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.

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.

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.

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.

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.

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.