Analysis of tympanosclerotic plaques via atomic force microscope and scanning electron microscope
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1 Basic Study / Temel Araflt rma J Med Updates 2012;2(3):89-93 doi: /jmu Analysis of tympanosclerotic plaques via atomic force microscope and scanning electron microscope Atomik kuvvet mikroskobu ve taray c elektron mikroskobuyla timpanosklerotik plaklar n analizi Hale Aslan, Mehmet Sinan Baflo lu, Ali Ekber lknur, Can Özbay, Erdem Eren, Duygu O uz K l ç, Erkan Kulduk, Sedat Öztürkcan, Hüseyin Kat lm fl Department of ENT, Atatürk Training and Research Hospital, zmir Katip Çelebi University, zmir, Turkey Abstract Objective: To present new information to the literature by imaging the calcospherule forming tympanosclerotic plaque s structure with atomic force microscope and to analyze the elementary structure of it with energy dispersive X-ray detector adapted to the scanning electron microscope. Methods: Samples taken from 30 patients who underwent surgery for tympanosclerosis were retrospectively evaluated in our tertiary referral center. The surface topography and three-dimensional images of the hardest plaque was analyzed using atomic force microscopy and examined elemental composition of 5 different calcospherule in 5 different plaques using scanning electron microscopy energy dispersive X-ray spectroscopy. Results: Three-dimensional analysis of the tympanosclerotic plaque using atomic force microscopy showed calcium phosphate crystalline structures. Scanning electron microscopy with energy dispersive X-ray spectroscopy was used to quantify the calcium, phosphate, carbon, nitrogen, oxygen, sodium, and magnesium present. Conclusion: We believe that knowledge of the surface topography and elemental composition of tympanosclerotic plaque will contribute to understanding the etiology of tympanosclerosis and its treatment. Key words: Tympanosclerosis, plaque, analysis. Özet Amaç: Atomik kuvvet mikroskobu ile kalsiyum kürecikleri oluflturan timpanosklerotik plak oluflumunu görüntüleyerek literatüre yeni bilgiler sunmak ve taray c elektron mikroskobuna monte edilmifl enerji yay c X- fl n dedektörü ile temel yap s n incelemek. Yöntem: Timpanoskleroz cerrahisi geçirmifl 30 hastadan al nan örnekler üçüncü basamak sevk merkezimizde geriye dönük olarak incelendi. Atomik kuvvet mikroskobuyla en sert pla n yüzey topografisi ve üç boyutlu görüntüleri analiz edildi ve taray c elektron mikroskobu enerji yay c X- fl n spektroskopisiyle 5 farkl plak içindeki 5 farkl kalsiyum küreci inin temel bileflimi incelendi. Bulgular: Atomik kuvvet mikroskobuyla timpanosklerotik pla n üç boyutlu analizi kalsiyum fosfat kristalli oluflumlar göstermifltir. Var olan kalsiyum, fosfat, karbon, nitrojen, oksijen, sodyum ve magnezyum miktar n n tayini için enerji yay c X- fl n spektroskopisiyle taray c elektron mikroskobu kullan lm flt r. Sonuç: Timpanosklerotik pla n temel bileflimi hakk nda bilgi edinmek için yüzey topografisi kullan m n n timpanosklerozun etiyolojisi ve tedavisini anlay fl m za katk da bulunaca na inanmaktay z. Anahtar sözcükler: Timpanoskleroz, plak, analiz. Tympanosclerosis is an important sequela of otitis media. Pathologically, tympanosclerotic plaques are localized in the lamina propria between the ciliary cuboidal mucosa of the middle ear and the periosteum of the temporal bone or the squamous epithelium of the tympanic membrane. [1] Isolated calcified structures are also called calcospherules. They consist of calcium phosphate crystals. Many light and electron microscopic studies of the calcospherules in Correspondence: Mehmet Sinan Baflo lu, MD. zmir Katip Çelebi Üniversitesi Atatürk E itim ve Araflt rma Hastanesi, Kulak Burun Bo az Hastal klar Klini i, Polat Caddesi 353 Sokak No:53, Karaba lar, zmir. basoglusinan@gmail.com Tel: Received: August 12, 2012; Accepted: September 22, 2012; Published online: November 1, 2012 Online available at: doi: /jmu QR code: 2012 Sürekli E itim ve Bilimsel Araflt rmalar Derne i (SEBAD)
2 Aslan H et al. tympanosclerotic plaque have been conducted. [2] The plaques occur mostly in the tympanic membrane, around the ossicles and oval window. [3] The surfaces of materials can be characterized using scanning electron microscopy and atomic force microscopy. These techniques are commonly used in disciplines involving the characterization of materials, including physics, chemistry, biology, architecture, restoration, geology, and dentistry. With scanning electron microscopy, the topographic structure of materials can be visualized on a nanometer or micrometer scale and information on the amount of particles and crystal structure can be obtained. To determine the elemental composition of the surface, scanning electron microscopy with energy dispersive X-ray spectroscopy can be used. [4] With atomic force microscopy, the surface of materials can be seen in three dimensions and surface roughness, phase status, porosity, and size can be analyzed. [5] In our study, we obtained scanning electron microscopy images of plaques at 50,000x magnification and then determined the percentage elemental composition in its structure using energy dispersive X-ray spectroscopy and obtained surface and three-dimensioned images of the crystal structure using atomic force microscopy. Materials and Methods This study was approved by the local ethics committee. We examined tympanosclerotic plaques removed at surgery in 30 patients (13 females, 17 males) who underwent surgery for chronic otitis media. The patients ages ranged from 11 to 60 years. All of the patients had had chronic otitis media for at least 3 years. Approximately 100 plaques were removed from the 30 patients. The weight of the plaques ranged from 1 to 6 mg. We cleaned the mucosal tissues from around plaques and stored them in sterile tubes at 4 C. One plaque with most smooth surface was examined for atomic force microscope. One plaque with most large area was examined for scanning electron microscope. Five plaques with the hard and most compact structure were used for the elemental analysis for the scanning electron microscope with energy dispersive X-ray spectroscopy. Scanning Electron Microscope (Phillips, XL-30S FEG) A scanning electron microscope can display the structure of materials on micro- or nanometer scales. Using a scanning electron detector, a three-dimensioned image can be obtained, and the elemental composition of the structures can be determined using energy dispersive X-ray spectroscopy qualitatively and quantitatively. A piece of plaque with a smooth surface was selected, plated with gold/palladium, and then three-dimensioned images of both the outer and a broken surface were taken, and the elemental composition was determined. Scanning Terminal Microscope (Digital Instruments, MMSPM Nanoscope 4) This apparatus contains an atomic force microscope and was used to examine the surface properties at the atomic level. The three-dimensioned topography can be determined through the interaction between the surface and probe. For atomic force microscopy, measurements were taken from the smoothest place on the broken surface, because the maximum roughness of the surface in atomic force microscopy should be 5 μm. With both methods, the quantity of the plaque was not important; the microscopic images were independent of the quantity. Results In scanning electron microscopic images at 50,000x magnification, the presence of collagenous fibrils in the hardest plaques was noteworthy, although their three-dimensional network could not be determined. They contained oval or spherical lacunae (Fig. 1). We performed the elementary analysis of 5 different calcospherule in 5 different plaques by energy dispersive X-ray detector (Figs. 2-6). Accordingly, the main elements forming the structure of the plaques were calcium, phosphorus, C Fig. 1. Cut surface of tympanosclerotic plaque showing calcospherule (C) and irregular collagen fibrilles (F) x50,000. F 90 Journal of Medical Updates
3 Analysis of tympanosclerotic plaques via atomic force microscope and scanning electron microscope Fig. 2. Analysis of calcospherule using by detector of energy dispersive X-ray spectroscopy in the first plaque (red signed area). Fig. 3. Analysis of calcospherule using by detector of energy dispersive X-ray spectroscopy in the second plaque (red signed area). carbon, nitrogen, oxygen, natrium and magnesium. The results were given in percentages and all were similar. Calcium min max , phosphorus min max %, carbon min max , nitrogen min max , oxygen min max , natrium min max. 1.45, magnesium min max were determined. Accordingly, mean calcium was found 22.8±2.1%, phosphorus 15.3±2.1%, carbon 17.2± 4.02%, nitrogen 8.8±0.7%, oxygen 32.6±3.6%, natrium 0.9±0.3%, and magnesium 2.0±0.4% (Table 1). Fig. 4. Analysis of calcospherule using by detector of energy dispersive X-ray spectroscopy in the third plaque (red signed area). Fig. 5. Analysis of calcospherule using by detector of energy dispersive X-ray spectroscopy in the fourth plaque (red signed area). Cilt / Volume 2 Say / Issue 3 Aral k / December
4 Aslan H et al. For a 5x5 μm area of the plaque surface, we obtained a three-dimensional image of the calcium phosphate crystalline structure and a phase image (Fig. 7). The atomic force microscopy results cannot be quantified. Discussion The etiology of tympanosclerosis is unclear, although it is thought to be a disproportionate response of the middle ear to allergy, trauma, infections, or autoimmune reactions. [6] Tympanosclerosis is a dystrophic calcification, because pathological calcification is seen in the degenerate tissues. The mechanism of dystrophic calcification involves calcium uptake in damaged mitochondria and increased hydroxyl ions in the milieu as a result of ph changes, which leads to the formation of hydroxyapatite crystals when calcium ions join together. [7] The extracellular membranous matrix plays an important role in the mineralization of tympanosclerosis. Anderson et al. showed that the hydroxyapatite is localized in the matrix vesicles, which have calcium-binding activity and accumulate calcium and phosphate. [8,9] Four different phases in the development of tympanosclerosis can be observed by light microscopy histologically. During the early phase, vesicles nm in diameter are formed in the fibroblasts, inflammatory cells, and epithelium cells of the extracellular collagenous matrix. Then, crystalline-like inclusions appear in these vesicles, so-called calcospherules. Calcium and phosphate precipitate in these spherules and mineralization begins. During the last phase, masses that are completely mineralized appear and they are then called plaques. [3] Transmission electron microscopy images show an extensive collagenous fibrillary network among the calcium phosphate crystals. [7] These crystals form calcospherules, 1-5 μm in diameter, and can be seen in cross-sectional images taken at greater than 2000x magnification. Tympanosclerotic plaques examined using scanning electron microscope at 25,000x magnification showed fibrillar collagen endings and globular aggregates. [10] For the first time, we obtained 50,000x magnification images of a cross-section of a plaque and saw that the three-dimensioned network among the collagenous fibrils was lost, and there were oval or spherical lacunae in the interspaces. This was consistent with literature reports. Bonnaud was the first to analyze the chemical composition of tympanosclerotic plaques in He found that calcium averaged 4 g/100 g tissue. Similarly, Buyonover et al. found calcium 1-4 mg/100 mg tissue, using a chemical analy- Fig. 6. Analysis of calcospherule using by detector of energy dispersive X-ray spectroscopy in the fifth plaque (red signed area). Fig. 7. Three dimensioned images of the crystal structure of plaque by atomic force microscope in 5x5 micrometer. Table 1. Results of analysis in five different tympanosclerotic plaques. Elements SD: Standard deviation. Mean±SD (range) Ca 22.88±2.14 ( ) P 15.36±2.15 ( ) C 17.29±4.02 ( ) N 8.85±0.78 ( ) O 32.62±3.67 ( ) Na 0.99±0.36 ( ) Mg 2.00±0.46 ( ) 92 Journal of Medical Updates
5 Analysis of tympanosclerotic plaques via atomic force microscope and scanning electron microscope sis method. They attributed the differences to the fact that each tympanosclerotic plaque examined was in a different mineralization phase. [11] Döner et al. reported a biochemical analysis of tympanosclerotic plaques in 2003 and found calcium 2.5±2.6 mg/100 mg tissue, phosphate 0.16±0.11 mg/100 mg tissue, and protein 3.4±3.4 mg/100 mg tissue. [12] Using scanning electron microscopy with energy dispersive X-ray spectroscopy for the first time, we scanned five calcospherule from five plaque and determined that the elemental composition, in percentages, mean calcium was found 22.8±2.1%, phosphorus 15.3±2.1%, carbon 17.2±4.02%, nitrogen 8.8±0.7%, oxygen 32.6±3.6%, natrium 0.9±0.3% and magnesium 2.0±0.4% (Table 1). We found no other information on calcospherules in the literature. Our use of atomic force microscopy was also an innovation and there is no report on the three-dimensional images of the crystals in a calcospherule. Conclusions Our results provide new information that increases our understanding of the structure of calcospherules and consequently tympanosclerotic plaques using scanning electron microscopy with energy dispersive X-ray spectroscopy and atomic force microscopy. Acknowledgments We thank to Material Research Center, High Technology Institute, and zmir Founding source by society of zmir Atatürk Training and Research Hospital for their contributions in the study. Conflict of Interest: No conflicts declared. References 1. Gibb AG. Tympanosclerosis. Proc Roy Soc Med 1976;69: Friedmann I, Galey FR. The initiation and stages of mineralization in tympanosclerosis. J Laryngol Otol 1980;94: Zöllner F, Beck C. Tympanosclerosis. Zeitschrift für Laryngologie und Rhinologie 1955;34: Goldstein J, Newbury DE, Joy DC, et al. Scanning Electron Microscopy and X-Ray Microanalysis. 3rd ed. New York: Springer; p Unertl WN. The surface structure of crystalline solids. In: Bonnell D, editor. Scanning Probe Microscopy and Spectroscopy. Theory, Techniques, and Applications. 2nd ed. New York: Wiley; 2001: Schiff M, Poliquin JF, Catanzaro A, et al. Tympanosclerosis. A theory of pathogenesis. Ann Otol Rhinol Laryngol 1980;89(Suppl.): Friedman I, Galey R, Odnert S. The ultrastructure of tympanosclerosis: the source of the matrix vesicles and the pattern of calcospherules. Am J Otol 1981;3: Bonucci E. The locus of initial calcification in cartilage and bone. Clin Orthop Relat Res 1971;(78): Bonucci E. Fine structure and histochemistry of calcifying globules in epiphyseal cartilage. Z Zellforsch Mikrosk Anat 1970; 103: McKee GJ, Kerr AG. Tympanosclerosis: a scanning electron microscopic study. Clin Otolaryngol Allied Sci 1989;14: Buyanover D, Tietz A, Luntz M, Sadé J. The biochemical composition of tympanosclerotic deposits. Arch Otorhinolaryngol 1987;243: Doner F, Yariktas M, Dogru H, Uzun H, Aydin S, Delibas N. The biochemical analysis of tympanosclerotic plaques. Otolaryngol Head Neck Surg 2003;128: This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY- NC-ND3.0) Licence ( which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Please cite this article as: Aslan H, Baflo lu MS, lknur AE, Özbay C, Eren E, O uz K l ç D, Kulduk E, Öztürkcan S, Kat lm fl H. Analysis of tympanosclerotic plaques via atomic force microscope and scanning electron microscope. J Med Updates 2012;2(3): Cilt / Volume 2 Say / Issue 3 Aral k / December
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