Ultrastructural analysis of the submandibular sialoliths: Raman spectroscopy and electron back-scatter studies.

The aim of work was the epidemiological analysis of the occurrence of sialolithiasis of the submandibular gland in adults and the evaluation of the ultrastructure of salivary stones. The study sample consisted of 44 sialoliths. Analysis of the structure and chemical composition of sialoliths was performed using a Scanning Electron Microscope and Raman Spectroscopy. Comparing our results with the literature we can say that the epidemiology of sialolithiasis has not changed significantly over the past 50 years. A wide variety of sialoliths structure was observed. In 75% (33) cases a layered structure of salivary stones was observed, while in 25% (11) - homogeneous structure. The various distribution of organic and inorganic components was observed among all the analyzed sialoliths. Raman spectroscopy allows for preliminary analysis of the sialoliths structure with only a qualitative assessment of their composition, which significantly reduces the research value of this method. The presence of organic and inorganic compounds in the core and inner layers of the salivary glands stones confirms 2 basic theories of the formation of sialoliths: inflammation and deposition of the inorganic component as a result of disruption of saliva flow in the salivary glands.

Evidence of a microbial etiology for sialoliths.

OBJECTIVES: Sialolithiasis is the primary etiology for parotid and submandibular swelling, potentially resulting in discomfort, bacterial infections, and hospitalization. The etiology of sialolith formation is unknown. Currently, the proposed etiologies range from inflammation, coalescence of organic molecules, sialomicrolith formation, pH changes, and biofilm formation. In this study, we performed a descriptive analysis of images obtained through electron microscopy of sialoliths. Based on our findings and descriptive analysis, we hypothesize that sialolith formation is likely multifactorial and begins with biofilm formation. Biofilm formation then triggers a host immune response, and it is the interaction of biofilm with host immune cells and calcium nanoparticles that forms the nidus and creates a favorable environment for calcium precipitation. METHODS: Sialoliths were extracted from patients and imaged under light and scanning electron microscopy. Specimens for light microscopy were prepared using a diamond saw. Specimens for electron microscopy were freeze-fractured, thus providing an undisturbed view of the core of the sialolith. RESULTS: We were able to identify clear evidence of biofilm caves at the core of each sialolith. These biofilm caves were complex with the presence of bacteria and dehydrated extrapolysaccharide matrix, host cells (immune cells, platelets and erythrocytes), and calcium nanoparticles. CONCLUSION: The etiology of sialolith formation is likely multifactorial. We propose that biofilm formation within a single salivary gland or duct leads to local ductal injury, which results in the influx of host immune cells that interact with the biofilm and calcium nanoparticles, creating a scaffold upon which further calcium deposition can occur. LEVEL OF EVIDENCE: NA Laryngoscope, 130:69-74, 2020.

Ultrastructural and elemental analysis of sialoliths and their comparison with nephroliths.

AIM: Sialoliths are common in the submandibular gland and its duct system, although their exact cause of formation is still a matter of debate. The aims of this study were to: (a) analyze sialoliths ultrastructurally, and to determine the role of foreign bodies or organic materials in the formation of sialolith nuclei; and (b) compare nephroliths with sialoliths ultrastructurally. METHODS: Three sialoliths and two nephroliths were analyzed ultrastructurally by a scanning electron microscope and X-ray diffractometer. RESULTS: The main structures of the sialoliths were found to be hydroxyapatite crystals. No organic cores were observed in the central parts of the sialoliths. In nephroliths, calcium oxalate, calcium phosphate, and struvite crystals were found. The energy-dispersive X-ray microanalysis found that sialoliths and nephroliths were predominantly composed of elements comprising calcium, phosphorous, magnesium, sodium, chloride, silicon, iron, and potassium. CONCLUSION: Sialoliths in the submandibular salivary glands might form secondary to sialadenitis, but not via a luminal organic nidus.

Structure and growth of sialoliths: computed microtomography and electron microscopy investigation of 30 specimens.

Theories have been put forward on the etiology of sialoliths; however, a comprehensive understanding of their growth mechanisms is lacking. In an attempt to fill this gap, the current study has evaluated the internal architecture and growth patterns of a set of 30 independent specimens of sialoliths characterized at different scales by computed microtomography and electron microscopy. Tomography reconstructions showed cores in most of the sialoliths. The cores were surrounded by concentric or irregular patterns with variable degrees of mineralization. Regardless of the patterns, at finer scales the sialoliths consisted of banded and globular structures. The distribution of precipitates in the banded structures is compatible with a Liesegang-Ostwald phenomenon. On the other hand, the globular structures appear to arise from surface tension effects and to develop self-similar features as a result of a viscous fingering process. Electron diffraction patterns demonstrated that Ca- and P-based electrolytes crystallize in a structure close to that of hydroxyapatite. The organic matter contained sulfur with apparent origin from sulfated components of secretory material. These results cast new light on the mechanisms involved in the formation of sialoliths.

Proteomic and scanning electron microscopic analysis of submandibular sialoliths.

OBJECTIVES: Several theories have been proposed regarding the genesis of sialoliths, including the organic core theory, which suggests epithelial or bacterial etiology originating in the central core. Our aim was to use novel methodologies to analyze central areas (the core) of calculi from sialolithiasis patients. MATERIALS AND METHODS: The structures of the halves of six submandibular salivary stones were analyzed by scanning electron microscopy (SEM). After structural analysis, from the other six halves, samples from the central parts of the core and peripheral parts of the core were digested with trypsin and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The peptide mass fingerprints were compared with the results of in silico digestion. RESULTS: SEM analysis of the sialoliths showed that organic structures (collagen/fibrous-like structures, bacterial fragments) were visible only outside of the core in the concentric layers of external areas, but not in the core area. The mass spectrometry (MS)/MS post-source decay experiments were completed from the four, most intense signals observed in the MS spectrum and human defensin was proven to be present in three of the examined samples, originated from the peripheral region of three cores. CONCLUSIONS: Although proteomic analysis demonstrated defensin protein in the peripheral region of the core in three sialoliths, SEM failed to prove organic structures in the core. CLINICAL RELEVANCE: New investigation modalities still cannot prove organic structures in the core, henceforward challenging the organic core theory.

Scanning electron micrographic features of a giant submandibular sialolith.

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 microstructures 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 mechanisms of mineralization occur during growth and development of the sialoliths.

Sialolith characterization by scanning electron microscopy and X-ray photoelectron spectroscopy.

The objective of this study has been to characterize sialolith, a calcium phosphate deposit that develops in the human oral cavity, by high-resolution field emission scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The morphological and chemical data obtained helped in the determination of their formation mechanism in salivary glands. Sialoliths in the submandibular salivary glands may arise secondary to sialodenitis, but not via a luminal organic nidus. We believe this is the first study that characterizes a sialolith by XPS.

On the structural diversity of sialoliths.

Sialoliths from parotid and submaxillar glands have been characterized. Fractured and polished surfaces revealed an intrinsic structural diversity across the calculi sections. In general, the calculi presented highly mineralized amorphous-looking cores surrounded by concentric alternating mineralized and organic layers. The thickness of these layers decreased from the outer regions toward the center of the sialolith, illustrating a sequence of growth stages. Nevertheless, a significant variability could be detected among the specimens. In some cases, the calculi displayed multiple cores and lacked concentric laminated structures. In other instances, the specimens exhibited extensive regions of globular structures. In these cases, the globule diameter decreased across the radius toward the center of the sialoliths, and the globular structures tended to reorganize, forming bright and dark laminated layers surrounding the core. The participation of globular structures in the layer formation process points to morphogenetic mechanisms not previously described.

Micromorphology of sialoliths in submandibular salivary gland: a scanning electron microscope and X-ray diffraction analysis.

PURPOSE: Sialoliths are common in the submandibular gland and its duct system. The exact cause of formation of a sialolith is still a matter of debate. The aim of this study was to analyze 6 sialoliths ultrastructurally to determine their development mechanism in the submandibular salivary glands. MATERIALS AND METHODS: Six sialoliths retrieved from the hilus and duct of the submandibular salivary glands of 6 patients with sialadenitis were analyzed ultrastructurally by scanning electron microscope and x-ray diffractometer. RESULTS: Scanning electron microscope revealed mainly irregular, partly rudely hexagonal, needle-like and plate-shaped crystals. The cross-section from the surface to the inner part of the sialoliths showed no organic material. X-ray diffraction showed that the sialoliths were composed of hydroxyapatite crystals. Energy dispersive x-ray microanalysis showed that all of the samples contained high levels of Ca and P, and small amounts of Mg, Na, Cl, Si, Fe, and K. CONCLUSION: The main structures of the submandibular sialoliths were found to be hydroxyapatite crystals. No organic cores were observed in the central parts of the sialoliths. In accordance with these preliminary results, sialoliths in the submandibular salivary glands may arise secondary to sialadenitis, but not via a luminal organic nidus.

Ultrastructural analysis of salivary calculus in combination with X-ray microanalysis.

Ultrastructural studies of salivary calculi were performed. Scanning electron microscopic examination of the calculi revealed lamellar and concentric structures. Granular or globular structures and pyramid structures were found on the surface of the calculi, and in some cases a scaly structure corresponding to fiber and bacteria was recognized. X-ray microanalysis showed the main constitutes of the calculi to be Ca and P. Transmission electron microscopic examination revealed a fine fibrous structure near the degenerated organelles, and analyses of the structure by electron diffraction revealed hydroxyapatite. Calcification was found around the degenerative organelles in the form of lipid-like structures, mitochondria, lysosomes, and microbial structures. Judging from our results, as one of the processes leading to calculi formation, it is speculated that degenerative substances are emitted by saliva, by some phenomenon, and calcification around these substances then occurs, contributing to the formation of calculi.

[Scanning electron microscopy and light microscopy under polarized light of a submandibular salivary gland calculus]. / Analisi al SEM ed al microscopio ottico in luce polarizzata di uno scialolita della ghiandola sottomandibolare.

BACKGROUND: The salivary calculus is an ancient pathologic condition, called sublingual stone by Hyppocrates. It is well-defined from a anatomo-pathologic, diagnostic and topographic viewpoint, though some pathogenesis-related doubts still exist. This work aims at relating the micro-morpho-structural aspect of a salivary calculus of remarkable dimensions with the current calculogenetic hypotheses. METHODS: A 1.1 g salivary calculus removed from the initial section of Wrthon s duct in the right-hand submandibular gland of a 42 year old male has been studied. Following the fixation in 4% buffered formalin and the inclusion in resin, it was serially sectioned in 15-20 micron slices. Some sections were coloured with toluidine blue O and acid fuchsin. The exeresis of the calculus was carried out intraorally, with marsupialization under local anesthesia. The analysis was performed using a SEM with backscattered electrons and a polarised and transmitted light microscope. RESULTS: The investigations have outlined the presence of various organic cores and a concentric, stratiform architecture interrupted by radial elements. CONCLUSIONS: The formation of this calculus may be due to mixed secondary lithiasis resulting from the formation of colloids and crystalloids.

Crystalloid architecture of a sialolith in a minor salivary gland.

A sialolith from a minor salivary gland of the mucosa of the upper lip was studied morphologically and analytically. Under stereoscopic microscopic visualisation, no core or any concentric laminar structure was found in the sialolith and it had a transparent glassy appearance. Scanning electron microscopy revealed differences between the internal structure of the sialolith and its external structure. No microbes were observed but some mineralized inclusion bodies were seen. X-ray diffraction indicated the absence of inorganic crystals in the sialolith, while energy dispersive x-ray microanalysis revealed a high content of S followed by Ca, Si and Na and little and scarce P on the fracture surface, with S, Na, Ca and P being distributed throughout the external coating of the sialolith. The results suggest that the sialolith was young and consisted of a crystalloid body with an incipient coating undergoing calcification.

High-resolution electron microscopic study of salivary calculus.

The present study was performed to examine crystals in salivary calculus by high-resolution electron microscopy (HREM) and electron diffraction analyses. Microradiography (MR) showed that the calculus had three nuclei and showed complex shapes and mineralization. Hydroxyapatite (HA), in a variety of sizes and distributions, and the intermingling of HA and whitlockite (WH) at well-calcified areas, were observed.

Estudo estereológico dos ductos granulosos e estriados de glândulas submandibulares de ratos machos e fêmeas / Stereologic study of the convoluted granular tubules and striated ducts of male and female rat submandibular glands

Avaliou-se através de métodos estereológicos, as várias dimensöes dos ductos granulosos e dos ductos estriados de glândulas submandibulares de ratos, de ambos os sexos. Em média, os ductos granulosos representam uma estrutura cilíndrica de 6.096,2cm de comprimento e 37,7µm de diâmetro, com um volume compartimental de 68,4mmü e uma superfície externa total de 67,7cm², e constituída por 595,2x10(5) células com volume celular de 994,5µmü; enquanto que, os ductos estriados representam um túbulo de 1.322,9cm de comprimento e diâmetro de 32,0µm, tendo um volume total de 10,9mmü e uma superfície externa total de 11,8cm², e formado por 185,2x10(5) células com volume celular de 652,6µmü. A análise estatística näo mostrou diferenças entre os sexos, para nenhuma das dimensöes quantificadas

[The composition and structure of salivary calculi (sialoliths)]. / O sostave i strukture sliunnykh kamnei (salivolitov).

Composition and structure of more than 50 sialoliths were examined to optimize the treatment of sialolithiasis. Infrared spectroscopy, x-ray phase analysis, optic and electron microscopy were used in examinations. The concrements consist of organic and mineral substances, the former ones predominating. Mineral phase is represented mainly by carbonate-containing hydroxylapatite. The structure of the concrements is concentrically lamellar, in some of them the mineral phase predominates in the central parts of stones, in others in the peripheral parts; stones with regular distribution of the mineral phase along the stone section are quite frequent. The authors conclude that search for the optimal parameters of hypershock exposure to disintegrate the concrements is going to be rather difficult because of the complexity of their structure. They distinguish two trends of this research: (1) specification of the optimal parameters of hypershock exposure and (2) combination of hypershock wave exposure and litholytic solutions.

Ultrastructural localization of microliths in salivary glands of cat.

Although microliths occur in normal human salivary glands and may be an aetiological factor of sialadenitis, little is known of their natural history. In an attempt to remedy this, we investigated a large archival collection of normal and experimental feline parotid, submandibular and sublingual salivary glands. In submandibular and sublingual glands, microliths were detected ultrastructurally in: all types of acinar secretory cells; myoepithelial cells; ductal cells; lumina; intercellular spaces; basement membrane; stroma; macrophages; multinuclear giant cells; and neutrophils. Microliths were not detected ultrastructurally in parotid glands. Microliths appear to form in acinar cells during autophagy and in stagnant secretory material in lumina. Microliths appear to be removed by secretion in the saliva, discharge from cells laterally and basally, and engulfment by macrophages. There appears to be a turnover of microliths, which possibly is upset by secretory inactivity with a resulting accumulation that leads to localized obstruction and sialadenitis.

Analytical ultrastructural investigation of microliths in salivary glands of cat.

Microliths in Araldite-embedded pieces of submandibular and sublingual glands of cat were stained in semithin sections by Methylene Blue and Azure II followed by Basic Fuchsin, and were examined in ultrathin sections by electron-microscopical X-ray microanalysis. Calcium and phosphorus were detected in substantial aggregates of crystals that were stained by Basic Fuchsin and appeared to be hydroxyapatite, but were not detected in granular material that was stained by Methylene Blue and Azure II and appeared to be organic. The polychromatic stain thus appears to be a useful indicator of calcified material. The majority of microliths in acini contained substantial aggregates of crystals, whereas the majority of those in ducts did not. This corresponds to the distribution of the glandular calcium, and suggests that microliths are variously enriched with calcium according to its local level.

Scanning electron microscopy-energy dispersive spectroscopy and X-ray diffraction analyses of human salivary stones.

Ten salivary stones in the human submandibular gland were investigated by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and X-ray diffraction analyses. The stones usually showed a lamellar pattern. SEM observations revealed cubical, plate-like, granular, small and large granules, polyhedral or globular structures in these stones. By X-ray powder diffraction analysis, the main constituents of salivary stones were found to be apatite and whitlockite. SEM-EDS analyses showed that Ca and P were the major elements, frequently accompanied by Mg and S, and less frequently by Na, Al, Si, Cl, K, Fe, Cu and Zn. Ca/P molar ratios ranged from 1.00 to 2.00 with the average of 1.53, showing two maxima of about 1.50 and 1.60. The Ca/P molar ratio of about 1.50 corresponded to the value of whitlockite. The Ca/P molar ratio of 1.60 corresponded approximately to the value of apatite.