Scanning Electron Micrographic Featuresof a Giant Submandibular Sialolith

Constantino Ledesma-Montes

and Maricela Garces-Ortız

Oral Pathology Laboratory, Divisi�oon

de Estudios de Posgrado e

Investigaci�oon, UNAM, Circuito

Institutos, Col. Copilco-CU, Mexico,

DF, Mexico

Jose Reyes-Gasga

Electron Microscopy Laboratory,

Instituto de Fısica, UNAM, Circuito

Institutos. Col. Copilco-CU,

Mexico, DF, Mexico

Juan Francisco Salcido-Garcıa

Clinical Diagnosis Service, Divisi�oon de

Estudios de Posgrado e Investigaci�oon,

UNAM, Circuito Institutos, Col.

Copilco-CU, Mexico, DF, Mexico

Florentino Hern�aandez-Flores

Oral and Maxillofacial Surgery Clinic,

Divisi�oon de Estudios de Posgrado e

Investigaci�oon, UNAM, Circuito

Institutos, Col. Copilco-CU, Mexico,

DF, Mexico

ABSTRACT To recognize recently appearing mineralization phenomena,

one must study the external surface of the sialoliths, since it is not possible

to study them in the central portions of sialoliths. The authors examined the

external surface of a sialolith by scanning electron microscopy and analyzed

its microstructures. The study revealed the presence of numerous micro-

structures of different shapes (nodular, laminar, reticular, microgranular,

and multinodular) and variable size arranged in a haphazard fashion. The

diverse microstructures encountered strongly suggest that different mec-

hanisms of mineralization occur during growth and development of the

sialoliths.

KEYWORDS biomineralization, salivary glands, scanning electron microscopy,

sialoliths

Sialolithiasis is the most common disease of the salivary glands and its

estimated frequency is 1.2% in the adult population, with a slight male

predominance. More than 80% of the salivary gland calculi appear in the

submandibular gland. They can be located in the glandular parenchyma

and more frequently in the excretory ducts [10].

Commonly, sialoliths measure from 1 mm to less than 1 cm and rarely

they measure more than 1.5 cm. Giant sialoliths are exceedingly rare. A

recently published search in the literature showed that only 16 well-

documented cases measuring 3.5 cm or more have been published [7].

Scanning electron microscopic studies on sialoliths demonstrated that dif-

ferent microstructures compose their mineralized material [5,6,8,13,14].

These studies made a special emphasis on the structural features of the inter-

nal surface of the sialoliths and only incomplete descriptions on the mor-

phological appearance of the external surface were done. Giant sialoliths

represent unique opportunities to study a wide superficial and actively

mineralizing area in order to know the different microstructures forming

their external surfaces. In addition, the importance to study the external sur-

face morphology of the sialoliths is to recognize recently appearing and

active mineralization phenomena, which are not possible to analyze by

scrutinizing their central inactive portion.

Received 24 August 2007; accepted 17September 2007.

The authors are indebted toM. C. Jacqueline Ca~nnetas for her expertadvice in obtaining the photographicmaterial. Help from Carlos Flores,Pedro Mexia, Roberto Hernandez, andGilberto Mondrag�oon, all from theElectron Microscopy Laboratory(Instituto de Fısica, UNAM), isacknowledged. This work wassupported by a grant from the MexicanDirecci�oon General de Asuntos delPersonal Academico (UNAM). Grantnumber DGAPA-IN104209.

Address correspondence toDr. Constantino Ledesma-Montes,Cipres #169-2, Col. Vergel Coapa,Mexico, DF, 14320, Mexico. E-mail:cotita@avantel.net;cledezma@servidor.unam.mx

Ultrastructural Pathology, 31:385–391, 2007Copyright # Informa Healthcare USA, Inc.ISSN: 0191-3123 print=1521-0758 onlineDOI: 10.1080/01913120701686586

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The aims of this article are to present the findings

of a scanning electron microscopic study made on

the external surface of a giant sialolith and

to discuss the different mechanisms of biomineraliza-

tion present during sialolith development.

MATERIALS AND METHODS

The analyzed sialolith was oval, weighing 12.0 g

with a surface showing multiple nodules of different

size. After surgical excision, it was carefully cleaned

with saline solution under pressure to remove saliva,

cellular debris, and blood. The sialolith was carefully

bisected with a jewel saw and stored in a sterile, che-

mically clean polypropylene flask until analysis.

Later, one-half of the sialolith was dried and its exter-

nal surface coated with a 30-nm-thick carbon layer

by means of a vacuum and carbon coater system

(Ernest F. Fullam, Latham, NY). The entire external

surface was carefully examined with a JEOL 2000

scanning electron microscope (JEOL, Japan), the dif-

ferent microstructures were located, and electron

micrographs were taken.

RESULTS

Scanning microscopic review showed several

kinds of microstructures. The most common was a

mineralized formation with nodular configuration,

which was observed isolated or forming irregularly

arranged groups of different sizes and shapes

(Figure 1a). When they were seen isolated, these

nodules showed a smooth surface and their size var-

ied from 70 to 400 mm. Multiple coalescent nodules

formed elongated structures larger than 900mm (Fig-

ure 1b) or appeared to form groups of closely

arranged individual structures (Figure 1c). In some

areas, a microfibrilar, reticular, delicate pattern was

seen (Figure 2a). In other instances, accumulations

of microgranular structures of approximately

2–7mm were observed (Figure 2b). Sometimes,

several areas showed a combination of reticular and

microgranular arrangements (Figure 2c). In other

areas, lamino-nodular microstructures formed by

small, smooth surfaced plaques were seen growing

over smooth mineralized areas (Figure 3a). In some

zones, spherical nodules composed by concentric

laminar sheets of calcified material were seen

(Figure 3b). In other areas, these nodular microstruc-

tures showed a concentric laminar arrangement with

a rounded solid or an empty core (Figure 3c).

We were unable to find any structure suggestive of

bacteria or any other type of microorganism.

DISCUSSION

Sialolithiasis is an uncommon disease. Males are

more frequently affected than females and children

are rarely involved [2]. Submandibular salivary glands

are more commonly affected than parotids and sublin-

gual or minor salivary glands are involved in only 1–

2% of the cases. This disease occurs at any age, but it

appears more frequently in patients in the 3rd to 6th

decades of the life and it is rare in children [2, 5, 9].

Giant sialoliths are a very rare finding in clinical oral

pathology; their size varies from 3.5 to 7.0 cm, and

according to the Ledesma-Montes et al. review [7],

excluding one case, all the giant sialoliths (94.4%) they

analyzed were located in the submandibular gland

tissues. Clinico-pathological findings of these cases

were widely discussed in a previous paper [7].

Several points should be taken into account for

explaining the development and growth of salivary

calculi: (1) diameter and longitude of the excretory

duct, (2) speed of the salivary flow, (3) alkalinity of

the saliva, (4) quantity of mucin proteins within the

salivary secretion, and (5) Ca and P content of the

secreted saliva. In addition, several local, chemical,

and mechanical factors are involved in the precipi-

tation of the mineral salts. Infection, inflammation,

salivary stagnation, physical trauma, introduction of

foreign bodies, and the presence of desquamated

epithelial cells seem to be the initial events for the

formation of a nidus, which later will be the site for

the precipitation of mineral salts contained in the

salivary secretion. The presence of salivary proteins

plays an important role in the initial formation of

these phenomena. In the late 1950s, Harril et al. [4]

proposed that salivary mucins coalesce to form gels

that eventually form more or less denser particles

suitable for mineralization. Recently, Grases et al.

[3] suggested that phytate concentration in saliva

from patients with salivary calculus is an important

factor implied in the sialolith development. Tanaka

et al. [11] found that mineralization was present

around the degenerative organelles in the form

of lipid-like structures, mitochondria, lysosomes,

and microbial structures and speculated that

C. Ledesma-Montes et al. 386

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FIGURE 1 (A) An isolated, smooth surfaced nodule is shown. (B) Several coalescing nodules formed large, elongated, mineralized

structures with irregular surfaces. (C) Individual microstructures forming closely arranged laminae are seen in some areas. All scanning

electron photographs are 3100. Bar 100 lm.

387 Ultrastructure of a Giant Sialolith

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FIGURE 2 (A) This microfibrilar delicate pattern was observed in only a few zones of the analyzed specimen, 3500. Bar 50 lm.

(B) Multiple microgranules covering several areas are shown in this scanning electron photograph, 375. (C) Several zones showed this

reticular pattern contiguous to multiple microgranular structures, 3500. Bar 50 lm.

C. Ledesma-Montes et al. 388

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FIGURE 3 (A) Plaques with nodular structures are shown. Also, a smooth base of these structures can be observed, 350. Bar 500 lm.

(B) Numerous mineralized, spherules with laminar aspect can be seen, 350. Bar 500 lm. (C) This scanning electron photograph shows two

nodules. Both of them show an empty core with a laminated structure, 3150. Bar 100 lm.

389 Ultrastructure of a Giant Sialolith

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mineralization around these substances contributes

to calculi formation.

Giant sialoliths constitute unique opportunities for

research, since they provide a wide area for study. In

addition, the importance of studying the external sur-

face morphology of the sialoliths is to recognize

active, recently appearing mineralization phenomena,

which are not possible to study in the older, inactive,

central portion of these mineralized structures.

Results of our study showed that on the external sur-

face of a giant calculus different microstructures are

present. These formations show different shapes and

sizes. The main microstructures identified in this work

were smooth-surfaced nodules, which coalesce to form

irregular aggregates or cylindrical multinodular struc-

tures with several thousands of micrometers long.

According to Harril et al. [4], these nodules can develop

from abnormal mucoid material coalescing to form

gels, which gave rise to nuclei of dense configuration.

Yamamoto et al. [13] considered these mineralized

arrangements as structures corresponding to apatite

and that the long, cylindrical structures arose from

coalescence of multiple individual nodules. Other for-

mations found in this study were microfibrilar-appear-

ing structures. We suggest they could arise from the

mineralization of thin and delicate threads composed

of aggregates of mucin proteins.

In this study, we found several areas containing

microgranular nodules. We think these structures

may arise from the deposition of microparticles of

loosely arranged, early-mineralized mucous material.

Other formations found on the external surface of the

analyzed specimen were laminar-appearing struc-

tures. These structures seem to arise from coalescent

mucoid material forming a gel, which eventually

formed a laminated structure. According to Hiraide

and Nomura [5], formation of these laminae could

have occurred by deposition of a layer of loosely

aggregated particles followed by rearrangement of

the initial bonds to give a denser configuration. They

considered that this lamellated pattern represented

the morphological expression of a rhythmical depo-

sition of material, a similar phenomenon to that seen

in the Liesegang’s ring formation.

It is possible that the nodular structures found in

this study arise from repeated lamination of the

microgranules. An alternative explanation is that

introduced by Hiraide and Nomura [5], who pro-

posed that a homogeneous core was formed mainly

by a chemical reaction, producing a mineral mass

from the initial stage of calculi formation. In our

Figure 3c, we show two nodules with an empty core.

Development of those formations is more difficult to

explain. It is possible that these structures will

develop according to the Hiraide and Nomura pro-

posal [5]. In their study,they found two specimens

showing no microstructural evidence of core and

they attributed this to ‘‘an unknown condition in that

saliva changes its physicochemical properties to form

a gel, in this core is where mineralization begins

which matures to a core which is not necessarily

homogeneous.’’ According to their theory, this core

is not suitable for mineral deposition and an

unknown salivary phenomenon prevents deposition

of minerals from the environment within it, resulting

in a void in the centre of the nodule. Later, newly

deposited salivary substance permits accretion of

laminated mineralized material.

Our findings do not support the assumption that

bacteria provide the major bulk of organic material in

the later phase of the pathogenesis of the calculus. In

our study, despite our careful search of the whole sur-

face of the studied specimen and unlike the results of

other studies [1, 5, 6, 12, 14], we were unable to find

structures on the external surface of the analyzed speci-

men suggesting the presence of mineralized bacteria.

An explanation for the negative results on bacterial

absence in this study can be that bacteria might be mor-

phologically changed during the mineralization pro-

cess and eventually lose their contour [6].

Mineralization process in sialoliths is a matter of

debate among researchers, and the diversity of struc-

tures encountered in the external surface during our

study demonstrate that different biomineralization

mechanisms are involved in the development and

growth of these calcified structures. We encourage

publication of more studies on the external surface

of sialoliths in order to know more accurately the

biomineralization phenomena taking place in these

structures.

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391 Ultrastructure of a Giant Sialolith

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