Engineering Patterns to Study Vascular Biology

Proper growth of blood vessels is critical for development, wound healing and homeostasis. This process is regulated by a variety of microenvironmental cues including growth factor signaling, cell-cell contacts and mechanical and biochemical signals from the extracellular matrix. The work presented in this dissertation encompasses the application of engineering principles to the study of angiogenesis and vascular biology within the contexts of tissue engineering and vascular disease. In Chapter 2, we present a novel strategy for generating a spatially patterned vascular network in vivo. Future development of clinically viable engineered tissues hinges on the ability to generate functional vasculature capable of delivering blood to parenchymal cells deep within the tissue. The vascularization strategy described here utilizes tissue constructs that contain patterned ‘cords’ of endothelial cells. Implantation of these constructs into mice leads to the formation of stable capillaries in a spatially controlled geometry. The capillaries become perfused with host blood as early as 3 days post implantation, remain stable for at least 28 days in vivo, are largely comprised of implanted endothelial cells, and are invested by α-SMA positive pericytes. We further demonstrate that spatial patterning of vascular architecture improves the function of engineered hepatic tissues. Specifically, co-implantation of patterned endothelial cell cords with primary hepatocyte aggregates suggested that organized vascular architecture significantly improved albumin promoter activity within the tissues. In Chapter 3, we describe the development of an organotypic vascular wall model and show that pulmonary arterial smooth muscle cells (PASMCs) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) exhibit a hyperproliferative phenotype in culture. While normal control PASMCs display Rac1-mediated growth control, the higher proliferation in IPAH PASMCs is dependent on increased RhoA activity. We observed that focal adhesion assembly and focal adhesion kinase signaling are abnormally increased in IPAH PASMCs and show that antagonizing adhesion signaling by direct inhibition of FAK abrogates IPAH PASMC hyperproliferation in vitro. In summary, our strategy for rapidly inducing the formation of spatially controlled capillaries comprises a novel technique for spatial control of vessel growth in vivo. Functional studies with engineered hepatic tissues also demonstrate the potential of this technique to be used in vascularizing engineered solid organs. Findings from our investigation into aberrant IPAH SMC proliferation suggest that a mechanosensitive proliferative control mechanism underlies IPAH etiology

Continuous unitary transformation approach to the Kondo-Majorana interplay

We analyze a setup composed of a correlated quantum dot (QD) coupled to one metallic lead and one end of topological chain hosting a Majorana zero mode (MZM). In such a hybrid structure, a leakage of the MZM into the region of the QD competes with the Kondo resonance appearing as a consequence of the spin-exchange interactions between the dot and the lead. In the work, we use the nontrivial technique called the continuous unitary transformation (CUT) to analyze this competition. Using the CUT technique, we inspect the influence of the coupling between the QD and the chain on effective exchange interactions and calculate the resultant Kondo temperature.Comment: 6 Pages, 5 figure

Experimental study and numerical simulation of low calorific fuel combustion at different oxygen content in the oxidizer

Paper presented at the 8th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics, Mauritius, 11-13 July, 2011.This study presents results of gas fuels combustion - propane and low calorific fuel. Low calorific fuel is produced during e.g. biomass gasification, which has got of 13% H2, 4% CH4, 30% CO, 13% CO2, 40% N2. This composition is very similar to composition of fuel obtained from wood pellets gasification. Investigations have been performed at room temperature and at conditions typical for High Temperature Air Combustion, i.e. high temperature of the oxidizer (above the level of autoignition) and low oxygen content (lower than 21 % as for conventional combustion). Such environment has been created on the base of exhaust gases coming from conventional gas burner in small scale single fuel jet furnace. The temperature of the exhaust gas as well as oxygen content has been adjusted in order to achieve test conditions. Experimental results show difficulties with burning of low calorific fuel at room temperature and stable combustion at high temperature.mp201

Eksperimentalno ispitivanje i modifikacija sustava za odsis drvnih čestica na kružnoj pili

The article presents the results of experimental examination of the wood chip suction system in the existing sliding table saw before and after its modification. The studies focused on the extraction hood of the mentioned system. The methodical experimental research of the pressure distribution inside the hood during wood chip removal for the selected rotational speed of saw blades of 3500 and 6000 min-1 with a diameter of 300 mm and 450 mm were carried out. The analysis of the results allowed estimating the areas with insufficient vacuum pressure hindering the organized transport of wood chips in the sliding table saw. That pressure was the main factor influencing the decision to adjust the hood construction. To achieve the efficient performance, several changes in the hood geometry were implemented. The results obtained from the experiments were used at the stage of shape modification of the extraction hood. As a result, a new design of the chip suction system was obtained, vastly improving the chip extraction from the tool.U radu su prikazani rezultati eksperimentalnog istraživanja sustava za odsis drvnih čestica na stolnoj kružnoj pili prije njegove izmjene i nakon nje. Istraživanja su bila usmjerena na usisno ušće istraživanog sustava. Provedeno je metodološko eksperimentalno istraživanje raspodjele tlaka unutar usisnog ušća tijekom rada sustava za odsis drvnih čestica pri brzini vrtnje kružne pile od 3500 i 6000 min-1 i uz promjer lista pile od 300 i 450 mm. Analiza rezultata omogućila je procjenu područjâ s nedovoljnim podtlakom unutar kojih je onemogućen učinkovit odsis drvnih čestica na stolnoj kružnoj pili. To je i bio glavni razlog odluke o prilagodbi konstrukcije usisnog ušća. Kako bi se postigao učinkovit odsis drvnih čestica, napravljeno je nekoliko promjena u geometriji usisnog ušća. Rezultati dobiveni eksperimentalnim istraživanjem upotrijebljeni su u fazi modificiranja oblika usisnog ušća. Kao rezultat toga dobiven je novi dizajn usisnog ušća kojim je znatno poboljšan odsis drvnih čestica iz radnog prostora alata

Patterning Vascular Networks In Vivo for Tissue Engineering Applications

The ultimate design of functionally therapeutic engineered tissues and organs will rely on our ability to engineer vasculature that can meet tissue-specific metabolic needs. We recently introduced an approach for patterning the formation of functional spatially organized vascular architectures within engineered tissues in vivo. Here, we now explore the design parameters of this approach and how they impact the vascularization of an engineered tissue construct after implantation. We used micropatterning techniques to organize endothelial cells (ECs) into geometrically defined “cords,” which in turn acted as a template after implantation for the guided formation of patterned capillaries integrated with the host tissue. We demonstrated that the diameter of the cords before implantation impacts the location and density of the resultant capillary network. Inclusion of mural cells to the vascularization response appears primarily to impact the dynamics of vascularization. We established that clinically relevant endothelial sources such as induced pluripotent stem cell-derived ECs and human microvascular endothelial cells can drive vascularization within this system. Finally, we demonstrated the ability to control the juxtaposition of parenchyma with perfused vasculature by implanting cords containing a mixture of both a parenchymal cell type (hepatocytes) and ECs. These findings define important characteristics that will ultimately impact the design of vasculature structures that meet tissue-specific needs.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Award Number EB000262)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Award Number EB08396)National Institutes of Health (U.S.). National Research Service Awards (1F32DK091007)National Institutes of Health (U.S.). National Research Service Awards (5T32AR007132-35

Geometric control of vascular networks to enhance engineered tissue integration and function

Tissue vascularization and integration with host circulation remains a key barrier to the translation of engineered tissues into clinically relevant therapies. Here, we used a microtissue molding approach to demonstrate that constructs containing highly aligned “cords” of endothelial cells triggered the formation of new capillaries along the length of the patterned cords. These vessels became perfused with host blood as early as 3 d post implantation and became progressively more mature through 28 d. Immunohistochemical analysis showed that the neovessels were composed of human and mouse endothelial cells and exhibited a mature phenotype, as indicated by the presence of alpha-smooth muscle actin–positive pericytes. Implantation of cords with a prescribed geometry demonstrated that they provided a template that defined the neovascular architecture in vivo. To explore the utility of this geometric control, we implanted primary rat and human hepatocyte constructs containing randomly organized endothelial networks vs. ordered cords. We found substantially enhanced hepatic survival and function in the constructs containing ordered cords following transplantation in mice. These findings demonstrate the importance of multicellular architecture in tissue integration and function, and our approach provides a unique strategy to engineer vascular architecture.National Institutes of Health (U.S.) (Grant EB08396)National Institutes of Health (U.S.) (Grant EB00262)National Institutes of Health (U.S.) (National Research Service Award 1F32DK091007

Modeling of low calorific gas burning in a deficient oxygen environment and high-temperature oxidizer

It is planned to carry out a comprehensive experimental and theoretical study on the high temperature of low calorific gas combustion with oxygen-deficient oxidizers. The experimental research will be performed using the experimental facility with a combustion chamber. The oxygen concentration in combustion oxidizers will be varied from 21% by volume (normal) air to 2%. The test combustion chamber will be fed with propane or methane as the reference fuel, then with low calorific fuels as test gases obtained by mixing various combustible components, e.g. H2, CH4, CO, and neutral gases, e.g. N2, CO2. Gaseous fuels prepared in this way will be burned in the atmosphere of a deficient oxidizer with a temperature changing from 800 °C to 1100 °C. Oxidizers will be heated up to a certain temperature using two methods: by flue gas heat exchanger and kanthal rod electric preheater. Different burner geometry will be used. The burner will be equipped with annular swirl vanes for co-axial or under different angles, fuel, and oxidizers flow to have a high swirl number achieved by flow aerodynamics and mixing. Experimental data will be verified with numerical simulations with the use of ANSYS CFD Fluent code

Technical problems in shipped hepatic allografts: the UCL experience

Due to developments in surgical techniques and organ preservation, the shipping of renal and extrarenal organs is becoming increasingly more frequent. During the period from 1 January 1991 to 31 December 1992, 39 of 180 (21%) implanted liver allografts were shipped to our center by local harvesting teams. The fact that each of nine livers (23.1%) presented with minor and major (vascular and parenchymatous) problems stresses the need for better surgical training and standardization in procurement techniques. The introduction of a liver allograft feedback report could be an easy way to perform quality control

Bioactive Hydrogels Made from Step-Growth Derived PEG-Peptide Macromers

Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400 Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate–PEG–(peptide–PEG)m-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500 kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate–PEG–CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches