Xian Peng

Ecological Effect of Arginine on Oral Microbiota

Dental caries is closely associated with the microbial dybiosis between acidogenic/aciduric pathogens and alkali-generating commensal bacteria colonized in the oral cavity. Our recent studies have shown that arginine may represent a promising anti-caries agent by modulating microbial composition in an in vitro consortium. However, the effect of arginine on the oral microbiota has yet to be comprehensively delineated in either clinical cohort or in vitro biofilm models that better represent the microbial diversity of oral cavity. Here, by employing a clinical cohort and a saliva-derived biofilm model, we demonstrated that arginine treatment could favorably modulate the oral microbiota of caries-active individuals. Specifically, treatment with arginine-containing dentifrice normalized the oral microbiota of caries-active individuals similar to that of caries-free controls in terms of microbial structure, abundance of typical species, enzymatic activities of glycolysis and alkali-generation related enzymes and their corresponding transcripts. Moreover, we found that combinatory use of arginine with fluoride could better enrich alkali-generating Streptococcus sanguinis and suppress acidogenic/aciduric Streptococcus mutans, and thus significantly retard the demineralizing capability of saliva-derived oral biofilm. Hence, we propose that fluoride and arginine have a potential synergistic effect in maintaining an eco-friendly oral microbial equilibrium in favor of better caries management.

Heat-Polymerized Resin Containing Dimethylaminododecyl Methacrylate Inhibits Candida albicans Biofilm

The prevalence of stomatitis, especially caused by Candida albicans, has highlighted the need of new antifungal denture materials. This study aimed to develop an antifungal heat-curing resin containing quaternary ammonium monomer (dimethylaminododecyl methacrylate, DMADDM), and evaluate its physical performance and antifungal properties. The discs were prepared by incorporating DMADDM into the polymer liquid of a methyl methacrylate-based, heat-polymerizing resin at 0% (control), 5%, 10%, and 20% (w/w). Flexure strength, bond quality, surface charge density, and surface roughness were measured to evaluate the physical properties of resin. The specimens were incubated with C. albicans solution in medium to form biofilms. Then Colony-Forming Units, XTT assay, and scanning electron microscope were used to evaluate antifungal effect of DMADDM-modified resin. DMADDM modified acrylic resin had no effect on the flexural strength, bond quality, and surface roughness, but it increased the surface charge density significantly. Meanwhile, this new resin inhibited the C. albicans biofilm significantly according to the XTT assay and CFU counting. The hyphae in C. albicans biofilm also reduced in DMADDM-containing groups observed by SEM. DMADDM modified acrylic resin was effective in the inhibition of C. albicans biofilm with good physical properties.

Anti-Bacterial and Microecosystem-Regulating Effects of Dental Implant Coated with Dimethylaminododecyl Methacrylate

The effects of dimethylaminododecyl methacrylate (DMADDM) modified titanium implants on bacterial activity and microbial ecosystem of saliva-derived biofilm were investigated for the first time. Titanium discs were coated with DMADDM solutions at mass fractions of 0 mg/mL (control), 1, 5 and 10 mg/mL, respectively. Biomass accumulation and metabolic activity of biofilms were tested using crystal violet assay and MTT (3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 16S rRNA gene sequencing was performed to measure the microbial community. Live/dead staining and scanning electron microscopy (SEM) were used to value the structure of biofilm. The results showed that the higher mass fraction of DMADDM the coating solution had, the significantly lower the values of metabolic activity and accumulated biofilms got, as well as fewer live cells and less extracellular matrix. Moreover, 5 mg/mL of DMADDM was the most effective concentration, as well as 10 mg/mL. In microecosystem-regulation, the DMADDM modified titanium implant decreased the relative abundance of Neisseria and Actinomyces and increased the relative abundance of Lactobacillus, a probiotic for peri-implant diseases. In conclusion, via inhibiting growth and regulating microecosystem of biofilm, this novel titanium implant coating with DMADDM was promising in preventing peri-implant disease in an ‘ecological manner’.

Anti-Caries Effects of Dental Adhesives Containing Quaternary Ammonium Methacrylates with Different Chain Lengths

The objectives of this study were to investigate the effects of dental adhesives containing quaternary ammonium methacrylates (QAMs) with different alkyl chain lengths (CL) on ecological caries prevention in vitro. Five QAMs were synthesized with a CL = 3, 6, 9, 12, and 16 and incorporated into adhesives. Micro-tensile bond strength and surface charge density were used to measure the physical properties of the adhesives. The proportion change in three-species biofilms consisting of Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii was tested using the TaqMan real-time polymerase chain reaction. Lactic acid assay, MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, exopolysaccharide staining, live/dead staining, scanning electron microscopy (SEM), and transverse microradiography (TMR) were performed to study the anti-biofilm and anti-demineralization effects of the dental adhesives. The results showed that incorporating QAMs with different alkyl chain lengths into the adhesives had no obvious effect on the dentin bond strength. The adhesives containing QAMs with a longer alkyl chain developed healthier biofilms. The surface charge density, anti-biofilm, and anti-demineralization effects of the adhesives increased with a CL of the QAMs from 3 to 12, but decreased slightly with a CL from 12 to 16. In conclusion, adhesives containing QAMs with a tailored chain length are promising for preventing secondary caries in an “ecological way”.

Function of alanine racemase in the physiological activity and cariogenicity of Streptococcus mutans

The enzyme alanine racemase (Alr) has been a new target for the development of antibacterial drugs based on the involvement of D-Ala in bacterial cell wall biosynthesis. Our previous study noted that Alr is essential for the growth and interspecies competitiveness of S. mutans, the major causative organism of dental caries. However, physiological activity and cariogenicity of S. mutans affected by Alr remains unknown. The current study examined the biofilm biomass, biofilm structure, extracellular polysaccharide (EPS) synthesis, glucosyltransferase (gtf) gene expression, acid production and acid tolerance in the alr-mutant strain. We found that biofilm formation, biofilm structure, and EPS synthesis was in a D-Ala dose-dependent manner. Biofilm structure was loose in alr-mutant group and the ratio of EPS/bacteria was also elevated. Additionally, the expression levels of multiple gtfs were up-regulated, and acid tolerance was decreased. We also established in vivo models of dental caries and found that the incidence and severity of the caries were decreased in the alr-mutant group in comparison to the parental S. mutans group. Our in vivo and in vitro experiments demonstrate that Alr is essential for the cariogenicity of S. mutans and that Alr might be a potential target for the prevention and treatment of caries.

Research on oral microbiota of monozygotic twins with discordant caries experience - in vitro and in vivo study

Oral microbiome is potentially correlated with many diseases, such as dental caries, periodontitis, oral cancer and some systemic diseases. Twin model, as an effective method for studying human microbiota, is widely used in research of relationship between oral microbiota and dental caries. However, there were few researches focusing on caries discordant twins. In this study, in vitro assays were conducted combined with 16S rRNA sequencing analysis on oral microbiota sampled from twins who presented discordant caries experience and mice model was developed as well. Results showed that oral microbiota from caries-active twin possessed higher metabolic activity and produced more lactic production. 16S rRNA sequencing analysis showed that more than 80% of family taxa could be transferred into gnotobiotic-mice. Key caries-associated genera were significantly different between twins and the same difference in genus level could be found in mice as well (p < 0.05). This study suggested that oral microbiota of twins could be distinguished from each other despite the similarities in genetic make-up, living environment, and lifestyle. The difference in microbiota was applied to develop a mice model which may facilitate the investigation of core microbiota of dental caries.

Evaluation of Novel Anticaries Adhesive in a Secondary Caries Animal Model

We investigated the anticaries properties of an adhesive containing dimethylaminododecyl methacrylate (DMADDM) in vivo via a secondary caries animal model. Cavities were prepared in the maxillary first molars of Wistar rats. DMADDM-containing adhesives were applied on one side and commercial adhesives on the opposite side as a control. After a 3-week feeding period to induce secondary caries, the molars were harvested for the evaluation of the secondary caries. Lesion depth (LD) and mineral loss (ML) were measured via a micro-CT method, and a modified Keyes scoring method yielded scores for the caries lesions. Statistical analysis was divided into 2 parts: a correlation analysis between 2 evaluations with one-way ANOVA and a least-significant differences (LSD) test, and an evaluation of anticaries adhesives with a paired samples t test. The results showed that: (1) secondary caries was successfully produced in rats; (2) there was a correlation between the modified Keyes scoring method and micro-CT in the evaluation of the secondary caries; (3) the adhesive containing DMADDM significantly reduced both LD and ML (according to micro-CT), and also lowered the scores (based on the modified Keyes scoring method). This suggests that the novel DMADDM adhesive could perform an anticaries function in vivo via the secondary caries animal model which was also developed and testified in research. Secondary caries is one of the major reasons leading to the failure of caries restoration treatment. As a solution, anticaries adhesives perform well in biofilm inhibition in vitro. However, the lack of secondary caries animal models limits the evaluation of anticaries adhesives in vivo.

Effects of different substrates/growth media on microbial community of saliva-derived biofilm

Abstract The aims of this study were to investigate the effects of substrates (glass versus hydroxyapatite [HA]) and growth media (SHI medium versus a modified artificial saliva medium with cysteine) on the microbial community of saliva‐derived biofilm in vitro. 16S rRNA gene sequencing technology was used to analyze the microbial community of saliva‐derived biofilm cultured for 72 h anaerobically. The metagenomes of biofilms were predicted from the clusters of orthologous groups. No significant difference was found between the saliva‐derived biofilms grown on HA and glass in ACE, Chao, Shannon and Simpson indices. The abundances of only a few bacteria on HA were significantly different from those on glass with a low relative abundance (<0.5%). Compared with the biofilms developed in a modified artificial saliva medium with cysteine, biofilms in SHI medium were significantly higher (P < 0.05) in diversity. Linear discriminant analysis coupled with effect size measurement showed that some obligate anaerobic genera (Lactobacillus, Veillonella, Porphyromonas and Leptotrichia) were more abundant in SHI medium biofilms. The biofilms grown in different media were also significantly different in predicted gene categories. In conclusion, the growth media, not the substrates, have significant effects on the microbial community of saliva‐derived biofilm in vitro.

The anti-caries effects of dental adhesive resin influenced by the position of functional groups in quaternary ammonium monomers

OBJECTIVES A new quaternary ammonium monomer (QAM), triethylaminododecyl acrylate (TEADDA) was synthesized, in which the position of the functional groups was different from that of dimethylaminododecyl methacrylate (DMADDM). The objectives were to: (1) investigate the effect of the changed position of the functional groups on the mechanical properties, anti-biofilm activity and biocompatibility of adhesive resin, and (2) study the anti-bacterial mechanism of QAM to improve the performance of the adhesive system modified by QAM. METHODS TEADDA and DMADDM were added into adhesives. Microtensile bond strength and surface charge density were measured. Multi-species biofilms were incubated on specimens for 16h, 48h and 72h and analyzed via MTT assay, lactic acid measurement and confocal laser scanning microscopy. The ratio of different species of bacteria was measured by real-time polymerase chain reaction. Cytotoxicity and biocompatibility were analyzed by eluents cytotoxicity test and histological images of H&E staining via an animal study in rats. RESULTS The mass fraction of TEDDA allowed to be added into adhesive was higher than that of DMADDM. However, even 10% TEADDA did not yield a strong anti-biofilm effect on biofilm growth, lactic acid production and bacteria compositions. TEADDA added into adhesives showed better mechanical properties but weaker anti-bacterial effect. There was no significant difference on cytotoxicity and biocompatibility between DMADDM and TEADDA. SIGNIFICANCE The study could be helpful for the investigation of the anti-caries mechanism of QAMs, the design of new QAMs and the improvement of the anti-caries activity of the modified dental materials.

ERG3 and ERG11 genes are critical for the pathogenesis of Candida albicans during the oral mucosal infection

The hyphal development of Candida albicans (C. albicans) has been considered as an essential virulent factor for host cell damage. However, the missing link between hyphae and virulence of C. albicans is also been discovered. Here, we identified that the null mutants of ERG3 and ERG11, two key genes in ergosterol biosynthesis pathway, can form typical hyphae but failed to cause the oral mucosal infection in vitro and in vivo for the first time. In particular, the erg3Δ/Δ and erg11Δ/Δ strains co-cultured with epithelial cells significantly reduced the adhesion, damage, and cytokine (interleukin-1α (IL-1α)) production, whereas the invasion was not affected in vitro. Importantly, they were incapable of extensive hyphal invasion, formation of micro-abscesses, and tongue epithelium damage compared to wild type due to the decrease of the colonization and epithelial infection area in a murine oropharyngeal candidiasis model. The fluconazole (FLC), an antifungal targeted at ergosterol biosynthesis, relieved the epithelial infection of C. albicansin vitro and in vivo even under non-growth inhibitory dosage confirming the virulent contribution of ergosterol biosynthesis pathway. The erg3Δ/Δ and erg11Δ/Δ strains were cleared by macrophages similar to wild type, whereas their virulence factors including agglutinin-like sequence 1 (Als1), secreted aspartyl proteinase 6 (Sap6), and hyphal wall protein-1 (Hwp1) were significantly reduced indicated that the non-toxicity might not result from the change on immune tolerance but the defective virulence. The incapacity of erg3Δ/Δ and erg11Δ/Δ in epithelial infection highlights the contribution of ergosterol biosynthesis pathway to C. albicans pathogenesis and fluconazole can not only eliminate the fungal pathogens but also reduced their virulence even at low dosage.Infectious disease: A trigger for fungal toxicityThe damage from oral infection with the fungus Candida albicans can be contained by targeting two cell membrane-building genes. C. albicans cells transition from a rounded shape into long filamentous structures called hyphae prior to invading and damaging host epithelial cells. Researchers led by Lei Cheng at Sichuan University have now identified a key intermediate step between hyphae formation and virulence. They determined that fungal cells lacking either of two genes that manufacture ergosterol, a component of the C. albicans membrane, still form hyphae and attach to epithelial cells. However, these mutant fungi inflict no cellular damage, and did not cause disease in mice. Furthermore, treatment with low-dose fluconazole, a drug that inhibits ergosterol synthesis, rendered the fungus non-virulent without killing it, indicating that this pathway represents an important ‘missing link’ for fungal pathogenesis.