Newly discovered clouting interplay between matrix metalloproteinases structures and novel quaternary Ammonium K21: computational and in-vivo testing.

AIMS AND OBJECTIVES: To analyze anti-MMP mode of action of Quaternary Ammonium Silane (QAS, codenamed as k21) by binding onto specific MMP site using computational molecular simulation and Anti-Sortase A (SrtA) mode of action by binding onto specific site using computational molecular simulation. MATERIALS AND METHODS: In silico Molecular Dynamics (MD) was used to determine the interactions of K21 inside the pocket of the targeted protein (crystal structure of fibroblast collagenase-1 complexed to a diphenyl-ether sulphone based hydroxamic acid; PDB ID: 966C; Crystal structure of MMP-2 active site mutant in complex with APP-derived decapeptide inhibitor. MD simulations were accomplished with the Desmond package in Schrödinger Drug Discovery Suite. Blood samples (~ 0.5 mL) collected into K2EDTA were immediately transferred for further processing using the Litron MicroFlow® PLUS micronucleus analysis kit for mouse blood according to the manufacturer's instructions. Bacterial Reverse Mutation Test of K21 Molecule was performed to evaluate K21 and any possible metabolites for their potential to induce point mutations in amino acid-requiring strains of Escherichia coli (E. coli) (WP2 uvrA (tryptophan-deficient)). RESULTS: Molecular Simulation depicted that K21 has a specific pocket binding on various MMPs and SrtA surfaces producing a classical clouting effect. K21 did not induce micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus, in the peripheral blood reticulocytes of male and female CD-1 mice when administered by oral gavage up to the maximum recommended dose of 2000 mg/kg. The test item, K21, was not mutagenic to Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA in the absence and presence of metabolic activation when tested up to the limit of cytotoxicity or solubility under the conditions of the test. CONCLUSION: K21 could serve as a potent protease inhibitor maintaining the physical and biochemical properties of dental structures.

Biochemical changes and macrophage polarization of a silane-based endodontic irrigant in an animal model.

Silane-based/fully hydrolyzed, endodontic irrigant exhibiting antimicrobial properties, is prepared, and is hypothesized to control macrophage polarization for tissue repair. Albino wistar rats were injected with 0.1 ml root canal irrigant, and bone marrow cells procured. Cellular mitochondria were stained with MitoTracker green along with Transmission Electron Microscopy (TEM) performed for macrophage extracellular vesicle. Bone marrow stromal cells (BMSCs) were induced for M1 and M2 polarization and Raman spectroscopy with scratch assay performed. Cell counting was used to measure cytotoxicity, and fluorescence microscopy performed for CD163. Scanning Electron Microscopy (SEM) was used to investigate interaction of irrigants with Enterococcus faecalis. K21 specimens exhibited reduction in epithelium thickness and more mitochondrial mass. EVs showed differences between all groups with decrease and increase in IL-6 and IL-10 respectively. 0.5%k21 enhanced wound healing with more fibroblastic growth inside scratch analysis along with increased inflammation-related genes (ICAM-1, CXCL10, CXCL11, VCAM-1, CCL2, and CXCL8; tissue remodelling-related genes, collagen 1, EGFR and TIMP-2 in q-PCR analysis. Sharp bands at 1643 cm-1 existed in all with variable intensities. 0.5%k21 had a survival rate of BMSCs comparable to control group. Bacteria treated with 0.5%k21/1%k21, displayed damage. Antimicrobial and reparative efficacy of k21 disinfectant is a proof of concept for enhanced killing of bacteria across root dentin acquiring functional type M2 polarization for ethnopharmacological effects.

3D Clumps/Extracellular Matrix Complexes of Periodontal Ligament Stem Cells Ameliorate the Attenuating Effects of LPS on Proliferation and Osteogenic Potential.

BACKGROUND: The effects of lipopolysaccharide (LPS) on cell proliferation and osteogenic potential (OP) of MSCs have been frequently studied. OBJECTIVE: to compare the effects of LPS on periodontal-ligament-derived mesenchymal stem cells (PDLSCs) in monolayer and 3D culture. METHODS: The PDLSCs were colorimetrically assessed for proliferation and osteogenic potential (OP) after LPS treatment. The 3D cells were manually prepared by scratching and allowing them to clump up. The clumps (C-MSCs) were treated with LPS and assessed for Adenosine triphosphate (ATP) and OP. Raman spectroscopy was used to analyze calcium salts, DNA, and proline/hydroxyproline. Multiplexed ELISA was performed to assess LPS induced local inflammation. RESULTS: The proliferation of PDLSCs decreased with LPS. On Day 28, LPS-treated cells showed a reduction in their OP. C-MSCs with LPS did not show a decrease in ATP production. Principal bands identified in Raman analysis were the P-O bond at 960 cm-1 of the mineral component, 785 cm-1, and 855 cm-1 showing qualitative changes in OP, proliferation, and proline/hydroxyproline content, respectively. ELISA confirmed increased levels of IL-6 and IL-8 but with the absence of TNF-α and IL-1ß secretion. CONCLUSIONS: These observations demonstrate that C-MSCs are more resistant to the effects of LPS than cells in monolayer cell culture. Though LPS stimulation of C-MSCs creates an early pro-inflammatory milieu by secreting IL-6 and IL-8, PDLSCs possess inactivated TNF promoter and an ineffective caspase-1 activating process.

Biomarkers for Temporomandibular Disorders: Current Status and Future Directions.

Numerous studies have been conducted in the previous years with an objective to determine the ideal biomarker or set of biomarkers in temporomandibular disorders (TMDs). It was recorded that tumour necrosis factor (TNF), interleukin 8 (IL-8), IL-6, and IL-1 were the most common biomarkers of TMDs. As of recently, although the research on TMDs biomarkers still aims to find more diagnostic agents, no recent study employs the biomarker as a targeting point of pharmacotherapy to suppress the inflammatory responses. This article represents an explicit review on the biomarkers of TMDs that have been discovered so far and provides possible future directions towards further research on these biomarkers. The potential implementation of the interactions of TNF with its receptor 2 (TNFR2) in the inflammatory process has been interpreted, and thus, this review presents a new hypothesis towards suppression of the inflammatory response using TNFR2-agonist. Subsequently, this hypothesis could be explored as a potential pain elimination approach in patients with TMDs.

More Accurate Oral Cancer Screening with Fewer Salivary Biomarkers.

Signal detection and Bayesian inferential tools were applied to salivary biomarkers to improve screening accuracy and efficiency in detecting oral squamous cell carcinoma (OSCC). Potential cancer biomarkers are identified by significant differences in assay concentrations, receiver operating characteristic areas under the curve (AUCs), sensitivity, and specificity. However, the end goal is to report to individual patients their risk of having disease given positive or negative test results. Likelihood ratios (LRs) and Bayes factors (BFs) estimate evidential support and compile biomarker information to optimize screening accuracy. In total, 26 of 77 biomarkers were mentioned as having been tested at least twice in 137 studies and published in 16 summary papers through 2014. Studies represented 10 212 OSCC and 25 645 healthy patients. The measure of biomarker and panel information value was number of biomarkers needed to approximate 100% positive predictive value (PPV). As few as 5 biomarkers could achieve nearly 100% PPV for a disease prevalence of 0.2% when biomarkers were ordered from highest to lowest LR. When sequentially interpreting biomarker tests, high specificity was more important than test sensitivity in achieving rapid convergence toward a high PPV. Biomarkers ranked from highest to lowest LR were more informative and easier to interpret than AUC or Youden index. The proposed method should be applied to more recently published biomarker data to test its screening value.

Susceptibility of Human Oral Squamous Cell Carcinoma (OSCC) H103 and H376 cell lines to Retroviral OSKM mediated reprogramming.

Although numbers of cancer cell lines have been shown to be successfully reprogrammed into induced pluripotent stem cells (iPSCs), reprogramming Oral Squamous Cell Carcinoma (OSCC) to pluripotency in relation to its cancer cell type and the expression pattern of pluripotent genes under later passage remain unexplored. In our study, we reprogrammed and characterised H103 and H376 oral squamous carcinoma cells using retroviral OSKM mediated method. Reprogrammed cells were characterized for their embryonic stem cells (ESCs) like morphology, pluripotent gene expression via quantitative real-time polymerase chain reaction (RT-qPCR), immunofluorescence staining, embryoid bodies (EB) formation and directed differentiation capacity. Reprogrammed H103 (Rep-H103) exhibited similar ESCs morphologies with flatten cells and clear borders on feeder layer. Reprogrammed H376 (Rep-H376) did not show ESCs morphologies but grow with a disorganized morphology. Critical pluripotency genes Oct4, Sox2 and Nanog were expressed higher in Rep-H103 against the parental counterpart from passage 5 to passage 10. As for Rep-H376, Nanog expression against its parental counterpart showed a significant decrease at passage 5 and although increased in passage 10, the level of expression was similar to the parental cells. Rep-H103 exhibited pluripotent signals (Oct4, Sox2, Nanog and Tra-1-60) and could form EB with the presence of three germ layers markers. Rep-H103 displayed differentiation capacity into adipocytes and osteocytes. The OSCC cell line H103 which was able to be reprogrammed into an iPSC like state showed high expression of Oct4, Sox2 and Nanog at late passage and may provide a potential iPSC model to study multi-stage oncogenesis in OSCC.

The effect of epiregulin on epidermal growth factor receptor expression and proliferation of oral squamous cell carcinoma cell lines.

BACKGROUND: Epiregulin (EPR) is a novel member of the epidermal growth factor (EGF) family. It has been shown to promote wound healing in oral epithelium, enhance proliferation of other epithelial tissues, and is involved in several epithelial-related malignancies such as colorectal, lung, and bladder carcinoma. More recently, EPR transcripts were found to be high in a study on archival oral squamous cell carcinoma (OSCC) specimens. This implies that EPR may be responsible for the progression of OSCC. The aim of this was to elucidate the effects of EPR on (i) cell morphological changes, (ii) cell proliferation and (iii) receptor expression of the H-series OSCC cell lines. METHODS: The clinicopathological origin and the expression of the epidermal growth factor receptor (EGFR) and ErbB4 receptors of the H-series cell lines were initially characterised. Based on these parameters, two of the H-series cell lines, namely H103 and H357 were selected for downstream experiments. The cell lines were treated with 1 ng/ml, 10 ng/ml, and 20 ng/ml of EPR for 24 and 48 hours in all subsequent experiments. Untreated cells acted as the control which was used for comparison with each treated group. The cell morphological changes, cell proliferation and receptor expression of the OSCC cell lines were evaluated using phase contrast microscopy, 5-bromo-2'-deoxy-uridine (BrdU) assays and flow cytometry respectively. The results were compared and analysed using the student t-test. RESULTS: There were no appreciable morphological changes in the cells regardless of the dose of EPR tested nor between the different timelines. There were no significant changes in cell proliferation after EPR treatment. As for the effect of EPR on receptor expression, 20 ng/ml of EPR significantly reduced the density of EGFR expression (p value = 0.049) in the H103 cell line after the 24-hour treatment. No other statistically significant changes were detected. CONCLUSIONS: The results show that EPR had no effect on the morphology and proliferativity of OSCC cells. However, the significant decline in EGFR expression after EPR treatment suggests that EPR might play an important role in the regulation of EGFR expression and hence OSCC progression.