Calcinosis universalis with dermatomyositis occurred in a 58-year-old woman. Tissues removed from the patient\u27s sublingual region were studied mainly by analytical electron microscopic methods. According to an elemental analysis using a wavelength dispersive X-ray spectroscope, most of the mineral deposits contained the elements sodium, phosphorus, calcium, rhenium, and some deposits also contained sulphur and magnesium. The elements sodium, sulphur, chlorine, and calcium were found in the stromal tissues of the material. Energy dispersive X-ray spectroscopy demonstrated that the analysis-peaks of phosphorus and calcium were higher in the mineral deposits than in the stromal tissues. The mineral deposits throughout the specimen were mainly composed of hydroxyapatite, judging from field-limited area electron diffraction and X-ray diffraction examinations

EDA SHIGEO

HASEGAWA HIROMASA

KAWAKAMI TOSHIYUKI

NAKAMURA CHIHITO

application/pdfCalcinosis universalis with dermatomyositis occurred in a 58-year-old woman. Tissues removed from the patient's sublingual region were studied mainly by analytical electron microscopic methods. According to an elemental analysis using a wavelength dispersive X-ray spectroscope, most of the mineral deposits contained the elements sodium, phosphorus, calcium, rhenium, and some deposits also contained sulphur and magnesium. The elements sodium, sulphur, chlorine, and calcium were found in the stromal tissues of the material. Energy dispersive X-ray spectroscopy demonstrated that the analysis-peaks of phosphorus and calcium were higher in the mineral deposits than in the stromal tissues. The mineral deposits throughout the specimen were mainly composed of hydroxyapatite, judging from field-limited area electron diffraction and X-ray diffraction examinations.journal articl

KAWAKAMI, TOSHIYUKI

NAKAMURA, CHIHITO

HASEGAWA, HIROMASA

EDA, SHIGEO

Matsumoto Dental University Repository

(Original) Matsumoto Shigaku, 14 : 41--48, 1988           key words: analytical electron microscopy - calcinosis universalis - ultrastructure - hydroxyapatite                   - mineral componentAnalytical Electron Microscopic Study of Mineral in a Case of Calcinosis UniversalisDeposits TOSHIYUKI KAWAKAMI CHIHITO NAKAMURAHIROMASA HASEGAWA and SHIGEO EDADePartment of Oral Pathology, Matsumoto Dental College               (Chief : Prof S. Eda)LaboratorySHOJI AKAHANEof Electron MicroscoPe, Matsumoto Dental     (Chief: S. Akahane B. Sc.)CollegeSummary   Calcinosis universalis with dermatomyositis occurred in a 58-year-old woman.Tissues removed from the patient's sublingual region were studied mainly by analyticalelectron microscopic methods. According to an elemental analysis using a wavelengthdispersive X-ray spectroscope, most of the mineral deposits contained the elementssodium, phosphorus, calcium, rhenium, and some deposits also contained sulphur andmagnesium. The elements sodium, sulphur, chlorine, and calciurn were found in thestromal tissues of the material. Energy dispersive X-ray spectroscopy demonstrated thatthe analysis-peaks of phosphorus and calcium were higher in the mineral deposits thanin the stromal tissues. The mineral deposits throughout the specimen were mainlycomposed of hydroxyapatite, judging from field-limited area electron diffraction andX-ray diffraction examinations.                                   Introduction   Calcinosis universalis is a rare complication associated with collagen diseases, particularlyscleroderma, dermatomyositis, and systemic lups erythematosus, therefore few case reports areavailable and those consist mainly of radiographic features, a few histopathological findings, andclinical datai'5). We were fortunate enough to examine a case of calcinosis universalis associatedwith dermatomyositis occurring in a 58-year-old woman6}, and studied its ultrastructuralappearance'}. These ultrastructural features were summarized as follows : the calcification wasclosely related to the foci of fibrinoid degeneration and membranous structures appearing in the(accepted for publication on February 6, 1988)42 Kawakami, et al. : Analytical Electron Microscopy of Calcinosis Universalisstromal matrix seemed to have a matrix vesicle-like function, causing initial calcification. Thepurpose of the present study was to examine, by analytical electron microscopy, the mineralcomposition of such deposits to obtain more information about this disorder.Materials and Methods    The patient, a 58-year-old woman, was diagnosed as having calcinosis universalis secondary todermatomyositis6). Her medical history in brief is the following : at the age of 15, she had chronicarticular rheumatism. About 10 years ago, she noticed hard masses in the anterior neck region andboth elbow joints ; one year later, calcified materials in her abdomen were pointed out by her doctorduring consultation for stomachache. About 3 years ago, she noticed rock-hard masses on the floorof her mouth which gradually enlarged and caused pain during rnastication and was admitted to theClinic of Oral and Maxillofacial Surgery, Shinshu University Hospital, for treatment. Routineclinical laboratory data (EKG, CPK, etc. ) were within normal limits. Under X-ray photographs, alarge number of small radiopaque particles were found widely distributed throughout her body,mainly located along the large vessels; in the head and neck area, these were mainly in thesublingual and neck regions (Figs. 1, 2). The calcified materials were removed from the sublingualreglon to lmprove tongue movement.   The removed tissues were fixed immediately in Karnovsky's solution8) at 4"C, and then embed-ded in paraffin for histopathological study after being pretreated with or without demineralizationin 100/o formic acid with formalin. The sectioned specimens were stained with the followingreagents: hematoxylin-eosin (H-E), van Gieson's, Mallory's azan, and von Kossa's stains. Thestained specimens were then examined by a light microscope.   For transmission electron microscopy (TEM), the specimens fixed in Karnovsky's solution werepostfixed in 1906 osmium tetraoxide and embedded into Epon 812, having been pretreated with orwithout demineralization in 50% ethylene-diamine-tetra-acetic acid (EDTA) at 4"C. The ultrathinsections were stained with uranyl acetate and lead citrate (U-Pb), and then examined by an electronmicroscope (JEOL JEM 100-B). Field-limited area electron diffraction and energy dispersive X-rayanalysis were carried out on the unstained specimens by an analytical electron microscope (JEOLJEM 1200 EX).   For scanning electron microscopy (SEM) and electron probe microanalysis (EPMA), the frac-tured specimens were processed by critical-point drying and carbon-coating methods. The Karnovs-ky's solution fixed specimens without demineralization were dehydrated and then embedded intoEpon 812. The surface of the Epon 812 was polished and the polished surface was coated withcarbon. Both groups of specimens were observed by a JEOL JCXA 733 X-ray microanalyser.   Some specimens were ground, and examined by an XD-9 X-ray diffractometer (Shimadzu,Japan).ResultsLight microscopic findings :   A large number of calcified deposits strongly stained with hematoxylin were observed in theconnective tissues of undemineralized specimens (Fig. 3), they reacted positively to von Kossa'sstain. In the demineralized specimens, the stainability of these deposits to hematoxylin obviouslydecreased, and they stained yellow with van Gieson's, and weak orange with Mallory's azan stains.All specimens showed an amorphous globular pattern in these stainings.                                  IE} Jzlg ss ":i! -` 14Åq 1) 1988 43Transmission electron microscopic findings :   Mineral deposits were observed as uniform, electron-dense materials mainly in the areas offibrinoid degeneration. High-power magnification revealed that they were composed of columnarcrystals of varying size (Fig. 4). Field-limited area electron diffraction patterns of these crystals,identified as hydroxyapatite, were similar to the crystals in normal bone tissue (Fig. 4, insert).   Generally, these calcified materials showed variation in their electron density and, especially inlarge mineral deposits, an electron-dense layer was found in the periphery.Scanning electron microscopic and electron microanalytical findings :    In the composition images, the mineral deposits were observed as light structures demonstra-ting irregular surfaces surrounded by a dark matrix (Figs. 5, 6). The brightness of individual themasses varied from slightly dark to very bright, with a striped and!or amorphous globular pattern.   Electron probe microanalysis of the calcified masses with an energy dispersive X-ray spectro-scope (EDS), revealed that the mineral deposits mainly consisted of calcium and phosphorus (Fig.7-a, b), also the stromal matrices contained traces of the same elements and sulphur (Fig. 7-c, d). Thelevels of elements detected varied according to the point under analysis.    According to the elemental analysis using a wavelength dispersive X-ray spectroscope (WDS),most of the mineral deposits contained the elements sodium, phosphorus, calcium, and rhenium ;some of them sulphur and magnesium as well. The elements sodium, sulphur, chlorine, and calciumwere present in the stromal tissues (Fig. 8).X-ray diffraction examination :    According to the X-ray diffraction pattern obtained from the mineral deposits, the main peakoccurred at a diffraction angle of 32', and other peaks, at angles of 25.8e, 31.8e, 32.8e, and 49.4". Thispattern indicated that the mineral deposits were chiefly composed of hydroxyapatite.Discussion   Calcinosis universalis is a rare complication associated with collagen diseases. The few casereports consisted mainly of radiographic findings and a few histopathological features. Nielsen et 'al.9) studied the ultrastructure and the composition of the mineral elements of the lesion. However,as far as we know, there are no other studies at the electron microscopic level on this disease. Werecently described the ultrastructural appearance of initial calcification in a case of calcinosisuniversalis accompanying dermatomyositis.'} In our report, the ultrastructure was described asfollows: the calcified materials were distributed on collagen fibers and seemed to have a positiverelationship with foci of fibrinoid degeneration. Globular andlor membranous structures, consideredto have derived from the degenerated cells of the stroma, were observed in these calcified areas, andsome of them contained electron-dense materials, Therefore, we proposed that the globular andlormembranous structures might be involved in.the initial calcification in this case.   In the present paper, we examined the mineral deposits in the same case of calcinosis univer-salis by analytical electron microscopy. Through EDS analysis, mineral deposits of the specimenwere found to consist of sodium, phosphorus, calcium, rhenium, and some also contained sulphur andmagnesium. The deposits were mainly composed of hydroxyapatite according to the results of thefield-limited area electron diffraction and X-ray diffraction examinations. It was proposed by Loewiand Dorling 'O) that the mineral deposits in calcinosis universalis are built up from hydroxyapatite ;and Nielsen et al.9) reported the mineral part to be calcium apatite (calcium-hydroxyapatite orcalcium fluoroapatite). The above•mentioned reports are in accord with our present findings. Pinter44 Kawakami, et aL : Analytical Electron Microscopy of Calcinosis Universaliset al.ii) investigated the calcified masses in a case of calcinosis universalis using X-ray diffraction,and found them to be composed of a rare kind of carbonate apatite. Therefore, it seems that thecomposition of mineral deposits is different depending on the case investigated. However, webelieve the mineral deposits in most cases are composed of hydroxyapatite.   Regarding our results of the mineral composition of the stromal tissue, we have a strongsuspicion, especially in view of the quantitative relationship between stromal elements and thosein calcified deposits, that the mineral elements in the stromal tissue are involved in the calcificationmechanism. So, in our continuing work, the relationship between the mineral components and theultrastructure in both deposits and surrounding stroma should be investigated, which should give usa better understanding of mechanism of calcification in calcinosis universalis.                                  Acknowledgement   This study was suported in part by a grant (No. 61570901) from the Ministry of Education,Culture and Science, Japan. The authors wish to express their thanks to Dr. T. Yamazaki, ShinshuUniversity School of Medicine, for suplying of the surgical material from and clinical data on thepatlent.                                      References1 ) Arendt, E. C., Pattee, C. J. and Mitchell, H. S. (1955) Calcinosis universalis. Can. Med. Assoc. J. 73 : 269   -272.2 ) Finerrna, G. A. M., Phang, J. M. and Hahn, T. J. (1967) Studies in calcinosis universals. Surg. Foru. 18 :   487-489.3 ) Gould, S. E. and Raiford, F. T. (1938) Calcinosis universalis, report of case with autopsy findings. Am.   J. Roentogen. 37 : 741-746.4 ) Jarmillo, S., Goodman, C. R., Golin, A. M. and Benton, J. E. (1965) Calcinosis universals : report of two   cases. Arch. Phy. Med. Rehabit. 48 : 667-671.5 ) Johnson, R. H. (1971) A unique case of pathologic calcification ? Oral Surg. 32 : 66-74.6 ) Minemura, T., Yamazaki, T., Yajima, M., Tamura, M., Kurashina, K., Takeda, S., Kotani, A., Kawa-   kami, T. and Eda, S. (1985) A case of calcinosis universalis especially appearing in the sublingual   region. J. Jpn. Stomatol. Soc. 34 : 442-448.7) Kawakami, T.,Nakamura, C., Hasegawa, H., Eda, S., Akahane, S., Yamazaki, T. and Takasu, N.   (1986) Ultrastructural study of calcinosis universalis with dermatomyositis. J. Cutane. Pathol. 13 : 135   -143.8 ) Karnovsky, M. J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron   microscopy. J. Cell Biol. 27:137 A. •9) Nielsen, A.O., Johnson, E., Hentzer, B.and Kobayashi, T. (1979) Dermatomyositis with universal   calcinosis, a histopathological and electron optic study. J. Cutane. Pathol. 6 : 486-491.10) Loewi, G. and Dorling, J. (1964) Calcinosis. Histological and chemical analysis. Ann. Rheum. Dis. 23 : 272   -279.11) Pinter, J., Lenart, G. and Rischak, G. (1975År Ultrastructure of mixed form of calcinosis. Clin. Orthop.   107 : 295-297.l!Lf"Lptss[\-r 14(1) 1988 45eet,R' rk .sc,ee ag-  twgWFig. 1.Fig. 2.Fig. 3.ag.ge,• twewff bewspt,,tw-twwwaj-, $t. tstt tt.esofa, S     g...   twajire ':SscthX-ray photograph showing small radiopaque granules located in the sublingual regionX-ray photograph revealing radiopaque granules located in the neck regionCalcified globular masses strongly stained xx'ith hematoxylin (H-E. undemineralizedsection, X220)aj46 Kawakami, et al. : Analytical Eiectron Microscopy of Calcinosis UniversalisFig. 4.Fig. 5.Transmission electron microseopic photograph showing columnar crystals and theirfield-limited area electron-diffraction pattern (insert) indicating them as hydroxyapatite(unstained & undemineralized section, Å~150,OOO)Scanning electron microscopic composition image of polished surface of embedded cal-cified materials (Å~ 50)iAfkJli[de[llij 14(1) 1988 47as" twt  x•agE, ,ge.st/.,ee7 Ae24a H=4eKEU ltlQ AQ=4eKEU IQ,1 e,eeKEU l v.3 247cXESH=4eKE (.J t : I Q.ps eRle,,-'xl24KEUÅr ..AQ=4eKEU 10,CRL,!5U-AREp-s76224 'if.le.r.er, l" ,' .....N.5t.. ".eq.3in#q.. MGNA. ti . eeKEU XES  ie24 H=4gK.EV 1:IQ7dP s CACAL5U-•AREP-81e . 2-' 4 kll E l..1 .:•AQ=4eKEI.t IQeAL.15U-AREP-lecuÅq A- eeKEU XEf.• le. 24 K. EUÅr Åq g. P- Et kl' EV XE$ 1e.24KEUÅrFig. 6.Fig. 7.Scanning electron microscopic composition image of the fractured surface of a calcifiedmass showing the bright mass surrounded by a dark matrix (Å~200)EPMA by EDS of calcified material (a and b) and of stromal tissues Åqc and d)48 Kawakami, et al. : Analytical Electron Microscopy of Calcinosis Universalis8a  PROBE  STAGEEL ULV 6.45RE 6.73SR 6.86U 6.9SSt 7.13TA 7.25•RB 7.32HF 7.S4LU 7.B4YB e.15AL e.34BR 8.37ER 8.e2SE S.??HO 9.20DY 9.59AS 9.67MG 9.89TB 10.00GE 10.44GA 11.29NA tl.9t** 15,80F 18.32RESULTS:THE  NA MeTHE  F CRCURRENT : 2,230E-Oe (A)POS.: X 16973 V 25077 Z     CH(1) TAP  CeVNT INTENSrTY(LOG)FOLLOVING     PFOLLOUING     HN 39 ***I**t**370 *I********1+++ 31 **#**** 3e t*tt*ttt 2B l*tt**** 26 *******+ 26 ***l***+ 22 l****** IS **S*** 13 ****** 16 ****t** 13 ****# 15 **Itt+ 12 **#*+ 12 t***** 6 **I* 9 ****t 5J *****++++ 11 ****** 6 **t* 5 ****23 ***t+++ 2 *+ 4 *+++ ELEMENTS ARE PRESENT S CA RE ELEHENTS ARE PROBABLLY LA EU11670 EL WL TI 2.7S BA 2.7e cs 2.eg SC 3,03  1 3.15 TE 3.29 CA 3.36 SB 1.4- SN 3,60 K 3.74  IN 3.77 U 3.Vt cn 3.vs TH 4.1- AG 4.15 PD 4.37 RH 4.60 CL 4.73 RU -.S5 S 5.37  HO 5.41 NB 5.72 ZR 6.07 P 6.16PRESENT   CH(2)COUNT  15  12  12  t9  13  15J4ee   7   9  10   7   7   4   5   5   5   3   3   1  t3   2   2   1 273     PET    INTENSITY(Lee)*********t********t*"ttt+**************"***I***++++++++++***#***l#t**t*t***+****+t*************t+t*t**+'**++++*+*++****++++++++++EL WLel 1.14PB 1.17TL 1.2iHG 1,2-AU 1.2ePT 1,31IR 1.35OS t.39ZN 1.44CU 1.54NI 1,66TH 1.73CO t,7?FE 1.?4GP 2,05MN 2.10EV 2.t2SH 2.20CR 2.29ND 2.37PR 2,46V 2.SOCE 2.56LA 2.67COUNT CH(3) LIF       rNTENSITY{LOG)14 ****I*!1 l****+11 l*****12 ***S*+13 ******10 ****S11 l*****11 ******13 *****t12 *****+ 7 ***** s ***st 6 **t* 5 **t+ 4 ***+ 7 ***++ 4 *+++ 4 ***t 2++. 2 *+ 1+ t* o 2 ++8b   PROBE   STAGE EL UL Y 6.45 RE 6.73 SR 6.S6 u 6,7e Sr 7,13 TA 7.25RB 7.32HF 7.54LU 7.84YB e.t5AL 8.34BR 8.37ER S.S2SE 8,7VHO 9.20DV Y.59AS 9,67MG 9,e9Te to,oeGE tO,4AGA 11.29NA tt.91** 15.80F 18.32RESULTS:THE  NA PTHE  MG KCURRENT t 2.220E-Oe (A)POS. t X 17153 Y 25230     CH(t) TAP COUNT :NTENSITY(LOG)FQLLOWING    sFOLLDUING    FEFig. 8.55 ********"+57 #********43 *****t***2? ***t****37 *t*****I+J5 ********34 ***U*tt34 *******t•17 *t*t***23 ***t*ttIS **l***17 ***t**+t3 ******13 ******t2 ***t** 9 **t#t3 #****le *****++ 9 ***** 7 t**** 4 ****se l****+++++ t* 1* ELEMENTS ARE PRESENT CL CA ELEMENTS ARE PROBAELLV SH GDEPMA by WDS of composltlonZ lt72eEL WLTr 2.75BA 2,7aCS 2.S9sc 3.e31 3.15TE ].29CA 3.36SB 3.44SN ].60K 3.74IN. 3.77V 3.91CD 3.96TH 4.laAG 4.t5PD 4.37RH 4.60eL 4.73RU 4.S5S 5.37HO 5.41NB S.72ZR 6.07P 6.16   CH(2)COUNT  4  4  6  4  4  ]142 3 1 ` 2 2 J 1  1  t  1 e 1 7 2 1 o 13     PET     INTENSITY(LDG)***********t***+***+***tt*t++l-+++t*+"*+**++**t**tlt**tts**+++*+++++**+++++++EL WLBI 1.14PB 1.17TL 1.21HG 1.24AU 1.2BPT 1.3tJR t.35OS t.39ZN 1.44CV 1.S4NI 1,66TM 1.73CD 1.79FE 1,94GD 2.05MN 2,10EU 2.12SH 2.2eCR 2.29ND 2.37PR 2.46V 2.50CE 2,56LA 2.67PRESENTa mineral deposit (a) and stromal tissue (b)   CH{3)COUNT4 ****5 ****4 *"**5 ****5 ***t4 ****4 **t*3 ***2 **5 ***+4 **t+A **++3 **+5 *+++4 *+++s+] **+2 ++2 **2 **1*2 *+1*o LIFINTENSITY(LOG)demonstrating element

Analytical Electron Microscopic Study of Mineral Deposits in a Case of Calcionsis Universalis

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Analytical Electron Microscopic Study of Mineral Deposits in a Case of Calcionsis Universalis

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