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.

Traumatic dental injuries in young individuals are often exposed to the invasion of oral microorganisms that leads to pulp necrosis. Infective necrosis in permanent teeth not-fully-developed causes aberrant root formation. Regeneration endodontic treatments (RETs) have shown promising results by promoting continued root development by stem cells. Critical to the success of RET is the thorough disinfection of the pulpal space. To establish effective antimicrobial protocols for root canal disinfection, the invading microorganisms need to be identified. In the present study, we use a combination of culture-based and high-throughput molecular sequencing techniques to investigate the microbial profiles from traumatized teeth (30 cases) and controls, i.e., teeth with pulp infections not caused by trauma (32 cases). Overall, a high microbial diversity in traumatized necrotic teeth was observed. Eubacterium yurii subsps. yurii and margaretiae, as well as key ‘bridging oral species’ F. nucleatum sp., Polymorphum and Corynebacterium matruchotti, were highly associated with traumatized teeth. The microbial compositions of traumatized teeth differed considerably from those of infected teeth not caused by trauma. Age and tooth position also influence microbial compositions. In conclusion, we show that the root canal microflora of traumatized teeth is highly diverse, and it differs from root canal infections not caused by trauma.

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.

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.

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. 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). 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. 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.

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.