Objectives: To investigate basic structural and physical properties of saliva and to identify the presence and distribution of globular proteinaceous structures in human whole and parotid saliva.
Methods: Unstimulated (UWS), chewing-stimulated (SWS) whole and citric-acid stimulated parotid (SPS) saliva, as well as buccal biopsies samples, collected from 10 healthy individuals (mean age 24±3 years), were prepared for cryo-scanning and transmission electron microscopy (cryo-SEM and cryo-TEM) in addition to confocal laser scanning microscopy (CLSM) to visualize salivary structures. Biopsies were stained with immunofluorescent dyes (Nile Red Lipid Stain, anti-MUC5B antibody and DAPI), and visualized with CLSM ZEISS Airyscan super-resolution (LSM 980). Spinnbarkeit, viscosity, residual mucosal saliva, zeta-potential and surface tension were also measured.

Results: Cryo-SEM of UWS and SWS displayed a complex proteinaceous web-like multilayer network with globular knots present. Size and structure of pores in the networks depended on time of sublimation. Cryo-TEM revealed globular structures in SPS, size ranging from 20-200 nm, appearing as single structures or in clusters. Fluorescent staining yielded architectural details of the surface epithelium and subepithelial layers. Images of buccal mucosa revealed interlaced MUC5B immunoreactive network attached to the surface of the epithelial cells. Spinnbarkeit was below 10 mm in 62.5% of the UWS samples. Viscosity was higher in SWS than in UWS samples (1.46±0.23 vs. 1.38±0.34). The tongue dorsum was the moistest surface (65.69 μm), and the palate the least moist surface (32.91±18.40 μm). The zeta-potential valued -7.06 mV, -15.57 mV and -17.90 mV for SPS, SWS and UWS, respectively. The surface tension did not differ between UWS and SWS samples (53.45±6.33 n/m vs. 56.88±5.04 n/m).

Conclusion: Our findings using cryo-SEM and cryo-TEM indicate the presence of micelle-like globular proteinaceous structures in whole and parotid saliva, with potential lubricating properties. CLSM showed interlaced MUC5B attached to the buccal epithelial surface layers.