Multiphoton imaging for morphometry of the sandwich-beam structure of the human stapedial annular ligament.

BACKGROUND: The annular ligament of the human stapes constitutes a compliant connection between the stapes footplate and the peripheral cochlear wall at the oval window. The cross section of the human annular ligament is characterized by a three-layered structure, which resembles a sandwich-shaped composite structure. As accurate and precise descriptions of the middle-ear behavior are constrained by lack of information on the complex geometry of the annular ligament, this study aims to obtain comprehensive geometrical data of the annular ligament via multiphoton imaging. METHODS: The region of interest containing the stapes and annular ligament was harvested from a fresh-frozen human temporal bone of a 46-years old female. Multiphoton imaging of the unstained sample was performed by detecting the second-harmonic generation of collagen and the autofluorescence of elastin, which are constituents of the annular ligament. The multiphoton scans were conducted on the middle-ear side and cochlear side of the annular ligament to obtain accurate images of the face layers on both sides. The face layers of the annular ligament were manually segmented on both multiphoton scans, and then registered to high-resolution µCT images. RESULTS: Multiphoton scans of the annular ligament revealed 1) relatively large thickness of the core layer compared to the face layers, 2) asymmetric geometry of the face layers between the middle-ear side and cochlear side, and variation of their thickness and width along the footplate boundary, 3) divergent relative alignment of the two face layers, and 4) different fiber composition of the face layers along the boundary with a collagen-reinforcement near the anterior pole on the middle-ear side. CONCLUSION AND OUTLOOK: Multiphoton microscopy is a feasible approach to obtain the detailed three-dimensional features of the human stapedial annular ligament along its full boundary. The detailed description of the sandwich-shaped structures of the annular ligament is expected to contribute to modeling of the human middle ear for precise simulation of middle-ear behavior. Further, established methodology in this study may be applicable to imaging of other middle-ear structures.

Gene expression analysis of human otosclerotic stapedial footplates.

Otosclerosis is a complex disease that results in a common form of conductive hearing loss due to impaired mobility of the stapes. Stapedial motion becomes compromised secondary to invasion of otosclerotic foci into the stapedio-vestibular joint. Although environmental factors and genetic causes have been implicated in this process, the pathogenesis of otosclerosis remains poorly understood. To identify molecular contributors to otosclerosis we completed a microarray study of otosclerotic stapedial footplates. Stapes footplate samples from otosclerosis and control patients were used in the analysis. One-hundred-and-ten genes were found to be differentially expressed in otosclerosis samples. Ontological analysis of differentially expressed genes in otosclerosis provides evidence for the involvement of a number of pathways in the disease process that include interleukin signaling, inflammation and signal transduction, suggesting that aberrant regulation of these pathways leads to abnormal bone remodeling. Functional analyses of genes from this study will enhance our understanding of the pathogenesis of this disease.

Detection of osteoprotegerin and TNF-alpha mRNA in ankylotic Stapes footplates in connection with measles virus positivity.

HYPOTHESIS: Otosclerosis is a bone remodeling disorder of the otic capsule causing conductive and sensorineural hearing loss. Persistent measles virus infection of the temporal bone with increased tumor necrosis factor (TNF)-alpha and decreased osteoprotegerin mRNA expression is supposed to be the main etiologic factor in otosclerosis. BACKGROUND: Determinants of measles virus infection and reactive inflammation were studied in otosclerosis. The presence of measles virus was shown in otosclerotic patients by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the viral RNA. No report is available, however, about the role and interactions of bone-specific cytokines in otosclerosis. METHODS: : Nucleic acid was extracted from stapes footplates of clinically otosclerotic patients. Measles virus nucleoprotein RNA was amplified by seminested RT-PCR. TNF-alpha and osteoprotegerin mRNA coexpression was detected by RT-PCR in otosclerotic bone and was correlated to measles virus positivity. RESULTS: Among 154 clinically stapes fixation otosclerotic patients, 99 stapedes contained measles virus RNA. TNF-alpha mRNA was detectable in 88 virus-positive and in 6 virus-negative stapes footplates. Osteoprotegerin mRNA expression was significantly lower in the TNF-alpha-positive specimens (P < .0001) that was independent from virus positivity. CONCLUSION: Detection of TNF-alpha mRNA demonstrates activated osteoclast functions and inflammatory pathways in otosclerotic stapes footplates associated with measles virus presence. Increased expression of TNF-alpha and its action on RANK production inhibits the protective functions of osteoprotegerin on normal bone turnover in the otic capsule.

Activated osteoclasts with CD51/61 expression in otosclerosis.

HYPOTHESIS: Stapes ankylosis is supposed to be a disease with variable histopathology caused by otosclerosis or pseudo-otosclerosis. Persistent measles virus infection of the otic capsule could induce reactivation of quiescent embryonic osteoclasts in otosclerosis. BACKGROUND: Presence of measles virus RNA was demonstrated in the footplates of otosclerotic patients by reverse-transcription polymerase chain reaction (RT-PCR). Histology of active otosclerosis is featured by the presence of numerous osteoclasts with unknown phenotype. METHODS: Nucleic acid was extracted from stapes footplates of clinically otosclerotic patients (n = 261). Genomic RNA of measles virus was amplified by RT-PCR. Amplification results were correlated to postoperative histologic and CD51/61 specific immunohistologic findings. A parallel alcalic phosphatase activity assessment was performed to evaluate the metabolic activity of osteoclasts in each section. RESULTS: Among 261 stapes fixation cases, 175 otosclerotic stapes contained measles virus RNA. Histology for virus negative stapes (n = 86) represented nonotosclerotic, degenerative disorders. Histologically confirmed otosclerosis was featured by the presence of osteoclasts with renewed, embryonic phenotype. In otosclerosis, alcalic phosphatase activity was significantly higher compared with nonotosclerotic stapes ankylosis (P < .001). CONCLUSION: The presence of CD51/61 positive osteoclasts in otosclerotic bone containing viral sequences provides the basis for an inflammatory bone remodeling disorder. Otosclerosis is a disease caused by persistent measles virus infection and reactivation of resting embryonic osteoclasts in the otic capsule.

Observation of the morphology and calcium content of vestibular otoconia in rats after simulated weightlessness.

Reduction in bone formation may have been the main reason for the lower calcium content of the otoconia after simulated weightlessness in rats. The head-ward distribution of blood volume may explain the morphological changes observed in the middle and inner ears. To observe morphological changes in the vestibular organs and measure the calcium content of otoconia in rats after simulated weightlessness. We used a tail suspension model of simulated weightlessness and then investigated changes in the vestibular organs using scanning electron microscopy and X-ray microanalysis. In comparison to untreated rats, the vestibular otoconia of the rats subjected to simulated weightlessness were small, irregularly shaped or fissured, and were arranged loosely and out of order. In addition, the calcium content of the otoconia was markedly decreased.

Safety of gold in stapes surgery.

Gold prostheses in middle ear surgery were found to have a higher extrusion rate than prostheses made from titanium. Incidences of deafness after insertion of a gold piston into the vestibule during stapes surgery have been observed. The aim of this study was to find out to what degree gold cations can diffuse from the prosthesis piston into the perilymph. For this, gold prostheses were incubated in artificial perilymph for four months, after which the gold content of the perilymph was analysed. As gold exhibits a special behaviour in complex fluids like the perilymph, a new analysing method was developed. The results show that gold does leak out of the pistons, that it can be reliably measured and that the amount of gold reaching the perilymph depends on the contact area. As the amount of gold measured in the perilymph stays far below the toxic level, it is very unlikely that the gold cations diffusing from a stapes prosthesis into the perilymph have a toxic effect on the inner ear hair cells. Inflammatory or allergic reactions to gold induced by direct tissue contact, however, cannot be excluded.

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.

[Otosclerosis and Van der Hoeven's syndrome: a contribution]. / Aportación al estudio de la otosclerosis y del síndrome de Van der Hoeve.

Morphological and microchemical changes that effect to the otosclerotic stape in the Van der Hoeve's syndrome were examined with a scanning electron microscope equipped with an energy dispersive X-ray fluorescence. Using the Ca/P ratio as criterion--measured by the characteristic x-ray fluorescence--it was shown that the Van der Hoeve stape had a higher Ca/P ratio (2.6:1) as compared to the normal stape (2:1). The Van der Hoeve's syndrome lesions as poorly mineralized, with low calcium salt and apparent increase of phosphates. This finding indicates a possible change from hydroxyapatite (or apatite) to brushite, which imply an acidification of bone.

Scanning electron microscopy images and energy-dispersive X-ray microanalysis of the stapes in otosclerosis and Van der Hoeve syndrome.

OBJECTIVE/HYPOTHESIS: The objective of this study was to evaluate the morphological and microchemical changes that affect sclerotic stapes in otospongiosis and van der Hoeve syndrome. METHODS: A scanning electron microscope equipped with an energy-dispersive x-ray analyzer was used in the experiments. RESULTS: In otosclerosis, focal lesions are poorly mineralized, with low calcium salt and reduced calcium-to-phosphorus (Ca/P) ratio (1.9:1). This finding correlates with a spongiotic type of lesion and indicates unstable mineralization with possible change from hydroxyapatite to calcium triphosphate. In van der Hoeve syndrome the presence of magnesium in stapes suggests osteoclastic function stimulation. The osteoclasts secrete many protons, causing an acidified microenvironment. Brushite is formed, and Ca/P ratio decreases in comparison with that of control patients (2.0:1 vs. 2.6:1).

[Content of fluoride and calcium in stapedial bone in otosclerosis]. / Zawartosc fluorków i wapnia w kosci strzemiaczka w otosklerozie.

The study dealing with the content of fluoride and calcium in stapedial bone and canal wall bone in otosclerosis was performed in 69 subjects (48 females and 21 males) out of the patients, in whom bone rebuilding activity had earlier been studied isotopically. The control group comprised 20 normal stapedies taken during autopsies. Fluoride content was determined by means of fluoride ions meter, the content of calcium was assessed by resorting to atomic absorptiometer. The content of studied elements was compared with the bone rebuilding activity and some clinical features such as the patient's age and duration of the disease. High fluoride content in otosclerotic stapes was revealed, being several times greater than in the bone of normal stapes. Concurrently the stapedial bone in otosclerosis contained less calcium as compared with the bone of normal stapes. In principle, that referred to otosclerotic focus and next to stapedial crura. The bones of stapedial footplates and otosclerotic foci with rebuilding activity lover han the means value had statistically significant, higher content of fluoride and calcium than the bones with greater rebuilding activity. Fluoride content in stapedial bone during otosclerosis dramatically increased with the patient's age and the length of the disease duration period, however, the calcium content had the tendency to decrease.

The role and significance of chondroitin sulfate in the development of otosclerosis.

Chondroitin sulfate is a sulfated glycosaminoglycan that predominates in the ground substance of cartilage. Using monoclonal antichondroitin sulfate in 61 specimens of human otosclerotic lesions, we studied the distribution of this glucosaminoglycan in various stages of otosclerosis. Our findings show that chondroitin sulfate plays an important role in the development of otosclerosis. In addition, the distribution of chondroitin sulfate clearly delineates the stage of otosclerosis referred to as active into two distinct histologic stages. Dividing the active stage into "osteolytic" and "sponge-chondroid" would be reasonable based on our findings.

Composition of the otosclerotic stapes: electron microprobe analyses.

For the first time, otosclerotic stapes have been distinguished from unafflicted controls at a high level of significance by using a spectrum of elements measured by energy-dispersive spectrometer-electron probe microanalyses (EDS/EPMA). Discriminant analyses of the maximum concentration of 13 elements measured at several sites within each of 32 stapes differentiated otosclerotic from unafflicted individuals well above the 95% confidence level. Eight of the 9 control (unafflicted) and 21 of the 23 afflicted stapes were correctly classified. In descending order of contribution to the discriminant function, the elements are Zn > Cr > K > Ca > Si > Mn > Na > Al > Mg > P > Fe > S > Ti. Zinc and chromium account for much of the difference, but discriminant analyses excluding them still distinguish the two groups at the 95% confidence level. These results are consistent with previous reports of high levels of alkaline phosphatase, a zinc-containing enzyme, in afflicted stapes. But the broad spectrum of elements capable of distinguishing otosclerotic stapes warrants study of additional zinc-containing and other metal-containing or metal-activated moieties.

Oversulfated mucopolysaccharides in the otosclerotic bone.

Mucopolysaccharides (glucosaminoglycans, GAGs) were extracted from otosclerotic and non-otosclerotic human stapes footplate, stapes superstructure and guinea pig stapes footplate. Cellulose acetate electrophoresis revealed an unknown highly anionic band (Rf = 1.05) beside the conventional hyaluronic acid, chondroitin sulfate and heparan sulfate fractions. Chemical analysis resulted in a high sulfate/hexosamine molar ratio in the otosclerotic GAG extract (1.7 and 1.9) and a very low ratio in non-otosclerotic human stapes and normal guinea pig stapes footplate GAG extract (0.20 and 0.21, respectively). Hexuronic acid content was highest in guinea pig stapes GAG preparation. Our results serve as indirect evidence of the presence of an oversulfated GAG fraction in the otosclerotic bone, which can potentially initiate otosclerotic bone resorption.