Cytotoxicity and biocompatibility of direct and indirect pulp capping materials

There are several studies about the cytotoxic effects of dental materials in contact with the pulp tissue, such as calcium hydroxide (CH), adhesive systems, resin composite and glass ionomer cements. The aim of this review article was to summarize and discuss the cytotoxicity and biocompatibility of materials used for protection of the dentin-pulp complex, some components of resin composites and adhesive systems when placed in direct or indirect contact with the pulp tissue. A large number of dental materials present cytotoxic effects when applied close or directly to the pulp, and the only material that seems to stimulate early pulp repair and dentin hard tissue barrier formation is CH.

Evaluation of 2 endodontic techniques used to treat human primary molars with furcation radiolucency area: A 48-month radiographic study

Objective: To evaluate 2 techniques for the treatment of human primary molars with necrotic Pulp and bifurcation bone loss by means of radiographic examination for 48 months. Method and Materials: Fifty-one mandibular primary molars were evaluated in children ranging from 4.5 to 6.5 years of age. The teeth with necrotic pulp and bifurcation bone loss were diagnosed by radiographic examination. The teeth were divided into 2 groups: group 1 (28 teeth)-pulpotomy technique using formocresol as a temporary dressing between sessions and coronal chamber obturation with zinc oxide-eugenol cement; and group 2 (23 teeth)-pulpectomy technique with calcium hydroxide paste as a temporary dressing between sessions and root canal obturation with a dense Calcium hydroxide paste. Standardized radiographs were taken immediately after the fillings were completed and after 12, 24, 36, and 48 months. The radiographs were digitized and analyzed with software that outlined and measured the bifurcation radiolucency. Results: Bifurcation radiolucency reduced significantly or repaired completely for both treatnients in the first 12 months. Minor radiographic reduction of the lesion was observed from 12 to 24 months, and no significant reduction of the remaining radioulcent area was observed from 24 to 48 months after treatment. Conclusion: The 2 endodontic techniques evaluated showed similar results. The main effect of treatment on the lesion repair was obtained in the first year after treatment.

Biocompatibility of a resin-modified glass-ionomer cement applied as pulp capping in human teeth

Purpose: to evaluate the human pulp response following pulp capping with calcium hydroxide (CI-I, Group 1), and the resin-modified glass-ionomer Vitrebond (VIT, Group 2). Materials and Methods: Intact teeth with no cavity preparation were used as control Group (ICG, Group 3). Buccal Class V cavities were prepared in 34 sound human premolars. After exposing the pulps, the pulp capping materials were applied and the cavities were Filled using Clearfil Liner Bond 2 bonding agent and Z100 resin-based composite. The teeth were extracted after 5, 30, and from 120 to 300 days, fixed in 10% buffered formalin solution, and prepared according to routine histological techniques. 6-mu m sections were stained with hematoxylin and eosin, Masson's trichrome, or Brown gr Brenn technique for bacterial observation. Results: At 5 days, CH caused a large zone of coagulation necrosis, the mononuclear inflammatory reaction underneath the necrotic zone was slight to moderate. VIT caused a moderate to intense inflammatory pulp response with a large necrotic zone. A number of congested venules associated with plasma extravasation and neutrophilic infiltration was observed. Over time, only CH allowed pulp repair and complete dentin bridging around the pulp exposure site. VIT components displaced into the pulp tissue triggered a persistent inflammatory reaction which appeared to be associated with a lack of dentin bridge formation. After 30 days a few histological sections showed a number of bacteria on the lateral dentin walls. In these samples the pulp response was similar to those samples with no microleakage. VIT was more irritating to pulp tissue than CH, which allowed pulp repair associated with dentin bridge formation. These results suggested that VIT is not an appropriate dental material to be used in direct pulp capping for mechanically exposed human pulps.

Human pulpal response to direct pulp capping with an adhesive system

Purpose: To evaluate clinically and microscopically the human pulp response when directly capped with an adhesive system or calcium hydroxide over short (9-12 days) and long (53-204 days) experimental periods. Materials and Methods: Fifty-one sound human premolars scheduled for orthodontic extraction, had their pulp horns gently exposed with a diamond point. Debris in the pulp wound was washed out with a sterile saline solution. The pulps were then capped with either an adhesive system (Scotchbond Multi-Purpose Plus) or calcium hydroxide. All teeth were subsequently restored with resin-based composite (Z-100) according to the manufacturer's instructions. After the experimental periods, the teeth were extracted and processed for light microscopic examination. Results: Short-term: the pulp tissue capped with SBMP-P exhibited dilated and congested blood vessels associated with a mo;derate inflammatory response and blanching of pulp cell nuclei. Long-term: no evidence of healing and bridge formation was observed. A persistent mild inflammatory pulp response was present. Micro-abscesses were detected in three cases associated with bacterial infiltration. Calcium hydroxide stimulated early pulp repair and dentin bridging which extended into the longest period.

Response of human pulps following acid conditioning and application of a bonding agent in deep cavities

Objectives. The aim of this in vivo study was to evaluate the human dental pulp response when a one-bottle adhesive system was applied on etched or unetched deep dentine.Methods. Eighteen class V deep cavity preparations were divided in three groups: group 1-total etching + two coats of single bond (SB) + composite resin (Z-100); group 2-enamel etching + two coats of SB + Z-100, group 3-cavity floor lined with a calcium hydroxide liner (Dycal) + acid-etching of enamel and lateral walls + two coats of SB + Z-100. Two teeth were used as intact control group. After 30 days the teeth were extracted and processed through H and E, Masson's trichrome and Brown and Brenn staining techniques.Results. Moderate inflammatory response, disorganization of pulp tissue, as well as, deposition of thin layer of reactionary dentin were observed in group 1 teeth in which the remaining dentin thickness (RDT) was less than 300 mum. These histological findings appear to be related to long resin tags formation and bonding agent diffusion through dentinal tubules. In group 2, slight inflammatory response was observed only in one tooth in which the RDT was 162 mum. In group 3, all the teeth showed normal histological characteristics which were similar to the intact control group. Presence of bacteria was not correlated with the intensity of pulpal response. The patients reported no symptoms during the experiment. Radiographic evaluation showed no periapical pathology for any of the teeth,Significance and conclusions. Acid-etched deep dentin (RDT less than 300 mum) lined with SB causes more intense pulpal response than unetched deep dentin. Based on the results observed in the present study and the conditions in which it was carried out, we recommend the application of a biocompatible liner before etching deep dentin and applying SB. (C) 2002 Academy of Dental Materials. Published by Elsevier B.V. Ltd. All rights reserved.

Response of human pulps capped with different self-etch adhesive systems

The aim of this study was to evaluate the response of human pulps capped with a calcium hydroxide hard-setting cement or with two-step self-etch adhesive systems. Pulp exposures were performed on the occlusal floor, and the bleeding control was performed with saline solution. The exposed pulp tissue was capped with Clearfil LB 2V (2V) or Clearfil SE Bond (SE) and restored with a composite resin. In control group, the pulpal wound was capped with Ca(OH)(2) cement and restored with Clearfil LB 2V or Clearfil SE Bond + composite resin. After 30 and 90 days, the teeth were extracted, processed for hematoxylin and eosin, and categorized in a histological score system. The pulpal response was worse for groups capped with the self-etch adhesive systems (2V and SE) in both periods of evaluation, when compared to their respective control groups at 90 days (p < 0.05). For both self-etch systems evaluated, the pulp tissue exhibited moderate to severe inflammatory cell infiltrate involving the coronal pulp with chronic abscesses. Dentin bridging was observed in a few specimens. For the calcium hydroxide groups, almost all specimens showed dentin bridge formation, with few scattered inflammatory cells and normal tissue below the pulp exposure site. Calcium hydroxide should be used as the material of choice for pulp capping, and the use of two-step self-etch adhesives for human pulp capping is contraindicated.

pH changes after manual or ultrasonic instrumentation and smear layer removal with EDTA or ultrasonic

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Persistence of resinous cement residues in dentin treated with different chemical removal protocols

The aim of this study was to evaluate the persistence of resin cement residues after dentin surface cleaning with different alcohol-based solutions or an essential oil (eucalyptol). Forty bovine teeth were sectioned in order to expose pulp chamber dentin to be washed with 1.0 mL of 2.5% sodium hypochlorite (NaOCl), followed by 0.1 mL of 17% EDTA application for 3 min, and final irrigation with 2.5% NaOCl. The specimens were air dried and resin-based cement was rubbed onto the dentine surface with a microbrush applicator. After 15 min, the surface was scrubbed with a cotton pellet and moistened with different dentin cleaning solutions, compounding the following groups: G195% ethanol, G270% ethanol, G370% isopropyl alcohol, or G4eucalyptol. The dentin was scrubbed until the cement residues could not be visually detected. Sections were then processed for SEM and evaluated at x 500 magnification. Scores were attributed to each image according to the area covered by residual sealer, and data were subjected to KruskalWallis at 5% significance. Eucalyptol promoted the most adequate dentin cleaning, although no statistical difference was detected amongst the groups (P > 0.05), except between the eucalyptol and 70% ethanol groups (P < 0.05). All the evaluated dentin cleaning solutions were unable to completely remove the cement residues from the dentin surface. Microsc. Res. Tech., 2012. (C) 2012 Wiley Periodicals, Inc.

Response of mice connective tissue to intracanal dressings containing chlorhexidine

Substances containing chlorhexidine (CHX) have been studied as intracanal medicaments. The aim of the present study was to characterize the response of mouse subcutaneous connective tissue to CHX-containing medications by conventional optical microscopy. The tissue response was evaluated by implanting polyethylene tubes containing one of the substances evaluated: Calen paste + 0.5% CHX, Calen + 2% CHX, 2% CHX gel, and Calen paste (control). After experimental periods of 7, 21, and 63 days, the implants (n = 10) were removed along with the subcutaneous connective tissue. Tissue samples were subjected to histological processing, and sections were stained with hematoxylin and eosin. Qualitative and quantitative analyses of the number of inflammatory cells, blood vessels, and vascularized areas were performed. Results were analyzed by ANOVA and Tukey tests with the significance level set at 5%. We concluded that Calen + 0.5% CHX led to reparative tissue response in contrast with Calen + 2% CHX and 2% CHX gel, which induced persistent inflammatory response, pointing to the aggressive nature of this mixture. When Calen + 2% CHX and 2% CHX gel were compared, the latter induced more intense inflammatory response. Microsc. Res. Tech., 2012. (C) 2012 Wiley Periodicals, Inc.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

Background: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli ( Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.Results: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside ( IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.Conclusion: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Aerobic biodegradation of butanol and diesel oil blends

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vesicle-micelle transition in mixtures of dioctadecyldimethylammonium chloride and bromide with nonionic and zwitterionic surfactants

We have investigated the effect of mixing spontaneously formed dispersions of the cationic vesicle-forming dioctadecyldimethylammonium chloride and bromide (DODAX, with X being anions Cl- (C) or Br- (B)) with solutions of the micelle-forming nonionic ethylene oxide surfactants penta-, hepta-, and octaethyleneglycol mono-n-dodecyl ether, C12En (n = 5, 7, and 8), and the zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propane sulfonate (HPS). We used for this purpose differential scanning calorimetry (DSC), turbidity, and steady-state fluorescence spectroscopy to investigate the vesicle-micelle (V-M) transition yielded by adding C12En and HPS to 1.0 mM vesicle dispersions of DODAC and DODAB. The addition of these surfactants lowers the gel-to-liquid crystalline phase transition temperature (T-m) of DODAC and DODAB, and the transition becomes less cooperative, that is, the thermogram transition peak shifts to lower temperature and broadens to disappear when the V-M transition is complete, the vesicle bilayer becomes less organized, and the T., decreases, in agreement with measurements of the fluorescence quantum yield of trans-diphenylpolyene (t-DPO) fluorescence molecules incorporated in the vesicle bilayer. Turbidity data indicate that the V-M transition comes about in three stages: first surfactants are solubilized into the vesicle bilayer; after saturation, the vesicles are ruptured, and, finally, the vesicles are completely solubilized and only mixed micelles are formed. The critical points of bilayer saturation and vesicle solubilization were obtained from the turbidity and fluorescence curves, and are reported in this communication. The solubility of DODAX is stronger for C12En than it is for HPS, meaning that C12En solubilizes DODAX more efficiently than does HPS. The surfactant solubilization depends slightly on the counterion, and varies according to the sequence C12E5 > C12E7 > C12E8 > HPS.

Vesicle-to-micelle transition in dioctadecyldimethylammonium bromide and dioctadecyldimethylammonium chloride dispersions induced by octaethylene glycol n-dodecyl monoether. An isothermal titration calorimetry study

We have used isothermal titration calorimetry to investigate the vesicle-to-micelle transition in dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) vesicle dispersions induced by the nonionic surfactant octaethylene glycol n-dodecyl monoether (C12E8) at room temperature. Small and giant unilamellar vesicles were prepared by sonication and without sonication, respectively, of the pure cationic surfactants at low concentrations in water. The titration of 1.0 mM DODAX (X = Cl- and Br-) by a concentrated micellar solution of C12E8 shows that the enthalpy of interaction (DeltaH(obs)) of C12E8 in micellar form with DODAX is always endothermic. The titration curves are understood on the basis of superposition of the enthalpies of partitioning of C12E8 into the bilayer, of micelle formation and of vesicle-to-micelle transformation. The enthalpy, DeltaH(obs), initially increases owing to the incorporation of C12E8 into the vesicle bilayer until the C12E8/DODAX saturation ratio (R-sat) is reached, then DeltaH(obs) decreases, in different ways for DODAB and DODAC, owing to degradation of vesicles and formation of mixed micelles and intermediary structures up to the C12E8/DODAX solubilization ratio, R-sol. Above R-sol only mixed micelles exist. The surfactant solubilization takes place in three stages. All the critical ratios are lower for DODAB than for DODAC, meaning that C12E8 solubilizes more strongly in DODAB for example, R-sat is 0.8 for DODAB and 1.2 for DODAC. Sonication has no significant effect on the transition.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Synthesis, characterization and solution properties of amphiphilic N-isopropylacrylamide-poly(ethylene glycol)-dodecyl methacrylate thermosensitive polymers

Terpolymers of N-isopropylacrylamide, dodecyl methacrylate (DOMA) and poly(ethylene glycol) (PEG) methacrylate, were synthesized by random copolymerization, and the composition was controlled to achieve systems having different thermosensitivities. H-1 NMR spectra and gel permeation chromatography (GPC) were employed to characterize the different samples obtained. The solution properties were studied by employing spectrophotometry, fluorescence, and dynamic light scattering techniques. The chemical compositions in the final terpolymers are close to those in the feed. The polymers exhibited cloud point temperatures (T-es) varying from 17 to 52 degrees C. Micropolarity studies using I-1/I-3 ratio of the vibronic bands of pyrene show the formation of amphiphilic aggregates capable of incorporating hydrophobic drugs as the polymer concentration is increased. The critical aggregation concentration (CAC) increases from 3.6 x 10(-3) to 1 x 10(-2) g/l with the PEG content varying from 5 to 35 mol%. Anisotropy measurements confirm the results obtained by pyrene fluorescence and show that the aggregates resulting from intermolecular interactions present different organizations. The hydrodynamic diameters (Dh) of the aggregates determined by dynamic light scattering (DLS) vary from 40 to 150 nm depending on the terpolymer composition. The T-cs and Dh values decreased with the ionic strength, and this behavior was attributed to the dehydration of the polymeric micelles. The capacity of solubilization of the aggregates was evaluated by employing pyrene, and the obtained results confirm the ability to incorporate hydrophobic molecules. (c) 2005 Elsevier B.V All rights reserved.