Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds.

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30 mm diameter × 5 mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds were either maintained in static conditions or transferred to the bioreactor 3 days post-seeding and a continuous flow rate of 3.47 mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10 days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.

A new rapid resazurin-based microdilution assay for antimicrobial susceptibility testing of Neisseria gonorrhoeae.

Objectives: Rapid, cost-effective and objective methods for antimicrobial susceptibility testing of Neisseria gonorrhoeae would greatly enhance surveillance of antimicrobial resistance. Etest, disc diffusion and agar dilution methods are subjective, mostly laborious for large-scale testing and take ∼24 h. We aimed to develop a rapid broth microdilution assay using resazurin (blue), which is converted into resorufin (pink fluorescence) in the presence of viable bacteria. Methods: The resazurin-based broth microdilution assay was established using 132 N. gonorrhoeae strains and the antimicrobials ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and penicillin. A regression model was used to estimate the MICs. Assay results were obtained in ∼7.5 h. Results: The EC 50 of the dose-response curves correlated well with Etest MIC values (Pearson's r = 0.93). Minor errors resulting from misclassifications of intermediate strains were found for 9% of the samples. Major errors (susceptible strains misclassified as resistant) occurred for ceftriaxone (4.6%), cefixime (3.3%), azithromycin (0.6%) and tetracycline (0.2%). Only one very major error was found (a ceftriaxone-resistant strain misclassified as susceptible). Overall the sensitivity of the assay was 97.1% (95% CI 95.2-98.4) and the specificity 78.5% (95% CI 74.5-82.9). Conclusions: A rapid, objective, high-throughput, quantitative and cost-effective broth microdilution assay was established for gonococci. For use in routine diagnostics without confirmatory testing, the specificity might remain suboptimal for ceftriaxone and cefixime. However, the assay is an effective low-cost method to evaluate novel antimicrobials and for high-throughput screening, and expands the currently available methodologies for surveillance of antimicrobial resistance in gonococci.

A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes.

The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.

Assessment for revascularization of transplanted pancreatic islets at subcutaneous site in mice with a highly sensitive imaging system.

BACKGROUND: The subcutaneous space is one of the ideal sites for pancreatic islet transplantation, owing to the minimal invasiveness and easy access. However, the results of pancreatic islet transplantation in subcutaneous sites remain unsatisfactory. One of the main obstacles to successful pancreatic islet transplantation in subcutaneous sites is poor revascularization. Therefore, the aim of this study was to evaluate the revascularization process at subcutaneous sites with a highly sensitive imaging system combining a dorsal skinfold chamber (DSC) technique and multiphoton laser scanning microscopy (MPLSM). METHODS: A few pancreatic islets isolated from C57BL/6-Tg (CAG-EGFP) mice were syngeneically transplanted into nonmetallic DSCs mounted on the backs of C57BL/6J mice. Time-dependent changes in the newly formed vessels of pancreatic islets were imaged using MPLSM on days 1, 4, 7, 11, and 14 (n = 6). Texas Red was injected intravenously to visualize blood vessels. To evaluate islet graft revascularization, we measured vascular volume surrounding the islet using the Volocity system (Improvision). RESULTS: The percentages of vascular volume at days 1 and 14 were assumed to be 0 and 100%, respectively. The vascular volume on each day was 9.4 ± 6.5% (day 4), 34.9 ± 11.2% (day 7), and 21.1 ± 4.6% (day 11). CONCLUSIONS: The present study showed that a highly sensitive imaging system combining the DSC technique and MPLSM was a useful tool to analyze the revascularization process of pancreatic islets in a subcutaneous site.

Synthesis and spectroscopic properties of silica-dye-semiconductor nanocrystal hybrid particles.

We prepared silica-dye-nanocrystal hybrid particles and studied the energy transfer from semiconductor nanocrystals (= donor) to organic dye molecules (= acceptor). Multishell CdSe/CdS/ZnS semiconductor nanocrystals were adsorbed onto monodisperse Stöber silica particles with an outer silica shell of thickness 2-23 nm containing organic dye molecules (Texas Red). The thickness of this dye layer has a strong effect on the energy transfer efficiency, which is explained by the increase in the number of dye molecules homogeneously distributed within the silica shell, in combination with an enhanced surface adsorption of nanocrystals with increasing dye amount. Our conclusions were underlined by comparison of the experimental results with numerically calculated FRET efficiencies and by control experiments confirming attractive interaction between the nanocrystals and Texas Red freely dissolved in solution.

Ellman's-reagent-mediated regeneration of trypanothione in situ: substrate-economical microplate and time-dependent inhibition assays for trypanothione reductase.

Trypanothione reductase (TryR) is a key enzyme involved in the oxidative stress management of the Trypanosoma and Leishmania parasites, which helps to maintain an intracellular reducing environment by reduction of the small-molecular-mass disulphide trypanothione (T[S](2)) to its di-thiol derivative dihydrotrypanothione (T[SH](2)). TryR inhibition studies are currently impaired by the prohibitive costs of the native enzyme substrate T[S](2). Such costs are particularly notable in time-dependent and high-throughput inhibition assays. In the present study we report a protocol that greatly decreases the substrate quantities needed for such assays. This is achieved by coupling the assay with the chemical oxidant 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), which can rapidly re-oxidize the T[SH](2) product back into the disulphide substrate T[S](2), thereby maintaining constant substrate concentrations and avoiding deviations from rate linearity due to substrate depletion. This has enabled the development of a continuous microplate assay for both classical and time-dependent TryR inhibition in which linear reaction rates can be maintained for 60 min or more using minimal substrate concentrations (<1 microM, compared with a substrate K (m) value of 30 microM) that would normally be completely consumed within seconds. In this manner, substrate requirements are decreased by orders of magnitude. The characterization of a novel time-dependent inhibitor, cis -3-oxo-8,9b-bis-(N(1)-acrylamidospermidyl)-1,2,3,4,4a,9b-hexahydrobenzofuran (PK43), is also described using these procedures.