Submandibular Gland Sialolithiasis Sialographic and Pathologic Findings with Evaluation Using SEM and EPMA Analysis

J. Nihon Univ. Sch. Dent., Vol. 29, 112-123, 1987

Masahiko MIYAKE, Toshihiko ISHII, Megumi ANDOH, Yasuo TAKA YAMA, Yoshinari TOHYAMA, Minoru HORI, Toyoo FUJISAKI, Hiroyasu ASAHINA, Hiroshi TANAKA and Hiroshi SA TO*

(Received 12 February 1987)

Key words: sialolithiasis, submandibular gland, calculus, sialography, SEM, EPMA

Introduction Submandibular gland sialolithiasis is a common disease treated by oral surgery, and many case reports have been presented in the Japanese medical literature[1-31]. However, the observation of salivary stones by scanning electron microscopy and electron probe X-ray microanalysis has not been widely reported[25-31]. Recently, we treated four cases of submandibular gland sialolithiasis by removal of the submandibular gland, and examined these stones with a scanning electron microscope (SEM) and electron probe X-ray microanalyzer (EPMA), as well as performing standard clinical sialographic and histopathologic examinations of the extirpated glands. In the present report, we compare our findings from these various methods of examination. Report of cases The clinical aspects of the four cases are summarized in Table 1.

Table 1

In all cases, the chief complaint was swelling of the submandibular region. Palpation revealed that case 1 (with the longest history of complaint) showed re-

三宅正彦,石井俊彦,安藤惠,高山泰男,遠山良成,堀稔,藤崎東洋男,朝比奈浩康,田中博,佐藤廣: First and Second* Departments of Oral Surgery, Nihon University School of Dentistry (Directors: Prof. Itsuro KUDO and Prof. Tomio TAKIGA WA*) 113

markable hardness of the submandibular gland in comparison with the other three. Although salivary flow from Wharton's duct could not be confirmed in case 1, swelling of the mouth floor and pus discharge were observed. In cases 2 and 3, salivary flow was confirmed upon stimulation with acetic acid, but the quantity and rate of flow seemed to be less than that on the normal side. These two patients com- plained of swelling of the submandibular region and so-called salivary colic during or after meals. In case 4 there was scarcely any salivary flow on the affected side, and no complaint of swelling or meal-associated pain. In cases 1 and 3, orthopantomo- grams revealed calculus level with the inferior border of the mandible, while in the other cases the calculus was below the mandibular border (Figs. 1 and 2). In all four of these cases the salivary stones could not be palpated bi-manually from either the intraoral or extraoral side. Surgical removal of the submandibular gland was therefore chosen as the method of treatment. Sialographic findings Urografin (60%) was used as a contrast medium, and examination was conducted radiologically at "the ductal phase"[7,34] in all cases. Case 1: Interruption and irregular outline of the main duct and extensive disappearance of peripheral ducts were observed, so that the gland itself was not apparent (Fig. 3).

Fig. 1 Orthopantomogram (Case 1) Fig. 2 Orthopantomogram (Case 4) The calculus is on the inferior border The calculus is under the border of of the mandible. the mandible.

Fig. 3 Sialographic lateral view (Case 1) Fig. 4 Sialographic lateralview (Case 2) 114

Fig. 5 Sialographic lateral view (Case 3) Fig. 6 Sialographic lateral view (Case 4)

Case 2 : Poor ductal filling by the contrast medium was observed at the site of calculus, and the main duct inside the gland was slightly dilated. However, the dendriform peripheral ducts were demonstrated clearly (Fig. 4). Case 3: The contrast medium did not reach as far as the posterior site of the calculus, so that the duct inside the gland was not apparent (Fig. 5). Case 4 : A radiolucent shadow of the main duct was seen at a portion of the calculus, and the main duct inside the gland was dilated moderately. The peripheral ducts clearly demonstrated a dendric pattern (Fig. 6). Histopathologic findings In case 1, remarkably thick and fibrous connective tissues were seen around the excretory duct, together with severe inflammatory cell infiltration composed of lymphocytes and plasma cells. Dilation of the intralobular duct and atrophy or disappearance of acini were marked, while these pathologic alterations were not so obvious in the other cases (Figs. 7 and 8). The excretory duct containing the sialolith was dilated. Squamous cell metaplasia and erosion and ulcer formation were occasionally observed in the covering epithelium (Fig. 9).

Fig. 7 Histopathological finding of sub- Fig. 8 Histopathological finding of submandibular gland (Case 1 H-E) mandibular gland (Case 3 H-E) 115

Fig. 9 Histopathological finding of excretory duct, showing site of calculus (Case 4, H-E)

Gross sialolith specimens The size of the stone in case 1 was 6 •~ 3.5 •~ 3.5 mm; case 2 had 2 pieces measur- ing 5 •~ 5 •~ 3 mm and 6 •~ 5 •~ 3 mm; in case 3 the stone measured 10 •~ 6 •~ 6 mm and in case 4 it was 2.5 •~ 5 •~ 5 mm. The surface of the stone in case 1 was rough in appearance, but in the other 3 cases the stones were smooth. In every case the stone was located in the submandibular duct within the gland parenchyma near the junc- tion of the duct and gland. A lamellar pattern with one core was seen in the cross- sectioned specimens in all cases. Observation of calculus with SEM Outer surface structure: Small granular structures were scattered over the surface of the calculus in every case. The surface of the stone in case 1 was relatively rough with abundant tunnel-like fissures (Fig. 10). Relatively homogeneous structures were demonstrated in the other three cases (Fig. 11). Cross-sectioned structure (Low magnification): In case 1 the stone exhibited abundant fissures and had a pumice-like appearance, with a fairly small core at its center and an irregularly diffuse lamellar structure at the periphery. In cases 2 and 3, the stones had a single, round and relatively large core in the center and a tight lamellar pattern at the periphery (Fig. 12). In case 4, the stone exhibited no lamellar pattern throughout most of the peripheral area despite the presence of an obvious central core, so that it seemed to be homogeneous and structureless (Fig. 13). High magnification of the lamellar structure: Aspects of the lamellar structures were diverse in each case. Case 1 was rich in crevices among the lamellar areas

(Fig. 14). Case 2 showed different structures consisting of alternate layers of closely homogeneous and roughly diffuse areas (Fig. 15). The stone in case 3 was relatively homogeneous but rough in appearance (Fig. 16), while that in case 4 appeared amorphous and structureless (Fig. 17). Analysis of calculus with EPMA Line X-ray energy pulse analysis was performed with an electron probe X-ray microanalyzer (Type JXA-5, Nihon Denshi Co.) with a 15-kV acceleration voltage

(Mg-103 cps, Ca-3 •~ 103 cps, P-3 •~ 103 cps) to determine the distribution of P, Ca 116

Fig. 10 SEM view Outer surface (Case 1), Fig. 11 SEM view Outer surface (Case 3) showing abundant tunnel-like fissures showing small scattered granular structures

Fig. 12 SEM view Cross-section (Case 2, Fig. 13 SEM view Cross-section (Case 4, low magnification), showing a lamellar pattern low magnification) 117

Fig. 14 SEM view Cross-section (Case 1, Fig. 15 SEM view Cross-section (Case 2, high magnification) Crevices are abundant high magnification) among the lamellar parts.

Fig. 16 SEM view Cross-section (Case 3, Fig. 17 SEM view Cross-section(Case 4, high magnification) high magnification) 118

Fig. 18 EPMA findings (Cases 1 and 2)

Fig. 19 EPMA findings (Cases 3 and 4) 119 and Mg. P and Ca distributions almost parallelled each other, but their quantities of distribution were uneven in different regions of the stones. However, the distributions of P and Ca in cases 3 and 4 were more uniform compared with those of cases 1 and 2. The levels of Mg in the 4 cases were uneven in each region, and the line distribution of Mg did not always parallel that of either P or Ca (Figs. 18 and 19). Discussion Clinical findings Sialolithiasis is a disease characterized by the formation of calculus inside a salivary duct. Although this condition may arise in any of the salivary glands, there is a tendency for stones to form in Wharton's duct of the submandibular gland[12,14, 18, 21, 33]. It is claimed that submandibular gland sialolithiasis is more commonly found in males[3,8, 21], but this view has also been contested[14,18], and no definitive conclusion has yet been reached. With regard to age as a factor in sialolithiasis, many reports agree that the greatest incidence occurs between the ages of 20 and 50 years. As to this aspect[8, 14,18, 21], OTsuKI and UEDA[3] reported that stones lying within the submandibular gland were found in younger age groups. In our present cases, the sex and age incidence corresponded to these general opinions: three of the four patients were in their thirties. Many reports have indicated that swelling and pain in the submandibular region are the most common symptoms[14,18, 21], with salivary colic and swelling of the gland at mealtimes also being characteristic of this condition. A decrease of salivary flow is also commonly reported[4, 18]. Among our cases, pain and swelling at mealtime were observed in cases 2 and 3. In these two cases, salivary flow from the diseased side was observed with a delay after stimulation, and the flow rate was less than that on the normal side. We considered these findings to be characteristic of this condition. The generally accepted forms of treatment are removal of an intra-ductal stone by the intraoral approach, or removal of the submandibular gland for an intra- glandular stone[14,18, 21]. There are differences of opinion concerning the methods of treatment for stones which lie at the boundary of the duct and the gland. HARA [14] recommended removal of the submandibular gland if functional recovery of the gland was not expected or if recurrence of inflammation was feared after sialographic examination. On the other hand, SELDIN [32]emphasized that the intra-oral approach for removal of a stone was the treatment of choice in most cases of sialolith, including stones at the duct / gland boundary. SuzA[181also recommended intra- oral conservative surgery unless dysfunctional salivary flow or recurrent symptoms indicated otherwise. We have been trying to remove stones at the duct / gland boundary using the intra-oral approach if they are relatively large and palpable from the mouth floor side, irrespective of whether or not functional disorders are indicated. However, if a stone is not palpable from the mouth floor, such as in the four cases reported 120 herein, we usually perform extra-oral removal of the submandibular gland. Our rationale is that the intra-oral approach does not provide a sufficiently wide operat- ing field in the most posterior region of the mouth floor, so that there is the pos- sibility of injury to the lingual nerve, artery or vein. Furthermore, we have found that roentgenological evidence showing whether or not the stone is level with the mandibular border has not been useful for determination of our operative approach. Sialographic findings Sialography is valuable for the diagnosis and treatment of this condition. How- ever, it has been pointed out that sialograms are influenced not only by the softness and quantity of the contrast medium but also by the stage at which the X-ray is taken. FuKuDA [34]and ARISUE [20],who carried out continuous X-ray imaging during the infusion of 1.2 ml of 76% urografin, reported that sialoliths appeared as ductal radiolucent shadows within excessively dilated ducts. In addition, such findings as structure, interruption and coarseness of the duct as well as sialostasis and homo- geneity of the gland were observed. For our cases (except case 3), we used 0.8-1.2 ml of 60% urografin and took the X-rays at the ductal phase. Our finding in case 1 of interruption of peripheral ducts differed from that in cases 2 and 4. From a comparison of the clinical and sialographic findings, we suspect that the long history of the condition in case 1 had caused severe changes in the peripheral ducts. Histopathologic findings Many reports have been published on histopathological studies of the submandibular gland under conditions of sialolithiasis[1,19, 20]. Pathological changes in the ductal system such as dilation of the duct, and flattening or abrasion of the epithelium, especially at the site of calculus, were remarkable. Changes noted in the glandular system were degeneration and atrophy of the acini, periductal lymphocytic infiltration, fibrosis and fatty degeneration of the stromac[33,20]. Of our cases showing inflammatory cell infiltration with periductal fibrosis, marked dilation of the intra-lobular duct and acinal atrophy were observed in case 1. However, these changes were not so obvious in the other three cases. We therefore considered that the degree of pathologic change was correlated with the sialographic findings. SEM findings KURACHIet al. [29] categorized the cross-sectional structure of a stone into three parts: a central core, a lamellar portion and an outer surface. He reported that each of the lamellae had almost the same thickness, but that the structure was not uniform in that the layers were composed of alternately close and roughly fused granules. He also stated that the outer surface presented a granular appearance. MORINAGA[28], on the other hand, categorized the cross-sectional structure as follows : (A) a calculus having a relatively small single core and an apparently lamellar structure of concentric layers throughout; (B) a calculus having a relatively large single core and lamellar structure mainly at the periphery; (C) a calculus having multiple cores and otherwise appearing to be an aggregate of calculi ; (D) a calculus having an almost uniform structure with an only slightly apparent lamellar pattern, otherwise appearing homogeneous. MoRINAGA [28]also reported lamellar structures in which each lamella was not even in thickness, and other lamellar structures in 121 which lamellae were closely piled upon each other, or roughly piled up showing crevices. In the closely lamellated structures he also observed board-shaped, needle- shaped or globular crystals. In our present study, the outer surface in case 1 had abundant crevices, while in the other three cases the outer surface was uniform with small granules. The cross-sectional appearance in every case showed some variation in structure and lamellar thickness, so that we were unable to draw any particular conclusions from these observations. Results of EPMA A sialolith is composed of about 80% inorganic and about 20% organic substances. The inorganic substances are mainly calcium phosphate (about 70%) and calcium carbonate (about 20%), with very small amounts of metals accounting for the remainder [33].A zumA[24]analyzed a sialolith with EPMA and reported that face X-ray energy pulse analysis revealed Ca and P mainly distributed in the peripheral and core regions, while Mg was distributed uniformly throughout the specimen. He also reported that line X-ray energy pulse analysis revealed a parallel distribution of P and Ca, and a correlation of Mg with Ca, even though the quantity was small. HISANOet al. [23]also reported that the distribution of Ca and P was not uniform, but parallel. Line X-ray energy pulse analysis of the specimens from our cases revealed an almost parallel distribution of Ca and P in all four. However, the distribution of Mg was not always parallel to that of Ca and P. Comparing the results of SEM and EPMA, the stone from case 1 with a rough and lamellar structure abundant with crevices, and that from case 2 in which the lamellar structure had very pro- nounced layers, both exhibited remarkable variations in the quantity of Ca, P and Mg in each part of the calculus, relative to cases 3 and 4, which exhibited more uniform and homogeneous structures. Conclusion Recently, we treated four cases of submandibular gland sialolithiasis by removal of the submandibular gland, and examined these stones by scanning electron microscopy (SEM) and electron probe X-ray microanalysis (EPMA), in order to supplement the clinical sialographic findings and histopathologic findings of the extirpated glands. The results were as follows: I. Clinical findings: Two 36-year-old males, one 30-year-old male and a 52-year-old female, all com- plaining chiefly of swelling of the submandibular region. 2. Sialographic findings: Sialography with 0.8 to 1.2 ml of 60% urografin revealed stones hindering the flow in the ductal phase, and one case with the longest history of complaint exhibited severe alteration of the peripheral duct. 3. Histopathologic findings: Inflammatory cell infiltration with periductal fibrosis, dilation of intra-lobular duct and acinal atrophy were observed, corresponding to the sialographic find- 122

ings. 4. SEM findings: All cases exhibited abundant small granules on the outer surface of the stones. Three cases exhibited a lamellar pattern in the cross-sectional structure. 5. EPMA findings: Line X-ray energy pulse analysis revealed an almost parallel distribution of Ca and P. Distribution of Mg was not always parallel with that of Ca and P.

Acknowledgements We would like to express our sincere thanks to Prof. Yoshihisa Toda, Second Department of Anatomy, and Associate Prof. ltaru Moro, Department of Pa- thology, for the SEM, EPMA and histopathologic findings.

References [1] TAKAHAHSI,T.: Pathological study of sialolithiasis, J. Stomatol. Soc. Jpn., 13, 367-383, 1939 (in Japanese) [2] MUKAIYAMA,M.: On the structure and the mechanism of development of salivary calculus, Shika Gakuho (J. Tokyo Dent. Coll. Soc.), 48, 688-695, 1955 (in Japanese) [3] OTSUKI,Y. and UEDA, H.: Clinico-statistical study and choice of sialolithiasis in submandibular gland, Chiba Med. J., 36, 979-991, 1960 (in Japanese) [4] OKADA, M.: X-ray diffraction of living body calculi, Jpn. J. Oral Maxillofac. Surg., 8, 1-8, 1962 (in Japanese) [5] HORIKOSHI,T., TSUCHIYA,H., TOMIOKA,T., MAMIYA,H. and NOGUCHI, I.: Five sialolithiasis cases and X-ray fluorescence analysis, Jpn. J. Oral Maxillofac. Surg., 8, 164-198, 1962 (in Japanese) [6] KAJIYAMA,M.: Study on structures of salivary calculus, J. Kyushu Dent. Soc., 20, 143-179, 1967 (in Japanese) [7] FUKUDA, K.: Physicochemical studies on the nature of human salivary stones, J. Osaka Univ. Dent. Soc., 12, 235-242, 1967 (in Japanese) [8] TAKIGAWA,T., AIHARA,K., SUGAWA,I., TOKITA, N., KOSUGE,T., KUROSAWA,H., YAMAMOTO, R. and YAMAGUCHI,H.: The study of sialolithiasis in the submandibular gland, Nihon Univ. Dent. J., 44, 159-173, 1970 (in Japanese) [9] TAEN, A. and KODAMA,K.: Ten cases of sialolithiasis in the past three years, Jpn. J. Oral Maxil- lofac. Surg., 18, 341-346, 1972 (in Japanese) [10] MASUDA,M.: Calculi in Wharton's duct : Report of three cases and chemical analysis of the calculi, Otologia Fukuoka, 18, 249-254, 1972 (in Japanese) [11] ISHIKAWA,M., HIROSE, Y., HATTORI, C., YAMAMOTO,Y., SUMIDA,H., UTSUMI, N., WAN, I. and NAKAJIMA,T.: A case of sialolithiasis in the bilateral submandibular duct and glands, Bull. Josai Dent. Univ., 5, 331-335, 1976 (in Japanese) [12] KOBAYASHI,S., KOWATA,I., ONODERA,A. and SAKUMA,M.: Sialolithiasis, Otologia Fukuoka, 25, 253-257, 1979 (in Japanese) [13] TAKIGAWA,T., TANAKA,H., MATSUMOTO,M., IIJIMA, M., NAKAMURA,T., SAITOH,K., HENMI, M., NAGANUMA,S., CHIBA, R., OOKI, H., KUMAKAWA,A., KATOH, K., SHIMIZU,T. and INADA, M.: A case of large dendriform sialolith in the submandibular gland, Nihon Univ. Dent. J., 53, 942-945, 1979 (in Japanese) [14] HARA, T., FUKUDA, K., NAGUMO, M., SODA, T. and ITOH, H.: Clinico-statistical and pathological observations on sialolithiasis, Jpn. J. Oral Maxillofac. Surg., 25, 1066-1072, 1979 (in Japanese) [15] MAIZUMI, Y., KOMORI,Y., NISHIHARA,S., MURAKAMI,S., KUDOH, T., HASEGAWA,K., KANA- ZAWA, M., HARIYA,M., KOJIMA, M., MATSUO,T., NARITA, Y. and UCHIDA,Y.: Sialolithiasis in children: Report of two cases, Jpn. J. Oral Maxillofac. Surg., 26, 1598-1602, 1980 (in Japa- 123

nese) [16] KAMEYAMA,T., KITA, K., KAWASHIMA,R., KUMAGAI, K., SUJAKU, C. and TATEYAMA,H.: A case of bilateral sialolithiasis in the right submandibular duct and left submandibular gland -Comparison of crystalline difference by X-ray diffraction-, Jpn. J. Oral Maxillofac. Surg., 29, 1075-1079, 1983 (in Japanese) [17] KAMEYAMA,T., KITA, K., KITAGAWA,H., MORINAGA,F. and SUJAKU, C.: A sialolithiasis with renal calculus, J. Jpn. Stomatol. Soc., 30, 270-275, 1981 (in Japanese) [18] SUZA, El., SHINOHARA,M., TASHIRO,H. and OKA, M.: Clinico-statistical study of sialolithiasis, Jpn. J. Oral Maxillofac. Surg., 29, 1304-1309, 1983 (in Japanese) [19] SATO, Y.: Clinicopathological investigation of submandibular sialolithiasis-Comparison of sialogram and histopathology-, Jpn. J. Oral Maxillofac. Surg., 30, 274-289, 1984 (in Japanese) [20] ARISUE, M.: Histopathological study of submandibular sialolithiasis, J. Jpn. Stomatol. Soc., 34, 8-34, 1985 (in Japanese) [21] EMA, H., MIZUNO, A., NAKAMURA,T., TORII, S., NAKASHIMA,Y., KAWABATA T., SHIGENO, T., KAMIYA,H., SHIBATA,T., SUZUKI, S. and MOTEGI, K.: A clinicostatistic study of sialolithiasis in our clinic, J. Jpn. Stomatol. Soc., 35, 470-475, 1986 (in Japanese) [22] NAKAMURA,H.: Physicochemical comparative study of salivary and dental calculus, Shika Igaku (J. Osaka Odontol. Soc.), 25, 246-264, 1962 (in Japanese) [23] HISANO, Y., AZUMA, T., KOBAYASHI,K. and MISAKI, M.: A long and wide submandibular ductal stone analyzed by EPMA, Jpn. J. Oral Maxillofac. Surg., 16, 189-193, 1970 (in Japanese) [24] AZUMA, T.: Electron microprobe analysis of salivary calculus, Odontology (J. Nippon Dent. Univ.), 61, 124-164, 1973 (in Japanese) [25] HIRADE,V. and NOMURA,K.: Morphological study on salivary calculi, Otolaryngology (Tokyo), 50, 241-248, 1978 (in Japanese) [26] UCHIDA, M., ISHII, J., OGAWA, I., SAKUMA,N. and TOMITA,H.: A scanning electron microscopic study on a salivary stone suspectedly caused by active calcification with bacteria, Oto- laryngology (Tokyo), 51, 235-245, 1979 (in Japanese) [27] ENDOH, H.: Scanning electron microscopical and energy dispersive spectroscopical X-ray analyzer studies on salivary calculi-Comparative investigation with biliary calculi, renal calculi and vesical calculus-, J. Iwate Med. Assoc., 32, 837-848, 1980 (in Japanese) [28] MORINAGA,F.: Scanning electron microscopic observation of the microstructure of salivary calculi, J. Jpn. Stomatol. Soc., 29, 254-267, 1980 (in Japanese) [29] KURACHI, Y., MATSUMOTO,H., NAGUMO, M., ENOMOTO,S. and TAKIGUCHI, R.: Scanning electron microscopic study of salivary calculi, Jpn. J. Oral Maxillofac. Surg., 26, 945-951, 1980 (in Japanese) [30] HORI, M., YOKOKAWA,T., SUZUKI, A., ENOMOTO,T., TANAKA,H., KUDO, I. and KOMIYAMA, K.: Sialolithiasis of minor salivary glands in buccal mucosa: Report of two cases, Jpn. J. Oral Maxillofac. Surg., 28, 264-271, 1982 (in Japanese) [31] IWANARI, S., HORI, M., MIYASHITA,T., TAKAYAMA,Y., MIYAKE, M., MISAKI, T., TANAKA, H. and KUDO, I.: Bilateral submandibular sialolithiasis: Report of two cases, Jpn. J. Oral Maxillofac. Surg., 30, 1104-1113, 1984 (in Japanese) [32] SELDIN,H. M. and SELDIN,S. D.: Conservative surgery for removal of salivary calculi, J. Oral Surg., 6, 579-587, 1953 [33] ISHIKAWA,G.: Oral pathology, Vol. II, 425-429, Nagasue, Kyoto, Japan, 1982 [34] FUKUDA,H.: Sialography, 40-64, 69-90, Fuji, Sapporo, Japan, 1986