The influence of pore size on colonization of poly(l-lactide-glycolide) scaffolds with human osteoblast-like MG 63 cells in vitro

A degradable copolymer of l-lactide and glycolide (PLG) was synthesized by ring opening polymerization using zirconium acetylacetonate [Zr(acac)₄] as a biocompatible initiator. The structure of the copolymer was studied by nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). Porous scaffolds of defined microstructure were prepared by solvent casting/salt particulate leaching, which resulted in the creation of three types of scaffolds with the same porosity (87% ± 1%) but with different diameters of the pores (600, 200 and 40 μm) and degree of interconnectivity. The potential of the scaffolds for cell colonization was tested in a conventional static cell culture system using human osteoblast-like MG 63 cells. As revealed by conventional fluorescence and confocal microscopy on days 5 and 7 after seeding, the cells on the scaffolds of large or medium pore size infiltrated the inside part of the material, whereas on the scaffolds of small pore size, the cells were retained on the material surface. On day 7 after seeding, the highest number of cells was found on the scaffolds of the largest pore size (more than 120,000 cells per sample of the diameter 15 mm and thickness 2 mm), whereas on the scaffolds with medium and smallest pore diameter, the number of cells was almost three times lower and similar for both pore sizes. These results corresponded well with the incorporation of bromodeoxyuridine into newly synthesized DNA, which was significantly higher in cells on scaffolds of the largest pore size than on the material with medium and smallest pore diameter. As indicated by the MTT test, the mitochondrial activity in cells on scaffolds with medium pore size was similar to that on the material with the highest pore size, and significantly higher than on scaffolds of the smallest pore diameter. These results suggest that PLG scaffolds with the largest pore diameter (600 μm) and better pore interconnectivity are the most suitable for colonization with osteogenic cells.     

Development and critical evaluation of fluorescent chloride nanosensors

In this study, we describe the preparation and evaluation of new fluorescent sensor nanoparticles for the ratiometric measurement of chloride concentrations. Both a chloride-sensitive dye (lucigenin) and a reference dye (sulforhodamine derivative) were incorporated into polyacrylamide nanoparticles via inverse microemulsion polymerization and investigated for their response to chloride ions in buffered suspension as well as in living cells. The fluorescence intensity of lucigenin reversibly decreased in the presence of chloride ions due to a collisional quenching process, which can be described with the Stern-Volmer equation. The determined Stern-Volmer constant K SV for the quenching of lucigenin incorporated into particles was found to be 53 M (-1) and is considerably smaller than the Stern-Volmer constant for quenching of free lucigenin ( K SV = 250 M (-1)) under the same conditions. To test the nanosensors in living cells, we incorporated them into Chinese hamster ovary cells and mouse fibroblasts by using the conventional lipofectamin technique and monitored the response to changing chloride concentrations in the cell.     

Fluorescence and structure of methyl red-clay nanocomposites

Two types of clay minerals-montmorillonite and vermiculite have been chosen as a host matrix for the intercalation of methyl red (MR) in order to investigate a possible fluorescence tuning via dye-clay interactions. The effect of silicate layer charge on the structure and fluorescence of dye-clay intercalated hybrid nanostructures was investigated using combination of molecular modeling with experiment. Structure of both intercalates MR-vermiculite (MR-VER) and MR-montmorillonite (MR-MMT) exhibits high degree of structural disorder resulting in broaden emission band. The fluorescence wavelength range of MR intercalated in clays is shifted to lower wavelengths compared with the pristine MR polycrystalline sample (800 nm). Results showed the strong dependence of fluorescence band maximum on the silicate layer charge, lambda(max) = 565 nm for MR-MMT, 645 nm for MR-VER and 800 nm for the methyl red fine crystalline powder, whereas the structural disorder in the arrangement of dye molecules affects the emission band broadening.     

Are Changes in the Percentage of Specific Leukocyte Subpopulations Associated with Endogenous DNA Damage Levels in Testicular Cancer Patients?

Chemoresistance of germ cell tumors (GCTs) represents an intensively studied property of GCTs that is the result of a complicated multifactorial process. One of the driving factors in this process is the tumor microenvironment (TME). Intensive crosstalk between the DNA damage/DNA repair pathways and the TME has already been reported. This study aimed at evaluating the interplay between the immune TME and endogenous DNA damage levels in GCT patients. A cocultivation system consisting of peripheral blood mononuclear cells (PBMCs) from healthy donors and GCT cell lines was used in an in vitro study. The patient cohort included 74 chemotherapy-naïve GCT patients. Endogenous DNA damage levels were measured by comet assay. Immunophenotyping of leukocyte subpopulations was performed using flow cytometry. Statistical analysis included data assessing immunophenotypes, DNA damage levels and clinicopathological characteristics of enrolled patients. The DNA damage level in PBMCs cocultivated with cisplatin (CDDP)-resistant GCT cell lines was significantly higher than in PBMCs cocultivated with their sensitive counterparts. In GCT patients, endogenous DNA damage levels above the cutoff value were independently associated with increased percentages of natural killer cells, CD16-positive dendritic cells and regulatory T cells. The crosstalk between the endogenous DNA damage level and specific changes in the immune TME reflected in the blood of GCT patients was revealed. The obtained data contribute to a deeper understanding of ongoing interactions in the TME of GCTs.     

Elemental analysis of flour-based ready-oven foods by slurry sampling inductively coupled plasma optical emission spectrometry

A slurry sampling technique has been utilised for the determination of Na, K, Ca, Mg, S, P, Fe, Mn, Cu and Zn in wheat-flour and flour-based ready-oven foods using ICP-OES and a direct aqueous calibration technique. For 0.1% w/v suspension of flour in 0.1% w/v Triton X-100 and 6% v/v HNO₃, the procedural limits of detection (all in mg kg⁻¹) for Na, K, Mg, Ca, P, S, Fe, Zn, Mn and Cu were 7.51, 61.1, 0.645, 1.59, 5.25, 4.58, 1.50, 1.07, 0.867 and 1.58, respectively. The precision was expressed as the relative standard deviation (RSD) of 5–10% (n = 10) for 0.1% w/v suspension of wheat-flour. The accuracy was confirmed by the analysis of reference materials wheat-flour GBW 08503 and rice flour NIST 1568a.