Dental pulp-derived stem cells inhibit osteoclast differentiation by secreting osteoprotegerin and deactivating AKT signalling in myeloid cells.

Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non-immune cells help initiating activation of myeloid cells for differentiation, whereas hyper-activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp-derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone-related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co-cultured in a contact-free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP-9 assessed by quantitative RT-PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT-PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up-regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose-dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M-CSF phosphorylate AKT and activate PI3K-AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG-mediated inhibition of the downstream activation of PI3K-AKT signalling pathway was similar to the DPSC co-culture-mediated inhibition of OC differentiation. This study provides novel evidence of DPSC-mediated inhibition of osteoclastogenesis mechanisms.

Cigarette smoking promotes cancer-related transformation of oral epithelial cells through activation of Wnt and MAPK pathway.

Aim: Study aims to investigate the effect of cigarette smoking on cancer-related transformation in oral epithelial cells of smokers through evaluating the alteration in Wnt/ß-catenin and MAPK pathways. Materials & methods: Exfoliated oral epithelial cells were collected from 138 subjects and categorized into nonsmokers, smokers and clinically diagnosed precancer and cancer patients. Real-time quantitative PCR was performed to detect the fold changes of related genes. Expressions of biomarkers were assessed using immunofluorescence and western blot. Results: Study shows significant (p < 0.001) alteration in mRNA level of TNF-α, NF-κß, FZD1, ß-catenin, PARD 3, MAPK1 and vimentin genes under cigarette smoking. Conclusion: Results suggested the progression of oral cancer under cigarette smoking occurs through multiple events and activation of canonical Wnt/MAPK pathways.

Repositing honey incorporated electrospun nanofiber membranes to provide anti-oxidant, anti-bacterial and anti-inflammatory microenvironment for wound regeneration.

Topical application of honey for tissue regeneration, has recently regained attention in clinical practice with controlled studies affirming its efficacy and indicating its role in regeneration over repair. Parallely, to overcome difficulties of applying raw honey, several product development studies like nanofibrous matrices have been reported. However, one approach concentrated on achieving highest possible honey loading in the nanofiber membranes while other studies have found that only specific honey dilutions result in differential cellular responses on wound healing and re-epithelization. From these results, it can be suggested that high honey loading provides optimum external microenvironment, low-loaded membranes could provide a more conducive internal microenvironment for tissue regeneration. With this hypothesis, this paper sought to evaluate ability of low-honey loaded nanofibers to modulate the anti-oxidant, anti-biofilm and anti-inflammatory properties which are important to be maintained in wound micro-environment. A loading-dependent reduction of biofilm formation and anti-oxidant activity was noted in different concentration ranges investigated. After scratch assay, a certain honey loading (0.5%) afforded the maximum re-epithelization. Since there is lack of methods to determine anti-inflammatory properties of nanofiber membranes during epithelial healing process, we performed anti-inflammatory assessment of nano-fibers by evaluating the expressions of pro-inflammatory markers-Cycloxygenase-2 (COX-2) and Interleukin-6 (IL-6) and to confirm the optimized concentration. Considering the role of COX-2 and IL-6, the novel methodology used in this study can also be developed as an assay for anti-inflammatory matrices for wound healing.

Risk prediction for oral potentially malignant disorders using fuzzy analysis of cytomorphological and autofluorescence alterations in habitual smokers.

AIM: This study aims to develop a novel noninvasive method for early cancer trend diagnosis in habitual smokers by corroborating cytomorphological and autofluorescence alterations. MATERIALS & METHODS: A total of 120 subjects were included and categorized into nonsmoker, smoker and clinically diagnosed oral potentially malignant disorder (OPMD) patients. Oral exfoliative epithelial cells were studied through differential interference contrast and fluorescence microscopy. Fuzzy trend analysis was performed using measured parameters for determining the risk factors among smokers. RESULTS: The risk assessment in this study showed a positive correlation of smoking duration with early cancer risk factors with a correlation co-efficient of 0.86. CONCLUSION: Alterations in cellular morphology and autofluorescence intensities showed positive correlation with OPMD. The present study will benefit to investigate early prediction of OPMD among susceptible individuals.

Effect of cigarette smoke extract on mitochondrial heme-metabolism: An in vitro model of oral cancer progression.

Tobacco smoking is considered as one of the major risk factors for development of oral cancer. In vitro studies indicate that cigarette smoke initiates transformation of epithelial cells toward development of oral cancer through altering mitochondrial metabolic pathways. However the present in vitro models need to be improved to correlate these molecular changes with epithelial transformations. In present study, we investigated the association of mitochondrial metabolic events with oral cancer progression under cigarette smoke extract (CSE). In this regard, an in vitro model of oral keratinocyte cell line (MOE1A) was developed by exposing them with different concentrations of CSE. Alterations in cellular phenomena were confirmed by Fourier-transform infrared spectroscopy (FTIR) study, which indicated changes in important functional groups of CSE-induced oral cells. Enhanced reactive oxygen species (ROS) of exposed cells altered the mitochondrial metabolic activities in terms of increased mitochondrial mass and DNA content. Further, mitochondrial heme-metabolism was investigated and real-time PCR study showed altered expression of important genes like ALAS1, ABCB6, CPOX, FECH, HO-1. Both transcriptomic and proteomic studies showed up- and down-regulation of important biomarkers related to cellular cancer progression. Overall data suggest that CSE alters mitochondrial heme metabolic pathway and initiates cancer progression through modifying cellar biomarkers in oral epithelial cells.

Liaison between heme metabolism and bioenergetics pathways-a multimodal elucidation for early diagnosis of oral cancer.

Metabolic alterations of oral epithelial cells under oxidative stress are important signatures for early diagnosis of oral cancer. Amongst different metabolic alterations, non-invasive photo-diagnostic methods have been extensively used for determining cellular heme metabolism and accumulation of protoporphyrin IX (PpIX) under administration of suitable photosensitizer. In this study, we report these metabolic alterations by direct analysis of oral exfoliated cells obtained from individuals with prolonged smoking habit without the exogenous administration of any photosensitizer. The relative expression level of relevant biomolecules of study groups were compared with clinically diagnosed and histopathologically confirmed leukoplakia (OLPK) and oral squamous cell carcinoma (OSCC) patients. The energy imbalance and variation in 'redox ratio' were examined through spectroscopic studies which showed an increasing trend (p < 0.001) in smokers to OSCC groups in comparison to nonsmoker control. Gene expression of important intermediates of the heme metabolic pathway (viz. 5-aminolevulinate synthase 1 (ALAS1), Ferrochelatase (FECH), hemeoxygenase 1 (HO-1) and ATP binding cassette subfamily G member 2 (ABCG2)) which affect production of PpIX was assessed. Relative mRNA level of ALAS1 and HO1 was upregulated whereas mRNA level of other genes (viz. FECH and ABCG2) were found to be downregulated in smokers as well as in cancer groups. Outcome of different spectroscopic studies on exfoliated cells (viz. fluorescence, atomic absorption and Fourier transform infrared spectroscopy) corroborated with the expression of biomarkers related to cellular endogenous metabolism related to heme cycle. This study indicates significant alterations in endogenous metabolic products, and cellular functional groups in oral epithelial cells among the study groups. Our study reports a strong possibility of diagnosis of early cancer signatures amongst habitual smokers by direct and non-invasive assessment of metabolic status of oral epithelial cells without exogenous administration of photosensitizers. The knowledge accrued from the study may guide clinicians in precise detection of precancer trend in the susceptible population through a noninvasive rapid screening method.

Pre-cancer risk assessment in habitual smokers from DIC images of oral exfoliative cells using active contour and SVM analysis.

Habitual smokers are known to be at higher risk for developing oral cancer, which is increasing at an alarming rate globally. Conventionally, oral cancer is associated with high mortality rates, although recent reports show the improved survival outcomes by early diagnosis of disease. An effective prediction system which will enable to identify the probability of cancer development amongst the habitual smokers, is thus expected to benefit sizable number of populations. Present work describes a non-invasive, integrated method for early detection of cellular abnormalities based on analysis of different cyto-morphological features of exfoliative oral epithelial cells. Differential interference contrast (DIC) microscopy provides a potential optical tool as this mode provides a pseudo three dimensional (3-D) image with detailed morphological and textural features obtained from noninvasive, label free epithelial cells. For segmentation of DIC images, gradient vector flow snake model active contour process has been adopted. To evaluate cellular abnormalities amongst habitual smokers, the selected morphological and textural features of epithelial cells are compared with the non-smoker (-ve control group) group and clinically diagnosed pre-cancer patients (+ve control group) using support vector machine (SVM) classifier. Accuracy of the developed SVM based classification has been found to be 86% with 80% sensitivity and 89% specificity in classifying the features from the volunteers having smoking habit.

Simple Bisthiocarbonohydrazone as a Sensitive, Selective, Colorimetric, and Ratiometric Fluorescent Chemosensor for Picric Acids.

A bisthiocarbonohydrazone-based chemosensor molecule (R1) containing a tetrahydro-8-hydroxyquinolizine-9-carboxaldehyde moiety has been synthesized and characterized as a new ratiometric fluorescent probe for picric acid (PA). The ratiometric probe R1 is a highly selective and sensitive colorimetric chemosensor for PA. The association between the chemosensor and PA and the ratiometric performance enabled by the key role of excited state intramolecular proton transfer in the detection process are demonstrated. Selectivity experiments proved that R1 has excellent selectivity to PA over other nitroaromatic chemicals. Importantly, the ratiometric probe exhibited a noteworthy change in both colorimetric and emission color, and this key feature enables R1 to be employed for detection of PA by simple visual inspection in silica-gel-coated thin-layer chromatography plates. Probe R1 has been shown to detect PA up to 3.2 nM at pH 7.4. Microstructural features of R1 and its PA complex have been measured by a field emission scanning electron microscope, and it clearly proves that their morphological features differ dramatically both in shape and size. Density function theory and time-dependent density function theory calculations were performed to establish the sensing mechanism and the electronic properties of probe R1. Furthermore, we have demonstrated the utility of probe R1 for the detection of PA in live Vero cells for ratiometric fluorescence imaging.