A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

Drug Delivery through the Blood-Brain Barrier through Use of Vascular Cell Adhesion Molecule 1- Targeted Nanoparticles

The blood brain barrier (BBB) is a semi-permeable membrane separating the brain from the bloodstream, preventing many drugs that treat neurological diseases, such as Alzheimer’s and Parkinson’s, from reaching the brain. Our project aimed to create a novel drug delivery system targeting the brain during neural inflammation. We developed a cationic solid lipid nanoparticle (CSLN) complex composed of cationic nanoparticles, biotin, streptavidin, and anti-vascular cell adhesion molecule-1 (anti- VCAM-1) antibodies. The anti-VCAM-1 antibody is used to target VCAM-1, a cell adhesion protein found on the BBB endothelium. VCAM-1 expression is elevated in the presence of inflammatory molecules, such as tumor necrosis factor-alpha (TNF- α). Through the use of a simple BBB model, results showed that our novel drug delivery system experienced some level of success in targeting the brain inflammation due to increasing TNF-α concentrations. This is promising for drug delivery research and provides support for VCAM-1 targeting using more robust and complex BBB models.

NgBR is essential for endothelial cell glycosylation and vascular development.

NgBR is a transmembrane protein identified as a Nogo-B-interacting protein and recently has been shown to be a subunit required for cis-prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial-specific NgBR knockout embryos. Here, we show that endothelial-specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE-cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo-B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.

Human Vascular Microphysiological System for in vitro Drug Screening.

In vitro human tissue engineered human blood vessels (TEBV) that exhibit vasoactivity can be used to test human toxicity of pharmaceutical drug candidates prior to pre-clinical animal studies. TEBVs with 400-800 μM diameters were made by embedding human neonatal dermal fibroblasts or human bone marrow-derived mesenchymal stem cells in dense collagen gel. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses within 3 hours after fabrication. After 1 week of perfusion, TEBVs exhibited endothelial release of nitric oxide, phenylephrine-induced vasoconstriction, and acetylcholine-induced vasodilation, all of which were maintained up to 5 weeks in culture. Vasodilation was blocked with the addition of the nitric oxide synthase inhibitor L-N(G)-Nitroarginine methyl ester (L-NAME). TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α which had a transient effect upon acetylcholine-induced relaxation, and exhibited dose-dependent vasodilation in response to caffeine and theophylline. Treatment of TEBVs with 1 μM lovastatin for three days prior to addition of Tumor necrosis factor - α (TNF-α) blocked the injury response and maintained vasodilation. These results indicate the potential to develop a rapidly-producible, endothelialized TEBV for microphysiological systems capable of producing physiological responses to both pharmaceutical and immunological stimuli.

Investigation of wall-slip behaviour in lead-free solder pastes and isotropic conductive adhesives

Slippage due to wall depletion effect is well-known in rheological investigation. The aim of this study was to investigate the influence of the paste microstructure on slip formation for the paste materials (lead-free solder paste and isotropic conductive adhesives). The effect of different flowgeometries, gap heights and surface roughness on the paste viscosity was investigated. The utilisation of different measuring geometries has not clearly showed the presence of wall-slip in the paste samples. The existence of wall-slip was found to be pronounced when gap heights were varied using the parallel plate geometry. It was also found that altering the surface roughness of the parallel plate measuring geometry did not significantly eliminate wall-slip as expected. But results indicate that the use of a relatively rough surface helps to increase paste adhesion to the plates and to a certain extent inducing structural breakdown in the paste. Most importantly, the study also demonstrated on how the wall-slip formation in the paste material could be utilised for understanding of the paste microstructure and its flow behaviour.

The effect of wall-slip formation on the rheological behaviour of lead-free solder pastes

Wall-slip plays an important role in the flow behaviour of solder paste materials. The wall-slip arises due to the various attractive and repulsive forces acting between the solder particles and the walls of the measuring geometry. These interactions could lead to the presence of a thin liquid layer adjacent to the wall, which causes slippage. The aim of this study is to investigate the influence of the solder paste formulation on wall-slip formation and its effect on the printability of these pastes material. A wall slip model is utilised to calculate the true viscosity and slip velocity for the lead-free solder pastes samples used in this study. The difference in the measured viscosity and the true viscosity could indicate wall-slip formation between the solder pastes and the parallel plate. Sample P1 showed a higher slip velocity compared to sample P2. The slip velocity calculated for the solder pastes could be used as a performance indicator to understand the paste release characteristics in the stencil printing process.

Investigation of wall-slip effect on lead-free solder paste and isotropic conductive adhesives

Wall-slip plays an important role in characterising the flow behaviour of solder paste materials. The wall slip arises due to the various attractive and repulsive forces acting between the solder particles and the walls of the measuring geometry.These interactions could lead to the presence of a thin solvent layer adjacent to the wall, which gives rise to slippage. The wall slip effect can play an important role in ensure successfulpaste release after the printing process. Wall-slip plays animportant role in characterising the flow behaviour of solderpastes and isotropic conductive adhesives. The study investigates the wall-slip formation in solder paste andisotropic conductive adhesives using flow visualisation technique. The slip distance was measured for parallel plate with different surface roughness in order to quantify the wallslip formations in these paste materials. An ink marker line was drawn between the parallel plate and the free surface of the sample. The parallel was rotated slowly at a constant shear rate of 0.05 sec-1 and the displacement of the ink marker was observed using a video microscope and image capturing software was utilised to capture the displacement of ink marker. From this study, it was found that the wall-slip effect was evident in all the paste materials. In addition, the different surface roughness of the parallel plates did not prevent the formation of wall-slip. This study has revealed that the wallslip effect could used to understand the flow behaviour of the paste in the stencil printing process.

Wall-slip effects in SnAgCu solder pastes used in electronics assembly applications

Solder paste is the most important strategic bonding material used in the assembly of surface mount components in electronics manufacturing. As the trend towards miniaturisation of electronic products continues, there is an increasing demand for better understanding of the flow and deformation that is, the rheological behaviour of solder paste formulations. Wall slip plays an important role in characterising the flow behaviour of solder paste materials. The problem of wall slip arises due to the various attractive and repulsive forces acting between the solder particles and the walls of the measuring geometry. These interactions could lead to the presence of a thin solvent layer adjacent to the wall, which gives rise to slippage. In rheological measurements, slip effects can generally be avoided by using roughened surfaces for measuring geometries. In this paper, a novel technique is developed to study the effect of wall slip in the rheological measurements of lead-free solder paste. The viscosity and oscillatory data obtained for three different solder paste samples (from measuring geometries of different surface roughness) havebeen analysed and compared. In viscosity measurements, slip effects were dominant at low shear rates and the use of serrated surfaces was found to be quite effective in minimizing slip effects. Oscillatory measurements were also affected by roughening the surfaces of measuring geometries.

Effect of machine dependence on wall failure property measurements, Jenike versus an annular shear tester

Annular, ring or torsional shear testers are commonly used in bulk solids handling research for the purpose of powder characterisation or equipment design. This paper reports from a DEFRA sponsored project which aims to develop an industrial powder flow-ability tester, (based on the annular shear tester) that is economic to buy and quick and easy to use in trained but unskilled hands. This paper compares the wall failure loci measured with an annular shear cell with measurements obtained using the accepted standard wall friction tester, the Jenike shear cell. These wall failure loci have been measured for several bulk solids which range from fine cohesive powders to free-flowing granular materials, on a stainless steel 304 2B wall surface.

Investigation of wall-slip effect on paste release characteristic in flip-chip stencil printing process

As the trend toward further miniaturisation of pocket and handheld consumer electronic products continues apace, the requirements for even smaller solder joints will continue. With further reductions in the size of solder joints, the reliability of solder joints will become more and more critical to the long-term performance of electronic products. Solder joints play an important role in electronics packaging, serving both as electrical interconnections between the components and the board, and as mechanical support for components. With world-wide legislation for the removal/reduction of lead and other hazardous materials from electrical and electronic products, the electronics manufacturing industry has been faced with an urgent search for new lead-free solder alloy systems and other solder alternatives. In order to achieve high volume, low cost production, the stencil printing process and subsequent wafer bumping of solder paste has become indispensable. There is wide agreement in industry that the paste printing process accounts for the majority of assembly defects, and most defects originate from poor understanding of the effect of printing process parameters on printing performance. The printing of ICAs and lead-free solder pastes through the very small stencil apertures required for flip chip applications was expected to result in increased stencil clogging and incomplete transfer of paste to the printed circuit pads. Paste release from the stencil apertures is dependent on the interaction between the solder paste, surface pad and aperture wall; including its shape. At these very narrow aperture sizes the paste rheology becomes crucial for consistent paste withdrawal because for smaller paste volumes surface tension effects become dominant over viscous flow. Successful aperture filling and release will greatly depend on the rheology of the paste material. Wall-slip plays an important role in characterising the flow behaviour of solder paste materials. The wall- slip arises due to the various attractive and repulsive forces acting between the solder particles and the walls of the measuring geometry. These interactions could lead to the presence of a thin solvent layer adjacent to the wall, which gives rise to slippage. The wall slip effect can play an important role in ensuring successful paste release after the printing process. The aim of this study was to investigate the influence of the paste microstructure on slip formation for the paste materials (lead-free solder paste and isotropic conductive adhesives). The effect of surface roughness on the paste viscosity was investigated. It was also found that altering the surface roughness of the parallel plate measuring geometry did not significantly eliminate wall slip as was expected. But results indicate that the use of a relatively rough surface helps to increase paste adhesion to the plates, inducing structural breakdown of the paste. Most importantly, the study also demonstrated on how the wall slip formation in the paste material could be utilised for understanding of the paste microstructure and its flow behaviour

Lipid productivity and cell wall ultrastructure of six strains of Nannochloropsis: Implications for biofuel production and downstream processing

Microalgae are generating considerable interest for third generation biodiesel production. However, appropriate strain selection is proving challenging due to the significant variation in cellular physiology, metabolic potential and genetics observed even amongst strains deemed morphologically similar. Six strains of Nannochloropsis from the CCAP culture collection were assessed for their lipid productivity and cellular structure, as proxies for oil production and harvesting ease, to assess their suitability as biodiesel production platforms. Differences in growth rate and lipid accumulation across the strains were observed. Nannochloropsis oculata strain 849/7 showed significantly reduced doubling time compared to Nannochloropsis salina strain 849/3, whilst Nannochloropsis oceanica 849/10 produced the highest lipid content. In addition the six strains could be differentiated into 3 distinct classes based on their cell wall thickness, which varied across the strains from 63 to 119 nm and which is independent of both species and geographical isolation location. The importance of these variations in ultrastructure and physiology for biodiesel production is discussed.

Adiciones al catálogo de la flora vascular de Alicante (SE de España)

En este trabajo se aportan algunos datos florísticos, ecológicos y biogeográñcos sobre diversos táxones que suponen adiciones interesantes al catálogo de la flora vascular de Alicante.