Periodontitis is caused by a dysbiotic shift in the dental plaque microbiome. Fusobacterium nucleatum is involved in the colonization of Porphyromonas gingivalis, which plays a key role in dysbiosi...

Porphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation. To elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining. Comparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p < 0.001); however, the other ECF sigma factor mutants or the complemented strains did not enhance biofilm formation. These results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation by P. gingivalis.

Intracellular Ca²⁺ signaling engendered by Ca²⁺ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca²⁺ is exported by the Na⁺–Ca²⁺ exchanger (NCX) and plasma membrane Ca²⁺–ATPase (PMCA) to maintain Ca²⁺ homeostasis. We previously demonstrated a functional coupling between Ca²⁺ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca²⁺ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca²⁺]_i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1–4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca²⁺]_i in odontoblasts in both rat and human odontoblasts. The Ca²⁺ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca²⁺]_i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4–8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca²⁺]_i homeostasis in odontoblasts by Ca²⁺ extrusion following [Ca²⁺]_i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca²⁺ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Anti-microbial nanopatterns have attracted considerable attention; however, its principle is not yet fully understood, particularly for inorganic nanopatterns. Titanium nanosurfaces with dense and anisotropically patterned nanospikes regulate biological functions with multiple physical stimulations, which may be because of the nanopattern-induced alternation of surface physical properties. This study aimed to determine the antimicrobial capability of titanium nanosurfaces and their mechanisms. Two types of alkali-etched titanium nanosurfaces with isotropically or anisotropically patterned nanospikes had markedly denser surface protrusions, greater superhydrophilicity, and greater negative charge than machined or micro-roughened titanium surfaces. The crystallographic properties of anisotropic titanium nanosurfaces were similar to those of isotropic nanosurfaces, but markedly higher in electric reactivity at nanoscale. The maximum value of the contact potential difference on titanium surfaces was significantly correlated with the product of the density and anisotropy in the distribution pattern of surface protrusions. Isotropic titanium nanosurfaces did not inhibit the attachment of gram-positive cocci, such as Staphylococcus aureus, whereas anisotropic titanium nanosurfaces substantially inhibited gram-positive cocci attachment. Most gram-negative bacilli, Escherichia coli, died via swelling of the cell body on anisotropic titanium nanosurfaces within 6 h of incubation, in contrast to other titanium surfaces where most of the cells did not lose viability or undergo morphological changes. The extent of cell swelling was positively correlated with the electric reactivity of the titanium surfaces. Titanium nanosurfaces with anisotropically patterned dense nanospikes exerted anti-biofouling or mechano-bactericidal effects on gram-positive or negative bacteria with electrical cue induced by the anisotropy of the nanospike patterns.