Extracellular host DNA contributes to pathogenic biofilm formation during periodontitis.

Introduction: Periodontal diseases are known to be associated with polymicrobial biofilms and inflammasome activation. A deeper understanding of the subgingival cytological (micro) landscape, the role of extracellular DNA (eDNA) during periodontitis, and contribution of the host immune eDNA to inflammasome persistence, may improve our understanding of the mechanisms underlaying severe forms of periodontitis. Methods: In this work, subgingival biolfilms developing on biologically neutral polyethylene terephthalate films placed in gingival cavities of patients with chronic periodontitis were investigated by confocal laser scanning microscopy (CLSM). This allowed examination of realistic cytological landscapes and visualization of extracellular polymeric substances (EPS) including amyloids, total proteins, carbohydrates and eDNA, as well as comparison with several single-strain in vitro model biofilms produced by oral pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus gordonii, S. sanguinis and S. mitis. Fluorescence in situ hybridization (FISH) analysis was also used to identify eDNA derived from eubacteria, streptococci and members of the Bacteroides-Porphyromonas-Prevotella (BPP) group associated with periodontitis. Results: Analysis of subgingival biofilm EPS revealed low levels of amyloids and high levels of eDNA which appears to be the main matrix component. However, bacterial eDNA contributed less than a third of the total eDNA observed, suggesting that host-derived eDNA released in neutrophil extracellular traps may be of more importance in the development of biofilms causing periodontitis. Discussion: eDNA derived from host immunocompetent cells activated at the onset of periodontitis may therefore be a major driver of bacterial persistence and pathogenesis.

In Vitro Biofilm Removal From Human Enamel Using a Philips® Sonicare® Power Flosser.

The objective of this in vitro study was to quantify the removal of dental biofilm from human enamel surfaces after treatment with the Philips® Sonicare® Power Flosser. Dental biofilms were grown from pooled human saliva on human enamel disks for 4 days, according to an established academic model.* The biofilms (n = 6) were treated with the Philips Sonicare Power Flosser for 3 seconds using the Quad Stream nozzle. To quantify the number of bacteria before treatment, the biofilm volume was measured using optical coherence tomography (OCT) and the bacterial cell density was determined from untreated control samples (n = 6) using confocal laser scanning microscopy (CLSM). After treatment the number of remaining bacteria were counted using CLSM. Additionally, scanning electron microscope (SEM) images were recorded. While before treatment 0.2-mm thick dense biofilms were present, after treatment only scattered groups of bacteria remained (Figure 1 through Figure 4). Quantitative analysis showed 99.96% removal for the Quad Stream nozzle. The Philips Sonicare Power Flosser oral irrigator with Quad Stream nozzle removed over 99.9% of the bacteria in this established laboratory model of dental biofilm.

Bacterial sealing ability of calcium silicate-based sealer for endodontic surgery: an in-vitro study.

BACKGROUND: Apical surgery with standard retrograde maneuvers may be challenging in certain cases. Simplifying apical surgery to reduce operating time and streamline retrograde manipulation is an emerging need in clinical endodontics. AIM OF THE STUDY: The aim of the study was to compare the bacterial sealing ability of a calcium silicate-based sealer with the single cone technique combined with root end resection only, and calcium silicate-based sealer as a retrograde filling versus MTA retrofilling, and to analyze bacterial viability using confocal laser scanning microscope (CLSM). MATERIALS AND METHODS: In this in vitro experimental study, 50 extracted human maxillary incisor teeth were instrumented and randomly divided into five groups: three experimental groups, a positive control group, and a negative control group (n = 10/group). In the experimental groups, the roots were obturated using the single cone technique (SCT) and a calcium silicate-based sealer. In group 1, the roots were resected 3 mm from the apex with no further retrograde preparation or filling. In groups 2 and 3, the roots were resected, retroprepared, and retrofilled with either a calcium silicate-based sealer or MTA, respectively. Group 4 (positive control) was filled with a single gutta-percha cone without any sealer. In group 5 (negative control), the canals were left empty, and the roots were sealed with wax and nail varnish. A bacterial leakage model using Enterococcus faecalis was employed to assess the sealing ability over a 30-day period, checking for turbidity and analyzing colony forming units (CFUs) per milliliter. Five specimens from each group were examined using CLSM for bacterial viability. Data for the bacterial sealing ability were statistically analyzed using chi-squared and Kruskal-Wallis tests. RESULTS: The three experimental groups did not show significant differences in terms of bacterial leakage, or bacterial counts (CFUs) (P > 0.05). However, significant differences were observed when comparing the experimental groups to the positive control group. Notably, the calcium silicate-based sealer, when used as a retrofilling, yielded the best sealing ability. CLSM imaging revealed viable bacterial penetration in all the positive control group specimens while for the experimental groups, dead bacteria was the prominent feature seen. CONCLUSION: Within the limitations of this study, it could be concluded that the bacterial sealing ability of calcium silicate-based sealer with the single cone technique combined with root end resection only and calcium silicate-based sealer as a retrograde filling were comparable with MTA retrofilling during endodontic surgical procedures.

A new method of adhesive system application improves the bond strength between fiber post and root dentin.

This study evaluated a new method of adhesive system application on the bond strength between fiber post and root dentin using two adhesive systems. The canals of sixty bovine incisors were prepared and obturated. The roots were divided into six groups (n=10) according to the adhesive system (Clearfil SE - CSE and Single Bond Universal - SBU) and the application strategy (microbrush - MB; rotary brush - RB; and ultrasonic tip - US). The glass fiber posts were cemented with resin cement (RelyX ARC). The roots were sectioned perpendicularly to their long axis, and three slices per root were obtained. Previously to the push-out test, confocal laser scanning microscopy (CLSM) was performed to illustrate the interfacial adaptation of the cement to the root canal walls. Failure patterns were analyzed with 40x magnification. Shapiro-Wilk indicated a normal distribution of the data. The bond strength values were compared using one-way ANOVA and Tukey's tests. Student's T test analyzed the differences between the adhesive systems within each third and protocol. A significance level of 5% was used. CSE with RB showed higher mean bond strength values compared to MB (conventional technique) (P < 0.05). US application resulted in intermediate bond strength values for CSE (P > 0.05). The application of SBU using RB generated higher mean bond strength values compared to MB and US (P < 0.05). Adhesive failures were predominant (65.5%). CSE and SBU application with the new rotary brush improved the bond strength of fiber posts to root dentin compared to the conventional strategy.

Antibacterial ability of different activated irrigation after root canal preparation: intratubular analyses.

This study assessed the intratubular antibacterial ability of different activated irrigations after chemical mechanical preparation. Seventy-two palatal root canals of upper molars were infected with Enterococcus faecalis for 4 weeks, and then initial bacterial collection from the main root canal was performed. The root canals were prepared by using a WaveOne Gold large (45/.05) and distributed into 6 groups according to the activation of the final irrigation: ultrasonic activation (UA), XP-Endo Finisher (25/.00), XP Clean (25/.02), EasyClean (25/.04) in reciprocating motion and continuous rotary motion (ECRot), and conventional irrigation. After final irrigation, another bacterial collection from the main root canal was performed, and the root was sectioned transversely in three-thirds and stained for analysis by confocal laser microscopy. Intratubular bacteria were collected through dentin powder and plated for bacterial viability analysis. Intergroup and intragroup comparisons were performed by using analysis of variance and repeated measures analysis of variance, respectively, both at 5% significance. ECRot had higher antibacterial ability than UA (p<0.05), and both were superior to the other groups (p<0.05) in both methodologies. It can be concluded that activation of final irrigation enhances the disinfection of the root canal system, and activators have different efficacies.

Neuromuscular Junction Development Differs Between Extraocular and Skeletal Muscles and Between Different Extraocular Muscles.

Purpose: To determine whether development of neuromuscular junctions (NMJs) differs between extraocular muscles (EOMs) and other skeletal muscles. Methods: Mouse EOMs, diaphragm, and tibialis anterior (TA) were collected at postnatal day (P)0, P3, P7, P10, P14, and P21, and 12 weeks. Whole muscles were stained with α-bungarotoxin, anti-neurofilament antibody, and slow or fast myosin heavy chain antibody, and imaged with a confocal microscope. Images were quantified using Imaris software. Results: NMJs in the EOMs show a unique pattern of morphological development compared to diaphragm and TA. At P0, diaphragm and TA NMJs were oval plaques; EOM single NMJs were long, thin rods. NMJs in the three muscle types progress to mature morphology at different rates. At all ages, EOM single NMJs were larger, especially relative to myofiber size. The inferior oblique and inferior rectus muscles show delayed single NMJ development compared to other EOMs. NMJs on multiply-innervated fibers in the EOMs vary widely in size, and there were no consistent differences between muscles or over time. Incoming motor nerves formed complex branching patterns, dividing first into superficial and deep branches, each of which branched extensively over the full width of the muscle. Motor axons that innervate multiply-innervated fibers entered the muscle with the axons that innervate singly-innervated fibers, then extended both proximally and distally. EOM NMJs had more subsynaptic nuclei than skeletal muscle NMJs throughout development. Conclusions: EOMs show a unique pattern of NMJ development and have more subsynaptic nuclei than other muscles, which may contribute to the exquisite control of eye movements.

Intrathoracic non-tuberculous mycobacteriosis with endobronchial lesion in a child aged 11 with HIV infection diagnosed by bronchoscopic biopsy, EBUS-TBNA and confocal laser endomicroscopy.

The diagnosis of intrathoracic non-tuberculous mycobacteriosis (NTM) is challenging. We report a case of a pediatric pulmonary NTM with endobronchial lesion and lymphadenitis in a child with HIV infection diagnosed by bronchoscopic biopsy, EBUS-TBNA and probe-based confocal laser endomicroscopy (pCLE). The pCLE showed a large number of highly fluorescent cells and zones of density and disorganized elastin fibers at alveolar areas. A combination of diagnostic endoscopic procedures is required to establish the diagnosis of NTM.

In vitro biofilm formation of Gardnerella vaginalis and Escherichia coli associated with bacterial vaginosis and aerobic vaginitis.

Objective: To determine the optimum biofilm formation ratio of Gardnerella vaginalis (G. vaginalis) in a mixed culture with Escherichia coli (E. coli). Methods: G. vaginalis ATCC14018, E. coli ATCC25922, as well as five strains of G. vaginalis were selected from the vaginal sources of patients whose biofilm forming capacity was determined by the Crystal Violet method. The biofilm forming capacity of E. coli in anaerobic and non-anaerobic environments were compared using the identical assay. The Crystal Violet method was also used to determine the biofilm forming capacity of a co-culture of G. vaginalis and E. coli in different ratios. After Live/Dead staining, biofilm thickness was measured using confocal laser scanning microscopy, and biofilm morphology was observed by scanning electron microscopy. Results: The biofilm forming capacity of E. coli under anaerobic environment was similar to that in a 5% CO2 environment. The biofilm forming capacity of G. vaginalis and E. coli was stronger at 106:105 CFU/mL than at other ratios (P<0.05). Their thicknesses were greater at 106:105 CFU/mL than at the other ratios, with the exception of 106:102 CFU/mL (P<0.05), under laser scanning microscopy. Scanning electron microscopy revealed increased biofilm formation at 106:105 CFU/mL and 106:102 CFU/mL, but no discernible E. coli was observed at 106:102 CFU/mL. Conclusion: G. vaginalis and E. coli showed the greatest biofilm forming capacity at a concentration of 106:105 CFU/mL at 48 hours and could be used to simulate a mixed infection of bacterial vaginosis and aerobic vaginitis in vitro.

Quantification of C. neoformans Capsule Diameter.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

Spatial variations in the osteocyte lacuno-canalicular network density and analysis of the connectomic parameters.

Osteocyte lacuno-canalicular network (LCN) is comprised of micrometre-sized pores and submicrometric wide channels in bone. Accumulating evidence suggests multiple functions of this network in material transportation, mechanobiological signalling, mineral homeostasis and bone remodelling. Combining rhodamine staining and confocal laser scanning microscopy, the longitudinal cross-sections of six mouse tibiae were imaged, and the connectome of the network was quantified with a focus on the spatial heterogeneities of network density, connectivity and length of canaliculi. In-vivo loading and double calcein labelling on these tibiae allowed differentiating the newly formed bone from the pre-existing regions. The canalicular density of the murine cortical bone varied between 0.174 and 0.243 µm/µm3, and therefore is three times larger than the corresponding value for human femoral midshaft osteons. The spatial heterogeneity of the network was found distinctly more pronounced across the cortex than along the cortex. We found that in regions with a dense network, the LCN conserves its largely tree-like character, but increases the density by including shorter canaliculi. The current study on healthy mice should serve as a motivating starting point to study the connectome of genetically modified mice, including models of bone diseases and of reduced mechanoresponse.

Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network.

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

Focused Ultrasound as a Novel Non-Invasive Method for the Delivery of Gold Nanoparticles to Retinal Ganglion Cells.

Purpose: The blood-retinal barrier (BRB) restricts the delivery of intravenous therapeutics to the retina, necessitating innovative approaches for treating retinal disorders. This study sought to explore the potential of focused ultrasound (FUS) to non-invasively deliver intravenously administered gold nanoparticles (AuNPs) across the BRB. FUS-BRB modulation can offer a novel method for targeted retinal therapy. Methods: AuNPs of different sizes and shapes were characterized, and FUS parameters were optimized to permeate the BRB without causing retinal damage in a rodent model. The delivery of 70-kDa dextran and AuNPs to the retinal ganglion cell (RGC) layer was visualized using confocal and two-photon microscopy, respectively. Histological and statistical analyses were conducted to assess the effectiveness and safety of the procedure. Results: FUS-BRB modulation resulted in the delivery of dextran and AuNPs to the RGC and inner nuclear layer. Smaller AuNPs reached the retinal layers to a greater extent than larger ones. The delivery of dextran and AuNPs across the BRB with FUS was achieved without significant retinal damage. Conclusions: This investigation provides the first evidence, to our knowledge, of FUS-mediated AuNP delivery across the BRB, establishing a foundation for a targeted and non-invasive approach to retinal treatment. The results contribute to developing promising non-invasive therapeutic strategies in ophthalmology to treat retinal diseases. Translational Relevance: Modifying the BRB with ultrasound offers a targeted and non-invasive delivery strategy of intravenous therapeutics to the retina.

Assessment of sealing efficacy, radiopacity, and surface topography of a bioinspired polymer for perforation repair.

Background: Root perforation repair presents a significant challenge in dentistry due to inherent limitations of existing materials. This study explored the potential of a novel polydopamine-based composite as a root repair material by evaluating its sealing efficacy, radiopacity, and surface topography. Methods: Confocal microscopy assessed sealing ability, comparing the polydopamine-based composite to the gold standard, mineral trioxide aggregate (MTA). Radiopacity was evaluated using the aluminium step wedge technique conforming to ISO standards. Surface roughness analysis utilized atomic force microscopy (AFM), while field emission scanning electron microscopy (FESEM) visualized morphology. Results: The polydopamine-based composite exhibited significantly superior sealing efficacy compared to MTA (P < 0.001). Radiopacity reached 3 mm aluminium equivalent, exceeding minimum clinical requirements. AFM analysis revealed a smooth surface topography, and FESEM confirmed successful composite synthesis. Conclusion: This study demonstrates promising properties of the polydopamine-based composite for root perforation repair, including superior sealing efficacy, clinically relevant radiopacity, and smooth surface topography. Further investigation is warranted to assess its clinical viability and potential translation to endodontic practice.

A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis.

Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during T cell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.

Advancements in Neurosurgical Intraoperative Histology.

Despite their relatively low incidence globally, central nervous system (CNS) tumors remain amongst the most lethal cancers, with only a few other malignancies surpassing them in 5-year mortality rates. Treatment decisions for brain tumors heavily rely on histopathological analysis, particularly intraoperatively, to guide surgical interventions and optimize patient outcomes. Frozen sectioning has emerged as a vital intraoperative technique, allowing for highly accurate, rapid analysis of tissue samples, although it poses challenges regarding interpretive errors and tissue distortion. Raman histology, based on Raman spectroscopy, has shown great promise in providing label-free, molecular information for accurate intraoperative diagnosis, aiding in tumor resection and the identification of neurodegenerative disease. Techniques including Stimulated Raman Scattering (SRS), Coherent Anti-Stokes Raman Scattering (CARS), Surface-Enhanced Raman Scattering (SERS), and Tip-Enhanced Raman Scattering (TERS) have profoundly enhanced the speed and resolution of Raman imaging. Similarly, Confocal Laser Endomicroscopy (CLE) allows for real-time imaging and the rapid intraoperative histologic evaluation of specimens. While CLE is primarily utilized in gastrointestinal procedures, its application in neurosurgery is promising, particularly in the context of gliomas and meningiomas. This review focuses on discussing the immense progress in intraoperative histology within neurosurgery and provides insight into the impact of these advancements on enhancing patient outcomes.

Diagnostic utility of in vivo confocal microscopy in Acanthamoeba keratitis following corneal crosslinking.

Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening complication of corneal collagen crosslinking (CXL) for keratoconus. In this report, we describe an early adolescent male who underwent routine CXL for progressive keratoconus in his left eye. Preprocedural left visual acuity (VA) was 6/9. At day 5 postprocedure, multifocal corneal infiltrates were identified. Corneal scrape, bandage contact lens cultures and herpetic and Acanthamoeba PCR were negative. In vivo, confocal microscopy (IVCM) identified Acanthamoeba cysts within the corneal stroma. Intensive amoebicidal therapy was initiated, but recovery was complicated by significant inflammation, resulting in widespread aggressive corneal vascularisation necessitating topical steroids and steroid-sparing agents. At 10 months, his left VA was 6/24. This report emphasises the importance of maintaining a high index of suspicion for AK in cases of post-CXL microbial keratitis and highlights the diagnostic value of IVCM, particularly in culture-negative and PCR-negative cases.