Antimicrobial and Antiproliferative Effects of Zingiberaceae Oils: A Natural Solution for Oral Health.

Essential oils have been recognised for their potential benefits in oral care. The aim of this study was to evaluate the antibacterial and antiproliferative activity of essential oils derived from four Zingiberaceae species. A combination of GC/MS and GC-FID was employed to analyse these essential oils. The results showed that ß-myrcene (79.77 %) followed by ethyl-cinnamate (40.14 %), ß-curcumene (34.90 %), and alloaromadendrene (25.15 %) as the primary constituents of Curcuma mangga, Curcuma xanthorrhiza, Kaempferia galanga and Curcuma aeruginosa, respectively. The Zingiberaceae oils were tested for their antibacterial activity against oral bacteria using the disc diffusion test. Curcuma xanthorrhiza oil showed the largest inhibition zones against Streptococcus mitis (19.50±2.22 mm) and Streptococcus sanguinis (15.04±3.05 mm). Similarly, Curcuma mangga oil exhibited significant antibacterial activity against Streptococcus mutans (12.55±0.45 mm) and mixed oral bacteria (15.03±3.82 mm). Furthermore, the MTT viability assay revealed moderate inhibitory activity of these essential oils against H103 and ORL-204 oral cancer cells. The study findings demonstrate that Curcuma xanthorrhiza and Curcuma mangga essential oils have potent antibacterial properties, suggesting their potential use as natural alternatives to synthetic antibacterial agents in oral care products. However, further investigations are necessary to fully explore their therapeutic applications.

Interaction mechanism of a cysteine protease inhibitor, odanacatib, with human serum albumin: In vitro and bioinformatics studies.

Odanacatib (ODN) is a selective cathepsin K inhibitor that acts as an anti-resorptive agent to treat osteoporosis. ODN is also found effective in reducing the effect of severe periodontitis. The interaction between ODN and human serum albumin (HSA) was investigated using spectroscopic, microscopic, and in silico approaches to characterize their binding. The fluorescence intensity of HSA increased upon the addition of increasing concentrations of ODN accompanied by blueshift in the fluorescence spectrum, which suggested hydrophobic formation around the microenvironment of the fluorophores upon ODN binding. A moderate binding affinity was obtained for ODN-HSA binding, with binding constant (Ka) values of ∼104 M-1. Circular dichroism results suggested that the overall secondary and tertiary structures of HSA were both only slightly altered upon ODN binding. The surface morphology of HSA was also affected upon ODN binding, showing aggregate formation. Drug displacement and molecular docking results revealed that ODN preferably binds to site III in subdomain IB of HSA, while molecular dynamics simulations indicated formation of a stable protein complex when site III was occupied by ODN. The ODN-HSA complex was mainly stabilized by a combination of hydrogen bonding, hydrophobic interactions, and van der Waals forces. These findings provide additional information to understand the interaction mechanism of ODN in blood circulation and may help in future improvements on the adverse effects of ODN.

Microbial adherence on vacuum-formed retainers with different surface roughness as constructed from conventional stone models and 3D printed models: a randomized controlled clinical trial.

OBJECTIVE: This single center parallel, randomized controlled trial aimed to determine the propensity of microbial adherence on vacuum-formed retainers (VFRs) with different surface roughness imprints. MATERIALS AND METHODS: Thirty-six patients debonded from fixed appliances at a teaching institution were allocated by block randomization stratified for gender to three groups [VFRs fabricated on conventional, fused deposition modeling (FDM) or stereolithography apparatus (SLA) working models]. Participants wore the VFRs for three months full-time followed by three months part-time. VFRs were collected after each follow-up for Streptococcus and yeast counts. Surface roughness was measured indirectly on the working models using a 3D optical surface texture analyzer. Blinding was not feasible due to appliance appearance. The trial was registered [NCT03844425 ( ClinicalTrials.gov )] and funded by the Universiti Malaya Dental Postgraduate Research Grant (DPRG/14/19). RESULTS: Thirty participants (eleven conventional, ten FDM, and nine SLA) were analyzed after six dropped out. No harms were reported. Microbial counts between the groups were not significantly different. There were more microbes in the lower VFRs than upper VFRs (total count: p<0.05; effect size, 0.5 during full-time wear and 0.4 during part-time wear). SLA had significantly (p<0.05) smoother surface than FDM (effect size, 0.3) and conventional models (effect size, 0.5). Microbial adherence was not associated with working model surface roughness. CONCLUSION: Microbial adherence on VFRs was not influenced by degree of surface roughness imprints from working models. CLINICAL RELEVANCE: 3D printed models can be used to make VFRs. Lower VFRs tended to accumulate oral microbes, potentially increasing the oral health risk in the lower arch.

ACE2 expression in saliva of patients with COVID-19 and its association with Candida albicans and Aggregatibacter actinomycetemcomitans.

Background: A relationship between oral microbiota and susceptibility to SARS-CoV-2 infection has been extensively studied. However, the relationship between oral commensal flora and expression of angiotensin-converting enzyme 2 ( ACE2) remains to be established. In this observational study, we collected saliva from patients with COVID-19 and evaluated the relationship between ACE2 expression and Candida albicans as well as with selected gram-negative bacteria ( Aggregatibacter actin o mycetemcomitans, Fusobacterium nucleatum, and Veillonella parvula). We investigated how this may be directly or indirectly involved in oral dysbiosis in patients with COVID-19. Methods: We included 23 hospitalized patients admitted to Universitas Indonesia Hospital with PCR-confirmed COVID-19, with six healthy participants serving as controls. Saliva and tongue surface swabs were collected from patients with diabetes (DG) and without diabetes (NDG) and subject controls. Using quantitative PCR (qPCR) we assessed the mRNA expression of ACE2, the abundance of C. albicans, and the transcription levels of its biofilm-associated genes, agglutinin-like protein 3 ( ALS3), hyphal wall protein 1 ( HWP1), and yeast-form wall protein 1 ( YWP1). We also counted the relative proportion of the three selected gram-negative oral bacteria in saliva. All analyses were performed to determine the relationship between ACE2 expression and C. albicans and gram-negative bacteria. Results: ACE2 mRNA expression was significantly higher in tongue swab samples than in saliva. However, no significant difference was observed between the patient groups. Conversely, DG patients had a significantly higher abundance of C. albicans in saliva compared to NDG patients and control group patients. The correlation and sensitivity/specificity relationship between ACE2 expression and C. albicans or the selected oral bacteria were also observed. Conclusions: The data show that ACE2 expression can be detected in saliva of patients with COVID-19 and its association with C. albicans and gram-negative oral bacteria might contribute toward developing an oral dysbiosis based predictor for prognosis of COVID-19 severity.

The Role of Recombinant Secretory Leukocyte Protease Inhibitor to CD163, FGF-2, IL-1 and IL-6 Expression in Skin Wound Healing.

Background: The wound healing process can be optimized through the addition of a biomaterial such as recombinant secretory leukocyte protease inhibitor (rSLPI). The SLPI is a non-glycosylated proteomic material that inhibits protease enzymes and has anti-inflammatory properties, thus accelerating wound healing. This study analyzed the administration of rSLPI doses 0.04 cc and 0.06 cc in skin wound healing on the CD163 expression of macrophages and cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6) and fibroblast growth factor 2 (FGF-2). Materials and Methods: rSLPI produced from Escherichia coli TOP10 as the cloning host, BL21 (DE3) strains as the expression host and pET30a plasmids were used for the expression system construction. The wound was created on Wistar rat dorsal skin, then rSLPI 0.04 cc and 0.06 cc was administered. In the next four days, the back skin was biopsied and stained by immunohistochemistry to analyze the CD163, FGF-2, IL-1 and IL-6 expression. Results: The administration of rSLPI increased CD163 and FGF-2 expression dependent on dose (p<0.05). On the other hand, administration of rSLPI decreased IL-1 and IL-6 expression depending on dose (p <0.05). Conclusion: The administration of rSLPI is able to accelerate the wound healing process by increasing the CD163 and FGF-2 expression. The cytokines such as IL-1 and IL-6 decreased depending on rSLPI doses.

CD89/CD35 Expression Ratio in Salivary Neutrophil as an Early Detection Marker for Severe Early Childhood Caries.

OBJECTIVES: To analyze CD35/CD89 expression ratio on the surface of neutrophils as an early detection marker for S-ECC. MATERIALS AND METHODS: Saliva was collected from 4- to 6-year-old kindergarten students. Salivary neutrophils were obtained by instructing the subjects to rinse their mouth with 1 mL of sterile 1.5% NaCl for 30 seconds before expectorating it into a sterile glass. The expression of CFSE+CD35+ and CFSE+CD89+was measured and analyzed using flow cytometry. RESULTS: The expression of CFSE+CD89+ in the caries-free group (2.46 ± 0.39) was significantly lower than that in the S-ECC group (3.41 ± 1.11), with a p-value of 0.0001, while the expression of CFSE+CD35+ in the caries-free group was (2.35 ± 0.56) compared with (1.54 ± 0.35) (p = 0.0001) in the S-ECC group. CONCLUSIONS: The expression ratio of CFSE+CD89+ and CFSE+CD35+constitutes a marker for S-ECC.

Oxidizing Effect of Ozonated-Water on Microbial Balance in the Oral Ecosystem.

Application of ozone is recommended for sterilisation in dental procedures. This study explored the antimicrobial effect of 0.1 ppm ozonated-water on selected common oral commensals. Based on deviation of their growth curves pattern upon ozone treatment, the inhibitory effect of ozone was determined. SEM examination of the ozone-treated microbes recorded its possible morphological effect. Findings suggested a bacteriostatic action of ozone when microbes were treated at the early phase, while, it was bactericidal when treated during the active phase of the growth cycle. Hence, suggesting rinsing the oral cavity with ozonated-water at 0.1 ppm immediately after tooth brushing may suppress microbial growth and slow biofilm formation. While, rinsing on already developed biofilm may result in microbial cell lysis that halted microbial growth and reduce microbial population in the biofilm. Both justify the great potential of ozone (0.1 ppm) for use as antimicrobial agent for the control of biofilm development in the oral cavity.

Correlation between numbers of cells in human dental pulp and age: Implications for age estimation.

OBJECTIVE: The aim of this study was to investigate correlations between dental pulp cell count of odontoblasts, subodontoblasts and fibroblasts and age, within different age groups. Formulation of regression equations using the dental pulp cell count for predicting age was attempted. DESIGN: Eighty-one extracted teeth were grouped into two age groups (6-25 years, 26-80 years). The teeth were demineralized and histological sections were prepared for cell count. Regression equations were generated from regression analysis of cell count and tested for age estimation. RESULTS: The number of dental pulp cells were found to increase until around the third decade of life and following this, the odontoblasts and subodontoblasts cell numbers began to decline while the fibroblasts seemed to remain almost stationary. The Pearson correlation test revealed a significant positive correlation between the cell number for all type of cells and age in the 6-25 years group (r=+0.791 for odontoblasts, r=+0.600 for subodontoblasts and r=+0.680 for fibroblasts). In the 26-80 years age group, a significant negative correlation of the odontoblasts (r=-0.777) and subodontoblasts (r=-0.715) with age was observed but for fibroblasts, the correlation value was negligible (r=-0.165). Regression equations generated using odontoblasts and subodontoblasts cell number were applicable for age estimation. The standard error of estimates (SEEs) were around±5years for 6-25 years and±8years for 26-80 years age groups. The mean values of the estimated and chronological ages were not significantly different. CONCLUSIONS: A significant correlation between the cell count of odontoblasts and subodontoblasts with age was demonstrated. Regression equations using odontoblasts and subodontoblasts cell number can be used to predict age with some limitations.

The effect of selected plant extracts on the development of single-species dental biofilms.

OBJECTIVE: To determine the effect of a mixture of plant extracts on the adherence and retention of bacteria in dental biofilm. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Oral Biology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia, from December 2009 to December 2011. METHODOLOGY: For determination of adhering ability, experimental pellicle was first treated with the Plant Extracts Mixture (PEM) before inoculating it with individual bacterial species (S. mitis / S. sanguinis / S. mutans). For the determination of retention ability, the procedure was repeated with the experimental pellicle being inoculated first with the individual bacterial species and then treating it with the PEM. These two experiments were repeated with deionized distilled water(negative control) and Thymol (0.64%) (positive control). The bacterial populations in biofilms for the two experiments were expressed as Colony Forming Unit (CFU) / mL x 10(4) and the corresponding values were expressed as mean ± SD. RESULTS: The effect of the Plant Extracts Mixture (PEM) for the two experiments was compared with that of Thymol and deionized distilled water. It was shown that there is a reduced adherence of bacteria to PEM-treated and Thymol (0.064%) treated experimental pellicle compared with the negative control (p < 0.001). It was also found that the retention of bacteria in both treated biofilms is also lower than that of negative control (p < 0.001). CONCLUSION: Plant Extracts Mixture (PEM) may influence the development of dental biofilm by affecting the adhering and retention capacities of the bacterial species in the dental biofilms.

Apoptosis-inducing effect of three medicinal plants on oral cancer cells KB and ORL-48.

Brucea javanica, Azadirachta indica, and Typhonium flagelliforme are medicinal plants commonly used to treat conditions associated with tumour formation. This study aimed to determine the antiproliferative activity of these plants extracts on KB and ORL-48 oral cancer cell lines and to suggest their mode of cell death. The concentration producing 50% cell inhibition (IC50) was determined and the activity was examined under an inverted microscope. Immunohistochemistry fluorescent staining method (TUNEL) was performed to indicate the mechanism of cell death and the fragmented DNA band pattern produced was obtained for verification. Compared to Azadirachta sp. and Typhonium sp., the antiproliferative activity of Brucea sp. extract was the most potent on both KB and ORL-48 cells with IC50 of 24.37 ± 1.75 and 6.67 ± 1.15 µg/mL, respectively. Signs of cell attrition were observed 24 hr after treatment. Green fluorescent spots indicating cell death by apoptosis were observed in images of both cells following treatment with all the three extracts. DNA fragments harvested from Brucea-treated cells produced bands in a ladder pattern suggesting the apoptotic effect of the extract. It is thus concluded that Brucea sp. extract exhibited cytotoxic activity on ORL-48 cells and their action mechanism is via apoptosis.

The antimicrobial effect of 0.1 ppm ozonated water on 24-hour plaque microorganisms in situ.

Ozone is a known oxidant present in the atmosphere and is commercially produced by simple ozonizer machines. It is a powerful antimicrobial agent in its gaseous and aqueous forms. Ozone readily dissolves in water and retains its antimicrobial property even in the dissolved state. In this study, the effect of 0.1 ppm ozonated water was analyzed on 24-hour supragingival plaque (SP) samples in situ. SP was collected from the two most posterior teeth in the contra-lateral quadrants before and after a 30-second rinse with either distilled water (control group) or 0.1 ppm ozonated water (test group). The plaque was used to count the number of total bacteria, total anaerobic bacteria, Streptococcus mutans, and Candida albicans on selective agar media. The statistical analysis of the number of colony forming units (CFUs) obtained demonstrated a significant antimicrobial effect of ozonated water on the total bacteria (p = 0.01) and anaerobes (p = 0.02). A reduction in the post-rinse CFU count for Streptococcus mutans was also observed, but the effect was not statistically significant (p = 0.07). The Candida species was only grown from one sample. Ozonated water at the 0.1 ppm concentration was effective in reducing the load of 24-hour plaque bacteria, but it did not eliminate them completely.

The antimicrobial effect of 0.1 ppm ozonated water on 24-hour plaque microorganisms in situ

Ozone is a known oxidant present in the atmosphere and is commercially produced by simple ozonizer machines. It is a powerful antimicrobial agent in its gaseous and aqueous forms. Ozone readily dissolves in water and retains its antimicrobial property even in the dissolved state. In this study, the effect of 0.1 ppm ozonated water was analyzed on 24-hour supragingival plaque (SP) samples in situ. SP was collected from the two most posterior teeth in the contra-lateral quadrants before and after a 30-second rinse with either distilled water (control group) or 0.1 ppm ozonated water (test group). The plaque was used to count the number of total bacteria, total anaerobic bacteria, Streptococcus mutans, and Candida albicans on selective agar media. The statistical analysis of the number of colony forming units (CFUs) obtained demonstrated a significant antimicrobial effect of ozonated water on the total bacteria (p = 0.01) and anaerobes (p = 0.02). A reduction in the post-rinse CFU count for Streptococcus mutans was also observed, but the effect was not statistically significant (p = 0.07). The Candida species was only grown from one sample. Ozonated water at the 0.1 ppm concentration was effective in reducing the load of 24-hour plaque bacteria, but it did not eliminate them completely.

The effect of Piper betle and Psidium guajava extracts on the cell-surface hydrophobicity of selected early settlers of dental plaque.

The adhesion of early settlers of dental plaque to the tooth surface has a role in the initiation of the development of dental plaque. The hydrophobic surface properties of the bacteria cell wall are indirectly responsible for the adhesion of the bacteria cell to the acquired pellicle on the tooth surfaces. In this study, the effect of aqueous extract of two plants (Psidium guajava and Piper betle) on the cell-surface hydro-phobicity of early settlers of dental plaque was determined in vitro. Hexadecane, a hydrocarbon was used to represent the hydrophobic surface of the teeth in the oral cavity. It was found that treatment of the early plaque settlers with 1 mg/ml extract of Psidium guajava reduced the cell-surface hydrophobicity of Strep. sanguinis, Strep. mitis and Actinomyces sp. by 54.1%, 49.9% and 40.6%, respectively. Treatment of these bacteria with the same concentration of Piper betle however, showed a comparatively lesser effect (< 10%). It was also observed that the anti-adhesive effect of the two extracts on the binding of the early plaque settlers to hexadecane is concentration dependent.

The anti-adherence effect of Piper betle and Psidium guajava extracts on the adhesion of early settlers in dental plaque to saliva-coated glass surfaces.

The aqueous extracts of Piper betle and Psidium guajava were prepared and tested for their anti-adherence effect on the adhesion of early plaque settlers (Strep. mitis, Strep. sanguinis and Actinomyces sp.). The saliva-coated glass surfaces were used to simulate the pellicle-coated enamel surface in the oral cavity. Our results showed that the anti-adherence activities of Piper betle and Psidium guajava extracts towards the bacteria were different between the bacterial species. Psidium guajava was shown to have a slightly greater anti-adherence effect on Strep. sanguinis by 5.5% and Actinomyces sp. by 10% and a significantly higher effect on Strep. mitis (70%) compared to Piper betle. The three bacterial species are known to be highly hydrophobic, and that hydrophobic bonding seemed to be an important factor in their adherence activities. It is therefore suggested that the plant extracts, in expressing their anti-adherence activities, could have altered the hydrophobic nature of the bonding between the bacteria and the saliva-coated glass surfaces.