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Background: Gremlin1 is a multifunctional protein whose expression is demonstrated to be involved in a series of physiology and pathological processes. The association between Gremlin1 and apcial periodontitis (AP) has been established. M1-polarized macrophages are crucial immune cells that exacerbate the progression of apical periodontal inflammatory response, but the function of Gremlin1 during macrophages activation in periapical lesions is still unclear. This study attempts to explore the regulatory effects of Gremlin1 on macrophage polarization on apical periodontitis microenviroment. Methods: Clinical specimens were used to determine the expression of Gremlin1 in periapical tissues by immunohistochemical (IHC) staining. Then, the disease models of periapical inflammation in rats were established, and adenovirus- associated virus (AAVs) was used to blockade Gremlin1 expression. Lentivirus carrying sh-Gremlin1 particles were used to transfect THP-1 induced M1-subtype macrophages. To assess the expression of associated molecules, Western blot, immunofluorescence staining were performed. Results: Gremlin1 was significantly up-regulated in the periapical tissues of subjects with AP as identified by IHC staining, and positively correlated with levels of M1 macrophage-associated genes. Rats AP model with inhibition of Gremlin1 in periapical lesions exhibited limited infiltration of macrophages and decreased expression of M1 macrophage-related genes in periapical lesions. Furthermore, Gremlin1 blockade substantially decreased the Notch1/Hes1 signaling pathway activation level. The in vitro experiments confirmed the above results. Conclusion: Taken together, current study illustrated that the Gremlin1 suppression in periapical lesions inhibited M1 macrophage polarization through Notch1/Hes1 axis. Moreover, Gremlin1 may act as a potential candidate in the treatment of AP. [ABSTRACT FROM AUTHOR]

Considering the correlation between survival microenvironment of E. faecalis and acidic pH value, this study aimed to investigate the potential of utilizing pH-responsive DMAEM monomers and their copolymers with resin-based root canal sealers to inhibit E. faecalis. Broth microdilution assay, crystal violet staining and qPCR were performed to evaluate antibacterial effects of DMAEM monomers against E. faecalis at different pH. Methacrylate-resin based root canal sealers were prepared and copolymerized with DMAEM. The flow, solubility, water sorption, apical sealing ability and cytotoxicity of sealers were investigated to optimize formulation. The anti -E. faecalis effects of DMAEM copolymers with sealers were evaluated by direct contact test, colony-forming unit counting and live/dead staining. DMAEM monomers inhibited the growth, biofilm formation and virulence factors expression of E. faecalis in a concentration- and pH-dependent manner. Incorporation of 1.25 % and 2.5 % DMAEM into experimental sealers would not affect the flowability, solubility and periapical sealing ability (P > 0.05), but increased the water sorption of sealers (P < 0.01). Cells viability was higher than 90 % in both 1.25 % and 2.5 % DMAEM groups at pH 7.0. DMAEM copolymers with sealers reduced E. faecalis counts, inhibited biofilm formation and decreased live cells within the biofilm in response to pH values. DMAEM monomers and their copolymers with resin-based sealers possessed antibacterial and antibiofilm effects on E. faecalis in response to pH values. DMAEM is promising to inhibit intraradicular E. faecalis in response to its acidic survival environment and maintain low cytotoxicity under neutral conditions, ensuring their biosafety in case of inadvertent entry into periapical tissues. [Display omitted] • DMAEM monomers inhibit E. faecalis in response to acidic pH values. • DMAEM incorporated resin-based root canal sealers achieve good physical properties. • DMAEM incorporated sealers maintain good biocompatibilities at neutral pH. • DMAEM incorporated sealers inhibit E. faecalis in response to acidic pH values. [ABSTRACT FROM AUTHOR]

This study reported on the outcome of the nonsurgical root canal treatment/retreatment of teeth with large apical periodontitis lesions. The influence of some variables on the prognosis was also evaluated. The study included 199 teeth with large apical periodontitis lesions from 184 patients, treated/retreated by a single operator. Most teeth were managed in a single visit using NaOCl irrigation. Cases were followed up periodically from >1 to 8 years. Treatment/retreatment outcome was evaluated by clinical and radiographic/tomographic criteria and categorized as healed, healing, or diseased. For statistical analysis, data were dichotomized in such a way that healing cases were considered as success in a loose criterion or failure in a rigid one. Clinical/radiographic analyses revealed that 67% of the initial treatment cases were classified as healed, 22.5% as healing, and 11% as diseased. Treatment success rates were 89% (loose) and 67% (rigid). Variables that influenced the treatment outcome included previous abscess and antibiotic use, very large lesions (≥10 mm), and a sinus tract. The median follow-up time for treatment was 31.5 months. As for retreatment cases, 47% were healed, 32% were healing, and 21% were diseased. Retreatment success rates were 79% (loose) and 47% (rigid), in a median follow-up of 32 months. Cases evaluated by cone-beam computed tomography had lower healed rates. Findings demonstrate that high favorable outcome rates can be achieved by nonsurgical root canal treatment or retreatment of teeth with large apical periodontitis lesions. [ABSTRACT FROM AUTHOR]

This study characterized the bacteriome in primary endodontic infection (PEI) with apical periodontitis (AP), identified core and rare bacteriome species and community diversity metrics, and analyzed the relationship between the bacteriome composition, diversity and features, and patient variables. Twenty-seven patients with PEI and AP were sampled. The DNA was extracted and quantified using quantitative polymerase chain reaction. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants, and taxonomic assignment of the amplicon sequence variants up to the species level was done against the Human Oral Microbiome Database. Core bacteriome and differential abundance analyses were performed using ANCOM. Alpha diversity was determined using Chao1, Shannon, and Simpson indexes. LeFse analysis was used to identify abundant taxa. Sparse Estimation of Correlations among Microbiomes analysis estimated linear and nonlinear relationships among bacteria. Of 27, 24 root canal samples were analyzed, and 3 root canal sampling were filtered out with a low read count. The bacterial phyla with top mean relative abundance were Bacteroidetes, Firmicutes, Synergistetes, Fusobacteria, and Actinobacteria. A total of 113 genera and 215 species were identified. The samples were gathered into 3 clusters. LefSe analysis identified differences in abundant taxa between distinct age, gender, symptomatology, and lesion size groups. Sparse Estimation of Correlations among Microbiomes distance analysis indicated Slackia exigua as the node with the highest degree. The bacteriome in PEI with AP among the patients in this study was complex and displayed high microbial heterogeneity. Moreover, age, gender, symptomatology, and lesion size were associated with differences in bacteriome features in PEI with AP. [ABSTRACT FROM AUTHOR]

This prospective study evaluated the effect of immediate functional loading (IFL) of a full-coverage prosthesis on the clinical and radiographic outcome of nonsurgical endodontic therapy (NSET) performed on mandibular molar teeth with pulp necrosis and asymptomatic apical periodontitis. In 20 subjects, standardized 2-visit NSET was performed in bilateral mandibular first molar teeth (split-mouth model) with a diagnosis of pulp necrosis and asymptomatic apical periodontitis exhibiting a radiographic periapical index (PAI) score ≥3. In each subject, the nonvital bilateral mandibular first molar teeth were randomized to 1 of 2 groups: the IFL group or the functional loading after a time interval of 6 months group. Provisional full-coverage prostheses were provided within 7 days after the completion of endodontic therapy. The cases were followed up clinically and radiographically at 6 and 12 months. Radiographs were assessed for periapical healing based on PAI scores, which were dichotomized as healed (PAI score ≤2) or nonhealed (PAI score ≥3). The data were compared using chi-square and Fisher exact tests. A recall rate of 100% was achieved at the end of 12 months. All teeth in the IFL group and the functional loading after a time interval of 6 months group were clinically asymptomatic. According to an intergroup comparison between the proportion of teeth healed and nonhealed (radiographic), there was a significant difference (P <.05). IFL of a full-coverage prosthesis in endodontically treated mandibular molar teeth with pulp necrosis and asymptomatic apical periodontitis delayed periapical healing. [ABSTRACT FROM AUTHOR]

Dens invaginatus is a developmental dental anomaly that can predispose the tooth to pulp and periradicular disease. Management of this condition can be challenging because of anatomic and microbiologic issues. This case report describes the regenerative endodontic treatment using a strategic antimicrobial protocol for management of an immature maxillary lateral incisor with type-II dens invaginatus associated with apical periodontitis in a 13-year-old patient. The tooth presented with a complex anatomy and was associated with an active sinus tract. Because the true root canal was not negotiable in its coronal part due to the presence of the dens invaginatus, the closed end of the invagination (pseudocanal) was perforated to permit access to the apical segment of the true root canal for cleaning and disinfection. Both the invagination and the true canal were treated using an antimicrobial regimen based on chemomechanical preparation with sodium hypochlorite irrigation, supplementary disinfection with passive ultrasonic irrigation and interappointment calcium hydroxide medication. After 2 exchanges of calcium hydroxide medication, the sinus tract did not disappear, then the antimicrobial protocol was changed to include an antibiotic solution for irrigation and antibiotic paste for intracanal medication. After signs and symptoms disappeared, regenerative endodontic treatment was performed by inducing blood clot formation within the root canal. The coronal canal segment including the invagination was filled with Biodentine. Follow-up including cone-beam computed tomography examination showed complete healing of the apical periodontitis lesion and mineralized tissue formation at the apical portion of the true root canal. [ABSTRACT FROM AUTHOR]

Chronic apical periodontitis (CAP) is characterized by inflammation and destruction of the apical periodontium that is of pulpal origin, appearing as an apical radiolucent area, and does not produce clinical symptoms. Little is known about whether the PD-1/PD-L1 ratio is associated with the balance between RANKL and OPG in CAP. The relationship between PD-1/PD-L1 and RANKL/OPG in CAP is investigated in this study. A CAP rat model was established using Sprague-Dawley rats. The pulp chambers were exposed to the oral cavity to allow bacterial contamination. The apical tissues of the bilateral mandibular first molars were analyzed for histological morphology using hematoxylin and eosin (H&E) staining. Immunohistochemistry and qRT-PCR were used to determine the expression of PD-1, PD-L1, OPG, and RANKL mRNA and proteins in periapical tissues and mandibular samples, respectively. The radiological images indicated a poorly defined low-density shadow and alveolar bone resorption after periodontitis induction. Histological analysis revealed an infiltration of inflammatory cells and alveolar bone resorption in the periapical tissues. Mandibular mRNA and periapical protein expression of PD-1, PD-L1, and RANKL was upregulated 7–28 days after periodontitis induction, while the expression of OPG was downregulated. No significant relationship was observed between PD-1/PD-L1 and RANKL/OPG at either mRNA or protein levels in CAP. There is an increased expression of PD-1, PD-L1, and RANKL and a decreased expression of OPG, indicating progression of CAP. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the influence of systemic antibiotic therapy on the development and progression of induced apical periodontitis (AP) in Wistar rats. Fifty-six rats were submitted to pulp exposure of the lower left first molar for the induction of AP. On the same day, intraperitoneal antibiotic therapy was administered once a day, for 15 days, until euthanasia. The groups were formed according to the different treatments (n = 8): C—control; GEN—treated with gentamicin (10 mg/Kg); AC—treated with amoxicillin (100 mg/Kg); MZ—treated with metronidazole (40 mg/Kg); AMP—treated with ampicillin (100 mg/Kg); AMC group—treated with amoxicillin + clavulanic acid (100 mg/kg); CLI—treated with clindamycin (60 mg/kg). After euthanasia, the jaws were collected and processed for (1) histological and histometric analysis using hematoxylin and eosin staining, (2) analysis of collagen fibers using Picrosirius Red staining and (3) bacteriological analysis using Brown–Brenn staining. The data were analyzed statistically (p < 0.05). AP induction was confirmed in all groups. The AMC group had the lower intensity of inflammatory infiltrate (p = 0.028) and less periapical bone resorption compared to control (p = 0.006). Regarding collagen maturation, PSR staining revealed a predominance of mature collagen fibers in all groups. The AC and AMC groups had the lower amount of mature fibers and the highest amount of immature fibers, compared to all other groups (p < 0.001). All groups showed bacterial contamination; however, the AC and AMC groups showed a lower extent of bacterial contamination compared to the control (p < 0.001). It can be concluded that systemic antibiotic therapy influences the development and progression of induced AP. [ABSTRACT FROM AUTHOR]

This ex vivo study devised an analytical ex vivo method for infection/disinfection of simulated lateral canals located in the middle and apical segments of the root. The antibacterial effects of supplementary approaches were tested in this model. Extracted mandibular premolars had their main root canals enlarged and then two lateral canals (100 μm in diameter) were created in the root, one in the apical and the other in the middle portion. Micro-computed tomography was used for specimen selection and to confirm the quality of the simulated ramifications. The specimens were contaminated with a mixed bacterial culture from subgingival bacterial biofilm added to pure Enterococcus faecalis strain ATCC 29212 grown overnight, using special strategies to facilitate culture medium penetration within the lateral canals. The following procedures were tested for disinfection: NaOCl/passive ultrasonic irrigation (PUI), NaOCl/XP-endo Finisher, ozonated water/continuous ultrasonic irrigation (CUI), and NaOCl/conventional irrigation with 30-G needles (control). Bacteriological samples were taken from the main canal before (S1) and after (S2) each supplementary protocol, and also from each lateral canal after treatment (S3). DNA extracted from the samples was subjected to quantitative real-time polymerase chain reaction. All S1 main canal samples were positive for bacterial presence. Bacterial counts in the main root canal substantially decreased by 99.2% after PUI, 99.1% after ozone/CUI, 99% after XP-endo Finisher, and 96% in the control group (P < 0.01 for all groups). There were no significant differences between groups (P > 0.05). The same was observed when comparing the effects of the supplementary approaches in the apical and middle lateral canals (P > 0.05). Only a few lateral canals showed no detectable bacteria. The method proposed here proved effective for ex vivo infection/disinfection studies. All supplementary approaches induced a substantial bacterial reduction in the main canal, with no significant differences between them. However, in terms of lateral canal disinfection, none of the tested approaches showed significant effects when compared to the control group. [ABSTRACT FROM AUTHOR]

Aim Methods Results Conclusions The influence of hypercholesterolemia on the development of apical periodontitis (AP) is inconclusive. Recent studies revealed that cholesterol metabolite 27‐hydoxycholesterol (27HC) can affect cellular responses to bacterial infections and oestrogen status and raloxifene may influence its action. Herein, we aimed to examine the impact of 27HC on production of inflammatory mediators by macrophages and the regulatory function of raloxifene. The contribution of 27HC to AP development and the therapeutic effect of raloxifene were evaluated in a rat model.Murine macrophages J774 cells were used. The expression of inducible nitric oxide synthase (iNOS) was examined by Western blot. The concentrations of C‐C motif chemokine ligand (CCL) 2 and 27HC were assessed by enzyme‐linked immunosorbent assay. Colorimetric assay was used to evaluate cholesterol levels. Experimental AP was induced in ovariectomized (OVX) or un‐operated rats receiving high‐fat/high‐cholesterol diet (HFHCD) or normal diet (ND). Micro‐computed tomography and immunohistochemistry were employed to evaluate disease severity and the therapeutic effect of raloxifene.Cholesterol enhanced 27HC production in macrophages. 27HC induced iNOS and CCL2 synthesis by macrophages and estradiol suppressed the responses. In our animal model of AP, HFHCD plus OVX significantly augmented serum and lesion tissue levels of 27HC (p < .05 versus the ND group). Lesion size, infiltration of CD68+ cells, and iNOS+ monocytes were increased in parallel with 27HC accumulation. Raloxifene inhibited pro‐inflammatory effects of 27HC on macrophages and suppressed AP progression in HFHCD/OVX rats (p < .05 versus the vehicle control group).Our results suggested that 27HC contributes to AP aggravation associated with hypercholesterolemia. Oestrogen deficiency may both enhance 27HC production and exacerbate its downstream action. [ABSTRACT FROM AUTHOR]

Aim Methodology Results Conclusions To evaluate M1 and M2 macrophage polarization in radicular cysts and periapical granulomas through an immunohistochemical analysis and the correlation between macrophage polarization and histopathological diagnosis, clinical characteristics and lesion volume using cone‐beam computed tomography.Periapical biopsies diagnosed as radicular cysts (n = 52) and periapical granulomas (n = 51) were analysed by immunohistochemical method. Teeth with periapical lesion with no history of root canal treatment (primary lesion) and lesions persistent to root canal treatment (persistent lesions) were included. Pathological diagnosis, patients' age, gender and clinical characteristics were obtained from treatment records. A cone‐beam computed tomographic periapical volume index (CBCTPAVI) score was assigned to each periapical lesion based on the volume of the lesion. Immuno‐expressions of CD68 and CD163 were quantified. The CD68/CD163 ratio was adopted to represent M1 or M2 macrophage polarization. Mann–Whitney U test was used to determine the different CD68/CD163 ratio between groups of radicular cyst and periapical granuloma. Spearman's correlation test was performed to assess the correlation between the CD68/CD163 ratio and lesion volume and CBCTPAVI score.Radicular cysts and periapical granulomas had CD68/CD163 median of 2.05 (IQR = 1.33) and 1.26 (IQR = 0.81), respectively. A significantly higher CD68/CD163 ratio was observed in radicular cysts (p < .001). In contrast, periapical granulomas had significantly lower median of CD68/CD163 ratio. Larger lesions had a higher median of CD68/CD163 ratio, while smaller lesions had lower median of CD68/CD163 ratio (p = .007, rs = .262). CD68/CD163 ratio was significantly correlated with the CBCTPAVI score in the overall periapical lesions (p = .002, rs = .306). The higher CD68/CD163 ratio in larger lesions indicated a higher degree of M1 polarization compared to smaller lesions. Regarding the pathological diagnosis, there was a significant positive correlation between CBCTPAVI score and CD68/CD163 ratio in periapical granulomas (p < .001, rs = .453), whereas the negative correlation was observed for radicular cysts (p < .001, rs = −.471).Periapical granulomas are characterized by a M2‐dominant macrophage polarization, while radicular cysts have significantly higher M1 macrophages. The higher degree of M1 macrophage polarization was significantly correlated with larger volume and higher CBCTPAVI scores of overall periapical lesion and periapical granuloma. [ABSTRACT FROM AUTHOR]

The aim of the study was to collect data about the prevalence and risk factors of apical periodontitis in a population of endodontically treated patients. The study group included 151 patients (52 males, 99 females; mean age 48.36 ± 15.708 yrs.) with 391 endodontically treated teeth (mean follow-up of 5.25 ± 1.759 yrs.). According to the initial tooth diagnosis, root-filled teeth were divided into Group A, root-filled teeth treated for pulpitis or for the purpose of prosthetic pulpectomies (vital pulp group), and Group B, root-filled teeth with non-vital pulp (necrotic pulp). Clinical and radiographic evaluation of the root and its periapical area were performed to establish the success/failure of endodontic therapy, the quality of the root canal fillings (length, density, taper), and coronal restoration. The presence of recurrent caries, periodontal pathology, or endo-periodontal lesions were also recorded. Univariate and multivariate analyses were used to determine the risk factors for apical periodontitis and calculate their odds ratios (ORs). For the root-filled vital pulp tooth group, the highest risks for apical periodontitis are associated with inadequate homogeneity (OR 30.938), periodontitis (OR 9.226), and over-filling (OR 8.800). For the root-filled non-vital pulp tooth group, the highest risks are associated with periodontitis (OR 4.235) and age over 60 yrs. (OR 4.875). For the necrotic pulp tooth group, multivariate analysis identified an age > 60 yrs., filled molars, intracanal posts, poor coronal restoration quality, under-filling, and periodontitis as significant combined risk factors. Inadequate root canal filling and periodontitis in both groups were risk factors associated with most cases of apical periodontitis. Other risk factors include age > 60 yrs., poor coronal restoration quality, and the presence of intracanal posts in root-filled teeth with necrotic pulp. [ABSTRACT FROM AUTHOR]

Apical periodontitis (AP) is the most prevalent chronic inflammatory disease of the teeth. Bone resorption dynamics in symptomatic and asymptomatic AP are still unrecognized. This study examined different inflammatory markers within gingival crevicular fluid, including matrix metalloproteinases 8 (MMP8), tissue inhibitors of metalloproteinases 1 (TIMP1), receptor activator of nuclear factor κB (RANK), its ligand (RANKL), and osteoprotegerin (OPG), to be used in comparing symptomatic apical periodontitis (SAP) and asymptomatic apical periodontitis (AAP) versus healthy teeth. Subjects with SAP, AAP, and a control group were recruited and GCF samples were collected by Periopaper strips. Clinical and radiographical measures were used for diagnosing AP. Levels of MMP8, TIMP, RANK, RANKL, and OPG were determined by ELISA and their abilities to discriminate between examined sites were evaluated by receiver operator characteristic (ROC) curves. All examined biomarkers were statistically significant higher (p < 0.05) in SAP than AAP and the control group, apart from RANK. Significant positive correlations (p < 0.05) were identified between all SAP and AAP biomarkers except TIMP1 and RANK in AAP teeth. TIMP1 and OPG exhibited the highest ability to distinguish between SAP and AAP with areas under the curve of 0.824 and 0.763 in comparing SAP and the control group, and 0.732 and 0.73 when comparing AAP and the control group, respectively. Additionally, TIMP1 and OPG showed the highest AUC of 0.778 and 0.747 when SAP and AAP were compared, respectively. This study concluded that GCF levels of TIMP1 and OPG can be used to differentiate between SAP, AAP, and healthy teeth. [ABSTRACT FROM AUTHOR]

Aim: To elucidate whether mitochondrial biogenesis disorder and damage from oxidative stress promote refractory apical periodontitis (RAP) in rat and human. Methodology: Twenty Enterococcus faecalis‐induced RAPs were established in the maxillary first molars of male Wistar rats. Concurrently, 12 periapical lesion specimens from patients presenting with RAP were obtained by apicoectomy. Radiographic examination and histologic analysis were conducted to evaluate periapical bone tissue destruction and morphological changes. The expression of key regulators of mitochondrial biogenesis, PGC‐1α and Nrf2, were detected by immunohistochemistry and double immunofluorescence staining, Western blot and real‐time PCR were also assayed. Mitochondrial ROS (mtROS) was identified by MitoSOX staining. Mitochondrial function was detected by the quantification of ATP production, mitochondrial DNA (mtDNA) copy number and activities of mitochondrial respiratory chain complexes. Furthermore, mitochondrial oxidative stress was evaluated by the determination of 3‐nitrotyrosine (3‐NT), 4‐hydroxy‐2‐nonenal (4‐HNE) and 8‐hydroxy‐deoxyguanosine (8‐OHdG) expression levels, as well as malondialdehyde (MDA) expression and antioxidant capacity. Student's t‐test was performed to determine significance between the groups; p <.05 was considered significant. Results: In the maxilla, significantly more bone resorption, greater number of periapical apoptotic cells and Tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells were observed in the RAP group compared with the control group (p <.01). PGC‐1α and Nrf2 were significantly reduced in rat and human RAP lesions compared to the control group (p <.01) at both the mRNA and protein levels. Double immunofluorescence analysis of PGC‐1α or Nrf2 with TOMM20 also indicated that mitochondrial biogenesis was impaired in RAP group (p <.01). Additionally, mitochondrial dysfunction was observed in RAP group, as reflected by increased mtROS, decreased ATP production, reduced mtDNA copy number and complexes of the mitochondrial respiratory chain. Finally, the expression levels of mitochondrial oxidative stress markers, 3‐NT, 4‐HNE and 8‐OHdG, were significantly increased in the RAP group (p <.01). Consistent with this, systemic oxidative damage was also present in the progression of RAP, including increased MDA expression and decreased antioxidant activity (p <.01). Conclusions: Mitochondrial biogenesis disorder and damage from oxidative stress contribute to the development of RAP. [ABSTRACT FROM AUTHOR]