Microbiological assessment of success and failure in pulp revitalization: a randomized clinical trial using calcium hydroxide and chlorhexidine gluconate in traumatized immature necrotic teeth.

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.

Exploring the impact of oral bacteria remnants on stem cells from the Apical papilla: mineralization potential and inflammatory response.

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.

Transcriptome Analysis Reveals Modulation of Human Stem Cells from the Apical Papilla by Species Associated with Dental Root Canal Infection.

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.

Combined Transcriptomic and Protein Array Cytokine Profiling of Human Stem Cells from Dental Apical Papilla Modulated by Oral Bacteria.

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/ß-Catenin, TGF-ß, 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/ß-Catenin and NF-κB signaling pathways: ß-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.

Endodontic pulp revitalization in traumatized necrotic immature permanent incisors: Early failures and long-term outcomes-A longitudinal cohort study.

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, ß 0.649), [CI 0.431; 0.867], trauma to the soft tissues (p = .026, ß -0.012), [CI -0.0225; -0.015], and pre-operative dentinal wall thickness (p = .009, ß -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.

New Insights into the Microbial Profiles of Infected Root Canals in Traumatized Teeth.

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.

Cytokine Secretion, Viability, and Real-Time Proliferation of Apical-Papilla Stem Cells Upon Exposure to Oral Bacteria.

The use of stem cells from the apical papilla (SCAPs) has been proposed as a means of promoting root maturation in permanent immature teeth, and plays a significant role in regenerative dental procedures. However, the role of SCAPs may be compromised by microenvironmental factors, such as hypoxic conditions and the presence of bacteria from infected dental root canals. We aim to investigate oral bacterial modulation of SCAP in terms of binding capacity using flow cytometry and imaging, real-time cell proliferation monitoring, and cytokine secretion (IL-6, IL-8, and TGF-ß isoforms) under anaerobic conditions. SCAPs were exposed to key species in dental root canal infection, namely Actinomyces gerensceriae, Slackia exigua, Fusobacterium nucleatum, and Enterococcus faecalis, as well as two probiotic strains, Lactobacillus gasseri strain B6 and Lactobacillus reuteri (DSM 17938). We found that A. gerensceriae, S. exigua, F. nucleatum, and E. faecalis, but not the Lactobacillus probiotic strains bind to SCAPs on anaerobic conditions. Enterococcus faecalis and F. nucleatum exhibited the strongest binding capacity, resulting in significantly reduced SCAP proliferation. Notably, F. nucleatum, but not E. faecalis, induce production of the proinflammatory chemokine IL-8 and IL-10 from SCAPs. Production of TGF-ß1 and TGF-ß2 by SCAPs was dependent on species, cell line, and time, but secretion of TGF-ß3 did not vary significantly over time. In conclusion, SCAP response is compromised when exposed to bacterial stimuli from infected dental root canals in anaerobic conditions. Thus, stem cell-mediated endodontic regenerative studies need to include microenvironmental conditions, such as the presence of microorganisms to promote further advantage in the field.

Host Determinants of Expression of the Helicobacter pylori BabA Adhesin.

Expression of the Helicobacter pylori blood group antigen binding adhesin A (BabA) is more common in strains isolated from patients with peptic ulcer disease or gastric cancer, rather than asymptomatic colonization. Here we used mouse models to examine host determinants that affect H. pylori BabA expression. BabA expression was lost by phase variation as frequently in WT mice as in RAG2-/- mice that do not have functional B or T cells, and in MyD88-/-, TLR2-/- and TLR4-/- mice that are defective in toll like receptor signaling. The presence of other bacteria had no effect on BabA expression as shown by infection of germ free mice. Moreover, loss of BabA expression was not dependent on Leb expression or the capacity of BabA to bind Leb. Surprisingly, gender was the host determinant most associated with loss of BabA expression, which was maintained to a greater extent in male mice and was associated with greater bacterial load. These results suggest the possibility that loss of BabA expression is not driven by adaptive immunity or toll-like receptor signaling, and that BabA may have other, unrecognized functions in addition to serving as an adhesin that binds Leb.

Plasminogen is a critical regulator of cutaneous wound healing.

Wound healing is a complicated biological process that consist of partially overlapping inflammatory, proliferation and tissue remodelling phases. A successful wound healing depends on a proper activation and subsequent termination of the inflammatory phase. The failure to terminate the inflammation halts the completion of wound healing and is a known reason for formation of chronic wounds. Previous studies have shown that wound closure is delayed in plasminogen-deficient mice, and a role for plasminogen in dissection of extracellular matrix was suggested. However, our finding that plasminogen is transported to the wound by inflammatory cells early during the healing process, where it potentiates inflammation, indicates that plasminogen may also have other roles in the wound healing process. Here we report that plasminogen-deficient mice have extensive fibrin and neutrophil depositions in the wounded area long after re-epithelialisation, indicating inefficient debridement and chronic inflammation. Delayed formation of granulation tissue suggests that fibroblast function is impaired in the absence of plasminogen. Therefore, in addition to its role in the activation of inflammation, plasminogen is also crucial for subsequent steps, including resolution of inflammation and activation of the proliferation phase. Importantly, supplementation of plasminogen-deficient mice with human plasminogen leads to a restored healing process that is comparable to that in wild-type mice. Besides of being an activator of the inflammatory phase during wound healing, plasminogen is also required for the subsequent termination of inflammation. Based on these results, we propose that plasminogen may be an important future therapeutic agent for wound treatment.