Light and scanning electron microscopic observations and electron probe microanalysis were carried out on granular calcified bodies that appeared in the wall of a postoperative maxillary cyst in a 59-year-old man. The granular bodies, stained slightly with hematoxylin, were scattered in the cyst wall. They reacted positively to von Kossa\u27s stain indicating calcification. Based on scanning electron microscopic observation, secondary electron images revealed them to be compact bodies, and they appeared as light spots in composition images. Electron probe microanalysis revealed that the bodies were ccomposed mainly of calcium and phosphorus. Some of the large bodies had a membranous calcification core, which was observed especially in von Kossa\u27s stained specimens. Therefore, we believe that these calcification core must be generated cell debris, cell membrane, nuclear membrane or subcellular organelles

ANTOH MOTOYOSHI

EDA SHIGEO

HASEGAWA HIROMASA

KAWAKAMI TOSHIYUKI

TAKEI NORIYUKI

UJI HIDEYO

application/pdfLight and scanning electron microscopic observations and electron probe microanalysis were carried out on granular calcified bodies that appeared in the wall of a postoperative maxillary cyst in a 59-year-old man. The granular bodies, stained slightly with hematoxylin, were scattered in the cyst wall. They reacted positively to von Kossa's stain indicating calcification. Based on scanning electron microscopic observation, secondary electron images revealed them to be compact bodies, and they appeared as light spots in composition images. Electron probe microanalysis revealed that the bodies were ccomposed mainly of calcium and phosphorus. Some of the large bodies had a membranous calcification core, which was observed especially in von Kossa's stained specimens. Therefore, we believe that these calcification core must be generated cell debris, cell membrane, nuclear membrane or subcellular organelles.journal articl

KAWAKAMI, TOSHIYUKI

TAKEI, NORIYUKI

UJI, HIDEYO

ANTOH, MOTOYOSHI

HASEGAWA, HIROMASA

EDA, SHIGEO

Matsumoto Dental University Repository

(Original) Matsumoto Shigaku 19: 10--16, 1993         key words : calcified bodies - histopathology - ultrastructure - electron probe microanalysis -                 postoperatiye maxillary cystObservations on Calcified Bodies in the Cyst Wall of a Postoperative Maxillary CystTOSHIYUKI KAWAKAMI NORIYUKI TAKEI HIDEYO UJIMOTOYOSHI ANTOH HIROMASA HASEGAWA and SHIGEO EDA   DePartment of Oral Pathology, Matsumoto Dental College                  (Chief .' Prof S. Eda)Summary    Light and scanning electron microscopic observations and electron probe mi-croanalysis were carried out on granular calcified bodies that appeared in the wall of apostoperative maxillary cyst in a 59-year-old man. The granular bodies, stained slightlywith hematoxylin, were scattered in the cyst wall. They reacted positively to von Kossa'sstain indicating calcification. Based on scanning electron microscopic observation, secon-dary electron images revealed them to be compact bodies, and they appeared as light spotsin composition images. Electron probe microanalysis revealed that the bodies wereccomposed mainly of calcium and phosphorus. Some of the large bodies had a membranouscalcification core, which was observed especially in von Kossa's stained specimens.Therefore, we believe that these calcification core must be generated cell debris, cellmembrane, nuclear membrane or subcellular organelles.Introduction   Pathological calcium deposition in various tissues mainly occurs in connection with necroticandlor degenerative changes'•2}. Therefore, these calcifications are generally characterized asdeposits that the needle like or amorphous in shape depending upon the underlying structures.Although granular calcified bodies commonly occur in meningiomas and adenocarcinomas, somepapers have also described microcalcifications, especially microcalculi in salivary glands. In oralneoplasms, this type of microcalcification appears mainly in salivary gland tumors3-"), particularlyin pleomorphic adenomas.   During our histopathological survey of surgically removed tissues at the Clinical Division ofMatsumoto Dental College Hospital, we discovered a case of postoperative maxillary cyst havinggranular calcified bodies scattered in the cyst wall. We describe herein the histopathological andultrastructural features of these bodies.Presented in part at the 35th meeting of the Matsumoto Dental College Society held on November 7, 1992. (acceptedfor publication on February 5, 1993)zaJ!Ntwnt]}=4 19(1) 1993 11Materials and Methods   Materials examined in this study were obtained from a postoperative maxillary cyst in a59-year-old man (MDC 082-91). The specimens were drawn from the patient during surgery , and forhistopathological analysis, selected portions were fixed in 10% formalin solution, dehydratedthrough a graded ethanol series, and then embedded in paraffin. After sectioning, the specimenswere stained with hematoxylin-eosin (H-E) and von Kossa's stain and then observed under a lightmlcroscope.   For scanning electron microscopy (SEM) and electron probe microanalysis (EPMA), sectionedspecimens were mounted on a carbon block and deparaffinized with a xylene solution. They werethen processed by critical-point drying, coated with carbon by the cathodic sputtering method, andthen examined with a JEOL JCXA 733 super probe.ResultsHistopathological findings :   Light microscopic observation revealed that the cyst wall consisted mainly of collagen bundleshaving limited lining epithelium (Figs. 1, 2), and in part of the wall signs of reactive bone formationwere evident (Fig. 1). The cyst wall connective tissue was partially hyalinized, which showed poorcellular areas, and in these areas some granular and irregular sized bodies stained slightly withhematoxylin were present (Figs. 3, 4). The large masses were globular in shape, and the small oneswere sand-like granules. These bodies seemed to have no relationship with the above mentionedreactive bone formation.   These globular masses and sand-like granules reacted strongly with von Kossa's stain, indica-ting the occurrence of calcification (Figs. 5, 6). Regarding the large globular bodies, their positivelystained portion appeared to be laminated, and the area around (Fig. 5 arrows), the periphery (Fig.6 arrows) and the central area of the bodies were strongly positive. In contrast, the entire smallsand-like granule was stained uniformly strong. No positive stainings were found in the surroundinghyalinized tissue.Scanning electron microscopic findings :   Examination by scanning electron microscopy under low magnification showed several lightspots in the composition images which corresponded to the von Kossa's-positive material and theirdistribution in various parts of the hyalinized collagen bundles (Figs. 7, 8); The secondary electronimages revealed that the sectioned surface of the cyst wall was relatively solid, and globular-shapedbodies were scattered in the cyst wall (Figs. 9, 10). These globular structures were approximately1 to 20 ptm in diameter (Figs. 9, 10).Electron probe microanalytic findings :   Analytically, these materials were calcified (Figs. 11, 12) as judged from their compositiondetermined from radiographic (Ca-Ka and P) images (Figs. 13, 14). EPMA demonstrated that thecalcified masses consisted mainly of calcium and phosphorus (Fig. 15), indicating calcium phos-phate ; in contrast, the surrounding tissues showed no trace of these elements (Fig. 16).Discussion   According to the literature, pathological calcification, which is composed mainly of hydrox-yapatite crystals, is often associated with membranous cellular debris, and these crystals are12 Kawakami, et al. : Observations on Calcified Bodies in the Cyst Wall of a Postoperative Maxillary Cyst.i,,--.#t=t2.#.I:.=]-.pttz-'.'.6ivÅre=!-ig-ex#yss.N3 ,sk,.a.Kxxg•i-, tl'•si•-ij -;••.k-...'-trt'k}.5,i,,g!yefSigis".,},.ym.S,iajiIlliili''"'` itS/H.Fig. 1fig. 2.Fig. 3.Fig. 4.Fig, 5.Fig. 6,1ec#.kF - .S!A,,i!tt l ''i  t"••ei.://"""1s.i''z  . }l/1'v { "- '1,y': ts ,//fi      1:..., '2f'1.,",-- xXL. ' 1"tt'I' -" t  .. :.ti •l •-;tt"' " '-''' 'iÅék X' '"'   hsl!N. ..1/kMl(of'tlsw's/liesiillllglts/wLfo.nt.i""\`/iMi4f,.,eg{g,tk  r-;".X. ,tit., ,k •'.',6ft"''.-.. .-`".Iles-t  Ig.  .g  1.+. . ,,   '.1'' iS' "'•i-,,i,,      -E,Å~40).  -E, Å~ 150).Xhl.l' 3,l,J':•I:'.}'tiJ:;'•""ti'seT3,;//,.i/r.titlt-•.i•,:l:l","•Fi';"i.'       t/   't ' -1.. .,.. .. .,       .t)tr -. .            t.           ,s      ',N ";•             tt"tt"').IJ. " . ,,;              '  tt             .t -   rl s' ,ofk ' s      }ge." ' '  " '-;)tiIE,'l•.,.,            ,g,..,si'      '''"/'i"'1.is }i"l " ',L "l' .rk"\'l,tS,,"1 ,"3s. yk l')Histopathological view of the cyst wall (HLining epithelium of the cyst wall (HCollagenous tissue of the cyst wall (H-E,Å~480).Globular bodies and sand-like materials in the tissue (HArrows showing the vont' .. ;g.-.J,,,     . 'e, •-v-gis•l.Pi.r- E,Å~480)-t- - .:V"' L. ,r. Sl,tl.::e:!;l •l'.   I-agJ "  -, :". '.".)':' 't-'i:t l l 't  /!"  ie.; .;pp •vl ' ;'t`j'   . Jt.;A} i- •.1    .i      '     -.     lr    '     ---bt  . ., . ,. F: -l;t.j/ t jvt' S,, S .ttt:tdi4',. },,,i,.l ,}S}!    • .'a      't- t tt/ttll/ . .J/ '                   Kossa's-p sitive stainings around the large body (Å~600).Positive stainings existing in the periphery (arrows) and the center of the large bodies (vonKossa's, Å~600).i![}Jztsiig':']tL'" 19(1) 1993 13Fig. 7.Fig. 8.Fig. 9.Fig. 10,Composition image showing numerous light spots in the tissue (Å~600).Composition image showing light spots in the tissue (Å~360).Enlarged photograph of the area shown in Fig. 8 (Secondary image,Å~1,200). Composition image of the same area of Fig. 9.14 Kawakami, et aL : Observations on Calcified Bodies15 c fiin the Cyst Wall of a Postoperative Maxillary Cyst16peAÅq e Åq e,16KEU XES te,4eKEuÅrFig. 11.Fig. 12.Fig. 13.Fig. 14.Fig. 15.Fig. 16.Enlarged graph of Fig. 9 demonstrating large globular bodyComposition irnage of same calcified body seen in Fig. 11.Ca-Ka image of the same area shown in Fig. 11.Phosphorus image of the same area shown in Fig. 11.Result of EPMA of calcified bodies.Result of EPMA of surrounding tissues.(Secondary image,Å~3,800).                                  iflrJ!XcaRt]i!.4 19(1) 1993 15thought to have started to form on these membranous structuresi). Regarding a case of calcinosisuniversalis, Kawakami et al. (1986)2) reported that the calcification site was closely related to fociof fibrinoid degeneration ; they proposed that the globular and/or membranous structures seen werederived from degenerating cells. They suggested, therefore, that these globular and/or membranousstructures might be involved in the initial calcification in this case. Furthermore, Kawakami et al.8•9)described membranous structures in the pathologic calcification sites elicited by calcium hydroxide-containing dental material, and discussed the relationship between the membranous structure andinitial calcification. We believe that some of these structures have a matrix vesicle-Iike function,although we think that the calcified bodies are caused mainly by dystrophic changes because of theirappearanceiO). Our previous papers described the ultrastructure of these calcifications in salivarygland tumor cases3"'}, and we suggested that the calcium binding capacity of these materials maybe closely related to these microcalcifications. Furthermore, in a case of central neurinoma thesemicrocalcifications were also observedii).    In our present case, the relationship between the calcium depositions and the vesicularmembranes was not evident, and we were unable to find obvious membranous structures in thepresent case despite careful observation. However, we believe that the microcalcifications areclosely related to cellular degeneration. In general, the cyst wall is invaded by inflammatory cellsat the early stage of its development but these cells disappear with the passage of time. At the timeof inflammatory cell disappearance, some of these cells may degenerate. Thus, we speculate that theinitial calcification will occur around these degenerating structures. In fact, we found core struc-tures on the periphery of large calcified bodies by light microscopic observation of vonKossa's-stained specimens and by scanning electron microscopic examination. We believe thesemembranous calcification cores to be that of generated cell debris, cell membrane or nuclearmembrane, as judged from their diameter. However, there were no core structures in the center ofthe small bodies, suggesting that the microcalcification in them occurred in close relation with thedegeneration of subcellular organelles, i. e. mitochondria, rough and smoothed surfaced-endoplas-mic reticulum, Golgi apparatus, and so on. According to the results of electron probe microanalysis,the elemental composition of the calcified bodies was similar to that in other cases alreadydescribedii•i2).                                   Acknowledgement    The authors wish to express their thanks to Professor M. Yamaoka, Department of Oral andMaxillofacial Surgery II, Matsumoto Dental College, for supplying the surgical material from thepatient. Chief Technologist S. Akahane, Laboratory of Electron Microscope, Matsumoto DentalCollege, is acknowledged for his technical assistance in electron microscopy.                                      References1) Kim, K. M. and Huang, S.(1971) UItrastructural study of calcification of human aortic valve. Lab.   Invest. 25 : 357-366.2) Kawakami, T., Nakamura, C., Hasegawa, H., Eda, S., Akahane, S., Yamazaki, T. and Takasu, N.   (1986) Ultrastructural study of calcinosis universalis with dermatomyositis. J. Cutan. Pathol. 13 : 135 -   143.3 ) Akahane, S., Kawakami, T., Nakamura, C., Hasegawa, H., Eda, S., Komatsu, M., Furusawa, K. and   Ideguchi, H. (1984) Electron microscopic studies on mucoepidermoid carcinoma, II calcified materials   appeared in the stroma. Matsumoto Shigaku, 10 : 29-41.16 Kawakami, et al. : Observations on Calcified Bodies in the Cyst Wall of a Postoperative Maxillary Cyst4 ) Kawakami, T., Nakamura, C, Hasegawa, H., Eda, S., Komatsu, M., Furusawa, K. and Akahane, S.(1986) Ultrastructure of stromal calcification in mucoepidermoid carcinoma. Jpn. J. Oral Biol. 28 : 217-222.5 ) Yamazaki, T., Kotani, A. and Kawakami, T. (1987) Basal cell adenoma of the sublingual gland. J. OralMaxillofac. Surg. 45 : 270-273.6 ) Hasegawa, H., Kawakami, T., Nakamura, C, Eda, S., Furusawa, K. and Kiga, M. (1987) Ultrastructuralstudy of varied calcified materials in the pleomorphic adenoma occurring in the soft palate. MatsumotoShigaku, 13 : 115-121.7 ) Nakamura, C, Kawakami, T., Hasegawa, H., Eda, S., Akahane, S. and Yamazaki, T. (1987) Light andelectron microscopic studies of microcalcifications appearing in monomorphic adanomas. MatsumotoShigaku, 13 : 329-336.8) Kawakami, T., Nakamura, C, Hayashi, T., Eda, S. and Akahane, S. (1979) Studies on the tissuereactions to the paste of calcium hydroxide added iodoform (root canal filling material : Vitapex ®),second report an electron-microscopic study. Matsumoto Shigaku, 5 : 161-170.9 ) Kawakami, T. (1984) An experimental study on tissue reactions to a paste made of calcium hydroxideand iodoform with an addition of silicone oil -with special reference to absorption of and calcificationby the paste-. Shikwa Gakuho, 84 : 1563-1593.10) Kawakami, T., Nakamura, C, Hasegawa, H., Akahane, S. and Eda, S. (1987) Ultrastructural study ofinitial calcification in the rat subcutaneous tissues. Oral Surg. 63 : 360-365.ll) Kawakami, T., Hasegawa, H., Nakamura, C, Eda, S., Yagasaki, T., Kitamura, Y., Chino, T. andAkahane, S. (1988) An electron microscopic observation of psammoma body-type microcalcifications ina case of intraosseous neurinoma. J. Clin. Electron Microsc, 21 : 167-171.12) Kawakami, T., Nakamura, C, Hasegawa, H., Eda, S. and Akahane, S. (1988) Analytical electronmicroscopic study of mineral deposits in a case of calcinosis universalis. Matsumoto Shigaku, 14 : 41-48.抄録：術後性上顎嚢胞の嚢壁にみられた石灰化物の観察川上敏行　武井則之　宇治英世　安東基善　長谷川博雅　枝重夫（松本歯大・口腔病理）59歳男性に発現した術後性上顎嚢胞で，その嚢壁に石灰化物の存在する1症例を経験したので，光顕的ならびに電子顕微鏡的に追究した．光顕的に嚢胞の裏装上皮は，高度の炎症により破壊されたためほとんど認められず，嚢壁の大部分は硝子化した線維性組織により構成されていた．同部にはへマトキシリソに淡染した構造物が介在しており，これらの構造物はvonKossa染色に強く陽性反応を示し，石灰化物であることが確認された．さらに大きな球状石灰化物の一部では，その周囲に膜様に石灰化した構造があった．なお，これらは，SEMの二次電子像では比較的平滑に，組成像では明るく，またEDSではCaとPが主たる成分であることを示した．したがって，今回の症例の検索では，その形成基盤となった嚢壁は裏装上皮がなくなるほど高度な炎症を起こしていたことなどから，大きな球状石灰化物の基盤となったものは変性・壊死した炎症性細胞などの核膜あるいは細胞膜であり，砂状の石灰化物では細胞内小器官であるものと推察された．

Observations on Calcified Bodies in the Cyst Wall of a Postoperative Maxillayr Cyst

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術後性上顎嚢壁にみられた石灰化物の観察

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