Blood vessel mimics (BVMs) are tissue engineered blood vessels that are intended as an intermediate testing environment for intravascular devices, such as stents. Specifically, Cal Poly’s Tissue Engineering Lab hypothesizes that BVMs can be used to test endothelial cell and smooth muscle cell responses to existing and new vascular stents. Characterization techniques are required for BVMs to be accepted as a valid testing model, prior to being employed as an in vitro model to determine the effects of medical treatments. Quantitative real-time polymerase chain reaction (qPCR) is one available option for evaluating gene expression of tissues. qPCR can be performed on DNA synthesized from RNA isolated from cells, and in this application, will provide quantitative information on what proteins where being transcribed within the cells at the time of RNA isolation. qPCR can be used to determine the proteins expressed in BVMs at baseline in order to then characterize changes in protein expression induced by stent deployment within the BVM. The aim of this thesis was to optimize existing qPCR protocols, and implement the optimized protocols to characterize gene expression of stented and unstented blood vessel mimics (BVMs) and cells from a donor with Diabetes grown in Cal Poly’s Tissue Engineering Laboratory. To accomplish this goal, existing qPCR protocols were evaluated and modified to ensure reproducible, valid results were produced. Standard operating procedures were created for RNA isolation, cDNA synthesis, qPCR and qPCR data analysis. Optimized qPCR methods were then applied to BVMs from umbilical and coronary cell sources to compare the models and to study the BVM responses to stent deployment. Additional primers were also identified for potential usage as reference genes and as diabetic markers for diseased BVMs.

Cerebral aneurysms occur due to the ballooning of blood vessels in the brain. Rupture of aneurysms can cause a subarachnoid hemorrhage, which, if not fatal, can cause permanent neurologic deficits. Minimally invasive neurovascular devices, such as embolization coils and flow diverters, are methods of treatment utilized to prevent aneurysm rupture. The rapidly growing market for neurovascular devices necessitates the development of accurate aneurysm models for preclinical testing. In vivo models, such as the rabbit elastase model, are commonly chosen for preclinical device testing; however, these studies are expensive, and aneurysm geometries are difficult to control and often do not replicate the variety of geometries found in clinical cases. A promising alternative for preclinical testing of neurovascular devices is an aneurysm blood vessel mimic (aBVM), which is an in vitro tissue-engineered model of a human blood vessel composed of an electrospun scaffold with an aneurysm geometry and human vascular cells. Previous work in the Cal Poly Tissue Engineering Lab has established a process for creating different aneurysm scaffolds based on the shape of different geometries, and this work aimed to further advance these aneurysm geometries in order to enhance the versatility of the in vitro model. The overall goal of this thesis was to customize the aBVM model through variations of different dimensions and to validate the scaffold variations for neurovascular device testing. First, a literature review was performed to identify critical ranges of aneurysm neck diameters and heights that are commonly seen in rabbit elastase models and in human clinical settings in order to set a foundation for creating new geometries. Based on the results, aneurysm geometries with varying neck sizes and heights were modeled and molded, and scaffolds were fabricated through electrospinning. Methods were developed to characterize scaffolds with internal measurements through imaging techniques using a scanning electron microscope. To validate these scaffolds for use as aBVMs for neurovascular device testing, constructs were created by dual-sodding human endothelial cells and smooth muscle cells into scaffolds with varying neck sizes. Finally, flow diverters were deployed in constructs with varying neck sizes in order to evaluate feasibility and initial healing. Customized aneurysm scaffolds can eventually be used with a variety of device studies for screening of neurovascular devices or as a predecessor for in vivo preclinical testing.

No description available.

The circulatory system is one of the first systems that develops during embryogenesis. Angiogenesis describes the formation of blood vessels as a part of the circulatory system and is essential for organ growth in embryogenesis as well as repair in adulthood. A dysregulation of vessel growth contributes to the pathogenesis of many disorders. Thus, an imbalance between pro- and antiangiogenic factors could be observed in infantile hemangioma (IH). IH is the most common benign tumor during infancy, which appears during the first month of life. These vascular tumors are characterized by rapid proliferation and subsequently slower involution. Most IHs regress spontaneously, but in some cases they cause disfigurement and systemic complications, which requires immediate treatment. Recently, a therapeutic effect of propranolol on IH has been demonstrated. Hence, this non-selective β-blocker became the first-line therapy for IH. Over the last years, our understanding of the underlying mechanisms of IH has been improved and possible mechanisms of action of propranolol in IH have postulated. Previous studies revealed that gap junction proteins, the connexins (Cx), might also play a role in the pathogenesis of IH. Therefore, affecting gap junctional intercellular communication is suggested as a novel therapeutic target of propranolol in IH. In this review we summarize the current knowledge of the molecular processes, leading to IH and provide new insights of how Cxs might be involved in the development of these vascular tumors

A gestação inicial da égua é um período fascinante que abrange numerosas e intensas mudanças em seu desenvolvimento, muitas das quais são únicas para a espécie equina. Esse desenvolvimento depende da manutenção da função lútea, do estabelecimento de um ambiente uterino e de uma interação precisa e orquestrada entre o concepto e o ambiente uterino. O objetivo deste estudo foi verificar as alterações histológicas do endométrio e a produção histotrófica em éguas cíclicas e prenhes nos dias 7, 10 e 13 pós-ovulação. No primeiro ciclo, biópsias endometriais de 30 éguas foram coletadas no dia 7 (n = 10), 10 (n = 10) e 13 (n = 10) constituindo o grupo éguas cíclicas. No segundo ciclo, as mesmas éguas foram cobertas por um garanhão fértil, acompanhadas diariamente até detectar a ovulação, considerada o dia 0. Foram coletadas biópsias endometriais nos dias7 (n 10), 10 (n 10) e 13 (n 10). Imediatamente após a coleta, o útero foi lavado e as éguas em que foi obtido embrião, foram inseridas no grupo de éguas prenhes. Um maior calibre dos vasos sanguíneos foi observado em prenhez comparados às éguas cíclicas do dia 7 aos 13. No sétimo dia pós-ovulação, uma grande perda de células ciliadas foi evidente no grupo de éguas prenhes, comparadas ao grupo de éguas cíclicas, as células do epitélio endometrial estavam mais protusas e uma pequena quantidade de secreção histotrófica entre as dobras endometriais foi observada. No décimo dia de prenhez, secreção histotrófica glandular e do epitélio luminal estavam mais presentes comparadas às éguas do grupo cíclico. No dia 13 de prenhes, foi observado um grande conteúdo de histotrofo nas aberturas glandulares que estavam cercadas por células ciliares. Ocorreram alterações no ambiente uterino logo após a entrada do embrião no útero. No estroma e no lúmen, essas modificações parecem visar fornecer a nutrição necessária para o desenvolvimento inicial do embrião e estas mudanças nas estruturas celulares irão interagir na sinalização embrionária, futura fixação, implantação e placentação. / The early pregnancy of mare is a fascinating period that encompasses numerous and intense changes in its development, many of which are unique to the equine species. This development depends on the maintenance of the luteal function, the establishment of a favorable uterine environment and a precise and orchestrated interaction between the concept and the uterine environment. The aim of this study was to evaluate histological changes in the endometrium in days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sample collection, the mare‟s uteri were flushed, and those mares with embryo recovery were assigned to the Pregnant group. A larger blood vessel caliber was observed in pregnant mares than in cyclic from day 7 to 13. On the 7th day a large loss of ciliated cells was evident in the group of pregnant mares in comparison with the Cyclic group and the superficial cells of the endometrium were more protruded, and a small amount of histotrophic material between the folds was observed. On the 10th day of pregnancy, the glandular histotrophic secretion and the secretion of luminal epithelium became more intense than the secretion of cyclic mares. On the 13th day of pregnancy, a very large amount of histotroph was observed within large glandular openings surrounded by ciliated cells. Changes occurred in the uterine environment thereupon the entry of the embryo into the uterus. In the stroma and in the lumen, these modifications seem aim to provide the necessary nutrition for the initial development of the embryo and to promote changes at cellular structures that will interact in the embryonic signaling and future fixation, implantation and placentation.

Background: Inadequate foetal growth cannot be remedied postnatally, leading to severe consequences for neonatal and adult development. Furthermore, sexual dimorphism in placental development has been suggested in humans although this remains poorly investigated in the pig. Hypotheses: Intrauterine Growth Restriction (IUGR) occurs due to aberrant conceptus attachment, which leads to alterations in angiogenesis and vascularity of the feto-maternal interface. Altered gene expression and vascularity will be observed at the feto-maternal interface in male foetuses compared to female foetuses. Increased apoptosis and decreased proliferation will be observed in the feto-maternal interface associated with the lightest foetuses compared to the closest to mean litter weight (CTMLW) foetuses. Aims: This thesis aimed to investigate the association between foetal size and sex and: integrin signalling; apoptotic and proliferation pathways; umbilical arterial (UA) blood flow; and angiogenesis and vascularity at the feto-maternal interface. This was performed by the collection of placental and endometrial samples associated with conceptuses or foetuses of different size (lightest and CTMLW) and sex at gestational day (GD) 18, 30, 45, 60 and 90. Conclusion This thesis has presented novel findings of associations between foetal size and sex, and placental and endometrial integrin signalling, apoptosis and proliferation, and angiogenesis and vascularity. Currently, this is the first suggestion in the literature that foetal size, and more intriguingly foetal sex, may have a strong influence on the activity of the endometrium. The mechanisms behind these findings warrant further investigation. Switches in the direction of differences at the feto-maternal interface between foetuses of different size were observed throughout gestation, notably between GD45 and 60, highlighting the dynamic nature of the feto-maternal interface and suggesting this as a potential window that could be manipulated by the industry to attempt to rescue the postnatal phenotype of IUGR piglets.

Ocular fundus image can provide information on pathological changes caused by local ocular diseases and early signs of certain systemic diseases. Automated analysis and interpretation of fundus images has become a necessary and important diagnostic procedure in ophthalmology. Among the features in ocular fundus image are the optic disc, fovea (central vision area), lesions, and retinal vessels. These features are useful in revealing the states of diseases in the form of measurable abnormalities such as length of diameter, change in color, and degree of tortuosity in the vessels. In addition, retinal vessels can also serve as landmarks for image-guided laser treatment of choroidal neovascularization. Thus, reliable methods for blood vessel detection that preserve various vessel measurements are needed. In this paper, we will examine the pathological issues in the analysis of retinal vessels in digital fundus images and give a survey of current image processing methods for extracting vessels in retinal images with a view to categorize them and highlight their differences and similarities. We have also implemented two major approaches using matched filter and mathematical morphology respectively and compared their performances. Some prospective research directions are identified. / Singapore-MIT Alliance (SMA)

It has been postulated that the control of thrombus formation on molecularly engineered surfaces is an important step in developing clinically durable small-diameter vascular prostheses. This has led to designing a membrane-mimetic assembly that contains physiological regulators of blood coagulation, thrombomodulin (TM) and heparin, to provide strategies for generating actively antithrombogenic surfaces. The membrane-mimetic construct contains polymeric phospholipid monolayer on an alkylated polyelectrolyte multilayer supported by planar substrate such as glass or silicone. When incorporated with TM, the model platform exhibited the biological function by catalyzing activation of protein C. Surface TM activity was extensively investigated at physiologic shear rates (50 sec-1 and 500 sec-1). Significantly, reaction rates become saturated at TM surface densities greater than or equal to ~ 800 fmole/cm2 due to due to a transport limitation. Based on the similar membrane-mimetic construct, a functional heparinized surface was designed as an alternative anticoagulant system. Immobilization of heparin onto membrane-mimetic surfaces was achieved through biotin-streptavidin binding specificity. Activity of surface heparin to facilitate thrombin inactivation was investigated at shear rates of 50 and 500 sec-1. Significantly, rate of thrombin decay becomes saturated when the surface coverage of heparin is higher than 4.4 pmole of heparin per cm2. We further investigated the effects of surface bound TM and heparin on tissue factor (TF) -induced thrombin generation in a flow model. Specifically, TF positioned over a 2 x 6 mm2 upstream region as a trigger for thrombin generation and TM and/or heparin positioned over the remaining downstream (34 x 6 mm2) portion of the test film. Compared to TF alone surface, thrombin generation was profoundly reduced in the presence of surface bound TM and/or heparin. Significantly, thrombin production was maximally inhibited more than 85% in the presence of TM and heparin, possibly due to anticoagulant synergism of both anticoagulants. We believe that current membrane-mimetic systems can potentially create actively antithrombogenic surfaces.

One of the major causes of death in developed countries is cardiovascular disease that affects small and medium sized blood vessels. In most cases autologous grafts have to be used which have limited availability. A functional tissue engineered vessel can be the ultimate solution for vascular reconstruction. Tissue engineered constructs with cells growing in an organized manner have been shown to have improved mechanical properties. In the present study collagen scaffolds with 650 nm, 500 nm and 332.5 nm wide channels and ridges were seeded with human vascular smooth muscle cells (VSMC) and human endothelial cells seperately and then co-cultured on tubular scaffolds. When the films were seeded with endothelial cells it was observed that nanopatterns do not affect cell proliferation or initial cell alignment / however, they significantly influenced cell retention under shear (fluid flow). While 35 &plusmn / 10 % of the cells were retained on unpatterned films, 75 &plusmn / 4 % was retained on 332.5 nm patterned films and even higher, 91 &plusmn / 5 % was retained on 650 nm patterned films. It was shown that nanopatterns as small as 332.5 nm could align the vascular smooth muscle cells (VSMC) and that alignment significantly improved mechanical properties. Presence of nanopatterns increased the ultimate tensile strength (UTS) from 0.55 &plusmn / 0.11 on Day 0 to as much as 1.63 &plusmn / 0.46 MPa on Day 75, a value within the range of natural arteries and veins. Similarly, Young&amp / #8217 / s Modulus values were ca. 4 MPa, again in the range of the natural vessels. Since the films would be ultimately rolled into tubes of collagen, nutrient transfer through the films is quite crucial. Diffusion coefficient for 4-acetaminophenol and oxygen through the collagen films were found to be 1.86 &plusmn / 0.39 x 10-7 cm2.s-1 and 5.41 &plusmn / 2.14 x 10-7 cm2.s-1, repectively in the unseeded form, and increased by 4 fold after cell seeding, which is comparable to that in natural tissues. When both cell types were co-cultured on the nanopatterned tubes (a both-side nanopatterned collagen tube), it was shown that on the outside of the tube VSMCs proliferated in an oriented manner and on the inside endothelial cells proliferated as a monolayer. Therefore, this study showed that cell guidance enhances the mechanical properties of engineered vessels, and help overcome the two most important challenges in vascular tissue engineering / the need for adequate mechanical properties and continuous lining of endothelial cells even under physiological shear stress.

Recombinant synthesis of elastin-mimetic proteins has been employed for several decades, however, long-term biocompatibility and biostability of such proteins was not fully defined. We present virtually crosslinked elastin-mimetic proteins which exhibit exceptional biocompatibility and long-term biostability over a period of at least seven months. This report is the first evidence of a non-chemically or ionically crosslinked system that exhibits long-term in vivo stability. Although, physically crosslinked protein-based materials possess a number of advantages over their chemically crosslinked counterparts, physical crosslinks and the related domains so formed may be deformed or damaged at applied stresses lower than those required to disrupt covalent crosslinks. In this regard, we have synthesized a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. We have demonstrated that chemical crosslinking provides an independent mechanism for control of protein mechanical responses. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. A number of reports have described the design of synthetic genes, which encode elastin-like proteins for bacterial expression in Escherichia coli. Although advantages with this expression system exist, significant limitations including the lack of eukaryotic post-translational systems, the tendency to sequester mammalian proteins into inclusion bodies, difficult purification protocols, and endotoxin contamination have been noted. We demonstrate the expression of a recombinant elastin-mimetic protein from P. pastoris. A novel synthetic strategy, monomer library concatamerization, was utilized in designing non-repetitive elastin genes for highly repetitive protein sequences. It is likely that this strategy will be useful for creating large, repetitive genes for a variety of expression systems in order to more closely approach the genetic diversity inherent to native DNA sequences. All told, elastin-based protein polymers are a promising class of material characterized by high degree of biocompatibility, excellent biostability, and a tunable range of mechanical properties from plastic to elastic. A variety of options facilitate the processing of these biopolymers into chemically crosslinked or non-crosslinked gels, films, or nanofibers for any of a number of implant applications including structural components of artificial organs and engineered living tissues, carriers for controlled drug release, or biocompatible surface coatings.