Effect of Bone Powder/Mesenchymal Stem Cell/BMP2/Fibrin Glue on Osteogenesis in a Mastoid Obliteration Model.

BACKGROUND/AIM: This study aimed to prospectively compare the osteogenesis of bone powder (BP) substances with and without mesenchymal stem cells (MSCs) and evaluate the synergistic effect of topically applied recombinant human bone morphogenic protein-2 (BMP2) on MSC-loaded BP using fibrin glue in a mastoid obliteration model. MATERIALS AND METHODS: To determine the expression of osteocyte-specific genes, total RNA was isolated from three MSC groups: Untreated MSCs, MSCs cultured with BP, and MSCs cultured with BP and BMP2. Real-time polymerase chain reaction was carried out with specific primers of osteogenesis-related genes runt-related transcription factor 2, osteocalcin, osteoprotegerin, osterix, alkaline phosphatase, transforming growth factor beta, and type I collagen. Live/dead staining was also performed. To observe the adhesion of MSCs to the BP, MSCs were treated with BP for 2 days and the surface was observed by scanning electron microscopy (SEM). Under general anesthesia, mastoid obliteration was performed in rats using three groups: treated with BP alone, BP/MSCs, and BP/MSC/BMP2. Before decapitation at 8 weeks post operation, in vivo micro computed tomography (micro CT) was performed. The bullae were dissected, fixed, and decalcified. followed by dehydration, paraffin embedding, and staining by hematoxylin and eosin and Masson's trichrome. RESULTS: SEM showed the MSCs to be well-attached to the superficial area of the BP. The expression of osteocyte-specific genes was the highest in the MSCs cultured with BP and BMP2, followed by cultured with BP only, and untreated MSCs. The BP/MSC/BMP2 group showed the highest radiodensity of bullae in microCT analysis. The microCT findings revealed that the BP/MSC/BMP2 group showed the most enhanced osteogenesis of the scaffold compared to the other two groups. No significant difference was found in osteoconductive osteogenesis between the control and BP/MSC groups. However, the BP/MSC/BMP2 group showed significantly enhanced osteoconductive osteogenesis and osteoinductive change of the BP as shown by hematoxylin and eosin staining. Histomorphometry of osteogenesis revealed that the difference between the BP/MSC/BMP2 group and the other two groups was statistically significant. CONCLUSION: A small amount of BMP2 is necessary during MSC loading to enhance the osteogenesis of BP and avoid complications associated with high doses of BMP2. These results may be applicable to mastoid obliteration in clinical practice.

Effect of ageing on the myosin heavy chain composition of the human sternocleidomastoid muscle.

The myosin heavy chain (MyHC) composition of ageing limb muscles is transformed into a slower phenotype and expresses fast-twitch fibre type atrophy, presumably due to age-related motor unit remodelling and a change in the patterns of physical activity. It is not known if ageing affects the sternocleidomastoid muscle (SCM) in a similar way. The goal of the study was to analyze the MyHC composition and the size of muscle fibres in the ageing SCM by immunohistochemical methods and quantitative analysis and stereology using our own software for morphometry. We hypothesize that with ageing the MyHC composition of SCM transforms similarly as in ageing limb muscles, but the size of the muscle fibres is less effected as in limb muscles. The study was performed on the autopsy samples of the SCM in 12 older males. The results were compared with those published in our previous study on 15 young adult males. An ageing SCM transforms into a slower MyHC profile: the percentage of slow-twitch fibres is enhanced (numerical proportion 44.6 vs. 31.5%, P<0.05; area proportion 57.2 vs. 38.4%, P<0.05). The share of hybrid 2a/2x fibres is diminished (numerical proportion 14.1 vs. 26.8%, P<0.05), the area proportion of all fast-twitch fibres expressing MyHC-2a and 2x is smaller (50.6 vs. 63.5%, P<0.05), and the area proportion of fibres expressing the fastest myosin isoform MyHC-2x is smaller too (19.0 vs. 34.5%, P<0.05). The slower phenotype with the preferential reduction of the fibres expressing the fastest MyHC-2x provide circumstantial evidence for: (i) more fast-twitch than slow-twitch motor units being lost; and (ii) reinnervation by the surviving motor units. There appears to be no significant influence on muscle fibre size, which is congruent with relatively unchanged SCM activity during life.

In-Vivo Electrical Impedance Measurement in Mastoid Bone.

Nerve monitoring is a safety mechanism to detect the proximity between surgical instruments and important nerves during surgical bone preparation. In temporal bone, this technique is highly specific and sensitive at distances below 0.1 mm, but remains unreliable for distances above this threshold. A deeper understanding of the patient-specific bone electric properties is required to improve this range of detection. A sheep animal model has been used to characterize bone properties in vivo. Impedance measurements have been performed at low frequencies (<1 kHz) between two electrodes placed inside holes drilled into the sheep mastoid bone. An electric circuit composed of a resistor and a Fricke constant phase element was able to accurately describe the experimental measurements. Bone resistivity was shown to be linearly dependent on the inter-electrode distance and the local bone density. Based on this model, the amount of bone material between the electrodes could be predicted with an error of 0.7 mm. Our results indicate that bone could be described as an ideal resistor while the electrochemical processes at the electrode-tissue interface are characterized by a constant phase element. These results should help increasing the safety of surgical drilling procedures by better predicting the distance to critical nerve structures.

Expression of the epidermal growth factor system in human middle ear cholesteatoma.

CONCLUSION: The detection of the HER4 receptor in 50% of cholesteatomas but never in the reference tissue, and the increased expression of its activating ligand EPI, suggest that EPI-mediated activation of HER4 might play a role in cholesteatoma growth. OBJECTIVE: To investigate the expression of the epidermal growth factor (EGF) system in human middle ear cholesteatoma. METHODS: Forty-seven patients referred for surgery due to cholesteatoma were included in the study. Clinical data were collected. Biopsies of cholesteatoma and skin from the external ear canal were obtained during surgery. mRNA expression was quantified with real-time PCR. The corresponding proteins were visualized using immunohistochemistry. RESULTS: A systematic investigation of all four receptors, HER1, HER2, HER3, and HER4, and the ligands EGF, transforming growth factor (TGF)-α, amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin (EPI) of the EGF system is presented. At the mRNA level, the study demonstrates an up-regulation of mRNA encoding EPI and AR. In contrast HER1 and EGF were down-regulated. HER4 mRNA could be detected in 50% of cholesteatoma and 20% of reference tissues, and the HER4 protein was detectable only in cholesteatoma tissue. HER1 and HER2 were also visualized by immunohistochemistry, whereas the ligands EPI, AR, and EGF were undetectable with our methods.

Matrix change of bone grafting substitute after implantation into guinea pig bulla.

BACKGROUND: Many different surgical techniques have been developed to remove open mastoid cavities. In addition to autologous materials, alloplastic substances have been used. A very slow absorption of these materials and extrusion reactions have been reported. We investigated a newly developed, highly porous bone grafting material to eliminate open mastoid cavities, in an animal model. To characterise the transformation process, the early tissue reactions were studied in relation to the matrix transformation of the bone material. MATERIAL AND METHODS: NanoBone (NB), a highly porous bone grafting material based on calcium phosphate and silica, was filled into the open bullae from 20 guinea pigs. The bullae were examined histologically. Energy dispersive X-ray spectroscopy (EDX) was used to investigate the change in the elemental composition at different sampling times. The surface topography of the sections was examined by electron microscopy. RESULTS: After 1 week, periodic acid-Schiffs (PAS) staining demonstrated accumulation of glycogen and proteins, particularly in the border area of the NB particles. After 2 weeks, the particles were evenly coloured after PAS staining. EDX analysis showed a rapid absorption of the silica in the bone grafting material. CONCLUSIONS: NanoBone showed a rapid matrix change after implantation in the bullae of guinea pigs. The absorption of the silica matrix and replacement by PAS-positive substances like glycoproteins and mucopolysaccharides seems to play a decisive role in the degradation processes of NB. This is associated with the good osteoinductive properties of the material.

Method for studying gas composition in the human mastoid cavity by use of laser spectroscopy.

OBJECTIVES: We evaluated a method for gas monitoring in the mastoid cavity using tunable diode laser spectroscopy by comparing it to simultaneously obtained computed tomographic (CT) scans. METHODS: The presented optical technique measures free gases, oxygen (O2), and water vapor (H2O) within human tissue by use of low-power diode lasers. Laser light was sent into the tip of the mastoid process, and the emerging light at the level of the antrum was captured with a detector placed on the skin. The absorption of H2O was used to monitor the probed gas volume of the mastoid cavity, and it was compared to the CT scan-measured volume. The ratio between O2 absorption and H2O absorption estimated the O2 content in the mastoid cavity and thus the ventilation. The parameters were compared to the grading of mastoid cavities based on the CT scans (n = 31). The reproducibility of the technique was investigated by measuring each mastoid cavity 4 times. RESULTS: Both O2 and H2O were detected with good reproducibility. The H2O absorption and the CT volume correlated (r = 0.69). The average ratio between the normalized O2 absorption and the H2O absorption signals was 0.7, indicating a lower O2 content than in surrounding air (expected ratio, 1.0), which is consistent with previous findings made by invasive techniques. All mastoid cavities with radiologic signs of disease were detected. CONCLUSIONS: Laser spectroscopy monitoring appears to be a usable tool for noninvasive investigations of gas composition in the mastoid cavity, providing important clinical information regarding size and ventilation.

A primary large cholesterol cyst of the mastoid presenting with dysgeusia.

The occurrence of postoperative secondary cholesterol cysts in the mastoid has been previously reported, however the occurrence of a primary large cholesterol cyst in the mastoid with bony destruction of the facial nerve has rarely been reported. The case report of a 17-year-old female patient with a primary large cholesterol cyst with dysgeusia is presented. Computed tomography and magnetic resonance imaging findings for the lesion distinguish a cholesterol granuloma, cholesteatoma and vascular tumor. The patient underwent a canal wall down mastoidectomy with mastoid obliteration. A dehiscent portion of the mastoid segment of the facial nerve was visible within the cavity; the gross finding of the facial nerve was edematous in appearance. Five years later, there has been no recurrence of disease.

Rate of nitrous oxide exchange across the middle ear mucosa in monkeys before and after blockage of the mastoid antrum.

OBJECTIVES: We tested the hypothesis that mastoid volume buffers the rate of change in middle ear pressure caused by transmucosal, inert gas exchange. STUDY DESIGN: Twelve monkeys were randomly assigned to group 1 or group 2. Right ears of group 1 had sham surgery and of group 2 had obstruction of the mastoid antrum. Before and after surgery, the time constant for transmucosal N(2)O exchange was estimated from N(2)O breathing experiments. The hypothesis predicts that the postoperative time constant measured for right ears of group 2 but not group 1 is greater than that measured before surgery. RESULTS: Mastoid antrum block significantly decreased right middle ear volume but did not affect the time constant for transmucosal N(2)O exchange. CONCLUSION: A mastoid gas-reserve function is not supported by the experimental data. SIGNIFICANCE: These results for monkeys and the theory developed to explain the effect of mastoid volume on transmucosal inert gas exchange suggest that the results for previous experiments in humans interpreted as evidencing a mastoid gas-reserve function are consistent with alternative explanations.

Structural changes and protein expression in the mastoid bone adjacent to cholesteatoma.

OBJECTIVE: Cholesteatoma of the mastoid and middle ear causes erosion of nearby bone. In this study we examined the mastoid bone adjacent to cholesteatoma and compared it with normal mastoid bone. In particular, noncollagenous proteins, which have a special structural and functional role in bone, were addressed. STUDY DESIGN: Nine mastoid specimens with cholesteatoma and four normal specimens obtained at surgery were examined. METHODS: Histological and immunohistochemical methods were employed to evaluate the nature of structure and noncollagenous protein content changes in the mastoid bone affected by cholesteatoma. RESULTS: The bone associated with cholesteatoma had structural changes as a noncontinuous periosteum, empty lacunae, irregular cement lines, and, specifically, the appearance of eosinophilic vesicles at the interface between the bone and cholesteatoma Immunohistochemistry demonstrated that noncollagenous proteins were apparently absent in the affected mastoid bone. Bone remote from the cholesteatoma seemed normal. CONCLUSIONS: These findings demonstrate for the first time the changes in the noncollagenous protein content in the mastoid bone affected directly by cholesteatoma These changes could be a result of a direct influence of cholesteatoma-derived products on the osteoblast.

Carbon dioxide exchange via the mucosa in healthy middle ear.

BACKGROUND: Recent studies have shown that gas exchange via the middle ear mucosa, which is performed between the middle ear cleft and capillaries in the submucosal connective tissue, has an essential role in ventilation and pressure regulation in the middle ear cleft. We speculated that gas exchange via the mucosa is induced by the gas diffusion caused by the partial pressure gradient of gas between the middle ear cleft and submucosal capillaries. OBJECTIVE: To evaluate the capacity of the gas exchange via the mucosa in the healthy middle ear of humans by examining the effect of the respiratory mode on middle ear pressure. SUBJECTS AND METHODS: We selected 13 volunteers ranging in age from 25 to 44 years with healthy ear drums and type A tympanograms. Middle ear pressure was measured in 1 ear of each subject every 2 minutes using tympanometry while the respiratory mode was altered, with the subject in the supine position. RESULTS: The partial pressure of carbon dioxide in the venous blood (PvCO2) and middle ear pressure were decreased by hyperventilation and increased by hypoventilation. The partial pressure of oxygen in the venous blood showed little change. CONCLUSIONS: Carbon dioxide diffused into the blood from the middle ear cleft in accord with the partial pressure gradient when the PvCO2 was reduced by hyperventilation, resulting in a decrease of middle ear pressure, whereas CO2 diffused into the middle ear cleft when the PvCO2 was elevated by hypoventilation, resulting in an increase of middle ear pressure. These findings suggest that a bidirectional CO2 exchange via the middle ear mucosa functions in the normal human middle ear.

Study of the distribution of oral ciprofloxacin into the mucosa of the middle ear and the cortical bone of the mastoid process.

This multicentre study evaluates the distribution of ciprofloxacin into the tissue structures of the middle ear following multiple dosing of one 500 mg tablet every 12 h. The samples were taken perioperatively from adult patients due to undergo surgery for chronic otitis. Administration of ciprofloxacin was instigated 9 days prior to the operation. The samples were taken at different intervals after the last dose in order to evaluate variations in concentration with time. The average peak concentrations recorded and the time taken to reach these concentrations were as follows: middle ear mucosa (n = 16): 5.54 +/- 3.46 micrograms/g (3-4 h): cortical bone of the mastoid process (n = 21): 1.07 +/- 1.29 micrograms/g (4 h). The measurements carried out 12 h after the last dose show that concentrations of ciprofloxacin in the middle ear mucosa were still at least as high as the minimum inhibitory concentration for this antibiotic for most of the pathogens implicated in acute exacerbations of chronic otitis. These results suggest that, administered as an oral dose of 500 mg every 12 h, ciprofloxacin may be an effective agent for the treatment of chronic suppurative otitis. These results now need to be backed up by clinical trials.

Tissue and serum concentrations of ofloxacin in the ear region following a single daily oral dose of 400 mg.

Even after a single daily dose of 400 mg, the concentrations of ofloxacin in infected middle ear tissues are within the therapeutic range. Even 20 h after a single dose of 400 mg ofloxacin, the tissue concentrations in cartilage and cholesteatomatous matrix are in excess of the minimal inhibitory concentration for a number of pathogens, such as Staphylococcus aureus and Haemophilus influenzae. The especially high concentrations in middle ear mucosa, auricular cartilage and cholesteatomatous matrix are striking. No complications or side effects were observed during ofloxacin treatment in this study. The study demonstrates that a dose of 400 mg ofloxacin per day is an effective and patient orientated therapeutic regime for the treatment of ENT infections, particularly of ear infections.

Distribution of topical antibiotic preparations in mastoid cavities.

Topical antibiotic preparations are widely prescribed in the treatment of infected mastoid cavities with no knowledge of the distribution of the preparation within the cavity. This study ascertains the distribution of drop and spray preparations within mastoid cavities and suggests methods of delivery to maximize coverage.

Pharmacokinetics of flomoxef in mucosal tissue of the middle ear and mastoid following intravenous administration in humans.

The pharmacokinetics of flomoxef in serum and in the mucosal tissue of the middle ear and mastoid were studied in 9 patients undergoing tympanoplasties. All patients received 1 g of flomoxef intravenously. Flomoxef levels in serum and in mucosal tissue were determined by a bioassay method. The peak value of mean concentrations of flomoxef in the mucosal tissue was 30.3 +/- 11.7 micrograms/ml at 10 min after the administrations. Pharmacokinetic analyses showed that the concentration of flomoxef in the mucosal tissue was over 1.56 micrograms/ml (which is the MIC90 for the common pathogens of otitis media) for more than 2 h and decreased parallel with serum concentration with a half-life of about 40 min.

[Otitis nigra: etiologic pathogenetic and clinical aspects (author's transl)]. / Otitis nigra: ätiologische, pathogenetische und klinische Gesichtspunkte.

The clinical picture of otitis nigra (idiopathic sero-hemmorrhagic otitis media) as independent clinical entity is shown, presenting 5 cases requiring mastoid surgery. Samples of mastoid mucosa were investigated by light- and electron-microscopy employing histochemical, immunofluorescent and virological techniques. Pathological changes of the small vessels in the subepithelial connective tissue with increased vascular permeability and extravasation of red blood cells and serum play a central role in the pathogenetic course of the disease. Disintegration of erythrocytes leads by catabolism of hemoglobin to formation of hemosiderin and ferretin, whereas by processing of phospholipids of the cell-membrane cholesterin cristals are deposited in the connective tissue. These cholesterin deposits in turn induce a foreign body reaction with formation of typical cholesterol granulomas. The mucosa shows secretory activity in intraepithelial secretory cells and glandlike structures in the subepithelial connective tissue. Neutral and acidic mucopolysaccharides are demonstrated in the secretory product. As possible cause for the vascular lesion that in turn sets off the pathomechanisme of otitis nigra a subclinical viral infection is discussed. This conjecture is supported by demonstration of virus-like inclusion bodies in macrophages and fibrocytes and of virus-like particles in antral smears. In addition myxoviruses of the parotitis-epidemica group could be identified in these smears by serological methods. Furthermore, the stimulation of the local immunological defense system, evidenced by the presence of numerous immunoglobulin-producing plasmacells in the tissue, is in concord with a viral infection. Therapy requires mastoidectomy in combination with posterior tympanotomy in order to eradicate completely the intractably diseased mastoid mucosa.