOB. All cultures expressed osteoblast-specific genes such as RUNX2, alkaline phosphatase, type I collagen, and osteocalcin. However, osteopontin was dramatically reduced, 9.4-fold at 14 days, in OSO compared to NSO. Receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG), important in bone resorption, was elevated in OSO with decreased levels of OPG levels. Alendronate had little effect on gene expression in HOB but in OSO increased osteopontin levels and decreased RANKL/OPG. CONCLUSIONS: OSO cultures displayed properties of hypermineralization due to decreased osteopontin (OPN) and also had increased RANKL/OPG, which were normalized by alendronate.

Gentamicin administration on the stapes footplate causes greater hearing loss and vestibulotoxicity than round window administration in guinea pigs.

Clinically, gentamicin has been used extensively to treat the debilitating symptoms of Mèniére's disease and is well known for its vestibulotoxic properties. Until recently, it was widely accepted that the round window membrane (RWM) was the primary entry route into the inner ear following intratympanic drug administration. In the current study, gentamicin was delivered to either the RWM or the stapes footplate of guinea pigs (GPs) to assess the associated hearing loss and histopathology associated with each procedure. Vestibulotoxicity of the utricular macula, saccular macula, and crista ampullaris in the posterior semicircular canal were assessed quantitatively with density counts of hair cells, supporting cells, and stereocilia in histological sections. Cochleotoxicity was assessed quantitatively by changes in threshold of auditory brainstem responses (ABR), along with hair cell and spiral ganglion cell counts in the basal and second turns of the cochlea. Animals receiving gentamicin applied to the stapes footplate exhibited markedly higher levels of hearing loss between 8 and 32 kHz, a greater reduction of outer hair cells in the basal turn of the cochlea and fewer normal type I cells in the utricle in the vestibule than those receiving gentamicin on the RWM or saline controls. This suggests that gentamicin more readily enters the ear when applied to the stapes footplate compared with RWM application. These data provide a potential explanation for why gentamicin preferentially ablates vestibular function while preserving hearing following transtympanic administration in humans.

Effect of angiotensin II on inflammation pathways in human primary bone cell cultures in otosclerosis.

INTRODUCTION: The aim of this study was to assess the expression and production of inflammation mediators in basal condition and after angiotensin II (AngII) in otosclerosis. MATERIALS AND METHODS: Human stapedial cell cultures (6 otosclerosis and 6 controls) were incubated with AngII (10(-7)M, 24 h) or vehicle. Cytokines and their mRNA expression were assessed by antibody and cDNA arrays. RESULTS: In basal conditions, otosclerotic cultures produced higher amounts of interleukin (IL)-1ß and interferon-inducible protein 10, and smaller amounts of tissue inhibitor of metalloproteinase 2. AngII promoted inflammation by increasing interferon γ and IL-10, and by decreasing macrophage inflammatory protein 1α and soluble tumor necrosis factor receptor II. CONCLUSIONS: Otosclerotic cultures produced higher proinflammatory cytokines in basal condition. AngII appeared to promote inflammation via these mediators in otosclerosis.

Bone conduction activation through soft tissues following complete immobilization of the ossicular chain, stapes footplate and round window.

Classically it has been thought that bone conduction activation at the mastoid leads to relative motion between the stapes footplate and the oval window due to inertial and to compression (distortion) mechanisms. However, several recent clinical findings and experimental manipulations may point to additional mechanisms. These manipulations were extended in the present study. In ten fat sand rats, following obliteration of one ear, auditory nerve brainstem evoked response (ABR) thresholds were recorded in response to broad band click stimuli, either air conducted (AC) through insert earphones or bone conducted (BC) delivered directly to the exposed skull bone. Following this, the entire ossicular chain, stapes footplate and round window were completely immobilized with super glue, leading to a mean AC threshold elevation of 44 dB, but to a mean BC threshold change (elevation) of only 3.5 dB. In this state of complete immobilization, the bone vibrator was applied to a pool of saline in the surgical area and ABR was elicited with a mean threshold which was not significantly different from that of the BC threshold. When the bone vibrator was then applied to the eye without touching the bone at the orbit, the resulting ABR threshold was about 20 dB greater than the BC threshold. In conclusion, BC stimulation can activate the cochlea without two mobile windows. Furthermore, the cochlea can be activated by a fluid pathway and by application of a bone vibrator to non-osseous sites (soft tissue conduction).

The effect of treatment with vincristine on transient evoked and distortion product otoacoustic emissions.

OBJECTIVE: Vincristine chemotherapy is mainly associated with neurotoxic effects. The ototoxicity of vincristine has been related to high dosage, while low and moderate doses do not seem to induce significant hearing impairment when measured by pure tone or speech audiometry. Otoacoustic emissions have been reported to be more sensitive in early detection of ototoxicity than conventional pure tone audiometry. The present study was directed at determining whether vincristine treatment interferes with outer hair cell function in the absence of measurable changes in pure tone audiometry. METHODS: We studied prospectively a cohort of ten children suffering from leukemia. All children were subjected to tympanogram, stapedial muscle reflex, pure tone audiometry, transient evoked (TEOAEs) and distortion product (DPOAEs) otoacoustic emissions on day 1 and on day 22 of treatment with vincristine. TEOAEs were analyzed in terms of emission level and reproducibility as a function of frequency. DPOAEs were obtained as DP-grams and were analyzed in terms of amplitude. RESULTS: The analyzed parameters of TEOAEs and DPOAEs revealed a declining tendency, although changes did not reach statistical significance. Pure tone audiometry and stapedial reflex thresholds were not altered. CONCLUSION: For the population of this study, vincristine did not seem to cause significant alterations of otoacoustic emissions' recordings and consequently significant outer hair cell damage.

[Biomaterial studies in cultures of human stapedial bone-like cells]. / Untersuchung unterschiedlicher Biomaterialien in einer Knochenzellkultur des menschlichen Steigbügels.

BACKGROUND: Cell culture studies may provide information on the behavior of biomaterials in the intended implant environment. Cell cultures from such an environment could be used for the development of middle ear implants. MATERIAL AND METHODS: Secondary bone-like cell cultures derived from human stapes were exposed to different materials [Al(2)O(3) ceramic, glass ceramic (Ceravital), gold and titanium]. Proliferation was studied for up to 40 days. RESULTS: The proliferation of cultured stapes bone-like cells did not differ significantly between the four tested biomaterials. The well known cytotoxic effect of copper, which was used as a control, was evident. CONCLUSIONS: Four biomaterials [Al(2)O(3) ceramic, glass ceramic (Ceravital), gold and titanium] have similar biocompatibility and no toxicity when tested in human stapes cell cultures. This in vitro model may be of considerable value for the further development of middle ear implants, e.g., when coated with bone morphogenetic proteins.

Effect of 17 beta-estradiol on diastrophic dysplasia sulfate transporter activity in otosclerotic bone cell cultures and SaOS-2 cells.

OBJECTIVE: Diastrophic dysplasia sulfate transporter (DTDST) is involved in the regulation of bone turnover, and its activity in otosclerosis has been shown to be abnormally high. Taking into account the role of estrogens in the progression of otosclerosis, the possible effect of estrogens on DTDST was investigated in otosclerotic bone cell cultures and in SaOS-2, a human osteoblastic cell line. MATERIAL AND METHODS: Primary bone cell cultures of stapes and external auditory canal (EAC) bone were obtained from 33 patients with otosclerosis and 18 control patients undergoing cerebellopontine angle tumor surgery. These cultures were assessed in parallel with SaOS-2 cells. Estrogen receptors (ERs) were detected using reverse transcriptase polymerase chain reaction. DTDST activity was assessed by sulfate uptake at baseline and after 24 h of incubation with 17 beta-estradiol at concentrations ranging from 10(-12) to 10(-6) M. RESULTS: Stapes and EAC cultures predominantly expressed mRNA of ER alpha, while ER beta expression was predominant in SaOS-2 cells. In stapes and EAC cultures no modification of DTDST activity was observed with 10(-8) M 17 beta-estradiol. In SaOS-2 cells, DTDST activity was inhibited by 17 beta-estradiol (93.5+/-9.21 vs 83.6+/-8.83 pmol/mg protein/5 min, n=29; mean of differences=10.0+/-3.22, paired t-test, p<0.01). CONCLUSION: DTDST activity is regulated by estrogens in SaOS-2 cells, but not in primary cell cultures from stapes and EAC. This difference in the regulation mechanisms may be related to the type of estrogen receptor expressed.

Comparing in vitro, in situ, and in vivo experimental data in a three-dimensional model of mammalian cochlear mechanics.

Normal mammalian hearing is refined by amplification of the motion of the cochlear partition. This partition, comprising the organ of Corti sandwiched between the basilar and tectorial membranes, contains the outer hair cells that are thought to drive this amplification process. Force generation by outer hair cells has been studied extensively in vitro and in situ, but, to understand cochlear amplification fully, it is necessary to characterize the role played by each of the components of the cochlear partition in vivo. Observations of cochlear partition motion in vivo are severely restricted by its inaccessibility and sensitivity to surgical trauma, so, for the present study, a computer model has been used to simulate the operation of the cochlea under different experimental conditions. In this model, which uniquely retains much of the three-dimensional complexity of the real cochlea, the motions of the basilar and tectorial membranes are fundamentally different during in situ- and in vivo-like conditions. Furthermore, enhanced outer hair cell force generation in vitro leads paradoxically to a decrease in the gain of the cochlear amplifier during sound stimulation to the model in vivo. These results suggest that it is not possible to extrapolate directly from experimental observations made in vitro and in situ to the normal operation of the intact organ in vivo.

Middle ear anomalies induced by hypertriazene administration in the mouse.

The middle ear is derived from various embryonic tissues. Many experiments using teratogens have been performed, employing the difference of each tissue's sensitivity to the teratogen and the tissue's critical time of development. Triazene, a foliate metabolism antagonist, produced anomalies in fetuses that resembled those associated with thalidomide in humans, the so-called the first and second branchial syndrome. In our experiment, we administrated 3,3-dimethyl-1-phenyltriazene, an inductor of triazene, to pregnant mice at 7 to 14 days of gestation resulting in unique fetal anomalies. We examined the development of the stapes, stapedial artery, facial nerve, and oval window using an optical microscopic and three-dimensional reconstruction. Middle ear and facial nerve anomalies in mice depend on the gestation day when triazene is administrated. The stapedial artery, oval window, facial nerve (horizontal segment), stapes footplate, and styloid process are affected on the 9th to 11th administration day, the annular stapedialis on the 10th to 11th day, and the malleus and incus on the 9th to 11th day. The use of the Vox View/Mac, allowed us to create three-dimensional pictures from two-dimensional slides providing an improved understanding of the relationships between anatomical structures.

Flavonoids alter bone-remodelling in auditory ossicle organ cultures.

The beneficial role of bioflavonoids in an otosclerosis-like bone-remodelling process can be implicated from its interference with bone resorption induced by prostaglandin E2 (PGE2) in cultured guinea pig ossicles. Ipriflavone (7-isopropoxy-isoflavon) and quercetin reduced PGE2-elevated collagenase-like peptidase (Cl-peptidase) activity and potentiated a PGE2-induced decrease in collagen synthesis. The fact that PGE2 effects are mediated through cyclic AMP in bone turnover and flavonoids act synergistically with PGE2 in collagen synthesis confirm a cyclic AMP phosphodiesterase inhibitory role of flavonoids. It has already been attempted to use Ipriflavone medical treatment of otosclerosis. Quercetin, which has a better than Ipriflavone water-solubility seems as promising as Ipriflavone in the control of the otosclerotic bone-remodelling disturbance.

The effect of flavone treatment on human otosclerotic ossicle organ cultures.

We studied the effect of 7-isopropoxy-isoflavone (Ipriflavone) on the collagen synthesizing activity in human otosclerotic auditory ossicle samples from whole organ cultures during incubation for 96 h, and compared this effect with that found in normal meatal cortical bone. Ipriflavone led to a dose-dependent increase in the collagen synthesizing activity in both the healthy and the otosclerotic bone samples. At the highest Ipriflavone concentration used (50 microM), collagen synthesis increased 6-fold in the cortical bone and 9-fold in the otosclerotic bone, as compared with untreated controls. These findings indicate that the otosclerotic bone cavities are filled in vitro with organic matrix.