Cellulose-based materials as scaffolds for tissue engineering

Two types of cellulose-based materials, 6-carboxycellulose with 2.1 or 6.6 wt% of –COOH groups, were prepared and tested for potential use in tissue engineering. The materials were functionalized with arginine, i.e. an amino acid with a basic side chain, or with chitosan, in order to balance the relatively acid character of oxidized cellulose molecules, and were seeded with vascular smooth muscle cells (VSMC). The cell adhesion and growth were then evaluated directly on the materials, and also on the underlying polystyrene culture dishes. Of these two types of studied materials, 6-carboxycellulose with 2.1 wt% of –COOH groups was more appropriate for cell colonization. The cells on this material achieved an elongated shape, while they were spherical in shape on the other materials. The number of cells and the concentration (per mg of protein) of contractile proteins alpha-actin and SM1 and SM2 myosins, i.e. markers of the phenotypic maturation of VSMC, were also significantly higher on this material. Functionalization of the material with arginine and chitosan further improved the phenotypic maturation of VSMC. Chitosan also improved the adhesion and growth of these cells. In comparison with the control polystyrene dishes, the proliferation of cells on our cellulose-based materials was relatively low. This suggests that these materials can be used in applications where high proliferation activity of cells is not desirable, e.g. proliferation of VSMC on vascular prostheses. Alternatively, the cell proliferation might be enhanced by another more efficient modification, which would require further research.     

Reaction Kinetics and Reduced Shrinkage Stress of Thiol-Yne-Methacrylate and Thiol-Yne-Acrylate Ternary Systems

Thiol-yne-methacrylate and thiol-yne-acrylate ternary systems were investigated for polymerization kinetics and material properties and compared to the analogous pure thiol-yne and (meth)acrylate systems. Both thiol-yne-methacrylate and thiol-yne-acrylate systems were demonstrated to reduce polymerization induced shrinkage stress while simultaneously achieving high glass transition temperatures (T(g)) and modulius. Formulations with 70 wt% methacrylate increased the T(g) from 51 ± 2 to 75 ± 1 °C and the modulus from 1800 ± 100 to 3200 ± 400 MPa (44% increase) over the pure thiol-yne system. Additionally, the shrinkage stress was 1.2 ± 0.2 MPa, which is lower than that of the pure methacrylate, binary thiol-yne and thiol-ene-methacrylate control systems which are all > 2 MPa. Interestingly, with increasing methacrylate or acrylate concentration, a decrease and subsequent increase in the shrinkage stress values were observed. A minimum shrinkage stress value (1.0 ± 0.2 MPa) was observed in the 50 wt% methacrylate and 70 wt% acrylate systems. This tunable behavior results from the competitive reaction kinetics of the methacrylate or acrylate homopolymerization versus chain transfer to thiol and the accompanying thiol-yne step-growth polymerization. The crosslinking density of the networks and the amount of volumetric shrinkage that occurs prior to gelation relative to the total volumetric shrinkage were determined as two key factors that control the final shrinkage stress of the ternary systems.     

Whole-cell optical biosensor for mercury — operational conditions in saline water

The present study demonstrates the influences of chlorides, fluorides and bromides of potassium and sodium on the growth and Hg²⁺-induced bioluminescence of bioreporter Escherichia coli ARL1. In a Luria-Bertani medium (LB), cell growth was inhibited by concentrations of sodium and potassium fluorides above 0.2 mol L^?1. The addition of NaCl increased cell tolerance to the toxic effects of fluorides and bromides. Lag periods of 10 h and more were observed for cultivations in LB without NaCl and with halides (NaCl, KCl, NaBr, KBr, NaF and KF) at concentrations lower than 0.06 mol L^?1. In a phosphate buffer (PB), the bioluminescence of E. coli ARL1, induced with HgCl₂, was increased by the addition of NaCl, KCl, NaBr, KBr, NaF and KF (concentration of 0–0.25 mol L^?1). In a saline phosphate buffer (PBS), the maxima of induced bioluminescence declined to 50 %, in the case of NaF (0.12 mol L^?1), and to zero for KF. An addition of tryptone to the induction medium increased induced light emission ten-fold. Concentrated artificial sea water (ASW) (70–100 % ASW) inhibited bioluminescence induction. The new detection assay with E. coli ARL1 made possible the detection of 0.57 µL^?1 of HgCl₂ in double-diluted artificial sea water (25 % ASW).     

Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy

The present work reports on in situ observations of the interaction of organic dye probe molecules and dye-labeled protein with different poly(ethylene glycol) (PEG) architectures (linear, dendron, and bottle brush). Fluorescence correlation spectroscopy (FCS) and single molecule event analysis were used to examine the nature and extent of probe-PEG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe-PEG interactions. Small probes are trapped by more dense PEG architectures and large probes interact more with less dense PEG surfaces. These results, and the tunable pore structure of the PEG dendrons employed in this work, suggest the viability of electrochemically-active materials for tunable surfaces.     

Probing electrostatic interactions and structural changes in highly charged protein polyanions by conformer-selective photoelectron spectroscopy

We have recorded the first conformer-selective photoelectron spectra of a protein polyanion in the gas-phase. Bovine cytochrome c protein was studied in 8 different negative charge states ranging from 5- to 12-. Electron binding energies were extracted for all charge states and used as a direct probe of intramolecular Coulomb repulsion. Comparison of experimental results with simulations shows that the experimental outcome can be reproduced with a simple electrostatic model. Energetics are consistent with a structural transition from a folded to an unfolded conformational state of the protein as the number of charges increases. Furthermore, the additional ion-mobility data show that the onset of unfolding can be assigned to charge state 6- where three conformers can be distinguished.     

DMSO-enhanced MALDI MS imaging with normalization against a deuterated standard for relative quantification of dasatinib in serial mouse pharmacology studies

Matrix-assisted laser desorption/ionization mass spectrometry imaging is an emerging powerful technique in drug metabolism and pharmacokinetics research. Despite recent progress in mass-spectrometry-based localization and relative quantification of small-molecule drugs and their metabolites in tissue, improved methods for drug extraction/ionization are required. Furthermore, relative quantification of drugs by mass spectrometry imaging in larger rodent cohorts is a necessary proof-of-concept study to demonstrate the utility of such a workflow in an industrial setting. Using as an example the tyrosine kinase inhibitor dasatinib, a leukemia drug, we demonstrate that inclusion of dimethyl sulfoxide in standard matrix solutions significantly improves ion intensity in mass spectrometry images and reveals enrichment of the drug in mouse kidney medulla. We furthermore show in a time-course study in multiple mice that normalization against a deuterated internal standard, dasatinib-D ₈, which is applied together with the matrix, makes possible relative quantification of the drug that correlates well with canonical liquid chromatography–tandem mass spectrometry based drug quantification.