19593124

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Med Mol Morphol (2005) 38:189–195 © The Japanese Society for Clinical Molecular Morphology 2005DOI 10.1007/s00795-005-0290-7

ORIGINAL PAPER

Masafumi Mimura · Nobuyuki Tanaka · Shizuko Ichinose

Yutaka Kimijima · Teruo Amagasa

Possible etiology of calculi formation in salivary glands: biophysical analysis

of calculus

Received: December 1, 2004 / Accepted: February 1, 2005

M. Mimura (*) · T. AmagasaMaxillofacial Surgery, Maxillofacial Reconstruction and Function,Division of Maxillofacial and Neck Reconstruction, GraduateSchool, Tokyo Medical and Dental University, 1-5-45 Yushima,Bunkyo-ku, Tokyo 113-8510, JapanTel./Fax +81-3-5803-5500e-mail: [email protected]

N. TanakaDepartment of Oral Surgery, Sapporo Medical University School of Medicine, Sapporo, Japan

S. IchinoseInstrumental Analysis Research Center for Life Science, TokyoMedical and Dental University, Tokyo, Japan

Y. KimijimaKimijima Dental and Oral-Surgery Clinic, Tokyo, Japan

Abstract Sialolithiasis is one of the common diseases of thesalivary glands. It was speculated that, in the process of calculi formation, degenerative substances are emitted by

saliva and calcification then occurs around these substances,and finally calculi are formed. However, the exact mecha-nism of the formation of calculi is still unclear. In this study,we identify some possible etiologies of calculi formation insalivary glands through biophysical analysis. Calculi from 13patients with submandibular sialolithiasis were investigatedby transmission electron microscopy, scanning electronmicroscopy, X-ray microanalyzer, and electron diffraction.Transmission electron microscopic observation of calculiwas performed in the submandibular gland (n = 13). In 3 of the 13 cases, a number of mitochondria-like structures andlysosomes were found near calcified materials. Scanningelectron microscopic examination of these materials re-

vealed that there were lamellar and concentric structuresand that the degree of calcification was different amongthe calculi. X-ray microanalysis disclosed the componentelements in the calculi to be Ca, P, S, Na, etc., and the mainconstituents were Ca and P. The calcium-to-phosphorusratio was 1.60–1.89. Analysis of the area including mito-chondria-like structures, lysosomes, and the fibrous struc-tures by electron diffraction revealed the presence of hydroxyapatite and calcified materials. It is speculated that

mitochondria and lysosomal bodies from the ductal systemof the submandibular gland are an etiological source forcalcification in the salivary gland.

Key words Salivary calculi · Ultrastructure · Mitochondria ·Ductal system

Introduction

Sialolithiasis is characterized by obstruction resulting froma calculus in the salivary glands and is associated with swell-ing, pain, and infection of the affected gland. The salivarystones are composed of calcium phosphate and carbonatein the form of hydroxyapatite (HA), with small amounts of

magnesium, potassium, and ammonium.1,2

The mechanismof calculi formation in the salivary glands is under investiga-tion, and a previous report noted that degenerative sub-stances might be emitted by saliva, by some phenomena,and that calcification around these substances might occur,contributing to the formation of calculi.3 However, the ori-gin of these substances has not been clarified.

The present study revealed that calculi contained mito-chondria-like materials recognized by transmission electronmicroscopy, and examination of the calculi was done withscanning electron microscopy, an X-ray microanalyzer, andelectron diffraction studies. Mitochondria-like features of salivary calculi under the ultrastructural level have not been

reported previously. The present article covers the particu-lar structure of the mitochondrial elements of the calculiand identifies some possible etiological factors in calculiformation.

Materials and methods

Calculi from 13 patients with submandibular sialolithiasiswere investigated by transmission electron microscopy. In 3of them, mitochondria-like structures were recognized, and

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these 3 calculi were examined by scanning electron micros-copy, an X-ray microanalyzer, and electron diffraction.

Each calculus was fixed with 2.5% glutaraldehyde in0.01M phosphate-buffered saline (pH 7.4) for several hoursand then cut into two pieces, one of which was used forscanning electron microscopic observation. The other piecewas cut into 1-mm3 pieces and used for transmission elec-tron microscopic observation. For scanning electron micro-

scopic observation, the fixed specimens were dehydrated ina graded ethanol series and then dried in a critical pointdrying apparatus (HCP-2; Hitachi, Tokyo, Japan) withliquid CO2. The samples were spatter-coated with platinumor carbon. They were observed by a scanning electronmicroscope (S-4500; Hitachi, Tokyo, Japan) and analyzedby an energy dispersive X-ray spectroscope (EMAX-7000;Horiba, Kyoto, Japan).

For transmission electron microscopic observation, thefixed specimens were postfixed with 2% osmium tetroxidein 0.01M phosphate-buffered saline (pH 7.4) for 2h, dehy-drated in a series of graded concentrations of ethanol, andembedded in epoxy resin. Before the transmission electron

microscopic observation, ultrathin sections were stainedwith toluidine blue and examined by light microscopy formorphologic orientation. Ultrathin nondecalcified sectionswere stained with uranyl acetate and lead citrate and then

examined by transmission electron microscopy (HitachiH-600; accelerating potential, 75kV; objective aperture,100mm in diameter; Hitachi). Some ultrathin sections werealso used for selected area electron diffraction to study HA.Atomic structure-related D-spacings of diffraction patternswere calibrated from the specimens using a gold controlevaluated under identical conditions.

Results

Scanning electron microscopic observation revealed thatthe cut surface of salivary calculi was composed of lamellarand concentric structures. The central parts of the calculiwere amorphous, and their electron density was high. Theinside of the central parts was smooth and the outside wasrelatively rough. On the outside of these central parts, therewere hill-like structures and small crater-like cavities, andthere were lamellar structures surrounding the hill-likestructures. The most external parts consisted of a number of

fibrous and round structures (Fig. 1).Backscattered scanning electron microscopic images

revealed that calcification of the external layer was not asprominent as in the other parts (Fig. 2).

Fig. 1. Scanning electron micrograph of the calculus shows concentric structures (case 1). C , central part; E, external layer

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X-ray microanalysis revealed the component elements inthe calculi to include Ca, P, S, and Na, and the main con-stituents were Ca and P (Fig. 3). The calcium-to-phosphorusratio ranged from 1.60 to 1.89, whereas it was 2.06 in

hydroxyapatite and 1.61–1.97 in the calculi in which nomitochondria-like structure was seen.

Transmission electron microscopic observation showedfibrous structures between mitochondria-like structuresand/or lysosomes. The mitochondria-like structures re-sembled intact mitochondria in shape and included highelectron density materials; however, cristae were rarelyfound. In the lysosomes, high electron density materialswere also found (Fig. 4A). In some parts, a few fibrousstructures were seen, and amorphous structures were alsofound surrounding the mitochondria-like structures andlysosomes (Figs. 4B–6). Analysis of the area including mito-

chondria-like structures, lysosomes, and the fibrous struc-tures by electron diffraction revealed that they includedhydroxyapatite and calcified materials. The area in whichthere were no fibrous structures was more crystallized than

that which included fibrous structures (Fig. 7).

Discussion

It is suggested that the biological process of calculi forma-tion in salivary glands is characterized by reduced secretoryactivity, alterations in electrolyte concentrations, and im-pairment of glycoprotein synthesis in the salivary glands, allof which could result from structural deterioration of thecell membranes during aging.4 More than 80% of salivary

Fig. 2. Scanning electron micrograph (A) and backscattered scanning electron microscopic image (B) show that calcification of the external layerwas not outstanding (case 1)

Fig. 3. X-ray microanalysis of the calculus shows that themain constituents are Ca and P(case 1)

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Fig. 4. Transmission electron micrographs show fibrous structures with high electron density (A) and calcified materials near mitochondria-likestructures and lysosomes (B) (case 2). Bar 3mm

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Fig. 5. Transmission electronmicrograph shows calcifiedmaterials near mitochondria-like structures and lysosomes(case 1). Bar 3mm

Fig. 6. Transmission electron micrograph showing calcified materials near mitochondria-like structures and lysosomes (case 3). Bar 3mm

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calculi occur in the submandibular gland or its duct.5 Thereasons are speculated to be the following: the submandibu-lar gland has a long duct with slow flow rates, the flow of saliva is against gravity, and saliva from the submandibular

gland contains more alkaline, mucin, and calcium than thatfrom the other glands.6,7

The genesis of calculi has been reported to be related toinflammation, bacterial organisms, stagnation of saliva, andforeign bodies8; however, the exact cause of the calculi re-mains unknown. The present study revealed calcificationaround the mitochondria-like structures and lysosomal bod-ies. Cristae of mitochondria were not clearly recognized inthe mitochondria-like structures. Mitochondrial cristaemight be lost during the degenerative change involving in-flammation in the striated duct. The existence of a numberof lysosomes is found in degenerative cells,9 and these may

possibly indicate some connection between degenerativecells and calculi formation in this study.

Numerous mitochondria in the basal infolding area isa specific characteristic of the salivary gland duct.

Triantafyllou et al.10 showed concentrations of mitochon-dria and a group of electron-dense lysososmes in the ductalcells of cat parasympathectomized submandibular glandthat had produced microliths. It is of interest that concen-trations of mitochondria and lysosomes appeared in thedegenerative ductal cells. These organelles seem to be acore structure of calcification. It is reported that in thecalcified odontogenic cyst the core of calcification might bedegenerative mitochondria and tonofibril bundles of ghostcells.11 Microliths are found in normal glands as well as insialadenitis, and it has been suggested that they are possiblythe origin of liths.12–14 However, on the basis of a clinico-

Fig. 7. Electron diffraction pattern of areas of Fig. 4A,B. Bothareas identified as hydroxyapatite; however, the latter (B) ismore crystallized than the former (A)

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pathological investigation of 154 cases with chronic sub-mandibular sialadenitis, Harrison et al.15 reported thatmicroliths and liths were unrelated, and that microlithswere related to the aging process in normal glands whereasliths occurred as a result of a duration of symptoms andappeared to be secondary to the sialadenitis.

In the present study, mitochondria-like structures andlysosomes were clearly evident in the calcified bodies, and

these findings may be helpful for diagnosis of the initialstage of calcification. Analysis by electron diffraction sug-gested that the area that included no fibrous structuresmight be more calcified than that the area which includedfibrous ones. It is also possible that the core substances of calcification are not limited to the degenerative mitochon-dria and lysosomal bodies. Previous reports have noted thatsialoliths contained a lipid-enriched matrix that was associ-ated with mineral deposition of sialoliths16–18 and also that amucoid element of saliva created the calculus by depositionof calcium.19

In the present analysis of the constituent element, thecalcium-to-phosphorus ratio was not so different from that

of the other calculi in which the mitochondria-like struc-tures could not be found, and the core substances were sodegenerative that original organelles were considered un-recognizable. The synthesis of degenerative materials intothe ductal system has an important role in the formation of calculi, and inflammation, bacterial organisms, abnormalstagnation of saliva, and foreign bodies are possible causesof the production of the degenerative organelles. In thepresent study, mitochondria-rich materials and lysosomesof the core substances of calcification were suggested to bepresent at the electron microscopic level in calculi of thesubmandibular glands.

References

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3. Tanaka N, Ichinose S, Adachi Y, Mimura M, Kimijima Y (2003)Ultrastructural analysis of salivary calculus in combination withX-ray microanalysis. Med Electron Microsc 36:120–126

4. Bonder L, Grosky M (1996) Parotid gland secretion of the agingrat. Arch Gerontol Geriatr 22:63–69

5. Escudier MP, Anthony N (1999) The management of sialolithiasisin 2 children through use of extracorporeal shock wave lithotripsy.Oral Surg Oral Med Oral Pathol Oral Radiol Endod 88:44–49

6. Iro H, Schneider HT, Fodra C, Waitz G, Nitsche N, Heinritz HH,Benninger J, Ell C (1992) Shockwave lithotripsy of salivary ductstones. Lancet 339:1333–1336

7. Iro H, Zenk J, Benzel W (1995) Minimally invasive therapy forsialolithiasis: the state of the art. In: Myers EN, Bluestone CD,Brackmann DE, Krause CJ (eds) Advances in otolaryngology:head and neck surgery, vol 9. Mosby, St. Louis, pp 31–48

8. Wakae H, Tomioka T (1998) Salivary calcus. In: Hashimoto K (ed)Electron microscopic atlas of oral disease. Nagasue Shoten, Kyoto,pp 52–53

9. Tanaka N, Sugihara K, Odajima T, Mimura M, Kimijima Y,Ichinose S (2002) Oral squamous cell carcinoma: electron micro-scopic and immunohistochemical characteristics. Med ElectronMicrosc 35:127–138

10. Triantafyllou A, Harrison JD, Garrett JR (1993) Production of salivary microlithiasis in cats by parasympathectomy: light andelectron microscopy. Int J Exp Pathol 74:103–112

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T (2002) An ultrastructural study of calcified odontogeniccyst – especially calcified material. Med Electron Microsc 35:109–116

12. Epivatianos A, Harrison JD, Dimitriou T (1987) Ultrastructuraland histochemical observations on microcalculi in chronic sub-manbibular sialadenitis. J Oral Pathol 16:514–517

13. Epivatianos A, Harrison JD (1989) The presence of microcalculi innormal human submandibular and parotid salivary glands. ArchOral Biol 34:261–265

14. Scott J (1978) The prevalence of consolidated salivary deposits inthe small ducts of human submandibular glands. J Oral Pathol7:28–37

15. Harrison JD, Epivatianos A, Bhatia SN (1997) Role of microlithsin the aetiology of chronic submandibular sialadenitis: a clinico-pathological investigation of 154 cases. Histopathology (Oxf)31:237–251

16. Anneroth G, Eneroth CM, Isacsson G (1977) The relation of lipidsto the mineral components in salivary calculi. J Oral Pathol 6:373–381

17. Boskey AL, Boyan-Salyers BD, Burstein LS, Mandel ID (1981)Lipid associated with mineralization of human submandibulargland sialoliths. Arch Oral Biol 26:779–785

18. Ennever J, Vogel JJ, Benson LA (1973) Lipid and calculus matrixcalcification in vitro. J Dent Res 52:1056–1059

19. Harirll J, King J, Boyce W (1959) Structure and composition of salivary calculi. Laryngoscope 69:482–492

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