Effects Of Sterilization Methods On The Physical, Chemical, And Biological Properties Of Silk Fibroin Membranes.

Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. Sterilization is a fundamental step in biomaterials processing and it must not jeopardize the functionality of medical devices. The aim of this study was to analyze the influence of different sterilization methods in the physical, chemical, and biological characteristics of dense and porous silk fibroin membranes. Silk fibroin membranes were treated by several procedures: immersion in 70% ethanol solution, ultraviolet radiation, autoclave, ethylene oxide, and gamma radiation, and were analyzed by scanning electron microscopy, Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction, tensile strength and in vitro cytotoxicity to Chinese hamster ovary cells. The results indicated that the sterilization methods did not cause perceivable morphological changes in the membranes and the membranes were not toxic to cells. The sterilization methods that used organic solvent or an increased humidity and/or temperature (70% ethanol, autoclave, and ethylene oxide) increased the silk II content in the membranes: the dense membranes became more brittle, while the porous membranes showed increased strength at break. Membranes that underwent sterilization by UV and gamma radiation presented properties similar to the nonsterilized membranes, mainly for tensile strength and FTIR results.

Viability of using enamel and dentin from bovine origin as a substitute for human counterparts in an intraoral erosion model

This study ascertained whether under dental erosion models that closely mimics the real-life situation enamel and root dentin from bovine origin would be reliable substitutes for human counterparts. Through a 2x2 crossover design, in a first trial, 14 volunteers wore a palatal device containing slabs of bovine and human enamel. Half of the participants ingested (4x daily, for 10 days) orange juice first, crossing over to mineral water, while the remainder received the reverse sequence. In a second trial, volunteers wore devices with slabs of bovine and human root dentin. Except for the duration of each intraoral phase, which lasted 2 rather 10 days, the experiment with root dentin run exactly as for enamel. Dental substrates were analyzed for surface microhardness. Two-way ANOVAs (α=0.05) indicated no difference between the microhardness values recorded for human and bovine enamel (p=0.1350), but bovine root dentin had lower microhardness compared to its human counterpart (p=0.0432). While bovine enamel can reliably substitute its human counterpart in in situ dental erosion models, bovine root dentin does not seem to be a viable alternative to the corresponding human tissue.

Rapid prototyping of polymeric electrophoresis microchips with integrated copper electrodes for contactless conductivity detection

A simple and easy approach to produce polymeric microchips with integrated copper electrodes for capacitively coupled contactless conductivity detection (CD) is described. Copper electrodes were fabricated using a printed circuit board (PCB) as an inexpensive thin-layer of metal. The electrode layout was first drawn and laser printed on a wax paper sheet. The toner layer deposited on the paper sheet was thermally transferred to the PCB surface working as a mask for wet chemical etching of the copper layer. After the etching step, the toner was removed with an acetonitrile-dampened cotton. A poly(ethylene terephthalate) (PET) film coated with a thin thermo-sensitive adhesive layer was used to laminate the PCB plate providing an insulator layer of the electrodes to perform CID measurements. Electrophoresis microchannels were fabricated in poly(dimethylsiloxane) (PDMS) by soft lithography and reversibly sealed against the PET film. These hybrid PDMS/PET chips exhibited a stable electroosmotic mobility of 4.25 +/- 0.04 x 10(-4) V cm(-2) s(-1), at pH 6.1, over fifty runs. Efficiencies ranging from 1127 to 1690 theoretical plates were obtained for inorganic cations.

High Solids Content, Soap-Free, Film-Forming Latexes Stabilized by Laponite Clay Platelets

High solids content film-forming poly[styrene-co-(n-butyl acrylate)] [poly(Sty-co-BuA)] latexes armored with Laponite clay platelets have been synthesized by soap-free emulsion copolymerization of styrene and n-butyl acrylate. The polymerizations were performed in batch in the presence of Laponite and a methyl ether acrylateterminated poly(ethylene glycol) macromonomer in order to promote polymer/clay association. The overall polymerization kinetics showed a pronounced effect of clay on nucleation and stabilization of the latex particles. Cryo-transmission electron microscopy observation confirmed the armored morphology and indicated that the majority of Laponite platelets were located at the particle surface. The resulting nanostructured films displayed enhanced mechanical properties.

Kinetics and Modeling of Fatty Alcohol Ethoxylation in an Industrial Spray Loop Reactor

The kinetics of the ethoxylation of fatty alcohols catalyzed by potassium hydroxide was studied to obtain the rate constants for modeling of the industrial process. Experimental data obtained in a lab-scale semibatch autoclave reactor were used to evaluate kinetic and equilibrium parameters. The kinetic model was employed to model the performance of an industrial-scale spray tower reactor for fatty alcohol ethoxylation. The reactor model considers that mass transfer and reaction occur independently in two distinct zones of the reactor. Good agreement between the model predictions and real data was found. These findings confirm the reliability of the kinetic and reactor model for simulating fatty alcohol ethoxylation processes under industrial conditions.

Liquid-Liquid Equilibrium of Aqueous Two-Phase Systems Containing Some Synthetic Polyelectrolytes and Polyethylene Glycol

Experimental results are presented for the liquid-liquid equilibrium of aqueous two-phase systems containing a synthetic polyelectrolyte (polysodium acrylate, polysodium methacrylate, and polysodium ethylene sulfonate) and polyethylene glycol at (298.2 and 323.2) K. A total of 40 phase diagrams were obtained, comprising data both of the binodal curve (obtained through cloud-point measurements) and of equilibrium compositions. The influences of temperature, the nature of the polyelectrolyte monomer unit, and the chain length of both types of polymers are analyzed and discussed.

An extension of the Pitzer equation for the excess Gibbs energy of aqueous electrolyte systems to aqueous polyelectrolyte solutions

Pitzer`s equation for the excess Gibbs energy of aqueous solutions of low-molecular electrolytes is extended to aqueous solutions of polyelectrolytes. The model retains the original form of Pitzer`s model (combining a long-range term, based on the Debye-Huckel equation, with a short-range term similar to the virial equation where the second osmotic virial coefficient depends on the ionic strength). The extension consists of two parts: at first, it is assumed that a constant fraction of the monomer units of the polyelectrolyte is dissociated, i.e., that fraction does not depend on the concentration of the polyelectrolyte, and at second, a modified expression for the ionic strength (wherein each charged monomer group is taken into account individually) is introduced. This modification is to account for the presence of charged polyelectrolyte chains, which cannot be regarded as punctual charges. The resulting equation was used to correlate osmotic coefficient data of aqueous solutions of a single polyelectrolyte as well as of binary mixtures of a single polyelectrolyte and a salt with low-molecular weight. It was additionally applied to correlate liquid-liquid equilibrium data of some aqueous two-phase systems that might form when a polyelectrolyte and another hydrophilic but neutral polymer are simultaneously dissolved in water. A good agreement between the experimental data and the correlation result is observed for all investigated systems. (c) 2008 Elsevier B.V. All rights reserved.

Thermodynamics of aqueous solutions of polyelectrolytes: Experimental results for the activity of water in aqueous solutions of some single synthetic polyelectrolytes

Experimental results for the activity of water in aqueous solutions of 10 single polyelectrolytes (two polysodium acrylates, two polysodium methacrylates, three polyammonium acrylates, two polysodium ethylene sulfonates, and one polysodium styrene sulfonate) at (298.2 and 323.2) K are reported. The isopiestic method was employed in these experiments with aqueous solutions of sodium chloride as references. The polyelectrolytes were characterized by three averaged molecular masses determined by gel permeation chromatography. Furthermore, the density and the refractive index increments of the aqueous polyelectrolyte solutions are reported. Although a similar pattern for the activity of water was observed for all systems (i.e., the osmotic coefficient increases with rising polyelectrolyte concentration), the experimental results show that this property depends on the monomer type as well as on the size of the polymer chain. The temperature (varied from (298.2 to 323.2) K) has only a small influence on the activity of water.

Biochemical and biophysical characterization of lysozyme modified by PEGylation

PEGylation is a strategy that has been used to improve the biochemical properties of proteins and their physical and thermal stabilities. In this study, hen egg-white lysozyme (EC 3.2.1.17; LZ) was modified with methoxypolyethylene glycol-p-nitrophenyl carbonate (mPEG-pNP, MW 5000). This PEGylation of LZ produced conjugates that retained full enzyme activity with glycol chitosan, independent of degree of enzyme modification; its biological activity with the substrate Micrococcus lysodeikticus was altered according to its degree of modification. The conjugate obtained with a low degree of mPEG-pNP/NH(2) modification was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), demonstrating a spectral peak at m/z 19,988 Da with 77% of its original enzymatic activity. Spectroscopic studies of Fourier transform infrared (FIR) and circular dichroism (CD) did not show any relevant differences in protein structure between the native and conjugate LZ. Studies of the effects of pH and temperature on PEGylated LZ indicated that the conjugate was active over a broad pH range, stable at 50 degrees C, and demonstrated resistance to proteolytic degradation. (C) 2010 Elsevier B.V. All rights reserved.

Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacological properties of proteins as well as their physical and thermal stability. Purified lysozyme obtained from hen egg white by batch mode was modified by PEGylation with methoxypolyethyleneglycol succinimidyl succinato (mPEG-SS, MW 5000). The conjugates produced retained full enzyme activity with the substrate glycol chitosan, independent of degree of enzyme modification, although lysozyme activity with the substrate Micrococcus lysodeikticus was altered according to the degree of modification. The conjugate with a low degree of modification by mPEG-SS retained 67% of its enzyme activity with the M. lysodeikticus substrate. The mPEG-SS was also shown to be a highly reactive polymer. The effects of pH and temperature on PEGylated lysozymes indicated that the conjugate was active over a wide pH range and was stable up to 50 degrees C. This conjugate also showed resistance to proteolytic degradation, remained stable in human serum, and displayed greater antimicrobial activity than native lysozyme against Gram-negative bacteria.

Biochemical and biopharmaceutical properties of PEGylated uricase

PEGylation is a successful strategy for improving the biochemical and biopharmaceutical properties of proteins and peptides through the covalent attachment of polyethylene glycol chains. In this work, purified recombinant uricase from Candida sp. (UC-r) was modified by PEGylation with metoxypolyethilenoglycol-p-nitrophenyl-carbonate (mPEG-pNP) and metoxypolyethyleneglycol-4,6-dichloro-s-triazine (mPEG-CN). The UC-r-mPEG-pNP and UC-r-mPEG-CN conjugates retained 87% and 75% enzyme activity respectively. The K(M) values obtained 2.7 x 10(-5) M (mPEG-pNP) or 3.0 x 10(-5) M (mPEG-CN) lot the conjugates as compared to 5.4 x 10(-5) M for the native UC-r, suggesting enhancement in the substrate affinity of the enzyme attached. The effects of pH and temperature on PEGylated UC-r indicated that the conjugates were more active at close physiological pH and were stable up to 70 degrees C. Spectroscopic study performed by circular dichroism at 20 degrees C and 50 degrees C did not show any relevant difference in protein structure between native and PEGylated UC-r. In rabbit and Balb/c mice, the native UC-r elicited an intense immune response being highly immunogenic. On the other hand, the PEGylated UC-r when injected chronically in mice did not induce any detectable antibody response. This indicates sufficient reduction of the immunogenicity this enzyme by mPEG-pNP or mPEG-CN conjugation, making it suitable for a possible therapeutical use. (C) 2009 Elsevier B.V. All rights reserved.

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses: therefore, the knowledge of the volumetric mass transfer coefficient (k(L)a) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k(L)a values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium. in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2(2) full factorial design. Both variables showed statistically significant effects on k(L)a, and the highest values of this parameter in both media for simple fermentation (241 s(-1)) and extractive fermentation with ATPS (70.3 s(-1)) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k(L)a values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N(3)D(2)) and superficial gas velocity (V(s)) determined in distilled water (alpha = 0.39 and beta = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (alpha=0.38 and beta=0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (alpha=0.50 and beta=1.0). A reasonable agreement was found between the experimental data of k(L)a for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions. (C) 2009 Elsevier B.V. All rights reserved.

Mesoporous dye-doped titanium dioxide for micro-optoelectronic applications

Optically transparent, mesostructured titanium dioxide thin films were fabricated using an amphiphilic poly(alkylene oxide) block copolymer template in combination with retarded hydrolysis of a titanium isopropoxide precursor. Prior to calcination, the films displayed a stable hexagonal mesophase and high refractive indices (1.5 to 1.6) relative to mesostructured silica (1.43). After calcination, the hexagonal mesophase was retained with surface areas >300 m2 g-1. The dye Rhodamine 6G (commonly used as a laser dye) was incorporated into the copolymer micelle during the templating process. In this way, novel dye-doped mesostructured titanium dioxide films were synthesised. The copolymer not only directs the film structure, but also provides a solubilizing environment suitable for sustaining a high monomer-to-aggregate ratio at elevated dye concentrations. The dye-doped films displayed optical thresholdlike behaviour characteristic of amplified spontaneous emission. Soft lithography was successfully applied to micropattern the dye-doped films. These results pave the way for the fabrication and demonstration of novel microlaser structures and other active optical structures. This new, high-refractive index, mesostructured, dye-doped material could also find applications in areas such as optical coatings, displays and integrated photonic devices.

Anaphylactic reaction induced by a polysulfone/polyvinylpyrrolidone membrane in the 10th session of hemodialysis with the same dialyzer

The majority of severe hypersensitivity reactions in hemodialysis patients have occurred due to sensitization to ethylene oxide or to nonbiocompatible membrane dialyzers. The use of polysulfone dialyzers rarely causes hypersensitivity reactions. In the present study, we describe a case of severe life-threatening reactions induced by polysulfone dialyzers (from different manufacturers subjected to a variety of sterilization methods), which occurred after 9 sessions of hemodialysis with the same prescription, exemplifying the complexity of such reactions.

Bacterial leakage in obturated root canals-part 2: a comparative histologic and microbiologic analyses

Objective. In this study, presence of dentin infection in root canals, obturated with 4 techniques submitted to the bacterial leakage test, was evaluated using histologic methods. Study design. The canals of palatal roots of 160 molars were instrumented and divided into different groups, according to the obturation technique used (lateral condensation, MicroSeal system, Touch `n Heat + Ultrafil, and Tagger`s hybrid technique) and extent of the remaining obturation material (5 mm and 10 mm). Ten additional roots were used as control samples. The roots were sterilized in ethylene oxide and mounted on a device for evaluation of bacterial leakage using the bacteria Enterococcus faecalis for 120 days. After the leakage test, roots were microscopically analyzed for the presence of dentin infection in the root canals and dentinal tubules. Results. A total of 154 specimens were analyzed using both methodologies in the experimental groups; 50 root canals (32.4%) showed bacterial leakage at the end of the experimental period, and 118 (76.6%) showed the presence of bacteria in the root canals using the histologic criteria. The lateral condensation technique allowed lower penetration of bacteria in the root canals and dentinal tubules, followed by Touch `n Heat + Ultrafil, MicroSeal, and Tagger`s hybrid technique, which allowed significantly greater penetration of bacteria. Root canals with 10 mm of remaining obturation material presented similar bacterial penetration as root canals with 5 mm. Conclusions. Even when an adequate seal of the apical foramen was shown by the absence of turbidity in the bacterial leakage test, E. faecalis dentin infection was present in a high percentage of the root canals after 120 days of root filling exposure to the bacteria. Tagger`s hybrid technique presented greater quantity of bacteria in histologic sections than root canals obturated with the other techniques. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 788-794)