The Lysyl Oxidase G473A Polymorphism Exacerbates Oral Cancer Development in Humans and Mice.

Oral cancer is primarily squamous-cell carcinoma with a 5-year survival rate of approximately 50%. Lysyl oxidase (LOX) participates in collagen and elastin maturation. The propeptide of LOX is released as an 18 kDa protein (LOX-PP) in the extracellular environment by procollagen C-proteinases and has tumor-inhibitory properties. A polymorphism in the propeptide region of LOX (rs1800449, G473A) results in a single amino acid substitution of Gln for Arg. Here we investigated the frequency of rs1800449 in OSCC employing TCGA database resources and determined the kinetics and severity of precancerous oral lesion development in wildtype and corresponding knockin mice after exposure to 4-nitroquinoline oxide (4 NQO) in drinking water. Data show that the OSCC is more common in humans carrying the variant compared to the wildtype. Knockin mice are more susceptible to lesion development. The immunohistochemistry of LOX in mouse tissues and in vitro studies point to a negative feedback pathway of wildtype LOX-PP on LOX expression that is deficient in knockin mice. Data further demonstrate modulations of T cell phenotype in knockin mice toward a more tumor-permissive condition. Data provide initial evidence for rs1800449 as an oral cancer susceptibility biomarker and point to opportunities to better understand the functional mechanism of LOX-PP cancer inhibitory activity.

Antimicrobial Activity and Biocompatibility of Antibiotic-Loaded Chitosan Hydrogels as a Potential Scaffold in Regenerative Endodontic Treatment.

INTRODUCTION: The objective of this study was to determine the effectiveness of several antibiotic-loaded hydrogel scaffolds against Enterococcus faecalis, as well as their ability to stimulate proliferation and mineralization of dental pulp stem cells. METHODS: Fibrin (Fg) or chitosan-fibrin hydrogels (Ch) were prepared using 12.5 mg/mL fibrinogen and 0.4% (w/v) chitosan. Triple antibiotics, clindamycin-modified triple antibiotic paste, or double antibiotics were loaded in gels (1 mg/mL). Antibacterial effect against E. faecalis biofilm was determined by using colony-forming units (CFUs) and confocal laser scanning microscope (CLSM). Cell viability and morphology were determined by loading cells into different gels at 7 and 14 days using the water-soluble tetrazolium salt-1 cell viability assay and Live & Dead cell analysis. Mineralization was detected by using alkaline phosphatase and alizarin red staining activity. RESULTS: Antibiotic-loaded Fg gel and Ch gel alone without antibiotics resulted in a significant reduction in CFUs compared with the positive control (P < .05). When antibiotics were loaded in Ch gel, there were no CFUs detected in any groups (P < .05). CLSM images showed dense red areas with mostly dead bacteria on the dentin surface in antibiotic-loaded Ch groups, which showed significantly less live bacteria compared with the other groups (P < .05). Triple antibiotic-loaded Fg and Ch gels resulted in a dramatic decrease in the mineralized nodule formation compared with all other gel groups (P < .05). Ch hydrogels resulted in round cell morphology up to 7 days. Ch alone or with double antibiotic paste showed more cell spreading with spindle-shaped morphology at 14 days and higher alkaline phosphatase activity compared with other antibiotic-loaded Ch groups (P > .05). CONCLUSIONS: Double antibiotic-loaded Ch gel appears to enhance the antibacterial properties while maintaining higher cell viability, cell spreading, and mineralization activity, compared with all the other scaffolds investigated.

Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB.

Extracellular lysyl oxidases (LOX and LOXL1-LOXL4) are critical for collagen biosynthesis. LOXL2 is a marker of poor survival in oral squamous cell cancer. We investigated mechanisms by which tumor cell secreted LOXL2 targets proximal mesenchymal cells to enhance tumor growth and metastasis. This study identified the first molecular mechanism for LOXL2 in the promotion of cancer via its enzymatic modification of a non-collagenous substrate in the context of paracrine signaling between tumor cells and resident fibroblasts. The role and mechanism of active LOXL2 in promoting oral cancer was evaluated and employed a novel LOXL2 small molecule inhibitor, PSX-S1C, administered to immunodeficient, and syngeneic immunocompetent orthotopic oral cancer mouse models. Tumor growth, histopathology, and metastases were monitored. In vitro mechanistic studies with conditioned tumor cell medium treatment of normal human oral fibroblasts were carried out in the presence and absence of the LOXL2 inhibitor to identify signaling mechanisms promoted by LOXL2 activity. Inhibition of LOXL2 attenuated cancer growth and lymph node metastases in the orthotopic tongue mouse models. Immunohistochemistry data indicated that LOXL2 expression in and around tumors was decreased in mice treated with the inhibitor. Inhibition of LOXL2 activity by administration of PXS-S1C to mice reduced tumor cell proliferation, accompanied by changes in morphology and in the expression of epithelial to mesenchymal transition markers. In vitro studies identified PDGFRß as a direct substrate for LOXL2, and indicated that LOXL2 and PDGF-AB together secreted by tumor cells optimally activated PDGFRß in fibroblasts to promote proliferation and the tendency toward fibrosis via ERK activation, but not AKT. Optimal fibroblast proliferation in vitro required LOXL2 activity, while tumor cell proliferation did not. Thus, tumor cell-derived LOXL2 in the microenvironment directly targets neighboring resident cells to promote a permissive local niche, in addition to its known role in collagen maturation.

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<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.

Outcomes of revascularization treatment in immature dog's teeth.

AIM: The purpose of the current study was to examine the success rate of a revascularization treatment protocol involving canal space disinfection using copious irrigation, a triantibiotic dressing, and induction of a blood clot matrix in immature dog's teeth. MATERIALS AND METHODS: Thirty-six immature mongrel dog's teeth were divided into two experimental and two control groups. The experimental groups included a necrotic-infected group (n = 20) and a vital group (n = 10). In the group with the necrotic-infected teeth, periapical lesions were induced, and disinfection of the canals was carried out using copious irrigation and a triple antibiotic medication (metronidazole, ciprofloxacin, and tetracycline). Subsequently, the periapical tissues were irritated to initiate bleeding, producing a blood clot. A double seal of the coronal access was then placed. In the vital group, the pulp was aseptically removed before the canal was irrigated and periapical tissues irritated to induce bleeding. The same protocol as that used for the necrotic-infected group was used to seal the coronal access. In the positive control group (n = 3), after pulp removal, sterile sponges soaked in plaque suspension were placed in the pulp chambers of the teeth, after which the chambers were sealed. In the negative control group (n = 3), one untouched 1st premolar tooth in each dog was assigned and left to develop naturally. Radiographic and histological findings were evaluated at 3 and 6 months. Data analysis was performed using Fisher's exact test. RESULTS: The necrotic-infected group radiographically demonstrated apical healing and apical closure in 70% of the cases and thickening of the walls in 40% after 6 months. The vital group showed apical closure in 77% and thickened walls in 44% of the cases after 3 months. Histological findings confirmed the radiographic findings. No significant difference was observed between the two groups (P > 0.05). CONCLUSIONS: If necrotic-infected canals are effectively disinfected and treated according to the protocol, the ensuing revascularization response is similar to that of vital immature teeth.

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.

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.

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 PGE2, IL-1ß, IL-6, IL-8, TNF-α, and TGF-ß1 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-ß1 in adult gingival fibroblasts than it did in the control group; whereas, the expression of IL-1ß and PGE2 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-1ß 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-1ß, IL-6, IL-8, and PGE2 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-ß1 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-1ß, TGF-ß1, and PGE2, 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.

Antibacterial effect of different concentrations of garlic (Allium sativum) extract on dental plaque bacteria.

BACKGROUND: Allium sativum, commonly known as garlic, exhibits antibacterial effects against a wide range of bacteria. AIM: The objective of this in vitro study was to assess the antibacterial effect of different concentrations of garlic extract against human dental plaque microbiota. MATERIALS AND METHODS: Antibacterial activities of four different concentrations of garlic extract (5%, 10%, 20%, and 100%) were evaluated against Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Pseudomonas aeruginosa, and lactobacillus spp. using the disk diffusion method. Papers soaked in 0.2% concentration chlorhexidine gluconate and saline were used as positive and negative controls, respectively. The data were subjected to one-way ANOVA and the Tukey multiple comparisons test at a 5% significance level. RESULTS: All bacterial strains were inhibited by all test materials. The inhibition zones of the different concentrations of garlic extract were not significantly different for S. mutans, S. sanguis, and S. salivarius. For P. aeruginosa and lactobacillus spp. the inhibition zones of 5%, 10% and 20% concentrations were not significantly different from one another, but they were significantly more than that of the 100% extract. CONCLUSION: The 5%, 10%, 20%, and 100% concentrations of garlic extract had similar effects, so further studies seem to be indicated on the usefulness of the 5% extract.

Assessment of antifungal activity of Proroot mineral trioxide aggregate and mineral trioxide aggregate-Angelus.

BACKGROUND: Fungi may play a key part in periradicular diseases. The aim of this study was to evaluate and compare the antifungal properties of two root-end filling materials, ProRoot Mineral trioxide aggregate (MTA) and MTA-Angelus, against Candida albicans using tube-dilution test. MATERIALS AND METHODS: The antifungal properties of ProRoot MTA and MTA-Angelus against C. albicans was assessed at 1, 24, and 48 hours following administration of two concentrations of the antifungal agents (50 and 100 mg/ml). A total of 50 culture wells were divided into four experimental groups (Freshly mixed MTA, Freshly mixed MTA-Angelus, 24-h set MTA, and 24-h set MTA-Angelus) and two control groups. Each well was prepared for one specific agent with a specific concentration. For the set groups, the mixture was prepared and left for 24 hours. One milliliter of suspension of fungal colonies with concentration of 10(4) CFU/ml was then added to the mixtures in each well. All wells were incubated at 37°C and assessed at 1, 24, and 48 hours. This observation was based on the turbidity of the suspension in the tubes. At each time point, 0.02 ml of each suspension was cultured on a Sabouraud dextrose agar plate to confirm C. albicans growth. The results were analyzed using Kruskal-Wallis test. RESULTS: Although all fresh and set samples were incapable of killing C. albicans at 1 hour, they demonstrated fungicidal ability on agar plates at 24 and 48-hour time points. CONCLUSION: MTA-angelus proved to be an effective antifungal agent compared to ProRoot MTA at concentrations of 50 mg/ml and 100 mg/ml.

The immunohistochemical characterization of MMP-2, MMP-10, TIMP-1, TIMP-2, and podoplanin in oral squamous cell carcinoma.

OBJECTIVES: The aim of this study was to immunohistochemically evaluate the expression of matrix metalloproteinase (MMP)-1, MMP- 2, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and podoplanin in oral squamous cell carcinoma (OSCC). Immunohistochemical staining of podoplanin-positive lymphatic vessel density (LVD) was also assessed. STUDY DESIGN: Forty cases of OSCC were analyzed by immunohistochemistry. RESULTS: MMP-2, MMP-10, TIMP-1, TIMP-2, and podoplanin were detected in each of the 40 OSCC cases. The expression of MMP-2 was significantly correlated with histologic grade. The expression of podoplanin was positively correlated with gender and negatively correlated with tumor size. A significant positive correlation was also detected between LVD and the presence of lymph node metastases, gender, age, and diameter of the lymph node (if involved), as well as histologic grade. CONCLUSIONS: The results are suggestive of important roles that MMP-2, MMP-10, TIMP-2, and podoplanin play in pathologic processes of OSCC, including invasion. Our findings also suggest that LVD may play a role in lymphatic metastasis and tumor progression.

Efficacy comparison of periapical infiltration injection of dexamethasone, morphine and placebo for postoperative endodontic pain.

OBJECTIVE: To evaluate the efficacy of periapical infiltration injection of dexamethasone and morphine in reducing postoperative endodontic pain. METHOD: Ninety patients participated in this double-blind randomised controlled clinical trial. They were referred to the dental school of Shahid Beheshti Medical University, Tehran, Iran for conventional endodontic treatment of molar teeth. The canals of each tooth were completely prepared with cleansing and shaping. The patients were randomly divided into three experimental groups to receive dexamethasone, morphine or normal saline (1 mL). Patients were then instructed to complete a pain diary 4, 8, 24 and 48 h after the appointment. Statistical analysis consisted of chi-squared test, analysis of variance and Kruskal-Wallis test. RESULTS: There was a statistically significant correlation between dexamethasone or morphine treatment and decreased levels and incidence of endodontic pain at 4, 8 and 24 h, but not at 48 h (P < 0.05). It was also observed that dexamethasone was significantly more effective (56.7% no pain) than morphine (43.3% no pain). CONCLUSION: Periapical infiltration of dexamethasone and morphine led to a considerable decrease in postoperative endodontic pain during the first 24 h after operation. Dexamethasone was more effective than morphine in pain reduction.

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.

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 AND 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.