Retreatability of NeoSEALER Flo obturated with warm vertical compaction versus single-cone technique using two different retreatment systems.

Aim: The aim of this study was to compare the retreatability of NeoSEALER Flo obturated with warm vertical compaction (WVC) and single-cone (SC) techniques using two different retreatment systems. Materials and Methods: Thirty-two root canals were shaped and obturated with NeoSEALER Flo either in an SC obturation technique or a WVC technique. Samples were retreated using ProTaper retreatment or EdgeFile XR retreatment system. The percentage of remaining debris after retreatment was analyzed under a scanning electron microscope using ImageJ software. The time taken to reach full working length (WL) and induce patency was recorded. Statistical Analysis: Statistical analysis was performed using an unpaired t-test and a one-way analysis of variance test. Results: The percentage of remaining debris after retreatment was significantly higher in the SC technique than in the WVC technique, regardless of the retreatment system used. EdgeFile XR system removed more filling material than the ProTaper retreatment system, regardless of the obturation technique. The apical region showed significantly higher remaining debris than other regions in all groups. The WL and patency were achieved faster in the SC group, while in the WVC group, the EdgeFile XR system was faster. Conclusions: The WVC technique showed better retrieval of the filling material; however, a longer time was taken for retreatment. EdgeFile XR system performed better in removing filling materials from inside the canals.

Comparative biological properties of resin-free and resin-based calcium silicate-based endodontic repair materials on human periodontal ligament stem cells.

OBJECTIVES: To investigate the effect of three different calcium silicate-based materials (CSBM) on the biological behavior of human periodontal ligament stem cells (hPDLSCs). METHODS: Eluates of Biodentine, NeoPutty and TheraCal PT prepared at 1:1, 1:2, and 1:4 ratios were extracted under sterile conditions. The cytotoxicity of the extracts to the hPDLSCs was assessed using the MTT assay. Scratch wound healing assay was utilized for assessing cell migration. Scanning electron microscopy was used to detect cell attachment and morphology. Calcium ion release was measured using inductively coupled plasma-optical emission spectrometry; the pH-value was evaluated with a pH-meter. ANOVA with post hoc Tukey test was used for statistical analysis. RESULTS: Cell viability was significantly higher for Biodentine and NeoPutty at day 1 with all dilutions (p < 0.05), while at day 3 and day 7 with dilutions 1:2 and 1:4; all materials showed similar behavior (p > 0.05). Biodentine had the highest percentage of cell migration into the scratched area at day 1 for all dilutions (p < 0.05). Stem cells were attached favorably on Biodentine and NeoPutty with evident spreading, and intercellular communications; however, this was not shown for TheraCal PT. Biodentine showed the highest pH values and calcium ion release (p < 0.05). CONCLUSIONS: The resin-free CSBM showed better performance and favorable biological effects on hPDLSCs and were therefore considered promising for usage as endodontic repair materials. CLINICAL SIGNIFICANCE: Proper selection of materials with favorable impact on the host stem cells is crucial to ensure outcome in different clinical scenarios.

Enterococcus faecalis in secondary apical periodontitis: Mechanisms of bacterial survival and disease persistence.

Enterococcus faecalis is a commensal bacterium commonly found in the human gastrointestinal tract. However, in individuals with compromised immune systems, the pathogen can lead to severe illness. This opportunistic pathogen is associated with secondary apical diseases and is adept at resisting antibiotics and other forms of treatment because of its numerous virulence factors. Enterococcus faecalis is capable of disrupting the normal functions of immune cells, thereby hindering the body's ability to eradicate the infection. However, intensive research is needed in further understanding the adverse immunomodulatory effects of E. faecalis. Potential strategies specific for eradicating E. faecalis have proven beneficial in the treatment of persistent secondary apical periodontitis.

Effect of rapamycin on human periodontal ligament stem cells that have been exposed to sodium hypochlorite.

AIMS: The present study investigated the effect of rapamycin on the viability and osteogenic differentiation potential of human periodontal ligament stem cells (hPDLSCs) in the presence of sodium hypochlorite (NaOCl). MAIN METHODS: After determining the minimum inhibitory concentration of NaOCl and optimum concentration of rapamycin, the viability of hPDLSCs was evaluated using the MTT assay subsequent to their exposure to NaOCl, rapamycin, or a combination of both. Osteogenic differentiation was evaluated by the cell mineralization assay performed by alizarin red S staining, alkaline phosphatase activity, and monitoring the expression of osteogenic genes markers Runt-related transcription factor 2, osteocalcin, and osteoprotegerin, using real-time quantitative polymerase chain reaction (RT-qPCR). The expression of autophagy-related genes PI3K, Akt, and mTOR, was also analyzed with RT-qPCR. KEY FINDINGS: Stem cells treated with rapamycin showed the highest percentage of viable cells in the presence of NaOCl. The same trend was observed for all osteogenic differentiation assays. The hPDLSCs treated with rapamycin demonstrated the highest calcium nodule deposition, alkaline phosphatase activity, and the expression of osteogenic gene markers. These effects were not adversely affected by the presence of NaOCl. Rapamycin significantly inhibited mTOR gene expression, while there were no differences in the gene expression of PI3K and Akt. SIGNIFICANCE: Rapamycin counteracts the cytotoxic effect of NaOCl by enhancing the viability and osteogenic differentiation potential of hPDLSCs. Rapamycin appears to accomplish these processes via autophagy activation, by inhibiting mTOR gene expression. The incorporation of rapamycin in regenerative endodontic therapy may encourage a higher success rate.

A microcomputed tomographic analysis of the morphological variabilities and incidence of extra canals in mandibular first molar teeth in an Egyptian subpopulation.

A well-protected microbial habitat may be present in the root and canal morphology, which is varied and complicated. Before initiating effective root canal treatment, a detailed knowledge of the root and canal anatomical variances in each tooth is a must. This study aimed to investigate the root canal configuration, apical constriction anatomy, location of the apical foramen, dentine thickness, and prevalence of accessory canals in mandibular molar teeth in an Egyptian subpopulation using micro-computed tomography (microCT). A total of 96 mandibular first molars were scanned using microCT, and 3D reconstruction was performed using Mimics software. The root canal configurations of each of the mesial and distal root were classified with two different classification systems. The prevalence and dentin thickness around middle mesial and middle distal canals were investigated. The number, location and anatomy of major apical foramina and the apical constriction anatomy analysed. The number and location of accessory canals were identified. Our findings showed that two separate canals (15%) and one single canal (65%) were the most common configuration in the mesial and distal roots, respectively. More than half of the mesial roots had complex canal configurations and 51% had middle mesial canals. The single apical constriction anatomy was the most common for both canals followed by the parallel anatomy. Disto-lingual and distal locations of the apical foramen are the most common location for both roots. Mandibular molars in Egyptians show a wide range of variations in root canal anatomy with high prevalence of middle mesial canals. Clinicians should be aware of such anatomical variations for successful root canal treatment procedures. A specific access refinement protocol and appropriate shaping parameters should be designated for each case to fulfil the mechanical and biological objectives of root canal treatment without compromising the longevity of treated teeth.

Effect of Different Sealers on the Cytocompatibility and Osteogenic Potential of Human Periodontal Ligament Stem Cells: An In Vitro Study.

BACKGROUND: There is tendency for unavoidable sealer extrusion in some clinical cases. This might adversely affect host stem cells and affect healing. This study aimed to investigate the effect of different sealers on the cytocompatibility and osteogenic potential of human periodontal ligament stem cells (hPDLSCs). METHODS: The cytotoxic effect of the extracted elutes of VDW.1Seal (VDW.1), Endosequence BC Sealer HiFlow (ES), GuttaFlow-2 (GF), and ADSeal (AD-S) on the hPDLSCs was determined using the MTT assay. Cell proliferation and migration were assessed by the scratch wound healing assay. Osteogenic differentiation potential was assessed. Measurement of pH values and calcium ions release was performed. RESULTS: GF had a significantly higher percentage of viable cells. The cell migration assay showed that GF demonstrated the lowest open wound area percentage. GF and AD-S showed the highest calcium nodule deposition. GF demonstrated higher ALP activity than ES. Expression of RUNX2 and OC genes was similar for all sealers, while OPG gene expression was significantly higher for VDW.1 and GF. ES and AD-S displayed the highest pH values on day 1. Calcium ion release of ES and VDW.1 was significantly the highest. CONCLUSIONS: GuttaFlow-2 and VDW.1Seal sealers have favorable behavior toward host stem cells.

Microbially-Induced Exosomes from Dendritic Cells Promote Paracrine Immune Senescence: Novel Mechanism of Bone Degenerative Disease in Mice.

As the aging population grows, chronic age-related bone degenerative diseases become more prevalent and severe. One such disease, periodontitis (PD), rises to 70.1% prevalence in Americans 65 years and older. PD has been linked to increased risk of other age-related diseases with more serious mortality and morbidity profiles such as Alzheimer's disease and cardiovascular disease, but the cellular and biological mechanisms remain unclear. Recent in vitro studies from our group indicate that murine dendritic cells (DCs) and T cells are vulnerable to immune senescence. This occurs through a distinct process involving invasion of DCs by dysbiotic pathogen Porphyromonas gingivalis (Pg) activating the senescence associated secretory phenotype (SASP). Exosomes of the Pg-induced SASP transmit senescence to normal bystander DC and T cells, ablating antigen presentation. The biological significance of these findings in vivo and the mechanisms involved were examined in the present study using young (4-5mo) or old (22-24mo) mice subjected to ligature-induced PD, with or without dysbiotic oral pathogen and injection of Pg-induced DC exosomes. Senescence profiling of gingiva and draining lymph nodes (LN) corroborates role of advanced age and PD in elevation of senescence biomarkers beta galactosidase (SA-ß-Gal), p16 INK4A p21Waf1/Clip1, IL6, TNFα, and IL1ß, with attendant increase in alveolar bone loss, reversed by senolytic agent rapamycin. Immunophenotyping of gingiva and LN revealed that myeloid CD11c+ DCs and T cells are particularly vulnerable to senescence in vivo under these conditions. Moreover, Pg-induced DC exosomes were the most potent inducers of alveolar bone loss and immune senescence, and capable of overcoming senescence resistance of LN T cells in young mice. We conclude that immune senescence, compounded by advanced age, and accelerated by oral dysbiosis and its induced SASP exosomes, plays a pivotal role in the pathophysiology of experimental periodontitis.

Proteomic Characterization, Biodistribution, and Functional Studies of Immune-Therapeutic Exosomes: Implications for Inflammatory Lung Diseases.

Dendritic cell (DC)-derived exosomes (DC EXO), natural nanoparticles of endosomal origin, are under intense scrutiny in clinical trials for various inflammatory diseases. DC EXO are eobiotic, meaning they are well-tolerated by the host; moreover, they can be custom-tailored for immune-regulatory or -stimulatory functions, thus presenting attractive opportunities for immune therapy. Previously we documented the efficacy of immunoregulatory DCs EXO (regDCs EXO) as immunotherapy for inflammatory bone disease, in an in-vivo model. We showed a key role for encapsulated TGFß1 in promoting a bone sparing immune response. However, the on- and off-target effects of these therapeutic regDC EXO and how target signaling in acceptor cells is activated is unclear. In the present report, therapeutic regDC EXO were analyzed by high throughput proteomics, with non-therapeutic EXO from immature DCs and mature DCs as controls, to identify shared and distinct proteins and potential off-target proteins, as corroborated by immunoblot. The predominant expression in regDC EXO of immunoregulatory proteins as well as proteins involved in trafficking from the circulation to peripheral tissues, cell surface binding, and transmigration, prompted us to investigate how these DC EXO are biodistributed to major organs after intravenous injection. Live animal imaging showed preferential accumulation of regDCs EXO in the lungs, followed by spleen and liver tissue. In addition, TGFß1 in regDCs EXO sustained downstream signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFß1 signaling require initial interaction of regDCs EXO with TGFß1R followed by internalization of regDCs EXO with TGFß1-TGFß1R complex. Finally, these regDCs EXO that contain immunoregulatory cargo and showed biodistribution to lungs could downregulate the main severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target receptor, ACE2 on recipient lung parenchymal cells via TGFß1 in-vitro. In conclusion, these results in mice may have important immunotherapeutic implications for lung inflammatory disorders.

Exacerbation of AMD Phenotype in Lasered CNV Murine Model by Dysbiotic Oral Pathogens.

Emerging evidence underscores an association between age-related macular degeneration (AMD) and periodontal disease (PD), yet the biological basis of this linkage and the specific role of oral dysbiosis caused by PD in AMD pathophysiology remains unclear. Furthermore, a simple reproducible model that emulates characteristics of both AMD and PD has been lacking. Hence, we established a novel AMD+PD murine model to decipher the potential role of oral infection (ligature-enhanced) with the keystone periodontal pathogen Porphyromonas gingivalis, in the progression of neovasculogenesis in a laser-induced choroidal-neovascularization (Li-CNV) mouse retina. By a combination of fundus photography, optical coherence tomography, and fluorescein angiography, we documented inflammatory drusen-like lesions, reduced retinal thickness, and increased vascular leakage in AMD+PD mice retinae. H&E further confirmed a significant reduction of retinal thickness and subretinal drusen-like deposits. Immunofluorescence microscopy revealed significant induction of choroidal/retinal vasculogenesis in AMD+PD mice. qPCR identified increased expression of oxidative-stress, angiogenesis, pro-inflammatory mediators, whereas antioxidants and anti-inflammatory genes in AMD+PD mice retinae were notably decreased. Through qPCR, we detected Pg and its fimbrial 16s-RrNA gene expression in the AMD+PD mice retinae. To sum-up, this is the first in vivo study signifying a role of periodontal infection in augmentation of AMD phenotype, with the aid of a pioneering AMD+PD murine model established in our laboratory.

Enterococcus faecalis shifts macrophage polarization toward M1-like phenotype with an altered cytokine profile.

Background: The macrophage is an innate immune defense cell involved in pathogen recognition and clearance. Aim: In view of the diversity of the macrophage phenotype and function, the present study investigated how Enterococcus faecalis infection affects the differentiation, phenotype and cytokine profile of macrophages. Methods: Murine bone marrow-derived stem cells were co-cultured with E. faecalis before and after differentiation. Macrophage M0 polarization towards M1 or M2 was initiated at day 6 by addition of LPS and INF-γ, or IL-4 and IL-13, respectively. Results: E. faecalis did not inhibit macrophage differentiation and were identified within macrophages. Viability of the macrophages infected with E. faecalis prior to differentiation was enhanced, evidenced by apoptosis inhibition, as was expression of CD38 and IRF5 proteins, indicators of M1-like polarization. These M1-like macrophages expressed an aberrant cytokine mRNA profile, with reduction in inflammatory cytokines IL-1ß and IL-12 and increase in regulatory cytokine IL-10. No changes in TNF-α or TGF-ß1 were detected, compared with the control groups. This atypical M1-like phenotype was retained even upon stimulation with growth factors that normally trigger their development into M2 macrophages. Conclusions: These findings suggested that E. faecalis infection of bone marrow-derived stem cells during differentiation into macrophages induces an atypical M1-like phenotype associated with intracellular bacterial survival.

Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo.

Chronic bone degenerative diseases represent a major threat to the health and well-being of the population, particularly those with advanced age. This study isolated exosomes (EXO), natural nano-particles, from dendritic cells, the "directors" of the immune response, to examine the immunobiology of DC EXO in mice, and their ability to reprogram immune cells responsible for experimental alveolar bone loss in vivo. Distinct DC EXO subtypes including immune-regulatory (regDC EXO), loaded with TGFB1 and IL10 after purification, along with immune stimulatory (stimDC EXO) and immune "null" immature (iDCs EXO) unmodified after purification, were delivered via I.V. route or locally into the soft tissues overlying the alveolar bone. Locally administrated regDC EXO showed high affinity for inflamed sites, and were taken up by both DCs and T cells in situ. RegDC EXO-encapsulated immunoregulatory cargo (TGFB1 and IL10) was protected from proteolytic degradation. Moreover, maturation of recipient DCs and induction of Th17 effectors was suppressed by regDC EXO, while T-regulatory cell recruitment was promoted, resulting in inhibition of bone resorptive cytokines and reduction in osteoclastic bone loss. This work is the first demonstration of DC exosome-based therapy for a degenerative alveolar bone disease and provides the basis for a novel treatment strategy.

Enterococcus faecalis Induces Differentiation of Immune-Aberrant Dendritic Cells from Murine Bone Marrow-Derived Stem Cells.

Enterococcus faecalis, long implicated in serious systemic infections and failure of root canal treatment, is a persistent inhabitant of oral periapical lesions. Dendritic cells (DCs) and other innate immune cells patrol the oral mucosa for infecting microbes. Dendritic cells are efficient at capturing microbes when immature, whereupon they can transform into potent antigen-presenting cells upon full maturation. Autophagy, a sophisticated intracellular process first described for elimination of damaged organelles, regulates DC maturation and other important immune functions of DCs. The present study examined how E. faecalis influences the differentiation of murine bone marrow-derived stem cells (BMSCs) into functional DCs in the presence of the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Although the viability and differentiation of DCs were not affected by E. faecalis, expression of the autophagy-related proteins ATG7, Beclin1, and LC3bI/II were significantly suppressed in an mTOR-dependent manner. Ultrastructurally, E. faecalis was identified in single-membrane vacuoles, some of which were in the process of binary fission. Bacterium-containing autophagosomes were absent within the cytoplasm. Accessory molecules (major histocompatibility complex class II [MHC-II], CD80, and CD86) and anti-inflammatory cytokine (transforming growth factor ß1 [TGF-ß1]) were suppressed in E. faecalis-induced DCs, while IL-1ß, tumor necrosis factor alpha (TNF-α), and IL-12 levels were upregulated. When pulsed with ovalbumin (OVA), the E. faecalis-induced DCs showed reduction in CD4+ OVA-specific OT-II T cell proliferation. It is concluded that E. faecalis promotes the differentiation of bone marrow stem cells into CD11c-positive DCs with aberrant immune functions while retaining the capability of proinflammatory cytokine induction.

Comparison of Shaping Ability of Different Single-File Systems Using Microcomputed Tomography.

OBJECTIVES: To evaluate the shaping ability of four different single-file systems using micro-computed tomography (micro-CT). MATERIALS AND METHODS: Eighty mesiobuccal and mesiolingual canals of permanent mandibular molars were randomly assigned to four groups according to the file used; WaveOne Gold, RECIPROC Blue, HyFlex EDM, and One Shape (n = 20). The samples were scanned using micro-CT for the preinstrumentation record; then the canals were instrumented and scanned for the postinstrumentation records. STATISTICAL ANALYSIS: The data were then analyzed statistically using one-way analysis of variance (ANOVA) with the level of significance set at α = 0.05. RESULTS: The results of canal centering ratio and canal transportation showed that no significant difference was found among the four file groups at the middle and the coronal levels. However, a significant difference was found at the apical level (p < 0.05), where WaveOne Gold showed the best centering ratio and the least amount of canal transportation. The change in the canal volume was significant at the coronal level (p < 0.05), whereas the change in the canal volume was higher for the HyFlex EDM group than the other groups. No significant difference was found between the other groups at the apical level, middle level, or in the total percentage of canal volume. CONCLUSION: WaveOne Gold produced a more centralized canal preparation with a lesser amount of transportation at the apical level. HyFlex EDM showed the highest canal volumetric change at the coronal level. The interim design of the endodontic files has a high impact on the behavior of the file inside the root canal.