1,721,038 research outputs found
Effects of ethanol and DMSO on TEGDMA solubility and cytotoxicity
No full textObjective: Several in vitro studies have been carried out to investigate the triethylenglycol-dimethacrylate (TEGDMA) cytotoxicity. However, these studies have never focused on how the solvents used to dissolve TEGDMA in the experimental conditions might influence monomer effective concentrations and in turn its cytotoxicity. Hence, the aim of this study was to evaluate the active concentrations of the TEGDMA over the routinely experimental conditions used in biocompatibility in vitro tests and to determine any changes in cytotoxicity depending on the TEGDMA solution composition.
Methods: TEGDMA dilutions were prepared directly in DMEM (in absence of cells) or were dissolved previously in DMSO or ethanol and then in medium. Monomer concentrations were quantified by an HPLC system. The cytotoxicity effects of TEGDMA dilutions (1 and 2 mmol/L, with and w/o solvents) were evaluated on 3T3-fibroblasts by MTT assay. ROS production (by FACScan flowcytometer) and intracellular and extracellular TEGDMA concentration (by HPLC) were also determined. Data were analyzed by ANOVA followed by Turkey’s test for multiple comparisons.
Results: Maximum solubility of TEGDMA in DMEM (in absence of cells) was 0.5 mmol/L both in the presence and absence of solvents. 2 mmol/L TEGDMA - solubilized in DMSO or ethanol and then dissolved in medium - caused a significant decrease in cell viability and an induction of ROS production compared to the same TEGDMA concentration dissolved in medium directly. Moreover when 2 mmol/L TEGDMA was added to the cells in presence of DMSO and ETOH, after 2h of incubation, TEGDMA concentration was reduced respectively 10% and 20%, while, TEGDMA added without vehicles remains constant.
Conclusions: Our results showed that TEGDMA solubilization in DMEM was not complete and that the cytotoxic effects of the monomer was influenced by the method of solubilization
In vitro Evaluation of the Cytotoxicity of Different Root Canal Filling Materials
No full textAbstract
OBJECTIVE:
Aim of the present study was to evaluate the cytotoxicity of Real Seal 1 compared to other commercially available endodontic filling materials: Real Seal (SybronEndo, Orange, CA, USA) and Thermafil (Tulsa Dental, Tulsa, OK, USA).
MATERIAL AND METHODS:
Periodontal ligament cells from healthy patients were cultured. The eluate of Real Seal 1(TM) (RS1), Real Seal (RS) and Thermafil (TF) samples was used for the cells viability tests, both diluted (50%) or undiluted (100%). Incubation of the specimens was performed in culture medium for 24 h, 48 h and 72 h at 37 °C under sterile conditions. The cellular mortality was evaluated by MTT test. Results were statistically analysed and the statistical significance was set at p< 0.05.
RESULTS:
None of the studied materials showed toxic effects during the period of observation (0 -72 h) when compared to the control group. Only RS induced a very modest increase in cell mortality (about 3% at both concentrations used, during the first 24 hours), when increasing the incubation time, however, only the lower concentration continued to show modest toxicity.
CONCLUSIONS:
Results of the present study showed that all tested materials did not exhibit cytotoxic effects when compared to the control grou
INTERACTIONS BETWEEN CHLORHEXIDINE AND SODIUM HYPOCHLORITE: CHEMICAL ANALYSIS
No full textEndodontic failure may occur in cases of persistent bacteria in the root canal system (RCS). Mechanical instrumentation alone is not able to obtain a complete cleaning of the RCS, so a commonly used method of disinfection in endodontic treatments consists in the use of sodium hypochlorite (NaOCl) followed by the use of chlorhexidine gluconate (CHX). After each irrigating solution, distilled water should be used to prevent possible reaction among the components. In fact, if NaOCl is present when CHX is added, a brown precipitate is formed with a possible negative effect on the outcome of the treatment. Some literature works reported the presence of 4-chloroaniline (PCA) in brown precipitate, other, on the contrary, didn’t observed its formation; in our study, we have tried to explain the reason of this discrepancy using HPLC-UV technique.
To 1.0 mL of 2% CHX were added different volumes of 6.0% NaOCl (from 0.01 to 0.12 mL). Also the reaction mixtures between 6.0% NaOCl and PCA (5 mg /mL in methanol) were prepared.
The specimens were centrifuged (13400 x g 5 min) and both supernatants (SNs) and precipitates (PTs) (re-suspended in 1 mL of methanol) were analyzed.
The column used was a Discovery HS C18 (250mm × 4.6mm, 5 μm) (SUPELCO, PA, USA), flow rate 0.7 mL/min, detection 214 nm. Water (A) and acetonitrile (B) were used for the elution: from 50% (B) to 70% (B) in 10 min and to 85 % (B) in 5 min.
The chromatograms of CHX showed a signal with a Retention Time (RT) 3.0 min. When NaOCl was added, many other signals appeared with RT between 20 and 25 min. Similar results were obtained in the PTs. The presence of PCA signal was not observed neither in PTs nor in SNs.
The HPLC analysis of mixture between NaOCl and PCA showed the presence of signal with RT 9.1 min (PCA), its intensity decreases when NaOCl was added, and completely disappeared with 0.12 mL of NaOCl. Simultaneously signals with RT between 20 and 25 minutes are produced, similarly to what was observed for the CHX. In PTs are present many peaks with RT around 2-3 min and at 25-30 min.
The obtained results showed that PCA is transformed in presence of NaOCl. Thus, the discrepancy of literature data could be due to the reaction conditions: the newly-formed PCA reacts with NaOCl and is transformed
NAC direct detoxification of TEGDMA cytotoxicity.
No full textObjectives: Various protective effects of N-acetylcysteine (NAC) against triethylene glycol dimethacrylate (TEGDMA)-induced cell damage have been demonstrated, but so far there is no evidence on NAC direct monomer detoxification mechanism. Here, we hypothesized that NAC might reduce TEGDMA cytotoxicity due to direct NAC adduct formation.
Methods: we measured the cytotoxic effects of TEGDMA in presence and in absence of NAC by MTT test. Then we analyzed the presence of TEGDMA-NAC adduct formation in extracellular and intracellular compartments by capillary electrophoresis-UV detection (CE-UV) and capillary electrophoresis–mass spectrometry (CE-MS) analytical techniques. Moreover, we quantified the effective intracellular and extracellular TEGDMA concentrations through HPLC in the presence and absence of 10 mmol/L NAC. Data from all experiments were summarized as means ± Standard Deviation (SD) and differences between means were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. The level of significance was set at p<0.05.
Results: TEGDMA reduced 3T3 cell vitality in a dose- and time-dependent manner, while NAC significantly decreased monomer cytotoxicity and extracellular monomer concentrations by a direct reaction with TEGDMA. The adducts between the two molecules were detected both in the presence and absence of cell.
Conclusion: Our results suggest that in vitro detoxification capability of NAC against TEGDMA-induced cell damage might occur also through the formation of NAC-TEGDMA adduct
BIOSYNTHESIS, CHARACTERIZATION AND BIOMEDICAL APPLICATIONS OF PEPTIDE-BASED HYDROGELS
No full textIn recent years, scientific as well as technological interest in the synthesis of peptide-based hydrogel materials have grown dramatically. Applications of such materials are mostly related to the biomedical field, thanks to their biocompatibility and biodegradability.
As it is well known, the solid component of a hydrogel can consist of a polymeric network or a supramolecular structure derived from the self-assembly of oligomers or non-polymeric molecules. The ability to control the assembly of such structures by the application of an external stimulus is extremely valuable. Enzyme-catalyzed reactions can be used as selective biological stimuli to trigger hydrogel assembly. In fact, the use of enzymes for the fabrication of peptide-based hydrogels has become an emerging area of scientific research.
Recently, we reported the possibility of using microbial lipases to catalyze the synthesis in water of self-assembling peptides. We employed different lipases to efficiently catalyze peptidic bond formation between F-moc phenylalanine and diphenylalanine in aqueous medium. Such biocatalysts were able to synthesize the reaction product F-moc triphenylalanine, with yields ranging from 15 to 33%.
The reaction products (Fmoc peptides) spontaneously self-assembled in water to form fibrils, that became entangled to form a three-dimensional structure of fibers with a diameter of approximately 7 nm, as evidenced by AFM measurements. Macroscopically, a stable, self-supporting hydrogel material was produced. Moreover, the influence of the chirality of the aminoacidic moieties on the structure and properties of the hydrogels was investigated. In fact, D aminoacids are interesting building blocks for biomedical applications as they are more stable in vivo.
The viscoelastic properties of the synthesized hydrogels were investigated and their biocompatibility with different mammalian cells was assessed.
Such hydrogel materials were used to entrap nanostructured drug delivery systems, with the aim of creating a biomaterial that may support cell growth and differentiation of human gingival fibroblasts (HGFs). The release kinetics of dexamethasone (DXM) from biopolymeric nanoparticles entrapped within the hydrogel was studied, confirming the possibility of using such system for the sustained release of a bioactive molecule that is able to promote HGFs differentiation into osteoblasts.
Our results suggest the possibility of using F-moc oligopetides as building blocks for a new class of injectable cell scaffolds that could play an important role in bone regeneration, i.e. to reconstruct anatomical defects caused by cancer surgery, malformations and trauma
Produzione di specie reattive dell'ossigeno, sindrome metabolica e parodontite.
No full textAim:
Metabolic syndrome (MetS) is often associated with obesity, impaired glucose tolerance, hyperinsulinemia and diabetes. In addition, recent clinical studies have provided evidence that MetS is associated with an increased risk of periodontitis; moreover literature data showed a link between obesity and periodontitis however, the underlying mechanisms remain largely unknown. Nevertheless, it is important to note that MetS is characterized by an alteration of reactive oxygen species (ROS) metabolism with a consequent cellular dysfunction. The association between obesity and periodontal diseases could be based on the effect of pro-inflammatory cytokines released by adipose tissue. An increased caloric intake with consequent higher metabolic activity, resulting in an increased production of ROS, could also be considered. All those conditions show increased serum levels of products derived from oxidative damage, promoting a proinflammatory state. For all these reasons, in this study, we investigated if differences in ROS metabolism of phagocytes isolated from (a) patients with MetS, (b) patients with both MetS and periodontitis, (c) patients with periodontitis and (d) healthy subjects, were present.
Methods:
Venous blood (10 mL), obtained from each volunteers, was diluted with physiological solution (10 mL). Dextran was added in physiological solution (6%, 4 mL) to enhance the sedimentation rate of erythrocytes at 1 × g. After 30 min, the white blood cells suspension was centrifuged on Lymphoprep (Pharmacia, Sweden), according to the manufacturer’s instructions, to remove contaminating mononuclear cells, and finally the pellet underwent hypotonic lysis to remove erythrocytes. The recovered PMNs were washed three times and resuspended in Krebs Ringer phosphate (KRP) solution (200.000/mL, pH 7,4).
Lympho-monocytes were isolated through Lymphoprep (Pharmacia, Sweden) density gradient centrifugation. Monocytes were then isolated from lympho-monocytes by adherence. Briefly, lympho-monocytes (1 × 106 cells/mL), resuspended in RPMI 1640 containing 2% inactivated fetal calf serum, were allowed to adhere directly to Chemiluminescence (CL) vials for 1 h at 37°C in a 5% CO2 humidified atmosphere. Non-adhered cells were then gently removed by three washes with modified KRP buffer and counted by Trypan blue exclusion test in order to calculate (by subtraction from the whole) the number of adhered cells.
ROS metabolism was studied by a CL technique: the system was made up of luminol (5-amino-2,3-dihydro-1,4- phthalazindione, 100 nmol/L) and cells (1 × 105) in the presence or absence of stimulus constituted by opsonized zymosan (0.5 mg). The final volume (1.0 mL) was obtained using modified KRP buffer. ROS production was measured at 25°C for 2 h, using a LB 953 luminometer (Berthold, EG&G Co, Germany). All the experiments were performed in triplicate.
All results are mean ± standard deviation (SD). The group of means were compared by analysis of variance (ANOVA), followed, when appropriate, by a multiple comparison of means by Student–Newman–Keulz test. A value of p < 0.05 was considered significant.
Results:
Results showed that basal ROS production (both from PMNs and from PBMs) of group A was increased respect to that obtained from group D (p <0.05).
Conclusion:
These results are congruent with literature data and represent a further link point between oxidative stress, MetS and periodontitis, although the actual clinical relevance of the phenomenon remains to be evaluated
METABOLIC ALTERATIONS INDUCED BY METHACRILIC MONOMERS IN HUMAN PULPAR FIBROBLASTS
No full textAim: Composite resins were introduced in the 1960s
for the restoring of anterior teeth in substitution
of the amalgam that presented both aesthetical
and biocompatibility problems. However, since the
polymerization of methacrylates is never complete, the
need to appraise the biocompatibility of composite resins
became evident. The incomplete conversion causes in
fact the release of monomers that may implement adverse
effects in the organism, i.e. allergic reaction, systemic
toxicity, cytotoxicity. Given that very little information
has been so far delivered on the effects of methacrylic
monomers on cell metabolism, the aim of this work was
the evaluation of the biochemical interactions between
methacrylates and human fibroblasts.
Methods: The effects of triethylenglycol-dimethacrylate
(TEGDMA), 2-hydroxyhethyl methacrylate (HEMA), and
1,4-butanediol dimethacrylate (BDDMA) on 1) cellular
energetic metabolism (oxygen consumption rate, glucose
consumption, G6PDH, lactate production) 2) cellular
redox status (GSH concentration, the activity of the
enzymes regulating glutathione metabolism and REDOX
status of cells) were analysed according to Nocca et al.
Data are expressed as the mean ± statistical error of
the mean (SEM). The means were compared by analysis
of variance (ANOVA) followed, when appropriate, by a
multiple comparison of means by Student-Newman-Keuls
test: p < 0.05 was considered significant.
Results: The results showed that TEGDMA, HEMA and
BDDMA induce a statically significant decrease of oxygen
consumption, an enhancement of glucose consumption,
lactate production and also GSH depletion.
Conclusions: The alterations in energy metabolism,
REDOX status and glutathione balance – produced in the
cellular metabolism by HEMA, TEGDMA and BDDMA –
can be considered among the mechanisms inducing the
clinical and sub-clinical adverse effects that have been
occasionally reported during the use of dental resins. In
our opinion, such investigations can therefore be helpful
to test the behavior of materials especially designed to be
applied inside the human body.
Nocca G, De Palma F, Minucci A, et al. Alterations of
energy metabolism and glutathione levels of HL-60 cells
induced by methacrylates present in composite resins. J
Dent 2007;35:187-94
Cytotoxicity of a new endodontic filling material
No full textIn vitro cell cultures have been widely used as a means of evaluating cytotoxicity of root canal filling materials. Following ANSI/ADA spec. no. 41, the aim of the present study was to investigate the biological compatibility of a new sealer (FibreFill) and compare it with some commercially available endodontic sealers (Bioseal and Acroseal). Mouse 3T3 fibroblasts were seeded and cultured and subsequently extracts of the cements were added. After 24 hours incubation, the cellular vitality of fibroblasts was evaluated by the neutral red uptake test (NRU), which measures the membrane permeability. Data were collected and statistically analysed. Results showed that all tested materials exhibited mild cytotoxic effects, which are compatible with normal clinical use, and no statistically significant difference was noted between FibreFill and the other tested materials. Therefore, selection amongst these sealers should be based on other factors
EFFECTS OF METHACRYLATES PRESENT IN COMPOSITE RESINS ON REDOX STATUS OF HUMAN PULP CELLS
No full textBackground: Composite resins, utilized in dentistry, are
complex mixed materials composed also by methacrylic
monomers like triethylenglycol-dimethacrylate (TEGDMA) and
2-hydroxyhethyl methacrylate (HEMA). Since the
polymerization reaction of monomers is uncomplete the
release of these compounds may implement adverse effects
in the organism. To understand the causes of this toxic action,
the effects of TEGDMA and HEMA on cellular redox status
were investigated in this study. The analytical techniques
utilized are usually applied in Clinical Biochemistry in patient’
s serum for the evaluation o oxidative stress.
Methods: Pulpal Cells were used in this study. Sub-cytotoxic
concentrations of monomers were identified by the MTT assay
and the following parameters were analysed: 1) Production of
reactive oxygen species (ROS), measured using the probe
2’,7’-dichlorodihydrofluorescin diacetate by a Glomax Multi
detection system fluorimeter (Promega, Milan, Italy) (490 nm
excitation and 526 nm emission wavelengths). 2) Reduced
Glutathione (GSH) and Total Glutathione (GSH+GSSG),
determined by Ellman method. 3) GSH metabolism,
investigated through the assay of glutathione reductase (GR)
and glucose-6-phosphate dehydrogenase (G6PDH) activity.
NADPH absorbance modifications at 340 nm were used to
determine the activity of both enzymes. 4) Superoxide
dismutase (SOD) and Catalase enzymatic activities, were
measured (Packard Spectracount; Packard BioScience) using
the appropriate SOD determination kit (19160 Fluka Analytical,
Sigma-Aldrich, Milan - Italy) and Catalase Assay kit (SigmaAldrich,
Milan - Italy) Statistical analysis Data are expressed as
the mean ± statistical error of the mean (SEM) and compared
by analysis of variance (ANOVA), P <0.05 was considered
significant.
Results: Monomers induced an increase (about 100%, P
<0.01) of ROS production with a consequent increase of SOD
(about 10% P <0.05) and catalase enzymatic activity (about
30% P <0.05). Moreover, monomers provoked a depletion
both of GSH and GSH+GSSG (40% P <0.01), no changes in
GR and G6PDH activity were observed.
Conclusions: These changes can then be considered as a
mechanism able to trigger clinical and sub-clinical adverse
effects, thus calling for further investigation on biocompatibility
of dental material
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