Confocal Microscopy Evaluation Research Articles

Bone cell differentiation and mineralization in wild-type and osteogenesis imperfecta zebrafish are compromised by per- and poly-fluoroalkyl substances (PFAS)

Perfluorinated compounds (PFAS) are well recognized toxic pollutants for humans, but if their effect is equally harmful for healthy and fragile people is unknown. Addressing this question represents a need for ensuring global health and wellbeing to all individuals in a world facing the progressive increase of aging and aging related diseases. This study aimed to evaluate the impact of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorohexanoic acid (PFHxA) exposure on development and skeletal phenotype using the osteogenesis imperfecta (OI) zebrafish model Chihuahua (Chi/+), carrying a dominant glycine substitution in the α1 chain of collagen I and their wild-type (WT) littermates. To this purpose Chi/+ and WT zebrafish expressing the green fluorescent protein under the early osteoblast marker osterix were exposed from 1 to 6 days post fertilization to 0.36, 1.5 and 3.0 mg/L PFOS, 0.005 and 0.5 mg/L PFOA and 0.01, 0.48 and 16.0 mg/L PFHxA, and their development and skeletal phenotype investigated. Morphometric measurements, confocal microscopy evaluation of operculum area delimited by the fluorescent preosteoblasts and mineral deposition analysis following alizarin red staining were employed. PFOS and the highest concentration of PFHxA significantly impaired standard length in both genotypes. Osteoblast differentiation was significantly compromised by PFOS and by PFOA only in Chi/+. Limited to WT exposed to PFOA a reduced mineralization was also observed. No effect was detected after PFHxA exposure. Apoptosis was only activated by PFOA, specifically in Chi/+ mutant operculum osteoblasts. Interestingly, an altered lipid distribution in both WT and mutant fish was revealed after exposure to both pollutants. In conclusion, our data demonstrate that PFAS impair operculum development mainly compromising cell differentiation in mutant fish whereas alter lipid hepatic distribution in both genotypes with a more severe effect on Chi/+ preosteoblast survival. These results represent a first warning sign of the negative impact of PFAS exposure in presence of genetically determined skeletal fragility.

Reflectance Confocal Microscopy Can Help in Detecting Clinically Silent Mammary Paget's Disease.

Early detection and comprehensive diagnostic approaches for breast cancer are essential for improving prognosis. When it comes to changes in the skin of the breast or the nipple-areola complex (NAC), particularly if they are unilateral, it is essential to be vigilant, as these changes could be an early sign of underlying malignancy or other pathologies. Primary breast malignancies, such as mammary Paget's disease (MPD), can manifest as erythema, scaling, or ulceration of the NAC, while secondary cutaneous metastases from other breast carcinomas may present as nodules, erythematous plaques, or inflammatory reactions. Non-malignant inflammatory conditions, including eczema or mastitis, can also mimic these changes; histologic evaluation is the gold-standard diagnostic tool. The usefulness of conventional diagnostic techniques breast lesions has been confirmed, but in recent years, reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) have emerged as additional tools to diagnose cases characterized by cutaneous changes; they may, therefore, result in new perspectives on the non-invasive diagnosis of MPD. RCM is a non-invasive diagnostic technique that allows high-resolution images of the skin at microscopic level in real time, offering a promising approach to the non-invasive diagnosis of MPD, particularly when a lesion is not clinically evident and may mimic other benign or inflammatory conditions. We describe an atypical clinical presentation of mammary Paget's disease diagnosed early by reflectance confocal microscopy evaluation and confirmed histologically.

Comparative evaluation of continuous and sequential chelation on the dentinal tubule penetration of bioceramic-based sealer - A confocal laser scanning microscopic study.

Effective endodontic treatment relies heavily on proper instrumentation, thorough disinfection, and precise three-dimensional obturation of the root canal system. This study aims to evaluate and compare continuous chelation (etidronic acid and sodium hypochlorite [NaOCl]) and sequential chelation (citric acid and NaOCl) on the dentinal tubule penetration of bioceramic-based sealer (Bio-C)-A confocal laser scanning microscopic study. In vitro experimental study. Sixty extracted permanent single-rooted teeth were selected and decoronated below cementoenamel junction to get a standardized length of 12 mm across all samples followed by enlargement of root canals till rotary ProTaper F3. The samples were randomly divided into three groups: Group I (control): Canals were irrigated with 3 mL 17% ethylenediaminetetraacetic acid/3 mL 3% NaOCl; Group II: Canals were irrigated with 10 mL etidronic acid + NaOCl solution; and Group III: Canals were irrigated with 3 mL 10% citric acid followed by 3 mL 3% NaOCl. 0.1% rhodamine B dye was mixed with Bio-C sealer followed by obturation. All the samples were subjected to confocal laser scanning microscopy evaluation. One-way analysis of variance with the Bonferroni post hoc test was used for the statistical analysis. The level of significance was set at P < 0.05. The pairwise comparison of depth of penetration showed statistically significant results in all the coronal, middle, and apical thirds. Group II showed better depth of penetration than Groups I and III. Continuous chelation protocol using etidronic acid and NaOCl showed greater and statistically significant sealer penetration depth when compared to the sequential chelation protocol, i.e., citric acid at all three levels.

53728 Reflectance confocal microscopy and clinical evaluation of a product containing Silybum marianum fruit extract (SMFE) in monotherapy for acne vulgaris treatment: a prospective study

53728 Reflectance confocal microscopy and clinical evaluation of a product containing Silybum marianum fruit extract (SMFE) in monotherapy for acne vulgaris treatment: a prospective study

Reflectance confocal microscopy and clinical evaluation of a product containing Silybum marianum fruit extract in monotherapy for acne vulgaris treatment: A prospective study

AbstractBackgroundAcne is a common pilosebaceous unit chronic inflammatory disease, with a multifactorial pathogenesis. It involves several processes: increased sebum production, alteration of the follicular keratinization process and bacterial colonization by Cutibacterium acnes.ObjectivesTo evaluate the effectiveness of reflectance confocal microscopy (RCM) in evaluating the reduction of mild to severe acne lesions in patients treated with an investigational product containing Silybum marianum fruit extract (SMFE).MethodsSubjects previously examined for counting acne lesions were topically treated in the face, with an investigational product containing SMFE in monotherapy over the course of 60 days (n = 31). They were examined for counting acne lesions. Efficacy and quality of life, tolerability, and safety assessments were performed. The infundibular diameter (ID) measurement of normal and acne skin areas was analyzed using RCM.ResultsThe RCM data statistical analysis allowed us to identify two ID patterns, 300–750 µm, with a reduction of 33.33% (p &lt; 0.05) and 900–1050 µm with 73.16% (p &lt; 0.05) reduction after 60 days. The noninflammatory lesions had a mean variation of −44.51% and −67.82% after 30 and 60 days, respectively (t test: p &lt; 0.05 and p &lt; 0.001 after Bonferroni's correction). The inflammatory lesions had a mean variation of −69.31% and −79.40% after 30 and 60 days (t test: p &lt; 0.05 and p = 0.118 after Bonferroni's correction).ConclusionsThe authors demonstrated the effectiveness of the monotherapy scheme with an investigational product containing SMFE through the statistically significant results obtained by the RCM in acneic and nonacneic skin, in addition to the reduction in the number of noninflammatory at the end of the 60 days of clinical follow‐up. Furthermore, the monotherapy scheme with the investigational product proved safe, with no serious adverse events during the entire clinical study. In summary, the use of RCM proved to be effective in assessing the therapeutic response in patients with moderate to severe acne.

Evaluation of Penetration Depth and Caries Progression using Resin Infiltrant in Natural White Spot Lesions of Primary Molars.

Management of white spot lesions (WSLs) using resin infiltrants, like ICON®, is a novel technique that emphasizes the concept of minimally invasive dentistry. Evaluate penetration depth and its effect on caries progression of ICON® in natural WSLs of primary molars. A total of 30 human primary molars with natural WSLs were selected. A total of 15 samples each were randomly selected to evaluate penetration depth and caries progression. To evaluate penetration depth, lesion surfaces were sectioned into two halves, wherein one half served as control, while the other half received ICON® infiltration treatment. To evaluate the effect on caries progression, samples were sectioned into two halves, wherein one half served as a control to its experimental counterpart, which received ICON® infiltration. The initial depth of the subsurface lesion (baseline data) was measured, and all the sample subsets underwent pH cycling. Confocal laser fluorescence microscope (CFLM) evaluation was performed, and penetration depth was assessed using a laser scanning microscopy (LSM) image reader, while caries progression was evaluated using PhotoScape® software. ICON® resin infiltrant penetrated to significant depths into the WSLs (p < 0.001) and demonstrated significant inhibition of caries progression (p < 0.001). The novel technique using ICON® could be an invaluable tool in the management of WSLs in primary teeth. Babu G, Mallikarjun S, Vijayan V, et al. Evaluation of Penetration Depth and Caries Progression using Resin Infiltrant in Natural White Spot Lesions of Primary Molars. Int J Clin Pediatr Dent 2024;17(4):390-394.

Formulation development of methotrexate lipid-based nanogel for treatment of skin cancer

Skin cancer (SC) represents a prevalent malignancy worldwide, characterized by its high drug resistance and limited therapeutic efficacy with conventional treatments. Nanoparticles have emerged as promising vehicles for targeted drug delivery, aiming to overcome these challenges and enhance treatment outcomes. The solid lipid nanocarriers (SLNs) provide improved drug absorption, encapsulation, and controlled release. These SLNs can be customized to meet unique needs, allowing for enhanced targeting, distribution to specific sites, and profiles of sustained release. This study was focused on the development and optimization of methotrexate loaded solid lipid nanoparticles (MTX-SLNs) for improving the drug delivery to the skin cancer therapy. The optimized MTX-SLNs exhibited a particle size of 203 nm, a zeta potential of −8.93 mV, entrapment efficiency of 87%, and drug loading of 62.92%. Subsequently, the optimized MTX-SLNs was incorporated into a gel system containing 1% Carbopol 934, which was further underwent comprehensive characterization, encompassing analysis of rheological properties, texture profile analysis, extrudability and spreadability, as well as uniformity of content. Notably, the dermatokinetic and confocal microscopy evaluations revealed excellent uptake and deposition of MTX-SLNs within the epidermal and dermal layers of the skin. These observations were complemented by the computation of the area under the curve (AUC0–8hr), which demonstrated values of 245.035 and 702.59 for the epidermal and dermal layers, respectively. These findings underscore the tremendous potential of our MTX-SLNs gel formulation as a promising candidate for skin cancer treatment compared to the conventional topical gel therapies, exhibiting superior topical pharmacokinetics, nanometric size, high drug loading capacity, and enhanced efficacy.

Secretome of bone marrow mesenchymal stromal cells cultured in a dynamic system induces neuroprotection and modulates microglial responsiveness in an α-synuclein overexpression rat model

Background aimsParkinson’s disease (PD) is the second most common neurodegenerative disorder. The etiology of the disease remains largely unknown, but evidence have suggested that the overexpression and aggregation of alpha-synuclein (α-syn) play key roles in the pathogenesis and progression of PD. Mesenchymal stromal cells (MSCs) have been earning attention in this field, mainly due to their paracrine capacity. The bioactive molecules secreted by MSCs, i.e. their secretome, have been associated with enhanced neuronal survival as well as a strong modulatory capacity of the microenvironments where the disease develops. The selection of the appropriate animal model is crucial in studies of efficacy assessment. Given the involvement of α-syn in the pathogenesis of PD, the evidence generated from the use of animal models that develop a pathologic phenotype due to the action of this protein is extremely valuable. Therefore, in this work, we established an animal model based on the viral vector-mediated overexpression of A53T α-syn and studied the impact of the secretome of bone marrow mesenchymal stromal cells MSC(M) as a therapeutic strategy. MethodsAdult male rats were subjected to α-syn over expression in the nigrostriatal pathway to model dopaminergic neurodegeneration. The impact of locally administered secretome treatment from MSC(M) was studied. Motor impairments were assessed throughout the study coupled with whole-region (striatum and substantia nigra) confocal microscopy evaluation of histopathological changes associated with dopaminergic neurodegeneration and glial cell reactivity. ResultsTen weeks after lesion induction, the animals received secretome injections in the substantia nigra pars compacta (SNpc) and striatum (STR). The secretome used was produced from bone marrow mesenchymal stromal cells MSC(M) expanded in a spinner flask (SP) system. Nine weeks later, animals that received the viral vector containing the gene for A53T α-syn and treated with vehicle (Neurobasal-A medium) presented dopaminergic cell loss in the SNpc and denervation in the STR. The treatment with secretome significantly reduced the levels of α-syn in the SNpc and protected the dopaminergic neurons (DAn) within the SNpc and STR. ConclusionsOur results are aligned with previous studies in both α-syn Caenorhabditis elegans models, as well as 6-OHDA rodent model, revealing that secretome exerted a neuroprotective effect. Moreover, these effects were associated with a modulation of microglial reactivity supporting an immunomodulatory role for the factors contained within the secretome. This further supports the development of new studies exploring the effects and the mechanism of action of secretome from MSC(M) against α-syn-induced neurotoxicity.

Study on the Quality Variation and Internal Mechanisms of Frozen Oatmeal Cooked Noodles during Freeze-Thaw Cycles.

Frozen staple food, attributed to its favorable taste and convenience, has a promising development potential in the future. Frequent freezing and thawing, however, will affect its quality. This study simulated several freeze-thaw cycles (FTC) that may occur during the cold chain process of frozen oatmeal cooked noodles (FOCN) production to consumption. The quality changes and their mechanisms were elucidated using methods such as differential scanning calorimetry (DSC), low-field nuclear magnetic resonance (LF-NMR), Fourier-transform infrared spectroscopy (FTIR), confocal laser scanning microscopy (CLSM), texture analysis, and sensory evaluation. The freezable water content of the FOCN decreased because of the FTC treatment, and the relative content of total water in FOCN also decreased accordingly. The increase in β-Turn after FTC induced disorder in the secondary structure of proteins, causing the protein microstructure to become loose and discontinuous, which in turn reduced the water-holding capacity of FOCN. Additionally, FTC reduced the chewiness and sensory score of FOCN. This research will contribute a theoretical foundation for optimizing the cold chain process.

In vivo confocal microscopy evaluation of infiltrated immune cells in corneal stroma treated with cell therapy in advanced keratoconus

PurposeThis study investigates immune cell (ICs) infiltration in advanced keratoconus patients undergoing autologous adipose-derived adult stem cell (ADASC) therapy with recellularized human donor corneal laminas (CL).MethodsA prospective clinical trial included fourteen patients divided into three groups: G-1, ADASCs; G-2, decellularized CL (dCL); and G-3, dCL recellularized with ADASCs (ADASCs-rCL). Infiltrated ICs were assessed using in vivo confocal microscopy (IVCM) at 1,3,6, and12 months post-transplant.ResultsInfiltrated ICs, encompassing granulocytes and agranulocytes, were observed across all groups, categorized by luminosity, structure, and area. Stromal ICs infiltration ranged from 1.19% to 6.62%, with a consistent increase in group-related cell density (F = 10.68, P < .0001), independent of post-op time (F = 0.77, P = 0.511); the most substantial variations were observed in G-3 at 6 and 12 months (2.0 and 1.87-fold, respectively). Similarly, significant size increases were more group-dependent (F = 5.76, P < .005) rather than time-dependent (F = 2.84, P < .05); G-3 exhibited significant increases at 6 and 12 months (3.70-fold and 2.52-fold, respectively). A lamina-induced shift in IC size occurred (F = 110.23, P < .0001), primarily with 50–100 μm2 sizes and up to larger cells > 300μm2, presumably macrophages, notably in G-3, indicating a potential role in tissue repair and remodeling, explaining reductions in cells remnants < 50μm2.ConclusionsADASCs-rCL therapy may lead to increased IC infiltration compared to ADASCs alone, impacting cell distribution and size due to the presence of the lamina. The findings reveal intricate immune patterns shaped by the corneal microenvironment and highlight the importance of understanding immune responses for the development of future therapeutic strategies.

Comparative evaluation of effect of nisin-incorporated ethylenediamine tetraacetic acid and MTAD on endodontic biofilm eradication, smear layer removal, and depth of sealer penetration.

To comparatively evaluate the nisin-incorporated ethylenediamine tetraacetic acid (N-EDTA) and MTAD on cytotoxicity, endodontic biofilm eradication potential, smear layer removal ability, and sealer penetration depth. N-EDTA was prepared and characterized using high-performance liquid chromatography (HPLC). Minimum inhibitory, minimum bactericidal, and minimum biofilm inhibitory concentration (MBC, MIC, and MBIC) were determined on Enterococcus faecalis (E. faecalis) strain. The cytocompatibility of N-EDTA and MTAD was evaluated using 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay. Dentin specimens (n = 88 for antibacterial analysis, n = 170 for sealer penetration depth) were prepared and subjected to the classical irrigating strategy and obturation, respectively. The scanning electron microscopic evaluation (SEM) was done for the evaluation of biofilm disruption and smear layer removal. Confocal laser scanning microscopy (CLSM) evaluation was done for determining percentage of bacterial viability and sealer penetration depth. Statistical analysis of one-way ANOVA and Tukey's HSD post hoc tests for bacterial viability and Kruskal-Wallis test and Mann-Whitney test for smear layer removal and depth of penetration were done with the significance level set at p < 0.05. MTAD and N-EDTA showed cytocompatibility without any statistical differences from each other. For N-EDTA, the MIC and MBC values were 12.5 μg/ml (1:8), and MBIC values were 36 μg/ml. Biofilm disruption and killed bacterial percentage of N-EDTA was statistically higher than MTAD, whereas both the materials showed similar efficacy in the removal of the smear layer and sealer penetration depth. N-EDTA had negligible cytotoxicity with similar smear layer removal ability, sealer penetration, and better antibiofilm potential than MTAD. N-EDTA can serve as a viable alternative endodontic irrigant.

In Vitro Evaluation of Viscosity, Depth of Penetration, Microleakage, and Shear Bond Strength of Conventional and Hydrophilic Sealants.

Sealants are effective in preventing and arresting pit and fissure caries. Newer brands of sealants continue to be developed despite the lack of scientifically based information addressing the caries-preventive properties of these materials. Hence, laboratory in vitro tests play a vital role in providing the necessary information regarding the efficacy of new products in a short period of time. To compare and evaluate the viscosity, resin tag length, microleakage, and shear bond strength between conventional and hydrophilic sealants. A total of 40 extracted third molars were selected. Only 20 were evaluated for microleakage and resin tag length and another 20 for shear bond strength evaluation between group I-ClinPro and group II-UltraSeal XT hydro. After sealant placement, specimens were immersed in 0.1% rhodamine dye, followed by thermocycling. Microleakage testing was done using a confocal laser scanning microscope and resin tag length evaluation using a scanning electron microscope (SEM) and shear bond strength using an Universal Instron machine. Viscosity was assessed using an Anton Paar viscometer. Results were subjected to statistical analysis using Statistical Package for the Social Sciences (SPSS) software version 20. Viscosity and mean microleakage scores for group I (0.92 MPa and 1.50) were found to be higher than for group II (0.72 MPa and 0.60). Mean resin tag length and mean shear bond strength for group I (7.46 ± 0.95 μm and 13.71 ± 0.94 MPa) were found to be less compared to group II (10.03 ± 1.00 μm and 20.39 ± 0.98 MPa). The results were found to be statistically significant (p < 0.05). Group II was found to be less viscous, which resulted in the formation of resin tags of sufficient length and showed less microleakage and higher shear bond strength than group I. Prabakar J, Jeevanandan G, Kengadaran S. In Vitro Evaluation of Viscosity, Depth of Penetration, Microleakage, and Shear Bond Strength of Conventional and Hydrophilic Sealants. Int J Clin Pediatr Dent 2023;16(5):745-750.

Breast carcinoma detection in ex vivo fresh human breast surgical specimens using a fast slide-free confocal microscopy scanner: HIBISCUSS project.

New generation ultra-fast fluorescence confocal microscopy allows the ex vivo intraoperative analysis of fresh tissue. The High resolution Imaging for Breast carcInoma detection in ex vivo Specimens after breast Conserving sUrgery by hiStolog Scanner (HIBISCUSS) project aimed to develop an online learning program to recognize the main breast tissue features on ultra-fast fluorescence confocal microscopy images and to evaluate the performance of surgeons and pathologists in diagnosing cancerous and non-cancerous breast tissue in ultra-fast fluorescence confocal microscopy images. Patients who underwent conservative surgery or mastectomy for breast carcinoma (invasive or in situ lesions) were included. The fresh specimens were stained with a fluorescent dye and imaged using a large field-of-view (20 cm2) ultra-fast fluorescence confocal microscope. One hundred and eighty-one patients were included. The images from 55 patients were annotated to generate learning sheets and images from 126 patients were blindly interpreted by seven surgeons and two pathologists. The time for tissue processing and ultra-fast fluorescence confocal microscopy imaging was between 8 and 10 min. The training program was composed of 110 images divided into nine learning sessions. The final database for blind performance assessment comprised 300 images. The mean duration for one training session and one performance round was 17 and 27 min respectively. The performance of pathologists was almost perfect with 99.6 per cent (standard deviation (s.d.) 5.4 per cent) accuracy. Surgeons' accuracy significantly increased (P = 0.001) from 83 per cent (s.d. 8.4 per cent) in round 1 to 98 per cent (s.d. 4.1 per cent) in round 7 as well as the sensitivity (P = 0.004). Specificity increased without significance from 84 per cent (s.d. 16.7 per cent) in round 1 to 87 per cent (s.d. 16.4 per cent) in round 7 (P = 0.060). Pathologists and surgeons showed a short learning curve in differentiating breast cancer from non-cancerous tissue in ultra-fast fluorescence confocal microscopy images. Performance assessment for both specialties supports ultra-fast fluorescence confocal microscopy evaluation for intraoperative management. NCT04976556 (http://www.clinicaltrials.gov).

In vivo imaging of prostate tumor-targeted folic acid conjugated quantum dots

Cancer is a major threat to human health; thus, early detection is imperative for successful management. Rapid diagnosis can be achieved by imaging primary (subcutaneous) tumors using fluorophores conjugated with tumor markers. Here, the application of biocompatible, quantum efficient, monodisperse, and photostable polymer-coated quantum dots (PQDs) is demonstrated for targeted prostate tumor imaging in living SCID mice. Briefly, PQDs (blue) are conjugated to folic acid (FA-PQDs) using DCC-NHS chemistry. Initially, in vitro targeted imaging via FA-PQDs is evaluated in LNCaP cells. The confocal microscopic evaluation demonstrates the uptake of FA-PQDs. To understand the dispersion of PQDs in vivo, the biodistribution of PQDs is assessed at different time intervals (1- 180 min) using whole-body fluorescence imaging and computed tomography (CT) scan. PQDs are seen to accumulate in organs like the liver, kidneys, spleen, lungs, and urinary bladder within 60 min, however, PQDs are not observed at 180 min indicating renal clearance. Further, to target the prostate tumor (~ 200 mm3) in mice, FA-PQDs are injected intravenously, and whole-body fluorescence imaging along with a CT scan is recorded. FA-PQDs are seen at the tumor site as compared to PQDs. The results confirm that the FA-PQDs function as excellent nanoprobes for targeted tumor imaging in vivo.

Bromelain: a candidate to enhance wound healing after endonasal surgeries.

In the present study, we investigated the topical bromelain's cytotoxic effects on mouse fibroblast NIH/3T3 cells via cell culture study. In this cell culture study, Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum (FBS, 10%) and penicillin/streptomycin (1%) was used as a cell growth medium for NIH/3T3 mouse fibroblast cells. MTT test was performed in 96-well plates seeded with NIH/3T3 cells 5x103/well and under standard cell culture conditions. Bromelain doses of 3.13 to 100 μM were administered to the wells and incubated for 24, 48, and 72 hours in the same cell culture conditions. For Confocal microscopic evaluation, NIH/3T3 cells were plated on cover slips in 6-well plates (105 cells/well) and treated with 100 μM concentration of bromelain for 24 h. Untreated cells were used as controls. MTT results showed that bromelain is not cytotoxic on mouse fibroblast NIH/3T3 cells. All three incubation times of 24, 48, and 72 hours bromelain initiated cell growth. A statistically significant rise in cell growth was detected in the only applied highest dose of 100 μM bromelain for all incubation times except for 24 hours. The nontoxic effect was further investigated by using confocal microscopy by applying the highest bromelain dose of 100 μM to NIH/3T3 mouse fibroblast cells. Confocal micrographs showed that bromelain did not change the morphology of mouse fibroblast cells at the incubation time of 24h. In untreated cells and bromelain-treated cells, the nucleus of NIH/3T3 cells was undamaged and compact, and the cytoskeleton was fusiform and non-fragmented. Bromelain is not cytotoxic on mouse fibroblast NIH/3T3 cells and enhances cell growth. If clinical trials will confirm this, it is possible that bromelain will be used topically in humans to enhance wound healing, in rhinosinusitis and chronic rhinosinusitis with nasal polyps and endonasal surgeries due to its anti-inflammatory effects.

A confocal laser scanning microscopic evaluation of nonthermal atmospheric plasma on the dentinal tubule penetration of bioceramic and epoxy resin-based root canal sealers.

Using confocal laser scanning microscopy (CLSM), the current study assessed the impact of nonthermal atmospheric plasma (NTAP) on the dentinal tubule penetration of bioceramic and epoxy resin-based root canal sealers (CLSM). Forty human mandibular premolar teeth with a single root that had just undergone extraction were chosen and biomechanical preparation of root canals was done with ProTaper Gold rotary Nickel-titanium instruments. Samples were divided into four groups (n = 10). Group 1: Bioceramic sealer (BioRoot RCS); Group 2: Epoxy resin-based sealer (AH Plus) without application of NTAP; Group 3: Bioceramic sealer (BioRoot RCS); and Group 4: Epoxy resin-based sealer (AH Plus) with the application of NTAP for 30 s. In Groups 3 and 4, all of the samples underwent obturation with the appropriate sealers following NTAP application. For determination of the sealer's dentinal tubule penetration values, slices with a thickness of 2 mm were taken from the middle third of sample's root and examined using CLSM. The acquired data were statistically analyzed with one-way analysis of variance and the Post hoc Tukey's test. The cutoff for statistical significance was P < 0.05. In comparison to other groups, the maximum sealer penetration values into dentinal tubules were significantly higher in Group 3 for Bioceramic sealer with NTAP application and Group 4 for Epoxy resin-based sealer with NTAP application. NTAP application increased the dentinal tubule penetration of bioceramic and epoxy resin-based sealers in comparison to groups without NTAP application.

Modulating the G-Quadruplex and Duplex DNA Binding by Controlling the Charge of Fluorescent Molecules.

Two fluorescent and non-toxic spirobifluorene molecules bearing either positive (Spiro-NMe3) or negative (Spiro-SO3) charged moieties attached to the same aromatic structure have been investigated as binders for DNA. The novel Spiro-NMe3 containing four alkylammonium substituents interacts with G-quadruplex (G4) DNA structures and shows preference for G4s over duplex by means of FRET melting and fluorescence experiments. The interaction is governed by the charged substituents of the ligands as deduced from the lower binding of the sulfonate analogue (Spiro-SO3). On the contrary, Spiro-SO3 exhibits higher binding affinity to duplex DNA structure than to G4. Both molecules show a moderate quenching of the fluorescence upon DNA binding. The confocal microscopy evaluation shows the internalization of both molecules in HeLa cells and their lysosomal accumulation.

Oral Peroxisome Proliferator-Activated Receptor-α Agonist Enhances Corneal Nerve Regeneration in Patients With Type 2 Diabetes.

Diabetic corneal neuropathy (DCN) is a common complication of diabetes. However, there are very limited therapeutic options. We investigated the effects of a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, fenofibrate, on 30 patients (60 eyes) with type 2 diabetes. On invivo confocal microscopy evaluation, there was significant stimulation of corneal nerve regeneration and a reduction in nerve edema after 30 days of oral fenofibrate treatment, as evidenced by significant improvement in corneal nerve fiber density (CNFD) and corneal nerve fiber width, respectively. Corneal epithelial cell morphology also significantly improved in cell circularity. Upon clinical examination, fenofibrate significantly improved patients' neuropathic ocular surface status by increasing tear breakup time along with a reduction of corneal and conjunctival punctate keratopathy. Tear substance P (SP) concentrations significantly increased after treatment, suggesting an amelioration of ocular surface neuroinflammation. The changes in tear SP concentrations was also significantly associated with improvement in CNFD. Quantitative proteomic analysis demonstrated that fenofibrate significantly upregulated and modulated the neurotrophin signaling pathway and linolenic acid, cholesterol, and fat metabolism. Complement cascades, neutrophil reactions, and platelet activation were also significantly suppressed. Our results showed that fenofibrate could potentially be a novel treatment for patients with DCN.

Evaluation of warm compresses and topical cyclosporine treatment in meibomian gland dysfunction by confocal scanning laser microscopy.

The aim of our study is to confirm the utility of warm compresses (WC) and artificial tears (AT) in meibomian gland dysfunction (MGD) management and to understand if its association with topical cyclosporine A (CsA) improves outcomes. Patients with diagnosis of MGD-related dry eye were evaluated. In this prospective, randomized, double-masked study, patients were randomized in two treatments: AT plus WC (group A), and AT plus WC plus CsA 0.05% ophthalmic emulsion (group B). At baseline and at 1, 3, and 6 months, Ocular Surface Disease Index (OSDI) questionnaire was completed, and tear evaluation (BUT, Schirmer and osmolarity test), ocular surface evaluation (fluorescein and lissamine green staining), clinical (Shimazaki grading) and in vivo confocal microscopy (IVCM) evaluation of rete ridges (RRs) were performed. A total of 40 eyes, 20 in each group, completed the study. Analysis of OSDI, tear test, ocular surface evaluation and clinical grading of MG showed significant improvement at 6 months, whereas no difference was found between the two groups at 6 months. The analysis of IVCM showed significant improvement in both groups, but significantly better results in group B were found compared to group A at 6 months. WC performed 3 times daily during the 1st month and once daily afterwards, in addition to AT, were useful to manage the obstruction of MG and related signs and symptoms. Additional effects of CsA were visible in IVCM only at 6 months. IVCM is an effective tool to monitor treatments in MGD.

Topical Treatment of Actinic Keratosis and Metalloproteinase Expression: A Clinico-Pathological Retrospective Study.

Actinic keratosis is an intraepithelial proliferation of atypical keratinocytes that could progress into invasive squamous cell carcinoma. Most evidence suggests an important role of the dermal matrix metalloproteinases in the progression of atypical skin epithelial lesions. We evaluated the clinical efficacy of three different therapeutic modalities (a medical device containing 0.8% piroxicam cream and 50+ sunscreen, photodynamic therapy, and ingenol mebutate gel) to treat suspicious actinic keratoses, which were biopsied for histopathological examination and then analyzed for the expression of matrix metalloproteinases by immunohistochemistry. Clinical, dermoscopic, and reflectance confocal microscopy evaluations revealed a gradual decrease in all standard scores validated for actinic keratosis assessment at the end of the treatments. From a histopathological point of view, we documented the substantial restoration of normal skin architecture, while the immunohistochemical evaluation of matrix metalloproteinases showed a reduction in expression in the treated skin lesions compared to the baseline. As actinic keratoses are considered the precursors of squamous cell carcinoma, their treatment is crucial to prevent the development of a more aggressive disease. Our study monitored the evolution of actinic keratoses subjected to three different topical therapies, with the value of correlating clinical and histopathological findings. Moreover, as the matrix metalloproteinases are largely recognized factors involved in the pathogenesis and evolution of actinic keratosis to squamous cell carcinoma, the demonstration by immunohistochemistry of a reduction in their expression after the treatments adds new valuable concern to the field.