Lysyl oxidase like-2 mediates tumor to stromal cell communication in oral cancer

INTRODUCTION: The lysyl oxidase family consists of 5 members and oxidizes specific lysine residues in biosynthetic collagen and elastin maturation. Lysyl oxidase like-2 (LOXL2) is elevated in oral cancer and promotes metastasis and correlates with poor prognosis. The objective of this study was to determine the mechanism by which LOXL2 promotes the progression and invasiveness of oral squamous cell carcinoma. METHODS: In vitro: The effects of LOXL2 inhibitor (PXS-S1C) on human gingival fibroblasts treated with tumor cell conditioned medium (CM) were investigated. Cell proliferation assays, signaling arrays, gene knock down and western blots were used to evaluate the effect of PXS-S1C on CM-treated fibroblasts. The effects of PXS-S1C on cancer cell expression of LOXL2 and proliferation were determined. To find potential LOXL2 substrates, carbonyl-containing proteins of gingival fibroblasts treated with CM +/- PXS-S1C were affinity-labeled and then purified by affinity chromatography and identified by western blot. In vivo: The effects of PXS-S1C on cancer cell growth and metastasis in vivo were investigated using orthotopic oral tongue cancer mouse models in both immunodeficient and immunocompetent mice. PXS-S1C at 10 mg/kg and 30 mg/kg was injected immediately following tumor cell injections. Tumor growth was monitored by both caliper measurement and in vivo imaging (IVIS). The mice were sacrificed and their organs were subjected to immunohistochemical staining with proliferation markers. RESULTS: PXS-S1C significantly inhibited gingival fibroblast proliferation stimulated by tumor cell CM and attenuated phosphorylation of PDGFRβ at the Y771 and Y857, but not Y751 residues in response to CM treatment. PXS-S1C inhibited ERK1/2-signaling in fibroblasts but not AKT pathway in response to CM treatment. PDGFR activation by oral tumor cells was mimicked by PDGF-AB but not PDGF-BB. PXS-S1C decreased the expression of LOXL2 in HSC3 oral cancer cells in vitro, suggesting the existence of a positive autoregulatory loop. Assessing for direct LOXL2 substrates in fibroblasts with functional consequences identified PDGFR. In vivo studies: Caliper measurements, IVIS, and immunohistochemistry demonstrated that inhibition of LOXL2 by injections of PXS-S1C significantly decreased both progression and metastasis of oral cancers in vivo in both mouse models. Mice without PXS-S1C treatment developed larger tongue volumes (p<0.05), and in immunocompetent mice larger lymph nodes (9 out 12) were observed compared to the PXS-S1C-treated mice (4 out of 12). IVIS imaging of immunodeficient mice revealed inhibition of metastasis by PXS-S1C treatment. The expression of proliferation marker (Ki-67 or PCNA) and LOXL2 was lower in tongue tumors treated with PXS-S1C in both in vivo models (p<0.05). CONCLUSIONS: LOXL2 secreted by cancer cells stimulates fibroblast proliferation by oxidizing PDGFR and thereby enhancing PDGF-mediated signaling. Inhibition of LOXL2 can be used as a therapeutic strategy to suppress the growth and metastasis of oral cancers by modulating tumor microenvironment.2019-10-24T00:00:00

Comparing microleakage in root canals obturated with nanosilver coated gutta-percha to standard gutta-percha by two different methods

INTRODUCTION: Favorable apical seal of root filling materials is a crucial factor for a successful root canal treatment. The aim of this in vitro study was to compare bacterial and dye microleakage of two root canal filling materials including standard gutta-percha and nanosilver coated gutta-percha, and to evaluate the agreement between results of these two methods.MATERIALS & METHODS: Fifty-eight extracted single-rooted teeth were randomly divided into two experimental groups of 26 each, and two control groups of three each. After decoronation, root canals were instrumented by crown-down technique. Obturation was conducted using standard gutta-percha in one of experimental groups and nanosilver-coated gutta-percha in another group. AH26 sealer was used as the sealer in both experimental groups. Bacterial leakage was investigated after 60 days using Enterococcus (E.) faecalis microbial strains, and dye leakage was assessed during 72 hours using 1% methylene blue. The data were statistically analyzed by Chi-square test, Kaplan-Meier survival analysis, and Cohen’s Kappa. RESULTS: There was 84% bacterial leakage in standard gutta-percha group and 76% in nanosilver gutta-percha group. Complete dye leakage occurred in 24% and 27% of standard and nanosilver gutta-percha groups, respectively. The above difference between groups was not significant. In the samples with leakage, recorded times of leakage were not significantly different. There was no significant measure of agreement between dye and bacterial penetration along root-end fillings.CONCLUSION: There was a poor agreement between dye and bacterial leakage methods. Leakage results produced by nanosilver gutta-percha were comparable to those by standard gutta-percha. Considering the antibacterial effects of nanosilver coated gutta-percha, use of this type of gutta-percha might be more efficacious in endodontic treatments.

The Effect of Orthodontic Forces on Tooth Response to Electric Pulp Test

Introduction: The current study investigated the pulp response to electric pulp testing (EPT), before, upon initiation and one month after the start of orthodontic tooth movement. Methods and Materials: A total of 402 anterior teeth from 39 patients (mean age of 16.8±2.7 years) were examined in this non-controlled prospective study. The aligning forces were administered using initial NiTi archwires ligated on fixed appliances by using the MBT straight wire technique. The electrical stimulation was provided by the EPT. The EPT readings were recorded at three time points: before bonding (EPT0), immediately upon initiation (EPT1) and 1 month post-treatment (EPT2). The data were statistically analyzed by the ANOVA and Bonferroni tests (P&lt;0.05). Results: Prior to bonding of the orthodontic brackets, the mean EPT value for all the experimental teeth was 3.42 EPT units. Upon initiation, the mean value of EPT1 for each tooth increased to 7.62 units. One month later, the mean EPT2 values dropped to 6.27 units. At this time point, 64 teeth (16%) of the experimental teeth failed to respond. The differences among EPT values at different time points were significant. There was no association between the EPT values and the location or the type of teeth. Conclusion: The physiological changes in the pulp affect the nerve fibers in the early stages of the orthodontic force application. As a result, thresholds to electrical stimulation would increase and the EPT may not initiate a response. Therefore results obtained by electrical pulp testing should be interpreted accordingly.Keywords:Electric Pulp Test; Orthodontic Treatment; Pulp Vitality

Proliferative and inductive effects of Cyclosporine a on gingival fibroblast of child and adult

Background: Gingival overgrowth is a serious side-effect that accompanies the use of Cyclosporin A (CsA). Up to 97% of the transplant recipient children, who were submitted to CsA therapy, have been reported to suffer from this side-effect. Several conflicting theories have been proposed to explain the fibroblast′s function in CsA-induced gingival overgrowth. The aim of this study is to assess the proliferation of gingival fibroblasts and levels of released cytokines after being exposed to CsA, in both adults and pediatric groups, and to make a comparison between the results of the two groups. Materials and Methods: The adult fibroblast samples were derived from four healthy adults, aged 35 to 42 years and pediatric samples were obtained from four healthy children, age between four and eleven years. Tissue samples were plated in Dulbecco′s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), Streptomycin and Penicillin. The samples were cultured in 25 cm 2 plates containing 5% CO2, and incubated at 37°C. The cells used for all the experiments were at the fourth passage. The concentration of PGE 2 , IL-1β, IL-6, IL-8, TNF-α, and TGF-β1 was determined by the enzyme-linked immunosorbent assay (ELISA) and the proliferation rate was assessed by the MTT assay. Alpha error levels were set as 0.05. Results: CsA stimulated significantly higher levels of IL-6, IL-8 and TGF-β1 in adult gingival fibroblasts than it did in the control group; whereas, the expression of IL-1β and PGE 2 in the fibroblasts exposed to CsA was significantly weaker (P < 0.05). The fibroblasts in the two groups did not reveal any noticeable difference in the production of TNF-α. Furthermore, cell proliferation in the CsA group was not significantly higher than that in the control group. No significant differences in cytokines TNF-α and IL-1β were noted between the two groups. The results indicated that CsA stimulated cell proliferation in the pediatric fibroblast cell line. Comparison between the results in the adult and pediatric groups demonstrated that the levels of IL-1β, IL-6, IL-8, and PGE 2 were significantly higher in the pediatric group than in the adult group; however, statistics showed no significant difference in the levels of TNF-α and TGF-β1 and CsA-induced proliferation between these two groups. Conclusions: The mechanism of a CsA-induced fibroblast overgrowth may converge on the steps involving fibroblast proliferation and cytokine network including IL-6, IL-8, IL-1β, TGF-β1, and PGE 2 , in both adults and pediatrics. As the prevalence and intensity of drug-induced gingival overgrowth is more serious in the pediatrics. As group than in adults, we suggest that more studies be conducted on the pediatric group

Root-end filling with cement-based materials: An in vitro analysis of bacterial and dye microleakage

Background: One ideal property of a root-end filling material is its apical sealing ability. The aim of this in vitro study was to assess bacterial and dye microleakage of white and gray mineral trioxide aggregate (WMTA and GMTA), Portland cement and calcium-enriched mixture (CEM) cement used as root-end filling material, and to assess the agreement between these two test methods. Materials and Methods: Fifty-four single-rooted teeth were used. The roots were randomly divided into four study and two control groups. After decoronation, root canals were instrumented and filled with gutta-percha and AH26 sealer. Root-ends were resected 3 mm above the root-end and 3 mm deep cavities were prepared. Root-end cavities were filled with each material. Enterococcus faecalis and methylene blue dye were used for determination of bacterial and dye leakage respectively. Data were analyzed using Fisher′s Exact Test, one-way ANOVA, Kaplan-Meier analysis, and Cohen′s Kappa. Results: There was 100% bacterial leakage in Portland cement and CEM cement, 58.3% in GMTA, and 91.7% in WMTA. GMTA showed significantly less bacterial leakage than Portland cement and CEM cement ( P < 0.05). In those samples with leakage occurrence, times of observation of leakage were not significantly different; however, by survival analysis, the results of the GMTA group were significantly different from those of the CEM cement and Portland groups. The difference in complete dye leakage was not significant. There was poor agreement between dye and bacterial leakage methods. Conclusion: CEM cement provides leakage results comparable to other commonly used root-end filling materials such as WMTA

Comparing the sealing properties of mineral trioxide aggregate and an experimental ceramic based root end filling material in different environments

Background: An apical seal is an important factor in achieving success in surgical endodontics. Aim: The purpose of this study was to compare the sealing properties of mineral trioxide aggregate (MTA) with a new ceramic based root end filling material (Cold Ceramic) in different environments. Materials and Methods: One hundred teeth were selected. The root canals were instrumented and obturated. Except for the apical 2 mm, the root surfaces were sealed. After root resection, 3 mm depth root-end cavities were prepared. For each material, roots were divided into 3 equal subgroups and the root-end filling was done in different environments (dry, saliva contaminated, blood contaminated). Five roots served as positive and 5 roots as negative controls. Samples were immersed in 2% methylene blue dye. Roots were sectioned longitudinally and examined under stereomicroscope to record the extension of dye penetration. Results: All experimental groups demonstrated dye penetration. The lowest linear leakage was seen in Cold Ceramic blood contaminated group while the highest leakage was observed in MTA blood contaminated group. The linear dye penetration of both MTA and Cold Ceramic (CC) groups did not show any significant differences among different environments. Also, the difference between MTA and CC was not significant in dry and saliva contaminated subgroups. Only the difference between dye penetration of MTA and CC in blood contaminated subgroups showed significant difference ( P = 0.008). Conclusion: The sealing property of this ceramic based root end filling material (Cold Ceramic) is better than MTA in blood contaminated condition and at least similar to MTA in other conditions

Cytotoxicity evaluation of three resin-based sealers on an L929 cell line

Background: Endodontic sealers usually come in contact with adjacent tissues and their biocompatibility is key in a successful treatment. The purpose of this study was to assess the cytotoxicity of three resin-based sealers, namely AH Plus, EndoREZ, and Epiphany in set and fresh states on an L929 cell line. Materials and Methods:In this in vitro experimental study, the materials were mixed according to the manufacturers′ instructions, and were divided into two groups, fresh and set. The elutes of materials were prepared separately and were incubated with L929 fibroblasts for 1 hour, 24 hours, and 72 hours. Pulp Canal Sealer and Dulbecco′s Modified Eagle Medium (DMEM) served as positive and negative controls respectively. Cell viability was evaluated by MTT assay ([3-4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide succinate), after 1 hour, 24 hours, and 72 hours. The data were analyzed by analysis of variance (ANOVA), and Tukey multiple comparison test. Results: After 1 hour, fresh Epiphany and fresh AH Plus were significantly more cytotoxic than their set samples. No significant difference was perceived between cytotoxicity of fresh state of sealers and positive control, or between set state and negative control. After 24 hours, both fresh and set samples of all materials were significantly more cytotoxic than the negative control group, and were less cytotoxic than the positive control group. After 72 hours, the fresh and set samples of all materials were as cytotoxic as the positive control group. At each time point, no significant difference was perceived among different materials in terms of cell viability. Conclusion: The observed differences among the cytotoxicity of AH Plus, EndoREZ, and Epiphany did not reach a significant level at comparable time points after exposure