Corrigendum to "The role of NMT induction on odontogenic proliferation and differentiation of dental pulp stem cells".

[This corrects the article DOI: 10.1016/j.heliyon.2021.e06598.].

Characterization of Oral Veillonella Species in Dental Biofilms in Healthy and Stunted Groups of Children Aged 6-7 Years in East Nusa Tenggara.

Impaired development that causes stunting is one of the most common health problems in Indonesia. In particular, the highest number of cases of stunting in Indonesia was reported in the East Nusa Tenggara (NTT) province. Previous studies have shown a tendency for deteriorating oral hygiene in children with a poor nutritional status. In addition, a higher proportion of oral Veillonella has been reported in children with poor oral hygiene. However, the relationship between populations of oral Veillonella and stunting has not been studied before. Therefore, this study aimed to analyze the oral Veillonella profile in the dental biofilms of healthy and stunted children aged 6-7 years. The participants were 60 elementary school students in the Nangapanda District, Ende, NTT, Indonesia. In this study, real-time polymerase chain reaction was used to examine dental biofilm samples from the healthy (n = 31) and stunted (n = 29) groups. The results revealed that seven oral Veillonella species were found in all groups. However, the number of four oral Veillonella species significantly differed between the healthy and stunted groups: V. denticariosi, V. infantium, V. rogosae, and V. tobetsuensis. This is the first study to demonstrate a potential association between oral Veillonella species and stunting in children.

Reuterin Isolated from Lactobacillus reuteri Indonesian Strain Affected Interleukin-8 and Human Beta Defensin-2 on Pathogens Induced-HaCat Cells.

Probiotic Lactobacillus reuteri has positive effects on health through inhibiting pathogenic bacteria and the ability to reduce inflammation. This study investigates the ability of reuterin isolated from L. reuteri Indonesian strain for increasing mRNA expression of interleukin (IL)-8 and human beta-defensin (hBD)-2 gene by epithelial cells, after exposure to oral bacteria. L. reuteri isolated from Indonesian's saliva, and species was confirmed by PCR, using 16S rRNA specific gene. To produce reuterin, the isolate was mixed in glycerol-containing MRS broth. Reuterin molecule's weight was counted by SDS-PAGE. Streptococcus mutans ATCC-25175 and Porphyromonas gingivalis ATCC-33277 were put in water (80°C) for 30 min, and each killed bacterial (107 CFU/mL) was inoculated into HaCat cell line (105 cell/mL). Reuterin was added in different concentrations (100%, 50%, 25%, 12,5%) and different incubation time at 37°C, 5% CO2. RNA was extracted, and a reverse transcription procedure was performed to obtain cDNA. Subsequently, a quantitative PCR method was performed to analyse the transcription level of IL-8 and HBD-2 mRNA expressed by inflamed HaCat cells. All results were statistically analysed by ANOVA test. PCR assays showed that clinical isolates were L. reuteri. Quantitative PCR results showed reuterin decreased the expression of IL-8 and increased the expression of hBD-2 in all concentrations and time periods set in this study (p < 0.05). Reuterin isolated from L. reuteri Indonesian strain increased expression of human beta defensin-2 as antimicrobial peptide and may be useful in combating inflammation.

Relationships between Solobacterium moorei and Prevotella intermedia in subgingival microbiota of periodontitis patients with halitosis: A preliminary study using qPCR.

Objective: Solobacterium moorei is suggested to be associated with the production of volatile sulphur compounds (VSCs) and can be found in subgingival plaques of deep periodontal pockets. We examined whether this bacterium's count was reduced in periodontitis patients with halitosis following non-surgical periodontal treatment, while the bacterial count of Prevotella intermedia was measured simultaneously as a control. Material & methods: This clinical study included 20 adults with chronic periodontitis who complained of halitosis. The bacterial relationship in the subgingival plaque sample was measured after 8 weeks post-treatment, including the probing pocket depth (PPD). Quantitative real-time PCR (qPCR) was used to measure the proportion of S. moorei, while the concentrations of H2S and CH3SH were determined using oral ChromaTM. Results: The presence of S. moorei was consistently observed in participants with periodontitis before and after non-surgical periodontal treatment and consistent showed a significantly lower proportion compared with P. intermedia. Solobacterium moorei showed a strong positive correlation with H2S and CH3SH concentrations, but a negative correlation with deep periodontal pocket measurements. Conversely, reduced P. intermedia may be more associated with a deep pocket, independent of the concentration of CH3SH. Conclusion: The study data showed that the proportion of S. moorei in the subgingival biofilm can be related to halitosis in periodontitis patients.

Association between Volatile Sulfur Compounds Prevotella intermedia and Matrix Metalloproteinase-8 Expression

Abstract Objective: To determine the correlation between levels of methyl mercaptan (CH3SH) hydrogen sulfide (H2S), the proportion of Prevotella intermedia (Pi), and matrix metalloproteinase-8 (MMP-8) gene expression levels in periodontitis patients accompanied by halitosis. Material and Methods: Samples were obtained from gingival crevicular fluid (GCF) in the deepest pocket and by swabbing in the tongue coating area in patients with periodontitis presenting with halitosis (n = 23) and healthy subjects as controls (n = 7). The values of CH3SH and H2S were obtained using Oral Chroma. The proportion of Pi and MMP-8 expression levels were evaluated using PCR-RT. All the result was statistically analyzed using SPSS software. Results: The levels of CH3SH and H2S in participants with PD ≥ 6 mm showed a robust negative correlation with the proportion of P. intermedia in GCF and tongue coating. No statistically significant association was detected between CH3SH and H2S levels and MMP-8 expression levels (p>0.05). Conclusion: There is no association between CH3SH and H2S levels, the proportion of P. intermedia, and MMP-8 expression in patients with periodontitis accompanied by halitosis (AU).

The Discrepancy between Clove and Non-Clove Cigarette Smoke-Promoted Candida albicans Biofilm Formation with Precoating RNA-aptamer.

This study explores the influence of precoating aptamer (Ca-apt1) on C. albicans viability while the fungus was growing in the presence of exposing condensed cigarette smoke (CSC), prepared from clove (CCSC) and non-clove (NCSC) cigarettes, for 48 h. Using qPCR, we found that mRNA expression of adhesion-associated genes ( ALS3 and HWP1) was impaired by precoating C. albicans yeast cells with the aptamer. Conversely, the gene transcription was upregulated when aptamer-uncoated yeast was pre-treated with either CSC. In addition, by analysing the result of MTT ([3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay, we found that the presence of added CCSC or NCSC in growth medium for 48 h was significantly enhanced C. albicans biofilm development. However, the presence of precoated aptamer was significantly impaired biofilm development accelerated by the NCSC. The inhibitory effect of the Ca-apt1 was not dependent on the precoated aptamer (1 and 10%). Interestingly, we noted that the enhancer effect of treated CCSC was no longer effective when the yeast had been precoated with 10% aptamer tested. Additionally, light microscopy analysis revealed that precoating aptamer alleviates morphological changes of C. albicans (from yeast to hypha formation) that are enhanced by adding CCSC or NCSC in the growth medium. In conclusion, these results suggest that administration on Ca-ap1 exhibits a significant protective effect on CSC-induced biofilm formation by C. albicans.

The role of NMT induction on odontogenic proliferation and differentiation of dental pulp stem cells.

This study was conducted to investigate the odontogenic proliferation and differentiation of dental pulp stem cells (DPSCs) after induction by nanoparticle mineral trioxide (NMT). DPSCs were isolated from permanent teeth and placed in tubes containing Dulbecco's modified Eagle's medium, followed by immunocytochemistry analysis. The viability of DPSCs exposed to NMT was measured using MTT assay with trypan blue dye exclusion. Alkaline phosphatase (ALP) activity was evaluated using ALP colorimetric reactions by reacting NMT supernatants with fluorescent-specific ALP substrates. The concentration of osteocalcin was determined using an instant human osteocalcin enzyme-linked immunosorbent assay (ELISA) kit. A human dentin sialophosphoprotein (DSPP) ELISA kit coated with anti-human DSPP antibody was employed to measure DSPP levels. There was a significant difference between ALP activity after exposing the cells to NMT and trioxide mineral aggregate on days 3, 7, and 21. Osteocalcin activity showed a significant difference on days 3, 7, 14, and 21. There was a significant difference in DSPP levels on days 7 and 21. DPSCs exposed to NMT and to trioxide mineral aggregate showed extracellular matrix formation on day 7 and 14, respectively. Furthermore, NMT may effectively increase the proliferation and differentiation of DPSCs as well as their maturation toward odontoblasts.

Metabolomics approach for determining potential metabolites correlated with sensory attributes of Melaleuca cajuputi essential oil, a promising flavor ingredient.

Melaleuca cajuputi subsp. cajuputi is one of the Australian Melaleuca species commonly found in Pulau Buru (Maluku, Indonesia). Its oil, the M. cajuputi essential oil (MCEO), has been utilized as the main flavor of the Indonesian functional food, Cajuputs Candy. However, the availability of MCEO is becoming limited. On the other hand, Indonesia has many other potential MCEO sources which can be developed as flavor ingredient. Thus, it is noteworthy to explore these new MCEO sources by studying their sensory characteristics and metabolite profiles. This study was conducted to identify potential metabolites that are correlated to sensory attributes of MCEO by using the metabolomics approach. The metabolite profiles of thirteen MCEOs from different origins were analyzed by gas chromatography-mass spectrometry while sensory analyses on Cajuputs Candy were conducted by difference-from-control and rate-all-that-apply tests. Sixty metabolites from the MCEO were annotated that includes 1,8-cineole, α-terpineol, caryophyllene, α-pinene, and γ-terpinene. Sensory analysis revealed cooling aftertaste and sweet taste as favorable attributes. Further analysis using Orthogonal Partial Least Square indicated that 1,8-cineole and γ-terpinene were correlated with cooling aftertaste, while 1,8-cineole and caryophyllene were also correlated with sweet taste. In contrast, linalool and nerolidol were associated with the feature of the most characteristic manufacturer's products which have unfavorable attributes such as floral, iodophor-like, metallic, and soapy attributes. The identification of these metabolites will be useful for the selection of MCEOs that can potentially be used as flavor.

Effect of Implantation Ti-6Al-4V ELI in femoral bone defect regeneration of Sprague Dawley rat.

Ti-6Al-4V ELI is one of the most commonly used dental implant restore function. The solution treatment temperature variation can significantly increase the strength, but it is not yet known the effect of these temperature variations on the alloy's biocompatibility properties. Twelve female Sprague Dawley rats were divided into six groups as follows: the treated group, the control group, and the defect group without implant material. In the treated group, the femur bone defect was implanted with as-cast Ti-6Al-4V ELI, 850°C, 950°C, and 1050°C heat-treated Ti-6Al-4V ELI implant material. The rats were euthanized after 30 days postimplantation and evaluated histologically. The results show that the histological scoring of the specimen for femur defect without implant material is 2 (fibrous union and fibrocartilaginous), score with implant as-cast is 2.5, the sample with 850°C heat treatment material is 2.5, 950°C is 2.5, and the temperature at 1050°C is 2.5. The score of 2.5 is between score 2 and score 3: hemorrhage, fibrous union, fibrocartilaginous microhemorrhage, and mineralized cartilage union. In conclusion, there is no effect of different heat treatment temperatures for Ti-6Al-4V ELI implant material in rat bone regeneration's maturation level.

Enhanced bone regeneration capability of chitosan sponge coated with TiO2 nanoparticles.

Chitosan has been a popular option for tissue engineering, however exhibits limited function for bone regeneration due to its low mechanical robustness and non-osteogenic inductivity. Here we hybridized chitosan with TiO2 nanoparticles to improve its bone regeneration capability. Morphology and crystallographic analysis showed that TiO2 nanoparticles in anatase-type were distributed evenly on the surface of the chitosan sponges. Degradation test showed a significant effect of TiO2 nanoparticles addition in retaining its integrity. Biomineralization assay using simulated body fluid showed apatite formation in sponges surface as denoted by PO4- band observed in FTIR results. qPCR analysis supported chitosan - TiO2 sponges in bone regeneration capability as indicated by DMP1 and OCN gene upregulation in TiO2 treated group. Finally, cytotoxicity analysis supported the fact that TiO2 nanoparticles added sponges were proved to be biocompatible. Results suggest that chitosan-50% TiO2 nanoparticles sponges could be a potential novel scaffold for bone tissue engineering.

Nanochitosan antimicrobial activity against Streptococcus mutans and Candida albicans dual-species biofilms.

OBJECTIVE: Chitosan nanoparticle (nanochitosan) has a broad antimicrobial spectrum against diverse pathogenic microorganisms. However, its effect on dental caries-associated microorganisms, such as Streptococcus mutans and Candida albicans is yet to be explored. These microorganisms are known for causing early childhood caries. Therefore, this study was aimed at investigating nanochitosan inhibition capacity against dual-species biofilms of S. mutans and C. albicans. In this study, nanochitosan antimicrobial activity is reported against mono and dual biofilm species of S. mutans and/or C. albicans at 3 and 18 h incubation time. Nanochitosan inhibition capacity was observed through biofilm mass quantity and cell viability. RESULTS: The present study successfully synthesized nanochitosan with average diameter of approximately 20-30 nm, and also established dual-species biofilms of S. mutans and C. albicans in vitro. With nanochitosan treatment, the cell viability of both microorganisms significantly decreased with the increasing concentration of nanochitosan. There was no significant decrease in biofilm mass both in the dual and single-species biofilms after 3 h of incubation. However, greater inhibition of biofilm was observed at 18 h incubation.

Expression of Osteopontin mRNA During Periodontal Healing Following Scaling and Root Planing

Objective: To analyze osteopontin mRNA expression levels in subjects with periodontitis prior to (baseline) and 7, 14, and 28 days following scaling and root planing (SRP). Material and Methods: Gingival crevicular fluid was collected as clinical samples from four subjects with periodontitis (pocket depth, 4-5 mm) aged 35-54 years old as well as from three healthy subjects (controls). The osteopontin mRNA expression levels were measured by quantitative real-time polymerase chain reaction. Spearman's rank correlation between osteopontin levels in gingival crevicular fluid and the modified gingival index (MGI) was also performed. Results: The Wilcoxon signed-rank test showed no significant difference in osteopontin mRNA expression levels between baseline and 28 days following SRP (p=0.068). The Friedman test showed no significant difference in osteopontin mRNA expression levels between baseline and following SRP (7, 14, or 28 days) (p>0.05). Spearman's rank correlation showed no significant correlation between osteopontin mRNA expression levels and MGI (r=0.087; p=0.749). Conclusion: Following SRP of periodontal tissue, there was a decreasing trend in osteopontin mRNA expression; however, this finding was not statistically significant. Nevertheless, osteopontin can be used as a biomarker to monitor the healing process; however, further studies are required to clarify our results.

Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study.

Background: Salivary nitric oxide plays an important role as an antibacterial agent in the oral cavity. Here, we analyze salivary nitric oxide, Simplified Oral Hygiene Index (OHI-S) scores and the salivary flow rate in smokers and non-smokers which has not been done previously. Methods: A cross sectional study included 25 smokers and 25 non-smokers. Their OHI-S results were categorized as "good," "medium," or "bad." Unstimulated saliva samples were collected, and their nitric oxide concentration was measured using the Griess method. Results: The salivary flow rate in smokers was lower, at 0.30 ml/minute, compared to non-smokers who had a salivary flow rate of 0.33 ml/minute. This was statistically insignificant. There was a significant difference in the concentrations of nitric oxide between smokers and non-smokers (p < 0.05). Smokers had higher concentrations than non-smokers (185.4 µM Vs 114.60 µM). In addition, there was a moderate positive correlation (r = 0.305) between the concentration of salivary nitric oxide level and the OHI-S results. Conclusions: It was concluded that salivary nitric oxide concentration was higher in smokers, and the oral hygiene condition of smokers was poor.

Cajuputs candy impairs Candida albicans and Streptococcus mutans mixed biofilm formation in vitro.

Background:Cajuputs candy (CC), an Indonesian functional food, utilizes the bioactivity of Melaleuca cajuputi essential oil (MCEO) to maintain oral cavity health. Synergistic interaction between Candida albicans and Streptococcus mutans is a crucial step in the pathogenesis of early childhood caries. Our recent study revealed several alternative MCEOs as the main flavors in CC. The capacity of CC to interfere with the fungus-bacterium relationship remains unknown. This study aimed to evaluate CC efficacy to impair biofilm formation by these dual cariogenic microbes. Methods: The inhibition capacity of CC against mixed-biofilm comprising C. albicans and S. mutans was assessed by quantitative (crystal violet assay, tetrazolium salt [MTT] assay, colony forming unit/mL counting, biofilm-related gene expression) and qualitative analysis (light microscopy and scanning electron microscopy). Result: Both biofilm-biomass and viable cells were significantly reduced in the presence of CC. Scanning electron microscopy imaging confirmed this inhibition capacity, demonstrating morphology alteration of C. albicans, along with reduced microcolonies of S. mutans in the biofilm mass. This finding was related to the transcription level of selected biofilm-associated genes, expressed either by C. albicans or S. mutans. Based on qPCR results, CC could interfere with the transition of C. albicans yeast form to the hyphal form, while it suppressed insoluble glucan production by S. mutans. G2 derived from Mojokerto MCEO showed the greatest inhibition activity on the relationship between these cross-kingdom oral microorganisms (p < 0.05). Conclusion: In general, all CC formulas showed biofilm inhibition capacity. Candy derived from Mojokerto MCEO showed the greatest capacity to maintain the yeast form of C. albicans and to inhibit extracellular polysaccharide production by S. mutans. Therefore, the development of dual-species biofilms can be impaired effectively by the CC tested.

Effect of Propolis on Streptococcus mutans Biofilm Formation

Abstract Objective: To evaluate the susceptibility of S. mutans during growth as a biofilm in the presence of different concentrations of propolis. Material and Methods: Three different concentrations of ethanolic extract of propolis (10%, 5%, and 2.5%) were used to evaluate its potential to attenuate the biofilm formation of S. mutans (ATCC 25175) on microplates. A crystal violet staining method was performed to measure the optical density (OD) of the biofilm biomass after 3 h and 18 h time periods. All the experiments were performed in triplicate, and the obtained data were expressed as mean ± standard deviation. A two-tailed Student's t-test was used to determine the different abilities of biofilm formation between the treated and control groups of the bacteria film in the presence of propolis. A p-value of <0.05 was taken as a significant value Results: The OD levels (determined using an ELISA reader) obtained after growing S. mutans as a biofilm in the presence of propolis were similar (p>0.05) to those of the control (S. mutans grown in tryptic soy broth + 1% sucrose) Conclusion: All the tested concentrations of propolis added to the growth medium did not inhibit the biofilm formation of S. mutans. Since biofilms consist of bacterial cells and extracellular matrices, we hypothesize that the extracellular matrix may have interfered with the antimicrobial properties of the tested propolis.

Expression of Toll-Like Receptor 4 and Matrix Metalloproteinase 8 in Gingival Crevicular Fluid in Patients with Periodontitis

Abstract Objective: To determine the expression of TLR4 and MMP8 in gingival crevicular fluid [GCF] in patients with periodontitis. Material and Methods: Clinical samples were collected from 23 gingival crevicular fluid of periodontal disease subjects (n = 14) and healthy periodontal subjects (n=9). Measurement of Clinical parameters of probing pocket depth (PPD), bleeding on probing (BOP), and clinical attachment loss (CAL) were included as diagnostic criteria. Pocket Depth (PD) and CAL were defined as present if the PPD was ≥ 4 mm and the CAL ≥ 1 mm. Expression of TLR4 and MMP8 in the gingival crevicular fluid of deep pockets (PD≥ 6mm), shallow pockets (PD 4-5 mm) and healthy periodontal sulcus (0-3 mm) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Statistical analysis to compare the pocket was using Independent t-test and Mann-Whitney test. Correlation between mRNA expression and clinical parameters was analyzed using Spearman's correlation test Results: Expression of TLR4 was higher in shallow pockets compared to the control group, but the difference was not statistically significant (p>0.05). The expression of MMP8 was higher in shallow pockets compared to the control group, but the difference was not statistically significant (p>0.05) either. There is no significant correlation between TLR4 and MMP8 with clinical periodontal parameters Conclusion: TLR4 and MMP8 mRNA expression levels should not be used as a clinical biomarker in periodontitis diagnostic tools.