Hydrogel-based microfluidics for vascular tissue engineering

In this work, we have explored 3-D co-culture of vasculogenic cells within a synthetically modified fibrin hydrogel. Fibrinogen was covalently linked with PEG-NHS in order to improve its degradability resistance and physico-optical properties. We have studied influences of the degree of protein PEGylation and the concentration of enzyme thrombin used for the gel preparation on cellular responses. Scanning electron microscopy analysis of prepared gels revealed that the degree of PEGylation and the concentration of thrombin strongly influenced microstructural characteristics of the protein hydrogel. Human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem cells (hASCs), used as vasculogenic co-culture, could grow in 5:1 PEGylated fibrin gels prepared using 1:0.2 protein to thrombin ratio. This gel formulation supported hASCs and HUVECs spreading and the formation of cell extensions and cell-to-cell contacts. Expression of specific ECM proteins and vasculogenic process inherent cellular enzymatic activity were investigated by immunofluorescent staining, gelatin zymography, western blot and RT-PCR analysis. After evaluation of the optimal gel composition and PEGylation ratio, the hydrogel was utilized for investigation of vascular tube formation within a perfusable microfluidic system. The morphological development of this co-culture within a perfused hydrogel over 12 days led to the formation of interconnected HUVEC-hASC network. The demonstrated PEGylated fibrin microfluidic approach can be used for incorporating other cell types, thus representing a unique experimental platform for basic vascular tissue engineering and drug screening applications. © 2016 by De Gruyter

Osteogenesis on surface selective laser sintered bioresorbable scaffolds

In this study we have used a novel surface selective laser sintering (SSLS) technique to develop CAD/CAM designed scaffolds for bone tissue engineering. SSLS polylactic acid scaffolds were evaluated in vitro and in vivo as templates for human fetal femur-derived cell and adult human bone marrow stromal cell osteogenesis. Both cell types were cultured successfully on SSLS scaffolds with an increase in expression of alkaline phosphatase activity. Cell in-growth and Alcian blue/Sirius red positive staining of matrix deposition were observed on SSLS scaffolds in vitro in basal medium and osteogenic culture conditions. Similar results were observed in vivo with type I collagen expressed by cells on the scaffolds. In the critical sized femur segmental defect, SSLS scaffolds seeded with the cells enhanced significantly bone tissue regeneratio

Fluorescence color diversity of Great Barrier Reef corals

A group of variously colored proteins belonging to the green fluorescent protein (GFP) family are responsible for coloring coral tissues. Corals of the Great Barrier Reef were studied with the custom-built fiber laser fluorescence spectrometers. Spectral analysis showed that most of the examined corals contained multiple fluorescent peaks ranging from 470 to 620 nm. This observation was attributed to the presence of multiple genes of GFP-like proteins in a single coral, as well as by the photo-induced post-translational modifications of certain GFP-like proteins. We isolated a novel photo-convertible fluorescent protein (FP) from one of the tested corals. We propose that two processes may explain the observed diversity of the fluorescent spectra in corals: (1) dark post-translational modification (maturation), and (2) color photo-conversion of certain maturated proteins in response to sunlight

Osteogenic differentiation of human mesenchymal stem cells in 3-D Zr-Si organic-inorganic scaffolds produced by two-photon polymerization technique

Two-photon polymerization (2PP) is applied for the fabrication of 3-D Zr-Si scaffolds for bone tissue engineering. Zr-Si scaffolds with 150, 200, and 250 μm pore sizes are seeded with human bone marrow stem cells (hBMSCs) and human adipose tissue derived stem cells (hASCs) and cultured in osteoinductive and control media for three weeks. Osteogenic differentiation of hASCs and hBMSCs and formation of bone matrix is comparatively analyzed via alkaline phosphatase activity (ALP), calcium quantification, osteocalcin staining and scanning electron microscopy (SEM). It is observed that the 150 μm pore size Zr-Si scaffolds support the strongest matrix mineralization, as confirmed by calcium deposition. Analysis of ALP activity, osteocalcin staining and SEM observations of matrix mineralization reveal that mesenchymal stem cells cultured on 3-D scaffolds without osteogenic stimulation spontaneously differentiate towards osteogenic lineage. Nanoindentation measurements show that aging of the 2PP-produced Zr-Si scaffolds in aqueous or alcohol media results in an increase in the scaffold Young's modulus and hardness. Moreover, accelerated formation of bone matrix by hASCs is noted, when cultured on the scaffolds with lower Young's moduli and hardness values (non aged scaffolds) compared to the cells cultured on scaffolds with higher Young's modulus and hardness values (aged scaffolds). Presented results support the potential application of Zr-Si scaffolds for autologous bone tissue engineering

Collagen structure deterioration in the skin of patients with pelvic organ prolapse determined by atomic force microscopy

We used atomic force microscopy (AFM) to diagnose pathological changes in the extracellular matrix (ECM) of skin connective tissue in patients with pelvic organ prolapse (POP). POP is a common condition affecting women that considerably decreases the patients' quality of life. Deviations from normal morphology of the skin ECM from patients with POP occur including packing and arrangement of individual collagen fibers and arrangement of collagen fibrils. The nanoindentation study revealed significant deterioration of the mechanical properties of collagen fibril bundles in the skin of POP patients as compared with the skin of healthy subjects. Changes in the skin ECM appeared to correlate well with changes in the ECM of the pelvic ligament tissue associated with POP. AFM data on the ECM structure of normal and pathologically altered connective tissue were in agreement with results of the standard histological study on the same clinical specimens. Thus, AFM and related techniques may serve as independent or complementary diagnostic tools for tracking POP-related pathological changes of connective tissue.10 page(s

Detecting phase transitions in supercritical mixtures:an enabling tool for greener chemical reactions

Detecting phase transitions in high-pressure CO(2) and supercritical fluids was first attempted in the nineteenth century. By contrast, Green Chemistry, the design and implementation of cleaner methods of manufacturing and processing chemicals, is barely 20 years old. Now, the use of CO(2) as an environmentally more acceptable replacement for traditional solvents for greener chemical reactions is creating the need for new, more rapid methods for elucidating high-pressure phase behaviour. This paper describes the advantages and limitations of a number of approaches, developed in Nottingham, to meet this need, including acoustic measurements, shear-mode quartz sensors, the fibre-optic reflectometer, the use of holey fibres, attenuated total reflectance infrared spectroscopy and pressure drop measurements