Med Mol Morphol (2005) 38:189195 DOI 10.

1007/s00795-005-0290-7

The Japanese Society for Clinical Molecular Morphology 2005

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

Abstract Sialolithiasis is one of the common diseases of the salivary glands. It was speculated that, in the process of calculi formation, degenerative substances are emitted by saliva and calcication then occurs around these substances, and nally calculi are formed. However, the exact mechanism of the formation of calculi is still unclear. In this study, we identify some possible etiologies of calculi formation in salivary glands through biophysical analysis. Calculi from 13 patients with submandibular sialolithiasis were investigated by transmission electron microscopy, scanning electron microscopy, X-ray microanalyzer, and electron diffraction. Transmission electron microscopic observation of calculi was performed in the submandibular gland (n = 13). In 3 of the 13 cases, a number of mitochondria-like structures and lysosomes were found near calcied materials. Scanning electron microscopic examination of these materials revealed that there were lamellar and concentric structures and that the degree of calcication was different among the calculi. X-ray microanalysis disclosed the component elements in the calculi to be Ca, P, S, Na, etc., and the main constituents were Ca and P. The calcium-to-phosphorus ratio was 1.601.89. Analysis of the area including mitochondria-like structures, lysosomes, and the brous structures by electron diffraction revealed the presence of hydroxyapatite and calcied materials. It is speculated that

mitochondria and lysosomal bodies from the ductal system of the submandibular gland are an etiological source for calcication in the salivary gland. Key words Salivary calculi Ultrastructure Mitochondria Ductal system

Introduction
Sialolithiasis is characterized by obstruction resulting from a calculus in the salivary glands and is associated with swelling, pain, and infection of the affected gland. The salivary stones are composed of calcium phosphate and carbonate in the form of hydroxyapatite (HA), with small amounts of magnesium, potassium, and ammonium.1,2 The mechanism of calculi formation in the salivary glands is under investigation, and a previous report noted that degenerative substances might be emitted by saliva, by some phenomena, and that calcication around these substances might occur, contributing to the formation of calculi.3 However, the origin of these substances has not been claried. The present study revealed that calculi contained mitochondria-like materials recognized by transmission electron microscopy, and examination of the calculi was done with scanning electron microscopy, an X-ray microanalyzer, and electron diffraction studies. Mitochondria-like features of salivary calculi under the ultrastructural level have not been reported previously. The present article covers the particular structure of the mitochondrial elements of the calculi and identies some possible etiological factors in calculi formation.

M. Mimura (*) T. Amagasa Maxillofacial Surgery, Maxillofacial Reconstruction and Function, Division of Maxillofacial and Neck Reconstruction, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan Tel./Fax +81-3-5803-5500 e-mail: mimuram@casio.co.jp N. Tanaka Department of Oral Surgery, Sapporo Medical University School of Medicine, Sapporo, Japan S. Ichinose Instrumental Analysis Research Center for Life Science, Tokyo Medical and Dental University, Tokyo, Japan Y. Kimijima Kimijima Dental and Oral-Surgery Clinic, Tokyo, Japan

Materials and methods

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

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these 3 calculi were examined by scanning electron microscopy, an X-ray microanalyzer, and electron diffraction. Each calculus was xed with 2.5% glutaraldehyde in 0.01 M phosphate-buffered saline (pH 7.4) for several hours and then cut into two pieces, one of which was used for scanning electron microscopic observation. The other piece was cut into 1-mm3 pieces and used for transmission electron microscopic observation. For scanning electron microscopic observation, the xed specimens were dehydrated in a graded ethanol series and then dried in a critical point drying apparatus (HCP-2; Hitachi, Tokyo, Japan) with liquid CO2. The samples were spatter-coated with platinum or carbon. They were observed by a scanning electron microscope (S-4500; Hitachi, Tokyo, Japan) and analyzed by an energy dispersive X-ray spectroscope (EMAX-7000; Horiba, Kyoto, Japan). For transmission electron microscopic observation, the xed specimens were postxed with 2% osmium tetroxide in 0.01 M phosphate-buffered saline (pH 7.4) for 2 h, dehydrated in a series of graded concentrations of ethanol, and embedded in epoxy resin. Before the transmission electron microscopic observation, ultrathin sections were stained with toluidine blue and examined by light microscopy for morphologic orientation. Ultrathin nondecalcied sections were stained with uranyl acetate and lead citrate and then

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

Results
Scanning electron microscopic observation revealed that the cut surface of salivary calculi was composed of lamellar and concentric structures. The central parts of the calculi were amorphous, and their electron density was high. The inside of the central parts was smooth and the outside was relatively rough. On the outside of these central parts, there were hill-like structures and small crater-like cavities, and there were lamellar structures surrounding the hill-like structures. The most external parts consisted of a number of brous and round structures (Fig. 1). Backscattered scanning electron microscopic images revealed that calcication of the external layer was not as prominent 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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Fig. 2. Scanning electron micrograph (A) and backscattered scanning electron microscopic image (B) show that calcication of the external layer was not outstanding (case 1)

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

X-ray microanalysis revealed the component elements in the calculi to include Ca, P, S, and Na, and the main constituents were Ca and P (Fig. 3). The calcium-to-phosphorus ratio ranged from 1.60 to 1.89, whereas it was 2.06 in hydroxyapatite and 1.611.97 in the calculi in which no mitochondria-like structure was seen. Transmission electron microscopic observation showed brous structures between mitochondria-like structures and/or lysosomes. The mitochondria-like structures resembled intact mitochondria in shape and included high electron density materials; however, cristae were rarely found. In the lysosomes, high electron density materials were also found (Fig. 4A). In some parts, a few brous structures were seen, and amorphous structures were also found surrounding the mitochondria-like structures and lysosomes (Figs. 4B6). Analysis of the area including mito-

chondria-like structures, lysosomes, and the brous structures by electron diffraction revealed that they included hydroxyapatite and calcied materials. The area in which there were no brous structures was more crystallized than that which included brous structures (Fig. 7).

Discussion
It is suggested that the biological process of calculi formation in salivary glands is characterized by reduced secretory activity, alterations in electrolyte concentrations, and impairment of glycoprotein synthesis in the salivary glands, all of which could result from structural deterioration of the cell membranes during aging.4 More than 80% of salivary

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

193 Fig. 5. Transmission electron micrograph shows calcied materials near mitochondrialike structures and lysosomes (case 1). Bar 3 mm

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

194 Fig. 7. Electron diffraction pattern of areas of Fig. 4A,B. Both areas identied as hydroxyapatite; however, the latter (B) is more crystallized than the former (A)

calculi occur in the submandibular gland or its duct.5 The reasons are speculated to be the following: the submandibular gland has a long duct with slow ow rates, the ow of saliva is against gravity, and saliva from the submandibular gland contains more alkaline, mucin, and calcium than that from the other glands.6,7 The genesis of calculi has been reported to be related to inammation, bacterial organisms, stagnation of saliva, and foreign bodies8; however, the exact cause of the calculi remains unknown. The present study revealed calcication around the mitochondria-like structures and lysosomal bodies. Cristae of mitochondria were not clearly recognized in the mitochondria-like structures. Mitochondrial cristae might be lost during the degenerative change involving inammation in the striated duct. The existence of a number of lysosomes is found in degenerative cells,9 and these may

possibly indicate some connection between degenerative cells and calculi formation in this study. Numerous mitochondria in the basal infolding area is a specic characteristic of the salivary gland duct. Triantafyllou et al.10 showed concentrations of mitochondria and a group of electron-dense lysososmes in the ductal cells of cat parasympathectomized submandibular gland that had produced microliths. It is of interest that concentrations of mitochondria and lysosomes appeared in the degenerative ductal cells. These organelles seem to be a core structure of calcication. It is reported that in the calcied odontogenic cyst the core of calcication might be degenerative mitochondria and tonobril bundles of ghost cells.11 Microliths are found in normal glands as well as in sialadenitis, and it has been suggested that they are possibly the origin of liths.1214 However, on the basis of a clinico-

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pathological investigation of 154 cases with chronic submandibular sialadenitis, Harrison et al.15 reported that microliths and liths were unrelated, and that microliths were related to the aging process in normal glands whereas liths occurred as a result of a duration of symptoms and appeared to be secondary to the sialadenitis. In the present study, mitochondria-like structures and lysosomes were clearly evident in the calcied bodies, and these ndings may be helpful for diagnosis of the initial stage of calcication. Analysis by electron diffraction suggested that the area that included no brous structures might be more calcied than that the area which included brous ones. It is also possible that the core substances of calcication are not limited to the degenerative mitochondria and lysosomal bodies. Previous reports have noted that sialoliths contained a lipid-enriched matrix that was associated with mineral deposition of sialoliths1618 and also that a mucoid element of saliva created the calculus by deposition of calcium.19 In the present analysis of the constituent element, the calcium-to-phosphorus ratio was not so different from that of the other calculi in which the mitochondria-like structures could not be found, and the core substances were so degenerative that original organelles were considered unrecognizable. The synthesis of degenerative materials into the ductal system has an important role in the formation of calculi, and inammation, bacterial organisms, abnormal stagnation of saliva, and foreign bodies are possible causes of the production of the degenerative organelles. In the present study, mitochondria-rich materials and lysosomes of the core substances of calcication were suggested to be present at the electron microscopic level in calculi of the submandibular glands.

References
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