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Aim: This case report explored the application of next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) to visualize and characterize microbial populations in a refractory endodontic infection with apical calculus-like deposits, a rarely reported phenomenon.

Summary: Histopathologic analysis revealed bacterial biofilms and calcifications on the root surface, with Gram-positive bacteria predominant in both hard and soft tissues. Microbial sequencing showed Pseudomonadota dominated hard tissues, whereas Bacillota were prevalent in soft tissues, with distinct genera like Lactibacterium and Streptococcus identified. FISH imaging confirmed spatially distributed bacterial taxa, including Actinomycetota and Chloroflexota, within the biofilm, aligning with NGS findings. Notably, Bacteroidota was exclusive to soft tissues, whereas Chloroflexota was detected only in hard tissues. The presence of extensive calculus-like deposits on the root surface provided new insights into the microbial complexity of persistent endodontic infections and their management.

Key learning points:

1. The combination of NGS and FISH provided unprecedented insights into the microbial composition of refractory endodontic infections, revealing a diverse and spatially organized ecosystem.
2. Distinct microbial compositions in hard and soft tissues emphasize the importance of targeted therapeutic strategies for endodontic infections.
3. The presence of unique bacterial taxa and biofilms in calculus-like deposits offers new avenues for research into the pathogenesis and persistence of endodontic infections.

Oral health in immature permanent teeth with traumatic injuries is particularly vulnerable, and regenerative endodontic treatment (RET) using stem cells from the apical papilla (SCAP) holds potential for root development and tissue regeneration. However, bacterial persistence, especially Enterococcus faecalis, poses a challenge to successful treatment outcomes. To address this, we evaluated the probiotic Lactobacillus gasseri for its co-aggregative and anti-adhesive properties against E. faecalis. An in vitro aggregation test demonstrated effective co-aggregation between the probiotic and opportunistic strains. Additionally, flow cytometry analysis revealed that E. faecalis binding to SCAP was significantly reduced when the L. gasseri concentration was nine times higher. To substantiate these findings, an in vivo Drosophila melanogaster gut model was used, where immunofluorescence imaging and culture-based methods confirmed decreased E. faecalis adhesion at both 1:1 and 9:1 probiotic-to-opportunistic ratios. These results highlight L. gasseri B16 as a promising probiotic strain to improve RET outcomes.

Introduction: Regenerative endodontic treatment (RET) aims to promote root maturation in necrotic immature teeth, where effective microbial disinfection is crucial for treatment success. This study evaluated the effect of calcium hydroxide (CH) and 2% chlorhexidine gluconate (CHD) as intracanal medicaments and their impact on bacterial loads and RET outcomes.

Methods: The material consisted of bacterial samples from 41 patients who participated in a previously conducted randomized controlled clinical trial comparing CH and CHD during RET. A total of 123 microbial samples were analyzed using real-time quantitative polymerase chain reaction (qPCR). Bacterial loads were assessed at three time points: before root canal disinfection (S1), after root canal disinfection (S2), and after intracanal dressing (S3). The microbial composition was evaluated at the kingdom (Eubacteria), phylum (Actinomycetota), and species (Enterococcus faecalis) levels.

Results: Significant reductions in bacterial loads were observed after root canal disinfection (S2) in both CH and CHD subgroups, regardless of treatment outcome. Further reductions after intracanal dressing (S3) occurred exclusively in the successful cases. Actinomycetota loads significantly decreased after root canal disinfection in the successful cases but remained unchanged after intracanal medication. The presence of E. faecalis after intracanal dressing was associated with failed RET (OR = 9.778; p = 0.0432), although no significant differences in the effectiveness of the intracanal medicaments were found.

Conclusion: Both CH and CHD effectively reduced bacterial loads, with greater reductions linked to successful outcomes. The association between E. faecalis and failed RET suggests that this species may play a role in treatment outcomes. Further research, including microbiome profiling, is desirable to identify potential prognostic markers for failed RET.

Stem cells from the apical papilla (SCAP) are essential for regenerative endodontic treatment. Although mineral trioxide aggregate (MTA) and Biodentine are widely used in regenerative endodontic treatment procedures, their effects on SCAP remain unclear. This study investigated the impact of ProRoot MTA and Biodentine on SCAP viability and mineralization in the presence of Enterococcus faecalis under aerobic and anaerobic environments. Stem cells from the apical papilla were isolated from three healthy donors and exposed to three different surface area-to-volume (SA:V) ratio extracts of ProRoot MTA and Biodentine for 21 days in aerobic or anaerobic conditions. Cell viability was assessed using a neutral red cytotoxicity assay, and mineralization was evaluated by measuring alkaline phosphatase (ALP) activity. No significant differences between ProRoot MTA and Biodentine regarding SCAP viability were detected; however, increased cytotoxicity was found (for both ProRoot MTA and Biodentine) at the highest SA:V ratio of extract used. Oxygen availability, as well as variability in responses of SCAP from the different donors, resulted in greater variation of ALP levels than did type of material. Both ProRoot MTA and Biodentine showed comparable effects on SCAP viability and mineralization, with high SA:V ratios of extracts resulting in increased cytotoxicity. Mineralization in SCAP is influenced by oxygen conditions and the presence of E. faecalis, elucidating the need for further in vivo studies to optimize regenerative endodontic treatment outcomes.

Aim: To compare differences in the disinfection efficacy of calcium hydroxide (CH) and chlorhexidine gluconate (CHD) dressings in pulp revitalization (PR) of traumatized immature necrotic teeth; to investigate the microflora in successful/failed PR and whether bacterial persistence influences the outcomes of PR.

Methods: Microbiological assessment of the average bacterial load (CFU/sample) and bacterial diversity (taxa/sample) was performed on 41 teeth at three timepoints (S2-before, S3-after debridement and S5- after root canal dressing).

Results: The primary microflora was more diverse in successful cases than in failed. Decreases in CFU/sample and taxa/sample occurred S2 - S3, though new increases occurred at S5 in the CHD subgroup (successful and failed) and CFU/sample in the CH subgroup (failed). At S5, the successful cases showed more bacterial decreases. No specific species was associated with the outcomes with no statistical differences between the disinfection efficacy.

Conclusions: There were no statistical differences in CH and CHD efficacy. At S5, microflora persisted in both successful and failed outcomes, but the abundance and diversity increased significantly only in the failed cases. The successful outcomes presented higher diversity and higher decreases of the primary microflora at S5 than the failed outcomes. The abundance and diversity increased significantly at S5 only in failed cases.

Introduction: Bacterial persistence is considered one of the main causal factors for regenerative endodontic treatment (RET) failure in immature permanent teeth. This interference is claimed to be caused by the interaction of bacteria that reside in the root canal with the stem cells that are one of the essentials for RET. The aim of the study was to investigate whether prolonged exposure of stem cells from the apical papilla (SCAP) to bacterial remnants of Fusobacterium nucleatum, Actinomyces gerensceriae, Slackia exigua, Enterococcus faecalis, Peptostreptococcaceae yurii, commonly found in infected traumatized root canals, and the probiotic bacteria Lactobacillus gasseri and Limosilactobacillus reuteri, can alter SCAP’s inflammatory response and mineralization potential.

Methods: To assess the effect of bacterial remnants on SCAP, we used UV-C–inactivated bacteria (as cell wall-associated virulence factors) and bacterial DNA. Histochemical staining using Osteoimage Mineralization Assay and Alizarin Red analysis was performed to study SCAP mineralization, while inflammatory and osteo/odontogenic-related responses of SCAPs were assessed with Multiplex ELISA.

Results: We showed that mineralization promotion was greater with UV C–inactivated bacteria compared to bacterial DNA. Immunofluorescence analysis detected that the early mineralization marker alkaline phosphatase (ALP) was increased by the level of E. coli lipopolysaccharide (LPS) positive control in the case of UV-C–inactivated bacteria; meanwhile, DNA treatment decreased the level of ALP compared to the positive control. SCAP’s secretome assessed with Multiplex ELISA showed the upregulation of pro-inflammatory factors IL-6, IL-8, GM-CSF, IL-1b, neurotrophic factor BDNF, and angiogenic factor VEGF, induced by UV-C–killed bacteria.

Discussion: The results suggest that long term stimulation (for 21 days) of SCAP with UV-C–inactivated bacteria stimulate their mineralization and inflammatory response, while DNA influence has no such effect, which opens up new ideas about the nature of RET failure.

Stem cells from the apical papilla (SCAP) are a promising resource for use in regenerative endodontic treatment (RET) that may be adversely affected by oral bacteria, which in turn can exert an effect on the success of RET. Our work aims to study the cytokine profile of SCAP upon exposure to oral bacteria and their supernatants—Fusobacterium nucleatum and Enterococcus faecalis—as well as to establish their effect on the osteogenic and immunogenic potentials of SCAP. Further, we target the presence of key proteins of the Wnt/β-Catenin, TGF-β, and NF-κB signaling pathways, which play a crucial role in adult osteogenic differentiation of mesenchymal stem cells, using the Western blot (WB) technique. The membrane-based sandwich immunoassay and transcriptomic analysis showed that, under the influence of F. nucleatum (both bacteria and supernatant), the production of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 occurred, which was also confirmed at the mRNA level. Conversely, E. faecalis reduced the secretion of the aforementioned cytokines at both mRNA and protein levels. WB analysis showed that SCAP co-cultivation with E. faecalis led to a decrease in the level of the key proteins of the Wnt/β-Catenin and NF-κB signaling pathways: β-Catenin (p = 0.0068 *), LRP-5 (p = 0.0059 **), and LRP-6 (p = 0.0329 *), as well as NF-kB (p = 0.0034 **) and TRAF6 (p = 0.0285 *). These results suggest that oral bacteria can up-and downregulate the immune and inflammatory responses of SCAP, as well as influence the osteogenic potential of SCAP, which may negatively regulate the success of RET.

Aim: This prospective cohort study evaluates clinical and radiographical outcomes of endodontic pulp revitalization (PR) of traumatized necrotic incisors.

Methodology: Pulp revitalization was performed in 75 traumatized necrotic immature incisors from 71 patients. The radiographic outcome measures were continued root formation (width and length), root resorption, apex closure, periapical index, and root development stage. The clinical outcome measures were percussion pain, palpation pain, pathological tooth mobility, swelling, sinus tract, ankylosis, crown discolouration, response to pulp sensitivity test, and subjective pain. Treatment outcomes were categorized as a success based on the absence of clinical symptoms and when radiographic evidence was present for apical healing and continued root development. The performed statistical tests were repeated measures anova, pairwise comparisons of interactions (t-test), McNemar's test, and linear regression model.

Results: In 45 of 75 teeth (60%), PR was successful with the resolution of clinical and radiographic signs and continued root development. PR failed due to the absence of bleeding (n = 19) and persistent infection (n = 11). PR showed statistically significant increases in root length (11%), and dentinal wall thickness (30%), root maturation (pre-operative 3.38 [CI 1.88; 4.88]; post-operative 4.04, [CI 2.56; 5.52]) apical closure (71.4%), healing of pre-operative apical periodontitis (100%), and healing of pre-operative inflammatory root resorptions (100%). Three predictive variables for continued root maturation were identified – root development stage at entry (p =.0001, β 0.649), [CI 0.431; 0.867], trauma to the soft tissues (p =.026, β −0.012), [CI −0.0225; −0.015], and pre-operative dentinal wall thickness (p =.009, β −0.001); [CI −0.001; 0.0001].

Conclusions: Our findings indicate that PR provides satisfactory clinical and radiographical outcomes in traumatized necrotic incisors. The failed cases were related to lack of bleeding and persistent infections, indicating that new techniques are needed to improve the predictability of PR.

Interaction of oral bacteria with stem cells from the apical papilla (SCAP) can negatively affect the success of regenerative endodontic treatment (RET). Through RNA-seq transcriptomic analysis, we studied the effect of the oral bacteria Fusobacterium nucleatum and Enterococcus faecalis, as well as their supernatants enriched by bacterial metabolites, on the osteo- and dentinogenic potential of SCAPs in vitro. We performed bulk RNA-seq, on the basis of which differential expression analysis (DEG) and gene ontology enrichment analysis (GO) were performed. DEG analysis showed that E. faecalis supernatant had the greatest effect on SCAPs, whereas F. nucleatum supernatant had the least effect (Tanimoto coefficient = 0.05). GO term enrichment analysis indicated that F. nucleatum upregulates the immune and inflammatory response of SCAPs, and E. faecalis suppresses cell proliferation and cell division processes. SCAP transcriptome profiles showed that under the influence of E. faecalis the upregulation of VEGFA, Runx2, and TBX3 genes occurred, which may negatively affect the SCAP’s osteo- and odontogenic differentiation. F. nucleatum downregulates the expression of WDR5 and TBX2 and upregulates the expression of TBX3 and NFIL3 in SCAPs, the upregulation of which may be detrimental for SCAPs’ differentiation potential. In conclusion, the present study shows that in vitro, F. nucleatum, E. faecalis, and their metabolites are capable of up- or downregulating the expression of genes that are necessary for dentinogenic and osteogenic processes to varying degrees, which eventually may result in unsuccessful RET outcomes. Transposition to the clinical context merits some reservations, which should be approached with caution.

Objective: To evaluate the impact of cone-beam computed tomography (CBCT) in endodontic therapeutic decision-making of immature traumatized teeth with suspected pulp necrosis.

Methods: Over two years, consecutive patients with a dental trauma in their front teeth (apex >0.5 mm) and with suspected pulp necrosis based on clinical and radiographic findings were referred to a specialist clinic in Sweden. Fifteen patients aged 6-13 (18 teeth) were included and clinically examined by an endodontist. Intraoral radiographs and CBCT examinations were obtained. Five practitioners, three endodontists and two residents in endodontics, used these examinations to determine the most appropriate treatment for the 18 cases (all central incisors) on two occasions scheduled 19 weeks apart. On the first occasion, the practitioners had access to clinical information and the intraoral radiographs ('before' CBCT); on the second occasion, the practitioners had also access to a radiologist report and the CBCT images ('after' CBCT). Their treatment plans - no treatment, watchful waiting, endodontic orthograde treatment, or extraction - were made anonymously and independently. Results were analysed using descriptive statistics and Wilcoxon signed-rank test.

Results: 'After' CBCT, practitioners changed treatment plans in 30% of the 90 assessments, 74% of which were more aggressive (p = 0.028). In 49% of the assessments, practitioners who chose the watchful and waiting treatment plan 'before' CBCT changed to a more aggressive therapy such as endodontic orthograde treatment and extraction 'after' CBCT (p = 0.005).

Conclusion: This study provides evidence that CBCT influences endodontic therapeutic decision-making regarding immature traumatised teeth with suspected pulp necrosis, chiefly when expectant management (i.e., watchful and waiting) was selected before access to CBCT.