Background The study aims to investigate Streptococcus cristatus, an oral commensal bacterium, as a probiotic for dental caries prevention by modulating the oral microbiome.Methods Saliva from four healthy donors was used to establish 24-h microcosm biofilms in an in vitro 96-well peg model. The preformed biofilms were then exposed to biofilm medium containing 0.2% sucrose (BM), with or without S. cristatus. They were grown for 48 h under two conditions: a constant pH-neutral regime (BM supplemented with 76 mM K2HPO4 and 15 mM KH2PO4, pH 7.0) or cariogenic pH-cycling regime (8 h pH-neutral and 16 h in BM containing 100 mM acetic acid, pH 5.5). Phosphate and acetate buffers were used to control pH. After 72 h, the biofilms were analyzed for biomass, lactic acid production, hydrogen peroxide (HP) concentrations, and microbial composition via 16S rRNA gene sequencing.Results S. cristatus successfully integrated into 24-h preformed microcosm biofilms derived from individual saliva. Under pH-neutral conditions, it reduced biofilm biomass and lactate production while increasing hydrogen peroxide (HP) generation in a donor-dependent manner. Conversely, under cariogenic pH-cycling conditions, these inhibitory effects on biomass and lactate production were consistent across all donors, although HP was undetectable. Microbiome analysis revealed that S. cristatus increased species richness and mitigated the compositional shifts caused by pH-cycling. This was achieved by inhibiting Streptococcus salivarius/vestibularis across all donors, while promoting Streptococcus mitis group and Streptococcus anginosus in a donor-dependent manner.Conclusions S. cristatus represents a promising microbiome modulator with the potential to substantially mitigate the cariogenicity of oral microcosms.

Aim or purpose: To investigate the impacts of Streptococcus oligofermentans, an oral commensal, on the cariogenicity of root surface plaque biofilms, and its inhibition on root caries in a rat model. Materials and methods: Root surface plaques from root-caries patients and caries-free individuals were inoculated into a polystyrene peg model for 24 h, then cultured with or without S. oligofermentans under cariogenic pH-cycling conditions to form 72-h biofilms. Subsequently, the biofilms were subjected to biomass assay, lactate and H₂O₂ quantification, and qPCR analysis for S. oligofermentans colonization. In a root caries model, rats were received the following treatments respectively: no treatment, S. oligofermentans alone, cariogenic microbes (Streptococcus mutans, Actinomyces viscosus and Candida albicans), S. oligofermentans followed by cariogenic microbes, cariogenic microbes followed by low- or high-frequency treatment of S. oligofermentans. After 6 weeks, biosafety was assessed via organ histology, and root caries severity evaluated using Doff’s score and micro-CT. Results: S. oligofermentans successfully colonized the pre-formed root-caries and caries-free plaque biofilms, significantly reducing biomass and acid production while increasing H₂O₂ generation (p<0.05). Pretreatment and treatment with low- or high-frequency S. oligofermentans significantly reduced root caries severity in rats (p<0.0001). Additionally, S. oligofermentans treatment alone displayed no toxicity to rat organs. Conclusions: S. oligofermentans effectively colonizes root surface plaque biofilms and suppresses their cariogenicity in vitro, as well as mitigates root caries severity in vivo, suggesting its inhibition on root caries.

Background Probiotics serve as a novel preventive or therapeutic approach for dental caries owing to their ability to reverse dysbiosis and restore a healthy microbiota. Here, we identified Burkholderia ambifaria AFS098024 as a probiotic candidate isolated from plants.Methods The safety of B. ambifaria was evaluated by hemolytic activity, D-lactic acid production and antibiotic susceptibility. In vitro biofilm model derived from the saliva of caries-free and caries-active donors and in vivo rat caries model were used to assess the efficacy of B. ambifaria in caries prevention and treatment.Results B. ambifaria was safe as a probiotic candidate and it could integrate with in vitro biofilm model. It significantly reduced the biomass and lactate production of biofilms from caries-active donors and disrupted biofilm structures. B. ambifaria effectively reduced the severity of carious lesions in rat molars, regardless of the inoculation sequence. Molars pretreated or treated with B. ambifaria demonstrated notably higher enamel volumes. Additionally, colonization of rat molars by B. ambifaria persisted for 6 weeks.Conclusion The B. ambifaria strain used in this study holds promise as a probiotic for inhibiting dental caries, both in vitro and in vivo.

Vital pulp therapy (VPT) is considered a conservative treatment for preserving pulp viability in caries-induced dental pulp infections. However, bacterial contamination negatively affects dentine-pulp complex repair. The common capping materials show limited antimicrobial effects against some microorganisms. To improve the VPT efficacy, capping materials with increased antibacterial properties and enhanced odontogenic and angiogenic activities are needed. Herein, a SrCuSi4 O10 /gelatin methacrylate(SC/Gel) composite hydrogel has been proposed for infected dental pulp treatment. SrCuSi4 O10 (SC) is a microscale bioceramic composed of assembled multilayered nanosheets that possesses good near-infrared photothermal conversion ability and multiple bioactivities due to sustained Sr2+ , Cu2+ , and SiO3 2- ion release. It is shown that the SC/Gel composite hydrogel efficiently eliminates Streptococcus mutans and Lactobacillus casei and inhibits biofilm formation under photothermal heating, while the ion extract from SC promotes odontogenesis of rat dental pulp stem cells and angiogenesis of human umbilical vein endothelial cells. The as-designed therapeutic effect of SC/Gel composite hydrogel-mediated VPT has been proven in a rat dental pulp infection model and yielded improved dentine-pulp complex repair compared with the commercially used iRoot® BP Plus. This study suggests that the SC/Gel composite hydrogel is a potential pulp-capping material with improved effects on dentine-pulp complex repair in infected pulp.

BACKGROUND:To investigate the effect of a rotary agitation method or ultrasonically activated irrigation on the antibiofilm effect of a mixture of sodium hypochlorite (NaOCl) and etidronate (1-hydroxyethylidene-1,1-bisphosphonate, HEBP) using a dual-species biofilm model in root canal system. METHODS:Mature dual-species biofilms of Enterococcus faecalis and Streptococcus gordonii were formed in root canals of mandibular premolars. Teeth were randomly allotted (n = 12) to group 1, XP-endo Finisher (XPF); group 2, ultrasonically activated irrigation (UAI); group 3, syringe-and-needle irrigation (SNI). In all groups, canals were instrumented with a rotary instrument (XP-endo Shaper) prior to irrigant agitation/activation. A mixture containing 2.5% NaOCl and 9% HEBP was used throughout the experiment. Bacterial counts from the canal were determined using qPCR before preparation (S1), after preparation (S2), and after final irrigation agitation/activation (S3). Bacterial viability within the dentinal tubules in the coronal, middle and apical root-thirds was quantified using confocal microscopy after Live/Dead staining. The bacterial counts and viability were compared between groups using one-way ANOVA and post-hoc Tukey's tests. Paired t-test was used to compare the bacterial counts within groups. RESULTS:Instrumentation alone could significantly reduce the microbial counts in all the groups (P < 0.0001). Subsequent agitation/activation resulted in significant microbial reduction only in XPF and UAI (P < 0.05), both of which reduced significantly more microbial counts than SNI (P < 0.05). Live/Dead staining revealed that XPF and UAI showed significantly greater percentage of dead bacteria within the dentinal tubules than SNI in the coronal third (P < 0.05); UAI resulted in the significantly highest percentage of dead bacteria in the middle third (P < 0.05); while there was no significant difference between the groups in the apical third (P > 0.05). CONCLUSIONS:When using the sodium hypochlorite/etidronate mixture for irrigation, final irrigant agitation/activation with XP-endo Finisher or ultrasonic can improve disinfection of the main root canal space and the dentinal tubules in the coronal third, while ultrasonically activated irrigation appears to exhibit better disinfection within dentinal tubules in the middle third.

In subgingival plaque biofilms, Fusobacterium nucleatum is closely related to the occurrence and development of periodontitis. Streptococcus gordonii, as an accessory pathogen, can coaggregate with periodontal pathogens, facilitating the subgingival colonization of periodontal pathogens. Studies have shown that F. nucleatum can coaggregate with S. gordonii and colonize the subgingival plaque. However, most studies have focused on monocultures or coinfection of species and the potential impact of coaggregation between the two species on periodontal interactions to human gingival epithelial cells (hGECs) remains poorly understood. The present study explored the effect of coaggregation between F. nucleatum and S. gordonii on subgingival synergistic virulence to hGECs. The results showed that coaggregation inhibited the adhesion and invasion of F. nucleatum to hGECs compared with that in the F. nucleatum monoculture and coinfection group. Coaggregation and coinfection with F. nucleatum both enhanced S. gordonii adhesion to hGECs, but neither of the two groups affected S. gordonii invasion to hGECs compared with S. gordonii monoculture. The gene expression levels of TLR2 and TLR4 in hGECs in the coaggregation group were higher than those in the monoculture groups but lower than those in the coinfection group. Compared with coinfection, the coaggregation inhibited apoptosis of hGECs and promoted the secretion of the proinflammatory cytokines TNF-α and IL-6 by hGECs, showed a synergistic inflammatory effect, while coaggregation inhibited the secretion of the anti-inflammatory cytokine TGF-β1. Coaggregation enhanced the phosphorylation of p65, p38, and JNK proteins and therefore activated the NF-κB and MAPK signaling pathways. Pretreatment with a pathway antagonist/inhibitor decreased the phosphorylation levels of proteins and the secretion of TNF-α and IL-6. In conclusion, coaggregation inhibited the adhesion and invasion of F. nucleatum to hGECs. However, it enhanced the adhesion of S. gordonii to hGECs. Compared with coinfection, coaggregation inhibited the apoptosis of hGECs. The coaggregation coordinately promoted the secretion of TNF-α and IL-6 by hGECs through the TLR/NF-κB and TLR/MAPK signaling pathways while inhibiting the secretion of TGF-β1, thus aggravating the inflammatory response of hGECs.

Contemporary endodontic microsurgery has emerged as a significant treatment modality in the retention of teeth with persistent apical periodontitis. This article proposes the concept of the full-cycle clinical management of endodontic microsurgery based on the condition of the patient and tooth, attempting to develop a comprehensive strategy for the examination, treatment and follow-up to save natural teeth. Full-cycle clinical management included preoperative consideration of the general condition and surgical site and selection of cases for endodontic microsurgery; intraoperative application of techniques such as lasers, "bone window" technique and targeted endodontic microsurgery to make the surgical approaches more varied and the operation minimally invasive; postoperative outcome assessment according to the history, clinical and radiographic examination; and analysis of the short- and long-term outcomes.

The black pigmentation‐related genes in Porphyromonas gingivalis are primarily involved in regulating gingipain functions. In this study, we identified a pigmentation‐related gene, designated as pgn_0361 . To characterize the role of pgn_0361 in regulating P. gingivalis ‐mediated epithelial cell detachment and inhibition of wound closure, PgΔ0361, an isogenic pgn_0361 ‐defective mutant strain, and PgΔ0361C, a complementation strain, were constructed using P. gingivalis ATCC 33277. The gingipain and hemagglutination activities, as well as biofilm formation, were examined in all three strains. The effect of P. gingivalis strains on epithelial cell detachment was investigated using the HO‐1‐N‐1 and Ca9‐22 epithelial cell lines. The inhibition of wound closure by heat‐killed P. gingivalis cells and culture supernatant was analyzed using an in vitro wound closure assay. Compared to the wild‐type strain, the PgΔ0361 strain did not exhibit gingipain or hemagglutination activity but exhibited enhanced biofilm formation. Additionally, the PgΔ0361 strain exhibited attenuated ability to detach the epithelial cells and to inhibit wound closure in vitro . Contrastingly, the culture supernatant of PgΔ0361 exhibited high gingipain activity and strong inhibition of wound closure. The characteristics of PgΔ0361C and wild‐type strains were comparable. In conclusion, the pgn_0361 gene is involved in regulating gingipains. The PGN_0361‐defective strain exhibited reduced virulence in terms of epithelial cell detachment and inhibition of wound closure. The culture supernatant of the mutant strain highly inhibited wound closure, which may be due to high gingipain activity.

Objectives The aim of this study was to investigate the accuracy of minimally invasive endodontic cavity preparation with the guidance of cone-beam computed tomography (CBCT) and three-dimensional printed templates through analyzing distance deviation and angle deviation. Methods Forty extracted human single-rooted maxillary first premolars with double root canals were selected. Preoperative CBCT scanning of all specimens was used for designing access cavities. All teeth were numbered and assigned into two groups according to the random number table. There were twenty teeth including forty root canals in each group. The minimally invasive endodontic cavity was designed as Truss access cavity. Preparation for access cavities in group A was guided by three-dimensional printed templates which were fabricated according to the CBCT images. Access cavities in group B were prepared according to the marks on occlusal surface after the analysis of CBCT images. Distance deviations and angle deviations in buccopalatal and mesiodistal directions as well as the areas of access cavities at occlusal and pulp horn levels were measured and calculated in the CBCT images. Statistical analysis for the data was performed by SPSS 20.0. Results The data were analyzed with Mann-Whitney U test, since they were not satisfied with the normal distribution and the homogeneity of variance. No statistic difference was observed between the two groups in the areas of access cavities at occlusal level [group A: (1.34 ± 0.18) mm2, group B: (1.30 ± 0.15) mm2; U=0.876, P=0.393] or at pulp horn level [group A: (1.74 ± 0.20) mm2, group B: (1.67 ± 0.24) mm2; U=1.290, P=0.194]. The mesiodistal distance deviations at occlusal level in group B [ (0.37 ± 0.26) mm] were less than that in group A [ (0.52 ± 0.30) mm], which had significant difference (U=2.237, P=0.024) . There was no significant difference between the two groups in the buccopalatal distance deviations of occlusal level [group A: (0.45 ± 0.40) mm, group B: (0.41 ± 0.28) mm; U=0.385, P=0.697], the buccopalatal distance deviations of pulp horn level [group A: (0.32 ± 0.25) mm, group B: (0.41 ± 0.30) mm; U=1.290, P=0.199], the mesiodistal distance deviations of pulp horn level [group A: (0.38 ± 0.24) mm, group B: (0.35 ± 0.26) mm; U=0.905, P=0.500], as well as the buccopalatal angle deviations [group A: (4.76 ± 3.04) °, group B: (4.72 ± 3.61) °; U=0.404, P=0.679] or the mesiodistal angle deviations [group A: (3.04 ± 1.97) °, group B: (3.05 ± 2.45) °; U=0.467, P=0.637]. Conclusions CBCT scanning was able to accurately guide the preparation of the minimally invasive endodontic cavities for the teeth with normal root canal anatomy. The guidance of CBCT scanning with templates did not significantly enhance the accuracy of access cavities, compared to that of CBCT scanning alone. Moreover, no significant difference was detected in the actual sizes of Truss access cavities prepared with the guidance of the two approaches. Key words: Endodontics, minimally invasive; Dental cavity preparation; Cone-beam computed tomography; Printing, three-dimensional; Deviation

Objective Relacin is a synthetic molecule that targets RelA, an essential protein in a conserved bacterial stress response system. It was shown to inhibit bacterial growth. The aims of this study were to evaluate the antimicrobial effect of relacin combined with sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilms and to evaluate the cytotoxicity of relacin. Material and Methods 48-h E. faecalis OG1RF biofilms were treated by various concentrations of relacin in order to determine its inhibitory concentration. Then, the 48-h biofilms were treated either with 1-min NaOCl (0.01%, 0.05%) alone, or in combination of relacin. As a means of comparison, the biofilms of ΔrelA were also treated by 1-min NaOCl (0.01%, 0.05%, 0.25%). The treatment efficacy was determined by agar plate count assays. The cytotoxicity of relacin was examined on human gingival epithelial cells Ca9-22 and murine fibroblasts NIH-3T3 by a methyl thiazolyltetrazolium (MTT) assay and a lactate dehydrogenase assay. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test and an independent Student’s t-test. A significance level of p<0.05 was used. Results Relacin inhibited the growth of OG1RF biofilms partially at 8 mM and fully at 14 mM. The relacin (14 mM) and NaOCl combined treatment resulted in significantly higher treatment efficacy than NaOCl treatment alone. At 0.05% NaOCl, the combined treatment resulted in 5.65 (±0.19) log reduction in biofilm viability. The ΔrelA biofilms were more susceptible to NaOCl treatment than the wild type biofilms at 0.25% NaOCl. Relacin at 14 mM was not toxic to host epithelial cells and fibroblasts. Conclusions The combination of relacin with a low concentration of NaOCl was effective and not cytotoxic.

Persistent apical periodontitis (PAP) is characterized by refractory inflammation and progressive bone destruction. Enterococcus faecalis infection is considered an important etiological factor for the development of PAP, although the exact mechanisms remain unknown. This study aimed at investigating the role of E. faecalis in cell proliferation, inflammatory reactions and osteoclast differentiation of macrophages using an in vitro infection model of osteoclast precursor RAW264.7 cells. A cell viability assay of cultured RAW264.7 cells exposed to live E. faecalis at a multiplicity of infection of 100 for 2h, indicated that the infection exhibited no cytotoxic effect. Transmission electron microscopy images revealed no apoptotic changes but a rise of metabolic activity and phagocytic features in the infected RAW264.7 cells. Confocal laser scanning microscopic and flow cytometric analysis indicated that the phagocytosis of RAW264.7 cells was activated by E. faecalis infection. Furthermore, quantitative real-time PCR assays demonstrated that the expression of inflammatory cytokines was remarkably elevated in infected RAW264.7 cells. Differentiation of infected RAW264.7 cells into osteoclasts was remarkably attenuated, and expression of osteoclast marker genes as well as fusogenic genes significantly dropped. In summary, E. faecalis appears to attenuate osteoclastic differentiation of RAW264.7 precursor cells, rather stimulates them to function as macrophages.

The cariogenic bacterium Streptococcus mutans can develop stable resistance to fluoride through chromosomal mutations in vitro. Fluoride-resistant S. mutans has seldom been isolated in clinical settings, despite the wide application of fluoride in oral-care products. One explanation is that the fluoride-resistant S. mutans strains have decreased fitness. However, so far, there has been no conclusive evidence to support this idea. The aim of this study was to investigate the fitness cost of 48-h biofilms of two fluoride-resistant S. mutans strains, UF35 and UA159-FR (UAFR), using the wild-type fluoride-sensitive strain UA159 as a reference. The engineered UF35 strain contains one point mutation, whereas UAFR, selected from NaF-containing agar plates, has multiple chromosomal mutations. All biofilms were formed for 48 h under a constantly neutral pH or a pH-cycling (8 h of neutral pH and 16 h of pH 5.5) condition in the absence of fluoride. The biomass of the biofilms was quantified with a crystal violet assay. The biofilms were also treated with chlorhexidine or solutions at pH 3.0, after which their lactic acid production was quantified. Compared to the UF35 and UA159 biofilms, the biomass of UAFR biofilms was two-four fold higher, and the UAFR biofilms were more resistant to chlorhexidine and low pH in terms of lactic acid production. No difference in biomass and lactic acid production was detected between UF35 and UA159 biofilms. The fluoride resistance of UAFR and UF35 strains in biofilms was further confirmed by treating the biofilms with NaF solutions. The level of NaF resistance of the three biofilms is generally ranked as follows: UAFR > UF35 > UA159. In conclusion, there is indeed a fitness consequence in UAFR, but surprisingly, this fluoride-resistant strain performs better than UF35 and UA159 under the described conditions. In addition, UF35 did not display a reduced fitness; it performed as well as the wild-type fluoride-sensitive strain.

Background/purpose Enterococcus faecalis plays a major role in continuing refractory apical periodontitis. The purpose of this study was to investigate the antibacterial effects of sodium hypochlorite (NaOCl) combined with MTAD [a mixture of tetracycline isomer (doxycycline), an acid (citric acid), and a detergent (Tween 80)] as an endosonic irrigation protocol against E. faecalis biofilm. Materials and methods The dentin blocks were incubated with E. faecalis and randomly assigned to five irrigation groups (n = 10): MTAD, 1.3% NaOCl with MTAD, 1.3% NaOCl, 5.3% NaOCl, and 5.3% NaOCl with 17% EDTA using Endoactivator oscillation for 30 seconds and 1 minute, respectively; saline without agitation as a control. The bacterial samples were measured by colony-forming counting. The percentage of viable cells and the thickness of the biofilms were evaluated using confocal laser scanning microscopy. Specimens were observed via scanning electron microscopy. Results When Endoactivator was applied for 30 seconds, viable counting of the 5.3% NaOCl/17% EDTA group and 1.3% NaOCl/MTAD group were lower than the MTAD group (P < 0.05), whereas the differences between the former two are not significant. There were no statistically significant differences in the number of colony-forming counting unit between 30 seconds and 60 seconds (P > 0.05). The 1.3% NaOCl/MTAD group and the MTAD group appeared to preserve the structure of the dentin surface and cause efficient removal of the smear layer. Conclusion Endoactivator could improve the synergistic antimicrobial action of 1.3% NaOCl and MTAD against E. faecalis biofilm and removal of the smear layer with less erosion of the dentin surface. The combination could be a safe and efficient irrigation regimen.

OBJECTIVE To compare the shaping quality in curved canals of two single-file technique systems with other two traditional full-sequential systems. METHODS Eighty mature molar canals with the curvature between 20 and 45 degrees were randomly divided into four groups. Specimens in each group were prepared to size 25 at working length using A (Reciproc), B (OneShape), C (MTwo) and D (Revo S), respectively. Each canal was scanned by micro-computed tomography before and after preparation. Parameters including changes in dentine volume, percentage of uninstrumented area, degree and tendency of transportation were analyzed. The operating time was also recorded. RESULTS In full canal length, there was no difference in canal dentine removal, instrumented percentage and transportation degree among four groups (P > 0.05). In the apical 4 mm region, group A removed more dentine [(2.14±0.76) mm(2) of canal surface area and (0.38 ± 0.15) mm(3) of canal volume] than groups B and C(P < 0.05). At 1 mm level, median of transportation degree of group A was 0.05 (0.03)mm, which was smaller than other groups (P < 0.05). Groups A and B took (86.3±24.6) s and (85.9±21.3) s, while groups C and D took (147.4±28.3) s and (126.3±27.7) s srespectively to finish preparation. Single file techniques were significantly faster than the two full-sequential systems (P < 0.01). CONCLUSIONS Compared with the continuous rotary systems, the reciprocating single-file system A showed better apical shaping ability. Both single-file techniques were more efficient than full-sequential systems for curved canal preparation. Single-file techniques appear to be the effective and efficient method for curved canal preparation.

Objective To investigate the expression of basic fibroblast growth factor (bFGF) in human dental pulp tissues of different state and the bFGF level in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS), and explore the potential role of bFGF in the progress of repairing pulp injury. Methods The messenger RNA and protein level of bFGF in normal and inflammatory pulp were detected by fluorescence quantitative-polymerase chain reaction (qPCR) and western blot. HDPCs were collected after stimulated by 1 mg/L LPS for 6, 12, 24 and 48 h. The expression of bFGF and HSP70 in hDPCs was evaluated by qPCR. In addition, the protein level of bFGF in in normal and LPS treated hDPCs were detected by western blot and immunofluorescence staining. Results The mRNA and protein level of bFGF was significantly up-regulated in the pulp with deep caries compared with healthy pulps, while inflamed dental pulp did not show significant difference. QPCR showed that bFGF and HSP70 mRNA were concomitantly increased from 0 h to 12 h after stimulated by LPS. Similarly, it was shown that bFGF was increased in LPS treated hDPCs compared with normal hDPCs by western blot. Moreover, immunofluorescence staining results demonstrated that bFGF was strongly positive stained in LPS treated hDPCs, while it was weakly expressed in normal hDPCs. Conclusion BFGF is up-regulated in pulp with deep caries and hDPCs induced by LPS, indicating that bFGF may participates in the progress of repairing pulp injury.