Petasin is the main component responsible for the anti-adipogenic effect of Petasites japonicus.

Petasites japonicus is one of the most popular edible wild plants in Japan. Many biological effects of P. japonicus have been reported, including anti-allergy, anti-inflammation, and anticancer effects. Although its anti-obesity effect has been reported in several studies, the most important component responsible for this activity has not been fully elucidated. On screening the components that suppress adipocyte differentiation in 3T3-F442A cells, we found that the extract of the flower buds of P. japonicus has anti-adipogenic effect. Among the known major components of P. japonicus, petasin exhibited a potent anti-adipogenic effect at an IC50 value of 0.95 µM. Quantitative analysis revealed that the active component responsible for most of the anti-adipogenic effects of P. japonicus extract is petasin. Petasin suppressed the expression of markers of mature adipocytes (PPARγ, C/EBPα, and aP2). However, as isopetasin and petasol, analogs of petasin, did not exhibit these effects, it indicates that a double bond at the C11-C12 position and an angeloyl ester moiety were essential for the activity. Petasin affected the late stage of adipocyte differentiation and inhibited the expression of lipid synthesis factors (ACC1, FAS, and SCD1). Additionally, it was revealed that petasin could be efficiently extracted using hexane with minimal amount of pyrrolizidine alkaloids, the toxic components. These findings indicate that P. japonicus extract containing petasin could be a promising food material for the prevention of obesity.

Antiproliferative activity of 3,5-disubstituted tetrahydro-2H-1,3,5-thiadiazine thione (THTT) derivatives and evaluation as potential prodrug.

Four series of tetrahydro-2H-1,3,5-thiadiazine thione derivatives were screened for their in vitro antiproliferative activities against two human cancerous PC3 and HeLa cell lines. The cytotoxicity of all the compounds (series A-D) was also determined on mammalian mouse fibroblast 3T3 cells. Most of the compounds showed significant anticancer potential against both cancer cell lines within the range of IC50 = 6.4-29.9 and 2.4-23.8 M respectively when compared with standard doxorubicin (IC50 = 0.3 M). All compounds demonstrated a notable selectivity for Hela cells and found either non-toxic or relatively less toxic for 3T3 cell lines model. The structure-activity relationship indicated that antiproliferative activity mainly influenced by the nature and position of substituents at thidiazine nucleus. In general, the presence of aryl groups for example 3,4-(OMe) 2.Bzl and CH(Ph)Me at N-3 position resulted in a significant activity. Under enzymatic hydrolysis, complete conversion (100%) of ester derivative of thiadiazine thione (10a) into its acidic counterpart (7c) was achieved during 20 min which indicated that these types of THTT ester derivatives can be a possible lead for future investigations as prodrug anticancer probes.

Targeted photodynamic therapy selectively kills activated fibroblasts in experimental arthritis.

OBJECTIVE: In RA, synovial fibroblasts become activated. These cells express fibroblast activation protein (FAP) and contribute to the pathogenesis by producing cytokines, chemokines and proteases. Selective depletion in inflamed joints could therefore constitute a viable treatment option. To this end, we developed and tested a new therapeutic strategy based on the selective destruction of FAP-positive cells by targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 coupled to the photosensitizer IRDye700DX. METHODS: After conjugation of IRDye700DX to 28H1, the immunoreactive binding and specificity of the conjugate were determined. Subsequently, tPDT efficiency was established in vitro using a 3T3 cell line stably transfected with FAP. The biodistribution of [111In]In-DTPA-28H1 with and without IRDye700DX was assessed in healthy C57BL/6N mice and in C57BL/6N mice with antigen-induced arthritis. The potential of FAP-tPDT to induce targeted damage was determined ex vivo by treating knee joints from C57BL/6N mice with antigen-induced arthritis 24 h after injection of the conjugate. Finally, the effect of FAP-tPDT on arthritis development was determined in mice with collagen-induced arthritis. RESULTS: 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. Accumulation of the anti-FAP antibody in arthritic knee joints was not affected by conjugation with the photosensitizer. Arthritis development was moderately delayed in mice with collagen-induced arthritis after FAP-tPDT. CONCLUSION: Here we demonstrate the feasibility of tPDT to selectively target and kill FAP-positive fibroblasts in vitro and modulate arthritis in vivo using a mouse model of RA. This approach may have therapeutic potential in (refractory) arthritis.

Resveratrol Attenuates Lipopolysaccharides (LPS)-Induced Inhibition of Osteoblast Differentiation in MC3T3-E1 Cells.

BACKGROUND LPS-inhibited osteoblastic differentiation plays an important role in the pathogenesis of osteomyelitis. Thus, searching for drugs that affect LPS-mediated osteoblastic differentiation may be crucial in developing therapies for osteomyelitis. The purpose of this study was to investigate the role and mechanisms of resveratrol, a natural polyphenol present in red wine, on LPS-inhibited osteoblastic differentiation. MATERIAL AND METHODS Cell viability was measured by MMT assay. Mitochondrial ATP levels, membrane potential, and superoxide production were measured to evaluate the effects of LPS and resveratrol on mitochondrial functions in osteoblast-like MC3T3-E1 cells. Osteoblast-related genes, including ALP, OCN, OPN, and RUNX2, were measured by ELISA analysis and RT-PCR in differentiated osteoblast cells treated with LPS and resveratrol. Cellular Sirt1 and PCG-1α levels were measured by Western blot to probe the impact of resveratrol treatment in LPS-stimulated MC3T3-E1 osteoblasts. RESULTS The results showed that LPS caused significant mitochondrial dysfunctions of MC3T3-E1 cells in a dose-dependent manner, which were attenuated by resveratrol. Furthermore, LPS markedly decreased the expression of ALP, OCN, OPN, and RUNX2 in MC3T3-E1 cells cultivated in osteoblast differentiation medium, suggesting that LPS inhibited the osteoblastic differentiation of MC3T3-E1 cells. However, resveratrol obviously alleviated the suppressive impact of LPS on osteoblast differentiation. In addition, resveratrol increased expression of Sirt1 and PGC-1α in MC3T3-E1 cells treated with LPS. CONCLUSIONS Taken together, these results show that resveratrol alleviated the suppression of LPS on osteoblast differentiation by improving, at least in part, mitochondrial function.

Cytotoxicity of a New Calcium Silicate Endodontic Sealer.

INTRODUCTION: Resin sealers with biocompatible and bioactive additives have been used in clinical practice. Recently, a calcium silicate root canal sealer was introduced under the name BioRoot RCS (Septodont, Saint Maur-des-Fossés, France). The aim of this study was to evaluate the effects of BioRoot RCS on cell survival and proliferation of cultured cells in parallel with an epoxy resin sealer with calcium phosphate and calcium oxide and a salicylate resin sealer with mineral trioxide aggregate filler. The tested hypothesis was that BioRoot RCS is significantly less cytotoxic than the other tested sealers. METHODS: The experiments were performed on NIH/3T3 cells (American Type Culture Collection, Manassas, VA) grown as monolayer cultures at 37°C in atmosphere containing 5% CO2 in air and 100% relative humidity. The sealers' extracts (24 hours and 1 week) were applied to cells at 1:1 and 1:2 dilutions. The effect was assessed by a modified sulforhodamine B staining assay in reference to controls after 24 and 72 hours of exposure. All experiments were performed at least twice in 6 replicates. Analysis of variance and post hoc comparison tests were used to evaluate the statistical significance of the results at a level of significance of P = .05. RESULTS: BioRoot RCS was significantly less cytotoxic than the other 2 sealers. MTA-Fillapex (Angelus, Londrina, Brazil) and SimpliSeal (Discuss Dental, LLC, Calver City, CA) exhibited a similar antiproliferative profile with no statistically significant differences in all settings. CONCLUSIONS: BioRoot RCS showed quite a positive biological behavior. Further investigation is needed in order to clarify the mechanism and the components that contribute to the beneficial results observed.

Synthesis of Oleanolic Acid Analogues and Their Cytotoxic Effects on 3T3 Cell Line.

BACKGROUND: Oleanolic acid (OA) is a known natural compound with many important biological activities. Thirteen oleanolic acid derivatives linked at C-3 and C-28 were synthesized and their structures were confirmed by 1H- and 13C NMR and mass spectral analyses. Among them, compounds 4, 6, 8-10, 12, 13 were synthesized for the first time. They were evaluated for their cytotoxic activity. They showed proliferative effect at low concentrations while cytotoxic effect was observed at high concentrations in a dose dependent manner. METHODS: We have first synthesized compounds 1 and 2 from the reaction of methyl iodide and OA. Compound 1 was reduced with LiAlH4 to give compound 3, and compound 2 gave compound 9 with MOMBr as a new compound. The compound 10 was then obtained from the reduction of compound 9 with LiAlH4 as a new oleanolic acid derivative. A diol derivative 11 was synthesized from OA and LiAlH4 at the room temperature. Compound 4 was obtained from the reaction of compound 3 with CBr4 as a new analogue of OA, and the reduction of compound 4 afforded compound 5 as a known product. In addition, we synthesized compounds 6-8 from compound 3 using MsCl, MeI and p-nitrobenzoyl chloride, respectively, in good yields. Compounds 6 and 8 are new analogues of OA. The new compounds 12 and 13 were also synthesized starting from OA using with MOMBr and TBDMSiCl as the reagents. The all synthesized compounds were purified by using column chromatography and/or crystallization. RESULTS: In the present study, thirteen OA derivatives linked at C-28 and (or) C-3 were synthesized and evaluated for their cytotoxic activity on 3T3 cell lines which are the standard fibroblast cell lines, derived from Swiss albino mouse embryo tissue. 3T3 cell viability was observed at low concentrations of the tested triterpenoids while they displayed anti-proliferative effect at higher concentrations. CONCLUSION: Oleanolic acid 28-methyl ester (2) showed fairly different behavior from all the other compounds tested and found to be the least cytotoxic compound. However, at 200 µM concentration, it exhibited the same cytotoxicity with compounds 3, 9 and 10 around 58-59%. Among the tested 13 compounds, 7 exhibited the most drastic decline for the viability from 12,5 µM to 25 µM concentration. Compound 6 displayed the most cytotoxic effect, almost in all concentrations, particularly at 6.25 and 25 µM concentrations while the highest cytotoxic effect at 50 µM was observed for compound 11 among all the tested triterpenoids. As a result, all the tested OA derivatives showed proliferative effect at 1,56 µM although no proliferative effect was observed for OA. Moreover, OA exhibited higher cytotoxic effect than its derivatives, particularly at higher concentrations (50, 100, 200 µM) with an exception for compound 11. Because, the latter showed highest proliferative effect at lowest concentration, and highest anti-proliferative effect at highest concentration which surpassed all the OA derivatives.

Re-establishment of Biocompatibility of the In Vitro Contaminated Titanium Surface Using Osteoconductive Powders With Air-Abrasive Treatment.

To achieve re-osseointegration on implant surfaces exposed to peri-implant infections, treatment should re-establish biocompatibility. The aim of this study was to test whether air powder abrasive treatment (APA) using osteoconductive powders can, in addition to cleaning, increase the biocompatibility of the contaminated implant surface. Ninety-six in vitro Ca-precipitated, organic film layer-coated sandblasted and acid-etched titanium discs were treated by APA using erythritol, hydroxylapatite (HA), and biocalcium phosphate (BioCaP) powders (n = 16 per group). Six treatment modalities were created (HA or erythritol cleaning with/without BioCaP coating). MC3T3-E1cells were seeded on discs, and cell attachment, viability, proliferation, and differentiation were evaluated. Pristine discs were used as control (control 1). Contaminated and nontreated discs were used as control (control 2). The cells were stretched and attached in all test groups. The cell viability and proliferation (DNA amount) in all test groups were significantly higher than in the pristine and contaminated disc groups. There was no significant difference between the test groups. The differentiation (alkaline phosphatase activity) of the cells on treated discs was significantly higher than on the contaminated discs but lower than in the pristine group. The cell viability in control 2 was significantly lower than the control 1. The APA with osteoconductive powder on contaminated titanium surfaces promoted the cell viability, proliferation, and differentiation of the MC3T3-E1 cells. The biocompatibility of the surface was higher than that of the contaminated discs. The tested aspects of cell response, with the exception of differentiation, reached to the level of the pristine surface. The in vitro results showed that APA with osteoconductive powders could be a promising method for implant surface treatment.

Efficacy and tolerability of Meratrim for weight management: a randomized, double-blind, placebo-controlled study in healthy overweight human subjects.

BACKGROUND: Meratrim is a blend of two plant extracts obtained from Sphaeranthus indicus flower heads and Garcinia mangostana fruit rinds. Previous studies have demonstrated that Meratrim is effective for weight management in obese individuals. The objective of this study was to assess the efficacy and tolerability of Meratrim in managing body weight in healthy overweight subjects. METHODS: Sixty participants with a mean BMI of 28.3 kg/m(2) were randomized into two groups receiving either 400 mg of Meratrim twice daily or two identical placebo capsules for a period of 16 weeks. Subjects were asked to consume about 2,000 kcal/day throughout the study period and walk 5 days a week for 30 min daily. The primary endpoint was defined as the change in body weight from baseline to end of week 16 for the Meratrim group versus placebo. Fifty seven subjects completed the trial. RESULTS: At study conclusion, statistically significant reductions in body weight (5.09 vs. 1.1 kg; p < 0.0001), BMI (1.91 vs. 0.43 kg/m(2); p < 0.0001), waist (9.97 vs. 3.71 cm; p < 0.001) and hip size (10.38 vs. 5.11 cm; p < 0.0001) were observed in the Meratrim versus the placebo group. Additionally, a significant change in serum LDL (-14.79 vs. 6.25 mg/dL; p < 0.0001), triglycerides (-43.62 vs. -13.68 mg/dL; p < 0.001) and total cholesterol (-20.0 vs. -0.75 mg/dL; p = 0.0002) was observed in the Meratrim cohort compared to the placebo. No supplementation related adverse events were noted during the study. CONCLUSIONS: The study findings suggest that Meratrim is well-tolerated and is an effective ingredient for weight management in healthy overweight subjects. TRIAL REGISTRATION: CTRI/2014/07/004727; www.CTRI.nic.in.

4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) promote adipogenesis in 3T3-L1 adipocyte cell culture.

4,4'-Dichlorodiphenyltrichloroethane (DDT), a chlorinated hydrocarbon insecticide, was extensively used in the 1940s and 1950s. DDT is mainly metabolically converted into 4,4'-dichlorodiphenyldichloroethylene (DDE). Even though most countries banned DDT in the 1970s, due to the highly lipophilic nature and very stable characteristics, DDT and its metabolites are present ubiquitously in the environment, including food. Recently, there are publications on relationships between exposure to insecticides, including DDT and DDE, and weight gain and altered glucose homeostasis. However, there are limited reports regarding DDT or DDE and adipogenesis, thus we investigated effects of DDT and DDE on adipogenesis using 3T3-L1 adipocytes. Treatment of DDT or DDE resulted in increased lipid accumulation accompanied by increased expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome-proliferator activated receptor-γ (PPARγ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), adipose triglyceride lipase, and leptin. Moreover, treatment of DDT or DDE increased protein levels of C/EBPα, PPARγ, AMP-activated protein kinase-α (AMPKα), and ACC, while significant decrease of phosphorylated forms of AMPKα and ACC were observed. These finding suggest that increased lipid accumulation caused by DDT and DDE may mediate AMPKα pathway in 3T3-L1 adipocytes.

A new method for the separation and purification of the osteogenic compounds of nacre Ethanol Soluble Matrix.

Nacre is able to induce bone-forming cells mineralization, and gains widely interest in bone regeneration. While, the osteoinductive compounds are not yet identified. ESM (Ethanol Soluble Matrix), a nacre extract from powder of Pinctada margaritifera pearl oyster shell, has been firstly proven having the capacity to induce mineralization and to restore mineralization defect in vitro. It is suitable to treat ESM as a source of osteoinductive compounds. Herein, we develop a new method for separating and purifying nacre extracts by an ionic approach. At first, cationic ESM (ESMc) and anionic ESM (ESMa) were achieved with ion-exchange resin. Then, ESM was separated and collected on cation exchange HPLC. Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectrometry (EDS) was used to reveal the concentrated elements in ESM fractions. A coupled cell models were used to test the ESM fractions. Alizarin Red staining was performed and quantified to evaluate the mineralization level. ESMc and 2 HPLC fractions stimulated the mineralization in both cells. EDS demonstrated the abundant presence of calcium and chloride in the osteogenic fractions. To validate, pure CaCl2 was tested and proven having an osteogenic effect in both cells, but less stable than ESM. The mineralization nodules induced by ESM fractions and CaCl2 differed in both cells. In conclusion, a new method was developed for separating and purifying nacre extracts by an ionic approach. By which, the osteoinductive compounds in ESM were proven cationic, and calcium in ESM was demonstrated to play a role in inducing the cell mineralization.

Various fractions of Hypericum x moserianum and Hypericum ericoides possess antiglycation, anti-lipid peroxidation, antioxidative activities and non-toxic effects in vitro.

In the present study, two species Hypericum x moserianum and Hypericum ericoides which belong to genus Hypericum were evaluated for their potential antiglycation, antioxidant, anti lipid peroxidation and cytotoxic activities. These species are widely used in folk medicine and to the best of our knowledge there were no previous reports regarding antioxidant, anti-glycation and cytotoxicity studies of these species. Among the crude methanol extracts and fractions of both the species, the ethyl acetate fraction of H. x moserianum exhibited promising antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 129.084±1.215µg/ml, followed by methanol extract (IC50=232.083 ± 1.215µg/ml) and aqueous fraction (IC50=266.962 ±2.213 µg/ml). The ethyl acetate fraction of H. ericoides exhibited IC50 value of 295.088 ± 2.320 µg/ml. In antiglycation assay, the ethyl acetate fraction of H. x moserianum showed 52.096% inhibition at 500µg/ml. For lipid peroxidation assay, the dichloromethane, aqueous and n-hexane fractions of H. x moserianum showed 67.241, 66.147 and 64.213% inhibition respectively, while aqueous fraction of H. ericoides exhibited 67.404% inhibition at 500µg/ml. In cytotoxicity assay, all fractions of both the species were found to be non-toxic on mouse fibroblast 3T3 cells with IC50 value greater than 30µg/ml as compared to cycloheximide with IC50 value 0.073±0.1µg/ml used as a standard. It was concluded from the study that among the two species, crude methanolic and ethyl acetate fractions were more active regarding the antioxidant, anti-glycation activities while dichloromethane, aqueous and n-hexane fractions possessed anti-lipid peroxidation activity.

Cell-targeted platinum nanoparticles and nanoparticle clusters.

Herein, we report the facile preparation of cell-targeted platinum nanoparticles (PtNPs), through the design of peptides that, as a single molecule added in small concentration during the synthesis, control the size of PtNP clusters during their growth, stabilise the PtNPs in aqueous suspension and enable the functionalisation of the PtNPs with a versatile range of cell-targeting ligands. Water-soluble PtNPs targeted respectively at blood group antigens and at integrin receptors are demonstrated.

Investigation of skin permeation, ex vivo inhibition of venom-induced tissue destruction, and wound healing of African plants used against snakebites.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation causes 5000-10,000 mortalities and results in more than 5-15,000 amputations in sub-Saharan Africa alone every year. The inaccessibility of antiserum therapy is a vast problem, and only about 2.5% of the actual need for antiserum in Africa is covered. Numerous plants have shown in vitro inhibitory activity against one or more of the hydrolytic enzymes involved in snakebite-induced necrosis. However, a more thorough examination of the plant species in ex vivo and in vitro cell assay models is needed to test their ability to inhibit necrosis. MATERIALS AND METHODS: Extracts which had previously shown in vitro inhibitory activity against necrosis enzymes, were tested in an ex vivo air-liquid-interface model, and a wound healing scratch assay as well as for their ability to permeate the skin barrier and inhibit venom induced cell death. RESULTS: Of the 14 water extracts and 16 ethanol extracts tested at a concentration of 10 µg/mL, only the ethanol extracts of Tamarindus indica and Paullinia pinnata resulted in a small but significant increase in cell migration of around 10% compared to treatment with buffer after 24h treatment. The remaining extracts showed no effect, or they even delayed the cell migration compared to the treatment with buffer. After 48 h treatment, 10 of the tested extracts showed a decreased cell migration compared to no treatment. At a 100 µg/mL concentration all the extracts inhibited cell migration and five extracts killed some of the cells, while four extracts killed all the cells. Ten of the thirty extracts were tested in a Franz cell set-up but none of the extracts tested did permeate the skin barrier over a 48 h period, and will therefore be of very limited use topically in the initial treatment of snakebites in its present form. None of the extracts were able to directly interact with the enzyme to lower the cell toxicity of the venom. Two extracts, Dichrostachys cinerea and Grewia mollis, were tested in the ex vivo model, but none of them inhibited the tissue destruction caused by venom. CONCLUSION: On the basis of this study, topical treatment with plant extracts for snakebite-induced tissue necrosis cannot be recommended.

Cytotoxic activity of chimeric protein PD-L4UWSCI(tr) does not appear be affected by specificity of inhibition mediated by anti-protease WSCI domain.

In a previously study, a type 1 ribosome inactivating protein (PD-L4) and a wheat subtilisin/chymotrypsin inhibitor (WSCI) were engineered into a chimeric protein (PD-L4UWSCI) that presented in addition to the same properties of both domains an intriguing selective cytotoxic action on murine tumor cells. This finding supported the idea that the protection of C-terminal region of PD-L4 could amplify its cytotoxic action by virtue of a greater resistance to proteases. Several authors indeed revealed that the cytotoxicity of RIPs depends not only on the intracellular routing, but also on the intrinsic resistance to proteolysis. In this regard in the present work we have produced a variant of chimeric protein, named PD-L4UWSCI(tr), changing the inhibitory specificity of WSCI domain. The purpose of this approach was to check if the cytotoxicity of the chimeric protein was altered depending on the properties of protease inhibitor domain or by a different fold of whole protein. Data collected supposedly indicate that WSCI domain contributes to cytotoxicity of chimeric protein exclusively from a structural point of view.

In vitro anti-inflammatory and wound-healing potential of a Phyllostachys edulis leaf extract--identification of isoorientin as an active compound.

Extracts prepared from the leaves of Phyllostachys edulis (bamboo) have received attention in pharmacological research due to their potent antitumor, anti-inflammatory, antimicrobial, and anti-ulcerogenic activities. In this study, anti-inflammatory effects of a bamboo leaf extract on tumor necrosis factor alpha-induced overproduction of interleukin 8, vascular endothelial growth factor, and interleukin 6 in immortalized human keratinocytes were investigated for the first time. In addition, wound-healing effects were evaluated in 3T3-swiss albino mouse fibroblasts. Bamboo leaf extract and isoorientin inhibited the tumor necrosis factor alpha-induced release of interleukin 8 and vascular endothelial growth factor. Furthermore, isoorientin dose-dependently reduced levels of interleukin 6 in tumor necrosis factor alpha-α-treated immortalized human keratinocytes cells. Wound healing was evaluated using a modification of the classical scratch assay. For evaluation of the wound gap, a new computerized method based on time-lapse microscopy was developed. It was shown that bamboo leaf extract (10 µg/mL) improved wound closure by 28 % (12 h) and 54 % (24 h), respectively. In concentrations of 50 µg/mL and above, bamboo leaf extract inhibited cell migration without affecting cell viability. Isoorientin (10 µM) improved wound closure by 29 % (12 h) and 56 % (24 h), respectively. Comparable to bamboo leaf extract, higher concentrations of isoorientin prevented cell migration. It is suggested that bamboo leaf extract as well as isoorientin have a dual activity - in higher doses, they show anti-inflammatory effects, and in lower concentrations, they exert anti-angiogenic activities.

Dithiol-PEG-PDLLA micelles: preparation and evaluation as potential topical ocular delivery vehicle.

Thiol-modified nanoparticles have potential applications in mucoadhesive drug delivery and have been examined in this regard for topical ocular delivery. In this paper we provide a simple method for the synthesis of a dithiol terminated amphiphilic diblock copolymer. Bidentate dithiol-poly(ethylene glycol)-poly(d,l-lactide) (SH2-PEG-PDLLA) was synthesized and micelles with dithiol-containing coronas were prepared from this block copolymer via the emulsion method. In vitro release studies indicated that the presence of the thiol groups at the surface did not affect the rate of release of dexamethasone, used as a representative ocular drug. The micelles also showed low cytotoxicity to human corneal epithelial cells (HCEC) and murine fibroblast cells (3T3 cells). A hydrophobic red fluorophore, Nile red, was loaded into the core of micelles and confocal microscopy was used to study HCEC uptake and retention of the micelles. The micelles were rapidly endocytosed by the HCEC, with intracellular micelle levels remaining unchanged with incubation times from 5 to 120 min. Interestingly, Nile red was eliminated significantly more slowly from HCECs treated with the thiolated micelles. These results suggest that these dithiolated micelles may be effective for topical ocular drug delivery.

[Smokeless tobacco extract affects biological properties of the pre-osteoblast cell line MC3T3-E1 ].

OBJECTIVE: To investigate the effects of smokeless tobacco extract (STE) on biological properties of osteoblast, and to identify possible pathological mechanisms of osseointegration. METHODS: MC3T3-E1 Sub-clone 14 cells were cultured in the presence of STE at 0 (control group),0. 01,0. 1,1,5,10 g/L. The cell proliferation was measured by MTT assay 1 d, 3 d, 5 d, and 7 d after exposure. The F-actin cytoskeleton of MC3T3 was stained with Rhodamine and DAPI, and then examined under a confocal laser scanning microscope 24 h after exposure to STE. The mRNA expressions of interleukin-6 (IL-6) and core-binding factor αl(Cbfαl) were quantified by real- time PCR (RT-qPCR) 48 h after exposure to STE. RESULTS: The MTT assay showed that 0. 01-10 g/L STE inhibited MC3T3 proliferation (P<0. 05). Prolonged time enabled 5-10 g/L STE to inhibit MC3T3 proliferation (P<0. 05). Network structure in F-actin cytoskeleton was demonstrated in the controls. In the cells exposed to STE, F-actin cytoskeleton started to change with disruptive structures. As the concentration of STE increased, the changes became more significant. STE increased the mRNA expression of IL-6 at the concentration of 5 g/L and 10 g/L (P<0.05), decreased the mRNA expression of Cbfα1 at the concentration of 0. 1-10 g/L (PO<0. 05). CONCLUSION: Tobacco may inhibit osteoblast proliferation, destroy F-actin cytoskeleton structure, increase the mRNA expression of IL-6 and decrease the mRNA expression of Cbfα1, and inhibit cell differentiation and adhesion accordingly. Smoking is a disadvantage to osseointegration.

In vitro study of the cytotoxicity and antiproliferative effects of surfactants produced by Sphingobacterium detergens.

The application of biosurfactants in the biomedical field is growing due to their antimicrobial activity, low cytotoxicity and ability to induce apoptosis in cancer cells. In the light of this therapeutic potential, as well as possible applications in cosmetics or as drug vehicles in pharmaceutical products, a new biosurfactant produced by Sphingobacterium detergens was investigated for its haemolytic activity and cytotoxic and antiproliferative effects in different cell lines. Fraction A showed 100% haemolysis in rabbit erythrocytes, but in Fraction B the rate was only 83%. When comparing cytotoxicity values (IC50) of the two fractions in model fibroblast and keratinocyte cell cultures, Fraction B was less cytotoxic, showing lower values than the reference compound SDS, indicating low skin irritability. Finally, in non-differentiated intestinal Caco-2 cultures, Fractions A and B reduced cell proliferation and induced apoptosis by 44% and 75%, respectively. According to these results, biosurfactants produced by S. detergens have potential application in cosmetic and pharmaceutical formulations.

Long-term cytotoxic effects of contemporary root canal sealers.

OBJECTIVES: The aim of the present study was to investigate the effects of root canal sealers on the cytotoxicity of 3T3 fibroblasts during a period of 5 weeks. MATERIAL AND METHODS: Fibroblasts (3T3, 1×105 cells per well) were incubated with elutes of fresh specimens from eight root canal sealers (AH Plus, Epiphany, Endomethasone N, EndoREZ, MTA Fillapex, Pulp Canal Sealer EWT, RoekoSeal and Sealapex) and with elutes of the same specimens for 5 succeeding weeks after immersing in simulated body fluid. The cytotoxicity of all root canal sealers was determined using the MTT assay. Data were analyzed using ANOVA and Tukey's test. RESULTS: RoekoSeal was the only sealer that did not show any cytotoxic effects (p<0.05). All the other tested sealers exhibited severe toxicity initially (week 0). MTA Fillapex remained moderately cytotoxic after the end of experimental period. Toxicity of the other tested sealers decreased gradually over time. The evaluated root canal sealers presented varying degrees of cytotoxicity, mainly in fresh mode. CONCLUSIONS: RoekoSeal had no cytotoxic effect both freshly mixed and in the other tested time points. MTA Fillapex was associated with significantly less cell viability when compared to the other tested root canal sealers.

The addition of nanostructured hydroxyapatite to an experimental adhesive resin.

OBJECTIVES: Was produced nanostructured hydroxyapatite (HAnano) and evaluated the influence of its incorporation in an adhesive resin. METHODS: HAnano was produced by a flame-based process and was characterized by scanning electron microscopy. The surface area, particle size, micro-Raman and cytotoxicity were evaluated. The organic phase was formulated by mixing 50 wt.% Bis-GMA, 25 wt.% TEGDMA, and 25 wt.% HEMA. HAnano was added at seven different concentrations: 0; 0.5; 1; 2; 5; 10 and 20 wt.%. Adhesive resins with hydroxyapatite incorporation were evaluated for their radiopacity, degree of conversion, flexural strength, softening in solvent and microshear bond strength. The data were analyzed by one-way ANOVA and Tukey's post hoc test (α=0.05), except for softening in solvent (paired t-test) and cytotoxicity (two-way ANOVA and Bonferroni). RESULTS: HAnano presented 15.096 m(2)/g of specific surface area and a mean size of 26.7 nm. The radiopacity values were not different from those of 1-mm aluminium. The degree of conversion ranged from 52.2 to 63.8%. The incorporation of HAnano did not influence the flexural strength, which ranged from 123.3 to 143.4MPa. The percentage of reduction of the microhardness after immersion in the solvent became lower as the HAnano concentration increased. The addition of 2% nanostructured hydroxyapatite resulted in a higher value of microshear bond strength than the control group (p<0.05). CONCLUSIONS: The incorporation of 2% of nanostructured hydroxyapatite into an adhesive resin presented the best results. CLINICAL SIGNIFICANCE: The incorporation of nanostructured hydroxyapatite increases the adhesive properties and may be a promising filler for adhesive resin.