Analysis and three-dimensional visualization of collagen in artificial scaffolds using nonlinear microscopy techniques

Extracellularly distributed collagen and chondro- cytes seeded in gelatine and poly-e-caprolactone scaffolds are visualized by two-photon excitation microscopy (TPEM) and second-harmonic generation (SHG) imaging in both for- ward and backward nondescanned modes. Joint application of TPEM and SHG imaging in combination with stereolog- ical measurements of collagen enables us not only to take high-resolution 3-D images, but also to quantitatively an- alyze the collagen volume and a spatial arrangement of cell-collagen-scaffold systems, which was previously im- possible. This novel approach represents a powerful tool for the analysis of collagen-containing scaffolds with applica- tions in cartilage tissue engineering. C 2010 Society of Photo-Optica

Thermus and the Pink Discoloration Defect in Cheese

peer-reviewedA DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch’s postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.Teagasc Walsh Fellowship Programm

Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH4:H2 gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000±14,000 and 152,000±10,000 cells/cm2, respectively, compared to 113,000±10,000 cells/cm2 on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells

Number of MG 63 cells on day 1, 3 and 7 (A, C, E), their spreading area (D) and their growth dynamics (B) on a standard polystyrene cell culture dish (PS), undoped NCD films (B_0) and NCD films doped with 133, 1000 and 6700 ppm of boron (B_133, B_1000 and B_6700, respectively).

<p>Mean ± S.E.M. from 3 experiments; each included 32 microphotographs (day 1 and 3) and 18 measurements in a hemocytometer (day 7) per experimental group). ANOVA, Student-Newman-Keuls method. Statistical significance: I, II, III, IV, V: <i>p</i>≤0.05 compared to the group labelled with the same Roman number.</p

Surface morphology of NCD samples without boron doping (A), with boron 133 ppm (B), 1000 ppm (C) and 6700 ppm (D).

<p>Field emission scanning electron microscope (e_Line, Raith). Scale bar is 200 nm.</p

Neutron depth profiling of boron over doped NCD samples.

<p>Neutron depth profiling of boron over doped NCD samples.</p

Raman spectra of undoped and boron-doped NCD samples.

<p>Raman spectra of undoped and boron-doped NCD samples.</p

Microscopic images of surface potential variations as detected by Kelvin force microscopy (left column), and surface topography as detected by atomic force microscopy (right column) on NCD samples without boron doping (A) and with nominal boron doping of 133 ppm (B), 1000 ppm (C), and 6700 ppm (D).

<p>The AFM oscillation amplitude was 100 nm, the setpoint was 50%. The KFM lift height was 30 nm. The detection frequency was 75 kHz.</p