Patterns of Bone Damage in Patients With Chronic Middle Ear Inflammation.

BACKGROUND: Despite different etiologies, chronic otitis media involves the damaging and restructuring of bone tissue. The inflammatory process destroys elements of the ossicular chain, and bone lesions may appear that allow the development of otogenous complications. AIMS/OBJECTIVES: A correlation between the degree of damage to the ossicular chain as well as the bony walls of the middle ear and the type of chronic inflammatory lesions was sought. Destructive changes to bones were observed using scanning microscopy. MATERIAL AND METHODS: The removed damaged fragments of the ossicles were prepared for evaluation with a scanning microscope. Preparations were sputter-coated with a thin layer of gold and subsequently evaluated. RESULTS: Of 220 surgeries carried out in the discussed period, destruction of the middle ear bone walls, opening the way for the development of intracranial complications, was found in 27 patients. Most of them had ongoing chronic otitis media with granulation. CONCLUSIONS: (1) Bone loss of the skull base was observed more frequently in patients with chronic otitis media with granulation than with cholesteatoma. (2) In chronic otitis media with cholesteatoma, damage to the ossicular chain was observed significantly more frequently than in the case of otitis media with granulation.

Macroscopic description of the external and middle ear of paca (Cuniculus paca Linnaeus, 1766) / Descrição macroscópica da orelha externa e media da paca (Cuniculus paca Linnaeus, 1766)

Paca (Cuniculus paca), one of the largest rodents of the Brazilian fauna, has inherent characteristics of its species which can conribute as a new option for animal experimantation. As there is a growing demand for suitable experimental models in audiologic and otologic surgical research, the gross anatomy and ultrastructural ear of this rodent have been analyzed and described in detail. Fifteen adult pacas from the Wild Animals Sector herd of Faculdade de Ciências Agrárias e Veterinárias, Unesp-Jaboticabal, were used in this study. After anesthesia and euthanasia, we evaluated the entire composition of the external ear, registering and ddescribing the details; the temporal region was often dissected for a better view and detailing of the tympanic bulla which was removed and opened to expose the ear structures analyzed mascroscopically and ultrastructurally. The ear pinna has a triangular and concave shape with irregular ridges and sharp apex. The external auditory canal is winding in its path to the tympanic mebrane. The tympanic bulla is is on the back-bottom of the skull. The middle ear is formed by a cavity region filled with bone and membranous structures bounded by the tympanic membrane and the oval and round windows. The tympanic membrane is flat and seals the ear canal. The anatomy of the paca ear is similar to the guinea pig and from the viewpoint of experimental model has major advantages compared with the mouse ear.

A paca (Cuniculus paca), um dos maiores roedores da fauna brasileira, possui características inerentes à sua espécie que podem contribuir como uma nova opção de animal experimental; assim, considerando-se que há crescente busca por modelos experimentais apropriados para pesquisas audiológicas e otológica cirúrgicas foram analisados e descritos em detalhes a anatomia macroscópica e ultraestrutural da orelha desse roedor. Para o estudo, utilizaram-se 15 animais adultos provenientes do plantel do Setor de Animais Silvestres da Faculdade de Ciências Agrárias e Veterinárias, Unesp-Jaboticabal, Jaboticabal/SP. Após anestesia e eutanásia, avaliou-se toda a composição da orelha externa, registrando-se e descrevendo-se os detalhes, também se dissecou a região temporal para melhor visibilização e detalhamento da bula timpânica e estas foram removidas e abertas a fim de expor as estruturas da orelha, as quais foram analisadas, macroscopicamente e ultraestruturalmente. O pavilhão auricular apresenta forma triangular e côncava com cristas irregulares e ápice pontiagudo; o conduto auditivo externo é sinuoso em seu trajeto até a membrana timpânica; a bula timpânica encontra-se na parte posterior-inferior do crânio; a orelha média é formada por uma região cavitária preenchida por estruturas ósseas e membranosas. É delimitada pela membrana timpânica e as janelas redonda e oval, sendo a membrana timpânica de forma plana e que veda todo o conduto auditivo. A anatomia da orelha da paca é semelhante à da cobaia e do ponto de vista de modelo experimental apresenta grandes vantagens em comparação com a orelha do rato.

Osteocyte apoptosis and absence of bone remodeling in human auditory ossicles and scleral ossicles of lower vertebrates: a mere coincidence or linked processes?

Considering the pivotal role as bone mechanosensors ascribed to osteocytes in bone adaptation to mechanical strains, the present study analyzed whether a correlation exists between osteocyte apoptosis and bone remodeling in peculiar bones, such as human auditory ossicles and scleral ossicles of lower vertebrates, which have been shown to undergo substantial osteocyte death and trivial or no bone turnover after cessation of growth. The investigation was performed with a morphological approach under LM (by means of an in situ end-labeling technique) and TEM. The results show that a large amount of osteocyte apoptosis takes place in both auditory and scleral ossicles after they reach their final size. Additionally, no morphological signs of bone remodeling were observed. These facts suggest that (1) bone remodeling is not necessarily triggered by osteocyte death, at least in these ossicles, and (2) bone remodeling does not need to mechanically adapt auditory and scleral ossicles since they appear to be continuously submitted to stereotyped stresses and strains; on the contrary, during the resorption phase, bone remodeling might severely impair the mechanical resistance of extremely small bony segments. Thus, osteocyte apoptosis could represent a programmed process devoted to make stable, when needed, bone structure and mechanical resistance.

Mice with vestibular deficiency display hyperactivity, disorientation, and signs of anxiety.

Previous studies revealed that vestibular cues are crucial for exploration in the absence of visual cues. The working hypothesis of this study was, accordingly, that mice with vestibular dysfunction would become disoriented or unable to globally explore an unfamiliar environment. In 2- and 3-month-old mutant headbanger (Hdb) mice, stereocilia of hair cells are abnormally elongated, yet maintain partial staircase arrangement, suggesting some spared vestibular function at these ages. Here we tested a group of 3-month-old mutant Hdb and a group of non-mutant mice obtained from the same litters (Wt mice). Each individual mouse was introduced into a dark 120 cm x 120 cm arena and its behavior was followed for 10 min. Hdb mice were hyperactive and appeared to engage in local exploration, traveling in a restricted zone for a while and then shifting to travel in another zone. In contrast, Wt mice traveled across zones incessantly with fewer visits to recently entered zones. Thus, Hdb seemed to display local compared with the global exploration of Wt mice, indicating that they were less oriented in the global environment. In addition, Hdb exhibited numerous stretch-attends, which is suggested as a sign of elevated anxiety. Altogether, the three comorbidities of hyperactivity, anxiety, and disorientation can be presented as a syndrome associated with vestibular deficiency in this animal model, and serve in studying vestibular deficiency in humans.

Scanning electron microscopic study of the human auditory ossicles.

The human mallei, incudes and stapedes from 34 cadavers were examined using scanning electron microscope (SEM) to compare the bone surface type among different regions of auditory ossicles for males and females. On the malleus of both males and females, almost all of the surfaces showed a smooth fibrous appearance, characteristic of resting surface. Limited bone-forming or resorbing surfaces were identified on the malleus. As compared with the malleus, the percentage area of the resorbing surface and the vascular canal openings were higher on the incus and stapes, especially on the long process (Crus longum) of the incus and the neck of the stapes for both males and females. The percentage area occupied by the resorbing surface of the long process of the incus and the neck of the stapes correlated with that of the vascular canal openings. We consider that the malleus maintained the stable condition, while the long process of the incus and the neck of the stapes demonstrated marked bone resorption. We suppose that the bone erosion may be related to the vascularization in these regions. Though the percentage area of the resorbing surface and the vascular canal openings had the tendency to be high in females, we did not find any significant differences between the males and females. There was no significant correlation between the age and the area of resorbing surface or vascular canal openings.

[The morphological study of eroded auditory ossicles by cholesteatoma].

OBJECTIVE: To observe the morphology of eroded auditory ossicles obtained in middle ear surgery for cholesteatoma and to investigate the mechanism of bone erosion in cholesteatoma. METHOD: The morphology of eroded auditory ossicles in 8 cholesteatoma cases and 2 normal cases were observed with light microscopy. The ultrastructure of eroded auditory ossicles in 5 cholesteatoma cases and the ultrastructure of control bones in external ear canal of 2 cases were observed and compared with transmission electron microscopy. RESULT: Osteomyelitis and multinucleate osteoclasts with ruffled borders were observed in the eroded auditory ossicles of cholesteatoma. Intramembranous and endochondral ossification were both observed. The obvious bone destruction and remodeling were observed consistently. CONCLUSION: Osteoclasts and Osteomyelitis are both responsible for bone destruction in cholesteatoma. Intramembranous and endochondral ossification may co-participate in bone remodeling. Osteogenesis is also a basic pathologic phenomena in cholesteatoma. The obvious bone destruction and remodeling can coexist in cholesteatoma cases.

Should we use ossicular remnants in ossicular reconstruction following cholesteatoma removal?

The remnants of the acoustic ossicles have been used in ossicular reconstruction during mastoid surgery for many decades. The present study assessed the status of the acoustic ossicles in 114 patients (57 with cholesteatoma and 57 without) during surgery for chronic otitis media using the operating microscope. In 52 cases, the ossicles (malleus and/or incus) were assessed using both the surgical and scanning electron microscope in order to reveal any erosions and compare the findings. From the 57 operated ears with cholesteatoma, 45 (79%) had ossicular erosion whereas 12 (21%) did not. In the group of 57 operated ears with chronic otitis media without cholesteatoma, 33 (58%) had ossicular erosion whereas 24 (42%) did not. This difference was statistically significant (p = 0.02). With regard to the 52 operated cases who were studied with both microscopes, in the cholesteatoma patients the surgical microscope was not able to reveal any ossicular erosions in 39% of the cases, whereas the scanning electron microscope revealed moderate or severe erosions in the same ears. This suggests that the operating microscope is not reliable enough to determine if ossicular remnants can be used in ossicular reconstruction following cholesteatoma surgery. There is a considerable risk that epithelia or other cholesteatoma particles remain in the areas of erosions that cannot be seen with the operating microscope. The use of such ossicular remnants may lead to cholesteatoma recurrence and failures in mastoid surgery. Therefore, autoclaving or alternative prosthesis may be considered in such cases.

Morphological effects of nanosecond- and femtosecond-pulsed laser ablation on human middle ear ossicles.

We evaluate the feasibility of nanosecond-pulsed and femtosecond-pulsed lasers for otologic surgery. The outcome parameters are cutting precision (in micrometers), ablation rate (in micrometers per second), scanning speed (in millimeters per second), and morphological effects on human middle ear ossicles. We examine single-spot ablations by a nanosecond-pulsed, frequency-tripled Nd:YAG laser (355 nm, beam diameter 10 microm, pulse rate 2 kHz, power 250 mW) on isolated human mallei. A similar system (355 nm, beam diameter 20 microm, pulse rate 10 kHz, power 160-1500 mW) and a femtosecond-pulsed CrLi:SAF-Laser (850 nm, pulse duration 100 fs, pulse energy 40 microJ, beam diameter 36 microm, pulse rate 1 kHz) are coupled to a scanner to perform bone surface ablation over a defined area. In our setups 1 and 2, marginal carbonization is visible in all single-spot ablations of 1-s exposures and longer: With an exposure time of 0.5 s, precise cutting margins without carbonization are observed. Cooling with saline solution result is in no carbonization at 1500 mW and a scan speed of 500 mms. Our third setup shows no carbonization but greater cutting precision, although the ablation volume is lower. Nanosecond- and femtosecond-pulsed laser systems bear the potential to increase cutting precision in otologic surgery.

Joint formation in the middle ear: lessons from the mouse and guinea pig.

The malleus, incus and stapes form an ossicle chain in the mammalian middle ear. These ossicles are articulated by joints that link the chain together. In humans and mice, fusion of the ossicles leads to hearing loss. However, in the adult guinea pig the malleus and incus are normally found as a single complex. In this report, we investigate how the malleus and incus form during mouse and guinea pig development. The murine malleus and incus develop from a single condensation that splits to form the two ossicles. Even before a morphological split, we show that the ossicles have distinct genetic identities and joint markers are expressed. In the guinea pig embryo, joint formation is initiated but no cavitation is observed, resulting in a single complex divided by a thin suture. The malleal-incudo complex in the guinea pig is, therefore, not caused by a defect in joint initiation.

Developing macroporous bicontinuous materials as scaffolds for tissue engineering.

Calcareous skeletal elements (ossicles) isolated from the seastar, Pisaster giganteus, were characterized and tested as potential biocompatible substrates for cellular attachment. These ossicles have a remarkably robust open-framework architecture with an interconnected network of ca. 10 microm diameter pores. Scanning electron and confocal microscopy was used to characterize the cell-substrate interaction. Cell culturing experiments revealed that the cells firmly attach to the ossicle surface, forming cell aggregates of several layers thick. The anchored cells extended to form 'bridges' between the openings in the bicontinuous framework and the degree of coverage increased as culture time progressed. Osteoblasts grown on the ossicles were found to be viable up to 32 days after initial seeding, as proven by assaying with AlamarBlue and FDA/PI staining indicating the ossicle's potential as an alternative highly effective tissue scaffold. Given the limitation in availability of this natural material, the results presented here should be seen as offering guidelines for future development of synthetic materials with physical and chemical properties strongly conducive to bone repair and restoration.

Histopathology of biocompatible hydroxylapatite-polyethylene composite in ossiculoplasty.

The biocompatibility of hydroxylapatite-polyethylene composite implants (HAPEX, Smith and Nephew) was investigated in this study. Eleven middle ear prostheses, removed during revision surgery, have been examined by light microscopy, transmission electron microscopy and scanning electron microscopy. The results show that the middle ear prostheses become overgrown by fibrous tissue inside a thin epithelial layer. In some cases the prosthesis is covered by fibrous tissue without an outer layer of epithelial cells. No arguments have been found for the accumulation of macrophages and foreign body giant cells associated with a foreign body reaction to the material. Therefore, hydroxylapatite-polyethylene composite implants (HAPEX, Smith and Nephew) would seem to be very suitable for reconstructive middle ear surgery.

Auditory ossicle abnormalities and hearing loss in the toothless (osteopetrotic) mutation in the rat and their improvement after treatment with colony-stimulating factor-1.

Osteopetrosis describes a group of skeletal metabolic diseases of heterogeneous etiology and varied severity that produces a generalized accumulation of skeletal mass, the result of reduced bone resorption. Inherited in a variety of species including humans, the most severe forms are lethal. Among common features are progressive blindness and deafness of controversial etiologies for which there are no universally effective treatments. We have studied the auditory responsiveness and auditory ossicle quantitative histomorphology and temporal bone vasculature in the toothless (tl) rat, a lethal osteopetrotic mutation with few osteoclasts, very low bone turnover, and limited angiogenesis in the axial skeleton. Compared with normal littermates, 3-week-old mutants showed significantly reduced auditory responsiveness, a hearing loss due to abnormalities in both form and tissue composition of the stapes, and little capillary sprouting in the vascular bed of the temporal bone. Treatment of mutants with colony-stimulating factor 1 (CSF-1), known to greatly reduce sclerosis in the axial skeleton, significantly improved hearing, stapedial form and tissue composition, and angiogenesis in the temporal bone. In normal rats, the stapes consisted of 89.3% bone, 9.1% mineralized cartilage, and 0.8% porosity. In osteopetrotic rats, the stapes consisted of 48.3% bone, 35.9% mineralized cartilage, and 15.9% porosity, while after CSF-1 treatment, the bone content increased to 55.2%, cartilage was decreased to 21.7%, and porosity increased to 23.0%, respectively. This is the first demonstration of an auditory abnormality in an osteopetrotic animal mutation and shows that the hearing loss in tl rats can be significantly improved following treatment with CSF-1.

Radiologic and mechanical properties of inactivated ossicle homografts.

OBJECTIVE: This study examined the effects of old and new inactivation (sterilization) techniques on the radiologic and mechanical properties of ossicle homografts. MATERIALS AND METHODS: Ninety normal incuses and malleuses received either treatment with 1) 5% formaldehyde/cialit, 2) 1N NaOH, 3) 0.9% LpH, or 4) autoclaving at 134'C, or no treatment. All ossicles were assessed radiologically by high-resolution computed tomography. After imaging, all ossicles underwent mechanical testing by destructive axial compression in a mechanical testing machine measuring force and displacement. RESULTS: Ossicles treated with cialit, NaOH, or autoclaving showed a significant decrease of ultimate force and stiffness compared with controls. LpH treatment caused no such changes in these structural properties. Material properties of yield strength, ultimate strength, and elastic modulus were also altered by cialit, NaOH, and autoclaving, but were much more difficult to assess because of uncertainty in parameter estimates. There was a significant increase in radiologic density in autoclaved ossicles, a reduction in cialit- and LpH-treated ossicles, and no change in NaOH-treated ossicles. CONCLUSIONS: All tested inactivation procedures changed the biomechanical and/or radiologic properties of ossicle homografts. However, the new procedures used to inactivate infectious agents produced changes similar to the older treatments with formaldehyde/cialit. Human allografts are able to withstand harsh but safe sterilization procedures. The NaOH treatment seems to be the most suitable method for the future. The biologic (osteogenic) potentials of ossicle homografts treated with these new preservation/inactivation methods are still unknown. Further investigations are necessary to re-evaluate the clinical use of ossicle homografts in middle ear reconstructive surgery.

Bone resorption in human cholesteatoma: morphological study with scanning electron microscopy.

The detailed mechanism of bone resorption in cholesteatoma was investigated by means of eroded ossicles obtained during middle ear surgery for cholesteatoma. In the light microscopic study, multinucleate osteoclasts with ruffled borders were found in contact with the eroded bone, which appeared to be osteoclastic lacunae. Scanning electron microscopy revealed the lacunae to be many absorption bays, 30 to 100 microns in diameter, clustered on the surface of eroded areas. Numerous cone-shaped stubs of digested collagen fiber bundles, consisting of scores of collagen fibrils and degenerated extracellular organic substances, were visible at higher magnification on the bottom of the absorption bay. The pattern of fusion and twining of the disarranged collagen fibrils at the top of the partly digested fiber bundles was clearly rendered by the alkali-water maceration method for scanning electron microscopy. We infer from the morphological evidence that osteoclastic resorption may be one of the major mechanisms of bone destruction in cholesteatoma, and that demineralization and degeneration of extracellular organic substances precede disruption of collagen fibrils at the front of bone resorption.

Bone tissue kinetics in the surgically disarticulated dog incus.

The incus of the right ear from 4 growing mongrel dogs was surgically disarticulated and left in the middle ear space. The external auditory canal was then filled with teflon paste and sutured. After 6 weeks (D-6 group) and 13 weeks (D-13 group) the animals were sacrificed. The right experimental incus and the left control one were embedded in methyl methacrylate and sectioned in single 50-microns-thick sections according to the principal axis of the two processes. On the microradiographs of each section we evaluated the thickness of the body and of both processes and the percentage area of the primary channels of the secondary osteons and that of the appositional bone tissue. The thickness of the body and of the two processes was more pronounced in all the experimental incuses, in which 6% (in D-6) and 8% (in D-13) of the total area were occupied by new appositional woven bone. In the body of the D-13 group, 9% of the pre-existing bone was substituted by secondary osteons. The results indicate that the incus react to the variations of mechanical stimuli.

The cellular mechanism of ossicular erosion in chronic suppurative otitis media.

This is the first report of the application of a new examination technique for the assessment of cellular activity during bone resorption in chronic suppurative otitis media (CSOM). A total of nineteen includes removed during the course of tympanomastoid surgery were studied (retraction pocket: 2; tubo-tympanic CSOM: 4; attico-antral CSOM: 13). The microscopic surface topography of each specimen was examined using the scanning electron microscopy (SEM), and the appearances are interpreted in terms of cortical cellular activity. The results suggest that the mechanism of ossicular erosion in CSOM is similar regardless of the exact type of disease. Extensively pitted areas were seen in all specimens. These pits are morphologically indistinguishable from those characteristic of osteoclastic activity (Howship's lacunae). We conclude that in all causes the surface topography of eroded incudes is consistent with the activity of osteoclasts.

Ossicular erosion by cholesteatoma: investigation by scanning electron microscopy utilizing a new preparation technique.

A new technique for removing the soft tissues from middle ear ossicles to prepare them for scanning electron microscopy is described. The technique involved use of a solution of hypochlorite. The bony surface of normal ossicles was studied as a control group after preparation by the technique and no morphological distortion was observed. Ossicles eroded by cholesteatoma were then studied, and we propose that the erosion occurs in three stages; pumicing, pitting and cavitating. The mucoperiosteum of normal ossicles and otosclerotic foci were also studied.

Use of the excimer laser in stapes surgery and ossiculoplasty of middle ear ossicles: preliminary report of an experimental approach.

Surgery of the stapes may cause a number of complications, including hearing deficits and balance disorders. This has made it necessary to look for improved techniques. Small-fenestra stapedotomy has recently been popularized. Lasers have been advocated for use in fenestrating the stapes footplate. On the other hand, sculpting the middle ear ossicles during tympanoplasty is often necessary for the reconstruction of the ossicular chain and the improvement of sound conduction. Using an excimer laser with a wavelength of 193 nm, fenestrations of the footplate and ossicular sculpting were performed on ossicles obtained during ear surgery and from human cadaver temporal bones. The results indicate that the excimer laser can be used effectively and accurately on an experimental basis and that further research is needed before this method can be used for clinical purposes.

Landmark structures to approach the internal auditory canal: a dimensional study related to the middle cranial fossa approach.

For the purpose of assisting the accurate topological identification of the internal auditory canal (IAC) during surgery through the middle cranial fossa, we conducted a dimensional study around the IAC using histologically prepared temporal bones. The anterior margin of the porus acoustics, the posterior margin of the porus acousticus, and the center of the fundus were located 19.7 mm, 16.8 mm, and 8.2 mm from the center of the malleus head, respectively. The angles formed by lines connecting those three points of the IAC and the ossicles were also measured. These values could be of help to identify the accurate localization of the IAC using the ossicles as surgical landmarks, when available. The common crus, i.e. a critical inner ear structure for hearing preservation, was also studied. The distance from the medial side of the common crus to the posterior wall of the IAC was relatively constant. However, the distance from the medial side of the common crus to the surface of the posterior cranial fossa was variable. Measurements in the vertical sections showed that the thickness of the superior bony wall of the IAC was similar to that of the cochlear otic capsule which should be kept unexposed for hearing preservation.

Comparative histological contributions to the development of the ear-ossicular joints and the temporomandibular joint in the mouse.

The genesis, differentiation, development and growth of the ear-ossicular joints and temporomandibular joint (TMJ) in the Slc-ICR mouse were studied by means of light and transmission electron microscopy. The differentiation of the ossicular joints and TMJ anlagen was initiated during the fetal stages. Although morphogenesis, development and growth of the TMJ were already evident in the prenatal stages, the joint-cleaving associated with the occurrence of interzones in the cartilaginous ossicular chain was observed during the neonatal stages. However, the simple diarthrodial ossicular joints were completed and became mature earlier than the double synovial TMJ having an articular disc in early postnatal life. TEM examinations revealed that the articulating epiphyseal cartilage of the ossicular joints consisted of hyaline cartilage, while the intracapsular structures in the TMJ were composed of fibrocartilage. The synovium of the diarthroidal joints was supplied by blood vessels, nerve fibers, collagen and elastic fibers. No distinct aging in the cellular and fibrous components of the synovial tissue of the completed TMJ and ossicular joints was evident in the present study.