Lactoferrin and the development of salivary stones: a pilot study.

Salivary stones (sialoliths) are calcified structures located in the ductal system of the major salivary glands. Their exact cause is not clear but in general they are characterized by concentric inorganic (hydroxyapatite) layers. The formation is a slow intermittent process which may result in enlargement of the sialolith causing obstruction of saliva secretion resulting in mealtime related pain and swelling of the affected salivary gland. Various studies reported the presence of organic material such as proteins and lipids in the core of sialoliths. In the present study the protein composition of twenty submandibular sialoliths was analyzed. It was found that proteins contributed on average 5% to the dry weight of submandibular stones whereby small salivary stones contained more extractable proteins than large salivary stones. Using a combination of SDS-PAGE gel electrophoresis and Western blotting, we identified α-amylase (in all stones; 100%), lysozyme (95%), lactoferrin (85%), secretory-IgA (75%), MUC7 (60%), complement C4 (60%) and C-reactive protein (35%). The presence, and the combinations, of lactoferrin, lysozyme, s-IgA and α-amylase in sialoliths was confirmed by ELISA. The gradually increasing size of a sialolith might provoke a local inflammatory response in the duct of the submandibular gland whereby the relatively low concentrations of lactoferrin and lysozyme may originate from neutrophils. The interaction of lactoferrin with s-IgA could contribute to the accumulation of lactoferrin in sialoliths. In summary, these results suggest a new pathophysiological role for lactoferrin, in the formation of sialoliths.

Trial Proteomic Qualitative and Quantitative Analysis of the Protein Matrix of Submandibular Sialoliths.

Our studies aimed to explore the protein components of the matrix of human submandibular gland sialoliths. A qualitative analysis was carried out based on the filter aided sample preparation (FASP) methodology. In the protein extraction process, we evaluated the applicability of the standard demineralization step and the use of a lysis buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). The analysis of fragmentation spectra based on the human database allowed for the identification of 254 human proteins present in the deposits. In addition, the use of multi-round search in the PEAKS Studio program against the bacterial base allowed for the identification of 393 proteins of bacterial origin present in the extract obtained from sialolith, which so far has not been carried out for this biological material. Furthermore, we successfully applied the SWATH methodology, allowing for a relative quantitative analysis of human proteins present in deposits. The obtained results correlate with the classification of sialoliths proposed by Tretiakow. The performed functional analysis allowed for the first time the selection of proteins, the levels of which differ between the tested samples, which may suggest the role of these proteins in the calcification process in different types of sialoliths. These are preliminary studies, and drawing specific conclusions requires research on a larger group, but it provides us the basis for the continuation of the work that has already begun.

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.

Mineralization of Sialoliths Investigated by Ex Vivo and In Vivo X-ray Computed Tomography.

The fraction of organic matter present affects the fragmentation behavior of sialoliths; thus, pretherapeutic information on the degree of mineralization is relevant for a correct selection of lithotripsy procedures. This work proposes a methodology for in vivo characterization of salivary calculi in the pretherapeutic context. Sialoliths were characterized in detail by X-ray computed microtomography (µCT) in combination with atomic emission spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. Correlative analysis of the same specimens was performed by in vivo and ex vivo helical computed tomography (HCT) and ex vivo µCT. The mineral matter in the sialoliths consisted essentially of apatite (89 vol%) and whitlockite (11 vol%) with average density of 1.8 g/cm3. In hydrated conditions, the mineral mass prevailed with 53 ± 13 wt%, whereas the organic matter, with a density of 1.2 g/cm3, occupied 65 ± 10% of the sialoliths' volume. A quantitative relation between sialoliths mineral density and X-ray attenuation is proposed for both HCT and µCT.

Biochemical composition of salivary stones in relation to stone- and patient-related factors.

BACKGROUND: Salivary stones are calcified structures most often found in the main duct of the submandibular or parotid salivary gland. They contain of a core surrounded by laminated layers of organic and inorganic material. MATERIAL AND METHODS: Submandibular and parotid sialoliths (n=155) were collected at the department of Oral and Maxillofacial surgery of a general hospital between February 1982 and September 2012. The weight of the sialoliths was determined and the consistency was subjectively classified. Subsequently, the biochemical composition of the stones was determined by wet chemical methods or FT-IR spectrometry. Age and gender of the patients were retrieved from their medical records. Data were statistically analyzed using Fisher's exact tests. RESULTS: Sialoliths are mainly composed of inorganic material. Carbonate apatite was identified in 99% of the stones, phosphate in 88%, calcium in 87%, magnesium in 68%, struvite in 44%, oxalate in 38% and carbonate in 35%. Solid salivary stones contain more frequently struvite than stones with a soft consistency (p=0.05). Larger stones (>100mg) contain more frequently carbonate (p=0.05). Stones from older patients (≥38years) showed an almost significant trend towards more frequent presence of phosphate (p=0.083). CONCLUSIONS: The biochemical composition of submandibular and parotid sialoliths is related to stone-related factors, probably to age but not to the gender of the patient.

Characterization of a submandibular gland sialolith: micromorphology, crystalline structure, and chemical compositions.

OBJECTIVE: The aim of this study was to understand the mechanism of mineralization and growth of a sialolith by analyzing its micromorphology, crystalline structure, and chemical compositions. STUDY DESIGN: A sialolith was removed along with submandibular salivary gland from a patient. After cross-cutting and polishing the sialolith, its morphology, chemical compositions, crystalline structure, and chemical states were analyzed by using optical camera, scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffractometer, Fourier transform infrared spectrophotometer, and transmittance electron microscopy. RESULTS: The sialolith had a core composed of organic material, such as lipid compounds, and the surrounding mineralized shell structure mostly consisted of hydroxyapatite. In the transition zone between the organic core and mineralized shell layers, inorganic layers were arranged alternately with organic layers. Congregated globular structures were calcified with hydroxyapatite and whitlockite crystallites. Analysis of crystalline structures and chemical compositions suggested that calcium phosphate minerals containing magnesium, such as whitlockite, were developed in the initial stage and gradually transformed into crystallites composed of hydroxyapatite during the growth of crystallites. CONCLUSIONS: Sialolith with an organic core grew as inorganic materials were deposited and calcified in alternate layers. The mineralization process might include the initial whitlockite development and successive transformation into more stable hydroxyapatite.

Local Response of Sialoliths to Lithotripsy: Cues on Fragmentation Outcome.

Lithotripsy methods show relatively low efficiency in the fragmentation of sialoliths compared with the success rates achieved in the destruction of renal calculi. However, the information available on the mechanical behavior of sialoliths is limited and their apparently tougher response is not fully understood. This work evaluates the hardness and Young's modulus of sialoliths at different scales and analyzes specific damage patterns induced in these calcified structures by ultrasonic vibrations, pneumoballistic impacts, shock waves, and laser ablation. A clear correlation between local mechanical properties and ultrastructure/chemistry has been established: sialoliths are composite materials consisting of hard and soft components of mineralized and organic nature, respectively. Ultrasonic and pneumoballistic reverberations damage preferentially highly mineralized regions, leaving relatively unaffected the surrounding organic matter. In contrast, shock waves leach the organic component and lead to erosion of the overall structure. Laser ablation destroys homogeneously the irradiated zones regardless of the mineralized/organic nature of the underlying ultrastructure; however, damage is less extensive than with mechanical methods. Overall, the present results show that composition and internal structure are key features behind sialoliths' comminution behavior and that the organic matter contributes to reduce the therapeutic efficiency of lithotripsy methods.

Rapid recurrent sialolithiasis: Altered stone composition and potential factors for recurrence.

The objective of this report was to identify potential factors associated with recurrent sialolithiasis after surgical intervention. This is a report of a woman with recurrent submandibular sialolithiasis after surgical intervention. Several characteristics of this patient indicate that she may have been predisposed to recurrent stone formation. Patient and disease factors leading to recurrent salivary stone formation are not well known. Notwithstanding, there may be stone factors and intraoperative findings that aid in determining whether a patient is likely to be successfully treated with a gland-sparing approach. Laryngoscope, 127:1365-1368, 2017.

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.

Analytical investigation of salivary calculi, by mid-infrared spectroscopy.

Sialolithiasis is common in salivary glands, especially in the submandibular and parotid ducts. X-Ray diffractometry was the principal technique used for their analysis, sometimes associated with scanning electron microscopy. Hydroxyapatite was the most frequently described constituent, in association with whitlockite and other calcium phosphates as brushite or octocalcium phosphate. Proteic matter was detected, as mucoproteins, albumin, nucleoproteins or as degenerative bacterial matter. This study presents the identification of constituents by mid-infrared spectrometry of 74 sialoliths. Their successive layers are analyzed from their crust to the nucleus, using absorbance measurements. Spectra are compared with reference mixtures of two or more constituents. Approximately 99% of sialoliths are constituted of calcium phosphates, under carbonated forms. More than three-quarters contain proteins, in which mucins represent the majority and albumin is found in 10% of all the specimens. Only 7% calculi are an association of two constituents, 66% are made of three and 27% have four or more components. For the 74 studied sialoliths, no specimen contains hydroxyapatite; but they are composed of carbonate apatites with irregular microcrystallized forms, even if proteins are present. Some of them have a pure protein nucleus, surrounded by carbonate apatite layers; the other stones are made of internal layers of apatites and covered with a dense and varnished crust of proteins.

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.

Submandibular duct sialolithiasis : an unusual presentation.

Multiple calculi in a submandibular gland duct is an uncommon occurrence. One such case occurring in a young boy of 25 years is reported here where thirteen very small stones of 1-3 mm of maximum diameter were lined up in the Wharton's duct in such a way that they were difficult to appreciate on inspection and palpation. Successful exploration was done in two steps.

Chronic sialolithiasis in a Trakehner mare.

An 11-year-old Trakehner mare was presented with a firm moveable mass over the left maxilla. Radiography revealed a discrete opaque ovoid mass. Sialolithiasis of left parotid duct was diagnosed. The sialolith was excised by using a transoral approach and found to contain an organic nidus. The mare recovered without complications.

Histological and histochemical observations on salivary microliths in ferret.

The fortuitous observation of salivary microliths in ferret was pursued in the present investigation. Major salivary glands obtained post-mortem from mature ferrets of either sex were examined with the use of histology and light microscopical histochemistry for calcium, protein, amino acids, mucosubstances and hydrolytic enzymes. Microliths were detected in most parotids, but were absent from submandibular and sublingual glands. The microliths were usually seen in lumens, and occasionally in parenchyma and interstices. They were variably stained for calcium, tryptophan, and neutral and acidic mucosubstances, similarly to acinar or ductal secretory granules. Unlike secretory granules, microliths showed autofluorescence, high levels of tyrosine and a low concentration of -SS- groups. Acid phosphatase and beta-glucuronidase reaction surrounded non-luminal microliths. The present data establish ferret as a new model for the investigation of salivary microliths and do not support the notion of microliths being almost absent from the parotid. Probably there is secretory inactivity in ferret parotid and this fosters the formation and accumulation of microliths containing calcium and disintegrated secretory material.

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.