The origin of the stapes and relationship to the otic capsule and oval window.

BACKGROUND: The stapes, an ossicle found within the middle ear, is involved in transmitting sound waves to the inner ear by means of the oval window. There are several developmental problems associated with this ossicle and the oval window, which cause hearing loss. The developmental origin of these tissues has not been fully elucidated. RESULTS: Using transgenic reporter mice, we have shown that the stapes is of dual origin with the stapedial footplate being composed of cells of both neural crest and mesodermal origin. Wnt1cre/Dicer mice fail to develop neural crest-derived cartilages, therefore, have no middle ear ossicles. We have shown in these mice the mesodermal stapedial footplate fails to form and the oval window is induced but underdeveloped. CONCLUSIONS: If the neural crest part of the stapes fails to form the mesodermal part does not develop, indicating that the two parts are interdependent. The stapes develops tightly associated with the otic capsule, however, it is not essential for the positioning of the oval window, suggesting that other tissues, perhaps within the inner ear are needed for oval window placement.

Femtosecond laser ablation of the stapes.

A femtosecond laser, normally used for LASIK eye surgery, is used to perforate cadaveric human stapes. The thermal side effects of bone ablation are measured with a thermocouple in an inner ear model and are found to be within acceptable limits for inner ear surgery. Stress and acoustic events, recorded with piezoelectric film and a microphone, respectively, are found to be negligible. Optical microscopy, scanning electron microscopy, and optical coherence tomography are used to confirm the precision of the ablation craters and lack of damage to the surrounding tissue. Ablation is compared to that from an Er:YAG laser, the current laser of choice for stapedotomy, and is found to be superior. Ultra-short-pulsed lasers offer a precise and efficient ablation of the stapes, with minimal thermal and negligible mechanical and acoustic damage. They are, therefore, ideal for stapedotomy operations.

[Ontogenic development of the incudostapedial joint]. / Desarrollo ontogénico de la articulación incudoestapedial.

OBJECTIVE: To study the development of the incudostapedial joint in human embryos and foetuses. MATERIAL AND METHOD: 46 temporal bones with specimens between 9 mm and newborns were studied. The preparations were sliced serially and dyed using the Martins trichrome technique. RESULTS: The incudostapedial joint takes on the characteristics of a spheroidal joint at 16 weeks of development. The cartilage covering the articular surfaces is formed by different strata that develop in succession: the superficial stratum at 19 weeks, the transitional between 20 and 23 weeks, and the radial from 24 weeks on. The subchondral bone develops after 29 weeks by the mechanisms of apposition and extension of the periosteal and endosteal bones, but it is not until week 34 that it completely covers the articular surfaces, following constitution of the bone fascicles transmitting the lines of force. The articular capsule is formed from the inter-zone, the surface zone develops the capsular ligament, and the internal surface develops the synovial membrane. CONCLUSIONS: At the time of birth, the incudostapedial joint is completely developed.

Relation between renin-angiotensin-aldosterone system and otosclerosis: a genetic association and in vitro study.

OBJECTIVE/HYPOTHESIS: Angiotensin II (Ang II) may be implicated in the regulation of bone remodeling, and its activity is related to several gene polymorphisms including AGT M235T for plasmatic and tissular concentrations of angiotensinogen (AGT), ACE I/D for the angiotensin-converting enzyme activity, and AT(1)R A/C(1166) for the Ang II receptor function. The objective of this study was to investigate the implication of this hormone in otosclerosis. STUDY DESIGN: Prospective case-control study. METHODS: The above-mentionedpolymorphisms were investigated in 186 patients with otosclerosis and 526 healthy controls, both groups originated from the French Caucasian population. Primary cell cultures of stapedial bone from patients with otosclerosis (n = 6) and control subjects (n = 5) were investigated for the messenger ribonucleic acid expressions of Ang II receptors (Types 1 and 2) and cellular AGT and the effect of Ang II (10(-7) mol/L, 24 h) on the alkaline phosphatase activity and the interleukin-6 secretion in the culture media. RESULTS: A significant association was found between otosclerosis and the AGT M235T and the ACE I/D polymorphisms. Higher proportions of TT (29% versus 16%; p < 0.01) and DD (50% versus 38%; p < 0.05) genotypes were observed in cases versus controls. No association was found between the AT(1)R A/C(1166) polymorphism and otosclerosis. Ang II receptor Types 1 and 2 and AGT were detected in the cultures. Ang II increased the in vitro secretion of interleukin-6 and decreased the alkaline phosphatase activity only in otosclerotic cells. CONCLUSION: These observations suggest a relation between the local renin angiotensin system activity and otosclerosis, opening new therapeutic insights.

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.

High-resolution X-ray computed tomographic scanning of the human stapes footplate.

OBJECTIVES: The mechanical behaviour of the footplate and its annular ligament depends critically on their shape and orientation in the oval window, but accurate measurements have been difficult to make owing to their small size. Our aims are to visualize the footplate at high resolution and understand its dynamics. METHODS: The human cadaver stapes footplate was dissected, and very high-resolution x-ray computed tomographic (CT) scans, with voxel sizes from 4 to 8 pm, were performed. Locally developed software was used to reconstruct the images. RESULTS: The data permit us to explore minor details of shape and orientation. The footplate looks like a footprint, and the annular ligament has variable thickness, with a cleft (groove) in its anterior attachment to the oval window. The CT data also permit us to create a three-dimensional finite-element model that can simulate footplate motion. CONCLUSIONS: The results obtained lead to further understanding of the mechanical behaviour of the footplate and the annular ligament.

Increased activity of the diastrophic dysplasia sulfate transporter in otosclerosis and its inhibition by sodium fluoride.

HYPOTHESIS: This study investigates the function of the diastrophic dysplasia sulfate transporter (DTDST) in otosclerotic bone and the effect on it of sodium fluoride (NaF). BACKGROUND: Otosclerosis is a localized bone dystrophy with increased bone turnover. DTDST is implicated in the regulation of the bone turnover. MATERIALS AND METHODS: Primary cultures of cells were obtained from the stapes and external auditory canal (EAC) of 26 patients with otosclerosis and from nine control patients. Sulfate uptake was quantified under basal conditions and with NaF. The NaF signaling pathways were investigated using forskolin and verapamil. RESULTS: The relative initial rates of sulfate uptake and the apparent Vmax values were: otosclerotic stapes > EAC > control stapes = control EAC. The sulfate uptake by the otosclerotic stapes was correlated with the loss of sensorineural hearing. The amounts of DTDST mRNA (RNase protection assay) in the four subgroups did not differ. NaF (10(-6)M, 1 hr) inhibited sulfate uptake by the otosclerotic stapes and EAC cells but not by control samples. CONCLUSION: The authors believe that whether the increased DTDST activity is a cause or an effect of otosclerosis, it appears to be a specific target for NaF treatment.

Osteoblast-like cell cultures from human stapes.

OBJECTIVE: In order to develop new middle ear prostheses for ossicular reconstruction it is important to study how the recipient middle ear tissues, especially the stapes footplate and superstructure, react to the implanted biomaterial. In this respect, animal studies and cell cultures using non-specific cells are of limited value. MATERIAL AND METHODS: The morphology and growth pattern of cells cultured from human stapes were studied. Cultured cells were examined for the presence of alkaline phosphatase and were processed for immunocytochemistry in order to detect the presence of osteocalcine. Fibroblast cultures served as controls. RESULTS: Cultured stapes cells proliferated in a polygonal-cubic shape and without any regular pattern in the culture. These cells were shown to contain alkaline phosphatase and osteocalcine. Cultured fascia fibroblasts proliferated in a spindle-shaped form and in a pattern resembling a shoal of fish. Cultured fibroblasts did not contain alkaline phosphatase or osteocalcine. CONCLUSIONS: It is possible to culture osteoblasts from human stapes. These cells can be characterized as osteoblast-like cells by means of their external shape and the presence of alkaline phosphatase and osteocalcine. Using these cultures, specific in vitro investigations concerning the interaction of biomaterials and middle ear ossicles could be performed.

[Some structural characteristics of articulation of the incus and stapes in man]. / Nekotorye osobennosti stroeniia nakovalnestremennogo sochleneniia cheloveka.

Structural characteristics of human incudostapedial articulation (ISA) have been studied on a series of histological sections from temporal bone decalcified pyramids in three planes and by means of macro-micropreparation of tympanal structural elements from 96 human fetuses at various terms of gestation. It was established that the surface of the lenticular process has a spherical form. Its length surpasses its width. The articular surface on the head of the stapes presents a dome-shaped lacuna. The capsule is usually thicker in its posterior part than in the anterior one. It can endure deviation of the long lenticular process up to 3 mm without rupture. When the deviation overruns 3.5 mm, the articular bursa and round ligament of the base of the stapes break. This property of the capsule should be kept in mind when operating on the stapes.

Study on normal and otosclerotic bone cell cultures: an advance in understanding the pathogenesis of otosclerosis.

The authors first reviewed the main theories concerning the pathogenesis of otosclerosis and studied the morphologic and functional characteristics of cell cultures derived from normal and otosclerotic bones. Light transmission and scanning electron microscopy did not permit definite identification of the cultured cells as predominantly osteoblasts, nor did these techniques show significant differences between cultured cells derived from normal and pathologic bone. Functional tests of the cell cultures proved more interesting. First, the bony nature of the cultured cells was demonstrated by studying the intracellular 45Ca++ uptake after stimulation with calcitonin and dybutryl-cAMP. Second, cell cultures derived from otosclerotic bone behaved differently from those derived from normal bone. Their peak uptake of calcium appeared later, and post-stimulatory values were higher, suggesting that cells derived from otosclerotic bone store a greater quantity of 45Ca++. Furthermore, after stimulation with calcitonin and propranolol, we observed an inhibition of the calcium uptake and decreased intracellular cAMP levels in normal bone cell cultures. In contrast, the cell cultures derived from otosclerotic bone exhibited an initial inhibition of calcium absorption followed by massive calcium penetration. The response of adenylate cyclase to the action of Mg++, Ca++, and F- ions was evaluated in cultures derived from normal bone, otosclerotic bone, and normal skin fibroblasts. The resulting data show that activation due to Mg++ is much lower in cultured cells derived from otosclerotic bone than in those from either normal bone or skin fibroblasts. No significant differences were found after Ca++ inhibition in any of the cell cultures. Moreover, in cell cultures derived from normal bone, F- ions induced a strong activation that was lower than the levels observed in cultures of otosclerotic bone or in normal fibroblasts. We hypothesize that an alteration at the calcitonin receptor site is responsible for the difference in calcium uptake and cAMP levels observed in the cells derived from otosclerotic bone as compared to those cultured from normal cells.