Antibacterial effects of surface pre-reacted glass-ionomer (S-PRG) filler eluate on polymicrobial biofilms.

PURPOSE: To analyze the effects of surface pre-reacted glass-ionomer (S-PRG) filler eluate on polymicrobial biofilm metabolism and live bacterial count. METHODS: Biofilm was formed using glass disks 12 mm in diameter and 150 µm in thickness. Stimulated saliva was diluted 50-fold with buffered McBain 2005 and cultured in anaerobic conditions at 37°C for 24 hours in anaerobic conditions (10% CO2, 10% H2, 80% N2) to form the biofilm on the glass disks. Following this, biofilms were treated with (1) sterilized deionized water (control), (2) 0.2% chlorhexidine digluconate (0.2CX), (3) S-PRG eluate diluted to 10% (10% S-PRG)，(4) 20% S-PRG，(5) 40% S-PRG，(6) 80% S-PRG，and (7) S-PRG for 15 minutes (n= 10 per group), and samples were subdivided into two groups for measuring live bacterial count immediately after treatment and after 48 hours of culturing after treatment. The pH of the spent medium collected at the time of culture medium exchange was tested. RESULTS: Immediately after treatment, the live bacterial count of samples treated with drug solutions was significantly lower than the control (8.2 × 108), and the counts of samples treated with 0.2CX (1.3 × 107) and S-PRG (1.4 × 107) were significantly lower than those treated with diluted S-PRG (4.4 × 107-1.4 x 108). When the medium was measured again after culturing for 48 hours, growth was continually inhibited in all treatment groups and the bacterial count of samples treated with S-PRG (9.2 x 107) was significantly lower than that of samples treated with 0.2CX (1.8 × 108). The pH of spent medium immediately after treatment was significantly higher in groups treated with drug solutions (5.5-6.8) than the controls (4.2), and it was highest in the S-PRG-treated group (6.8). Thereafter, when culturing was continued for 48 hours, the pH of all treated groups decreased; however, the pH of the S-PRG-treated group was significantly higher than groups treated with other drug solutions. CLINICAL SIGNIFICANCE: Surface pre-reacted glass-ionomer (S-PRG) filler eluate not only reduced the live bacterial count of polymicrobial biofilm, but also continuously inhibited the lowering of pH.

Existence of SARS-CoV-2 Entry Molecules in the Oral Cavity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin, which promote entry of the virus into the host cell, have been identified as determinants of SARS-CoV-2 infection. Dorsal tongue and gingiva, saliva, and tongue coating samples were examined to determine the presence of these molecules in the oral cavity. Immunohistochemical analyses showed that ACE2 was expressed in the stratified squamous epithelium of the dorsal tongue and gingiva. TMPRSS2 was strongly expressed in stratified squamous epithelium in the keratinized surface layer and detected in the saliva and tongue coating samples via Western blot. Furin was localized mainly in the lower layer of stratified squamous epithelium and detected in the saliva but not tongue coating. ACE2, TMPRSS2, and furin mRNA expression was observed in taste bud-derived cultured cells, which was similar to the immunofluorescence observations. These data showed that essential molecules for SARS-CoV-2 infection were abundant in the oral cavity. However, the database analysis showed that saliva also contains many protease inhibitors. Therefore, although the oral cavity may be the entry route for SARS-CoV-2, other factors including protease inhibitors in the saliva that inhibit viral entry should be considered.

Antimicrobial effects of carbamide peroxide against a polymicrobial biofilm model.

PURPOSE: To investigate the in vitro antimicrobial effects of carbamide peroxide (CP) and CP-based home bleaching agents against polymicrobial (PM) biofilms. METHODS: Using a high-throughput active attachment model, PM biofilms were cultured on glass coverslips by diluting the stimulated saliva of one healthy adult. All experiments were performed anaerobically in McBain medium, which was refreshed twice daily. After biofilm formation for 24 or 72 hours, the biofilms were treated with 0.5%, 2.5%, 5%, or 10% CP, 20-fold dilutions of HiLite Shade Up (HS) or Opalescence Regular (OR), 0.2% chlorhexidine digluconate (CHX), 0.2% NaF, or deionized water (n = 10 each). Biofilms were dispersed and the number of colony forming units (CFU) was measured on tryptic soy agar blood plates. Coverslips containing 72-hour biofilms treated with 0.5% and 10% CP and deionized water were stained and scanned by confocal laser scanning microscopy (CLSM). RESULTS: Treatment of 24- and 72-hour biofilms with HS, OR and CH yielded significantly fewer colonies than treatment with water or 0.2% NaF. No growing colonies were observed after treatment with 10% CP. CLSM showed that the percentage of dead bacteria increased as the concentration of CP increased.

Effect of High Fat and Fructo-Oligosaccharide Consumption on Immunoglobulin A in Saliva and Salivary Glands in Rats.

Consumption of indigestible dietary fiber increases immunoglobulin A (IgA) levels in saliva. The purpose of this study is to clarify the synergistic effect of the intake of a high amount of fats and indigestible dietary fiber on IgA levels in saliva and submandibular glands (SMG). Seven-week-old Wistar rats were fed a low-fat (60 g/kg) fiberless diet, low-fat fructo-oligosaccharide (FOS, 30 g/kg) diet, high-fat (220 g/kg) fiberless diet, or high-fat FOS diet for 70 days. The IgA flow rate of saliva (IgA FR-saliva) was higher in the low-fat FOS group than in the other groups (p < 0.05). Furthermore, the concentration of tyrosine hydroxylase (a marker of sympathetic nerve activation) in the SMG was higher in the low-fat FOS group (p < 0.05) and positively correlated with the IgA FR-saliva (rs = 0.68. p < 0.0001. n = 32) in comparison to that in the other groups. These findings suggest that during low-fat FOS intake, salivary IgA levels may increase through sympathetic nerve activation.

Low-Intensity Pulsed Ultrasound Prevents Development of Bisphosphonate-Related Osteonecrosis of the Jaw-Like Pathophysiology in a Rat Model.

We developed a rat model of bisphosphonate-related osteonecrosis of the jaw (BRONJ) by removing a maxillary molar tooth (M1) from ovariectomized rats after treatment with alendronate. To mimic periodontitis, some of the rats were administered Porphyromonas gingivalis (p. gingivalis) at the M1 site every 2 to 3 d for 2 wk. Rats pretreated with alendronate plus p. gingivalis showed delayed healing of socket epithelia, periosteal reaction of alveolar bone formation and lower bone mineral density in the alveolus above adjacent M2 teeth. These abnormalities were prevented by tooth socket exposure to 20 min/d low-intensity pulsed ultrasound (LIPUS), which restored diminished expression of RANKL, Bcl-2, IL-6, Hsp70, NF-κB and TNF-α messenger ribonucleic acids in remote bone marrow, suggesting LIPUS prevented development of BRONJ-like pathophysiology in rat by inducing systemic responses for regeneration, in addition to accelerating local healing. Non-invasive treatment by LIPUS, as well as low-level laser therapy, may be useful for medication-related osteonecrosis of the jaw patients.

13. Effect of Low-Intensity Pulsed Ultrasound (LIPUS) on Remote Bone Marrow in Rats With Healing Socket.

OBJECTIVE: We reported at the previous annual meeting that LIPUS treatment of the molar tooth sockets of retired breeder rats accelerated alveolar bone healing, and that associated humoral effects were seen with elevated blood flow. Namely, LIPUS induced VEGF/angiogenesis along with elevated baseline blood flow rate, which was further associated with a sudden depression of blood flow rate in the socket immediately after cessation of LIPUS treatment. Prior injection with EP4 PGE2 receptor antagonist, but not EP3 antagonist, abolished this LIPUS-induced depression, and topical application of PGE2 to the socket epithelium mimicked the LIPUS-induced depression. In fact, the serum level of PGE2 increased after LIPUS treatment, and significantly increased in the blood flow rate at remote sites on the foot dorsum and tail after 20 minutes. Therefore, in the current study, we examined the tibia bone marrow, which is likely to respond to circulating PGE2. METHODS: Right maxillary first molars were removed from retired female breeder rats in both the LIPUS and the control groups. LIPUS was applied extrabuccally to the socket every 24 hours for 2 weeks starting one day after extraction. Removed bone samples were fixed with 4% formaldehyde to prepare undecalcified frozen sections using Kawamoto's method for immunohistochemical or histochemical staining. Bone marrow samples dissected from the tibia were treated with RNAlater (Ambion) for later RT-PCR analysis. RESULTS AND DISCUSSION: Chemokine receptor CXCR4-positive bone marrow cells increased in the tibia of the LIPUS-treated rat. Together with ubiquitously expressed CXCL12(SDF-1), it is suggested that PGE2 released from the exposed socket is responsible for the recruitment, proliferation and mobilization of the precursors of bone forming cells. LIPUS is thought to exert humoral effects by recruiting bone marrow cells into the healing socket along with VEGF/angiogenesis induced by PGE2.