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Cardiovascular disease is the main cause of death in the United States. Many of these conditions require the grafting or bypassing of compromised blood vessels. To this effect, biological vascular grafts (autografts and allografts) are the first line of action. However, when the patient lacks vasculature suitable for grafting use, several synthetic grafting options are available. The search for an inert biomaterial for vascular grafts has proven to be unsuccessful. This makes the interaction taking place on the blood-biomaterial interface critical for the success of the grafts. This thesis introduces a new bio-inspired approach to tackle the mechanical and biological challenges of vascular material design. The hypothesis of this research is that recombinant fibronectin protein can be stably incorporated onto elastin-mimetic polymers to increase endothelialization. Recombinant elastin, designed to recreate the mechanical properties of natural elastin as a candidate material for vascular graft fabrication, was used as a model surface. Recombinant fibronectin-functionalized elastin-mimetic polymer displayed significant improvement in cell adhesion. Quantification of surface bound recombinant fibronectin verified the concentration dependence of this cell adhesive behavior. Modified elastin-mimetic polymer also demonstrated an enhanced ability to support endothelial cell proliferation. Furthermore, the stability of recombinant fibronectin-modified polymers was assessed. These studies provide the foundation for fabricating elastin-mimetic vascular grafts with improved endothelialization and subsequent biological performance.

Les matériaux à structure tridimensionnelle laissent entrevoir de nombreuses applications prometteuses dans le domaine de l'ingénierie tissulaire et de la médecine régénérative en fournissant un micro-environnement approprié pour l'incorporation de cellules ou de facteurs de croissance afin de régénérer des tissus ou organes endommagés. Dans ce contexte, une membrane a été élaborée à partir d'un mélange de poly (ε-caprolactone) PCL / chitosan CHT à partir d'une technique d'inversion de phase permettant un apport localisé de non solvent. La technique permet d'obtenir une double morphologie poreuse : (i) des macrovides en surface (gros pores) facilement accessibles pour l'invasion et la viabilité des cellules; (ii) un réseau macroporeux interconnecté (petits pores) pour transférer les nutriments, l'oxygène, le facteur de croissance à travers le matériau. Les propriétés physico-chimiques (taille des pores, chimie de surface et biodégradabilité) des matériaux ont été caractérisées. Il est montré comment il est possible d'ajuster les propriétés de la membrane en modifiant le rapport PCL / CHT. Des cultures de cellules souches mésenchymateuses humaines (CSMh) ont été réalisées sur la membrane. La viabilité et la prolifération cellulaires ont été étudiées par des essais de test au MTT et de taux d'absorption d'oxygène. Les expériences démontrent que la membrane est biocompatible et peut être colonisée par les cellules. La microscopie confocale montre que les cellules sont capables de pénétrer à l'intérieur des macrovides de la membrane. La prolifération cellulaire de CSM dans ce matériau pourrait être utile pour augmenter la longévité d'autres cellules primaires en modifiant les CSM pour produire des facteurs de croissance. Pour tester le comportement dynamique des cellules sur la membrane, un dispositif d'organe sur puce a été développé avec des cellules endothéliales ombilicales humaines ensemencées sur la membrane. Les résistances hydrauliques de la barrière cellulaire sur la membrane ont été quantifiées en temps réel pour une pression trans-endothéliale (PTE), 20 cm H2O à 37 ° C et avec des cellules vivantes après 1 jour et 3 jours après l'ensemencement. Les résultats suggèrent que ce type d'échafaudages polymères peut être utile à l'avenir comme patch in vivo pour réparer des vaisseaux endommagés. / Over the last decades, three-dimensional (3D) scaffolds are unfolding many promising applications in tissue engineering and regenerative medicine field by providing suitable microenvironment for the incorporation of cells or growth factors to regenerate damaged tissues or organs. The three-dimensional polymeric porous scaffolds with higher porosities having homogeneous interconnected pore network are highly useful for tissue engineering. In this context, a poly (ε- caprolactone) PCL/chitosan CHT blend membrane with a double porous morphology was developed by modified liquid induced phase inversion technique. The membrane shows: (i) surface macrovoids (big pores) which could be easily accessible for cells invasion and viability; (ii) interconnected microporous (small pores) network to transfer essential nutrients, oxygen, growth factors between the macrovoids and throughout the scaffolds. The physico-chemical properties (pore size, surface chemistry and biodegradability) of the materials have been characterized. This study shows how it is possible to tune the membrane properties by changing the PCL/CHT ratio. Human mesenchymal stem cell (hMSCs) culture was performed on the membranes and the cell viability and proliferation was investigated by MTT assay and oxygen uptake rate experiments. The experiments demonstrate that the membranes are biocompatible and can be colonized by the cells at micron scale. Confocal microscopy images show that the cells are able to adhere and penetrate inside the macrovoids of the membranes. Both cell proliferation and oxygen uptake increase with time especially on membranes with lower chitosan concentration. The presence of chitosan in the blend produces an increase of porosity that affect the entrapment of the cells inside the porous bulk of the membranes. Successful cellular proliferation of hMSCs could be useful to enhance longevity of other primary cells by production of corresponding growth factors. To test the dynamic behavior of cells on the membranes, an organ-on-chip (OOC) device has been developed with human umbilical endothelial cells (HUVECs) seeded on the membrane. The hydraulic resistance of the cellular barrier on the membrane has been quantified for real time trans-endothelial pressure (TEP) 20 cmH2O at 37 degree C and with living cells after 1 day and 3 day of post seeding. Results suggests this kind of polymeric scaffolds can be useful in future as an in vivo patch to repair disrupted vessels.

The aim of the presented experimental work was to study an acute cell and antibody- mediated immune response in recipients of abdominal aortic grafts treated by a new standardized clinical cryopreservation/slow thawing protocol used in the "Vascular graft transplant program in the Czech Republic" in a rat model. Another aim of our study was to compare the influence of two basic types of conservation protocols used in this program (cryopreservation/slow thawing protocol and cold-stored protocol) on the acute immune response after transplantation of such treated abdominal aortic grafts in rats. Cryopreserved abdominal aortic grafts were transplanted syngeneously between Lewis rats (CRYO-ISO group, cryopreservation period 172.6 days) and allogeneically between Brown-Norway and Lewis rats (CRYO-ALO group, cryopreservation period 179.3 days). The grafts were explanted on day 30 after transplantation and subsequently examined by histological and immunohistochemical methods, focusing on typical signs of acute rejection in the three basic layers of the aortic wall. We monitored the presence of endothelial cells, signs of intimal hyperplasia, tunica media thickness, the presence of necrosis and deposition of imunoglobulin class G in this layer, the number of CD4+, CD8+ and LEW MHC II+ immunocompetent cells...

Les taux élevés de mortalité et de morbidité associés aux maladies vasculaires en font des pathologies dont les conséquences physiopathologiques, chirurgicales et socio-économiques sont d’une importance majeure pour le système de santé. Malgré leurs avantages, la disponibilité limitée des vaisseaux autologues a conduit au développement de prothèses synthétiques. Cependant, leur surface hautement thrombogène limite leur utilisation dans la substitution des vaisseaux de petit calibre (< 6 mm). De ce fait, à cause de leur obstruction précoce, la reconstitution d’une surface luminale proche de l’endothélium natif est incontournable. Pourtant, les revêtements de surface actuellement disponibles possèdent de médiocres qualités de rétention des néo-endothélium lorsqu’ils sont soumis à des contraintes de cisaillement physiologiques. Dans ce travail, nous proposons un substitut vasculaire de petit calibre endothélialisé réalisé à partir de trois éléments : 1) une matrice préparée à partir d’une artère ombilicale désendothélialisée, 2) un recouvrement de surface innovant constitué du film multicouche de polyélectrolytes (MPE) (PAH-PSS)3-PAH, et 3) un néo-endothélium constitué de cellules endothéliales matures ou progénitrices. Les études in vitro menées sur ces substituts ont montré que la formation, la rétention sous contraintes de cisaillement et la fonctionnalité du néoendothélium élaboré sur la surface luminale étaient améliorées par le film MPE. L’implantation du substitut par pontage termino-latéral sur le lapin a montré que le cahier des charges imputé aux substituts de petit calibre était rempli, principalement en termes de perméabilité et de diamètre, mais aussi de résistance à la suture et aux infections. En conclusion, le film MPE favorise le développement d’un substitut vasculaire de petit diamètre perméable à « long » terme et qui pourrait répondre aux exigences des chirurgiens / Vascular diseases with their high rate of mortality and morbidity belong to the pathologies involving important socio-economic factors for health system. Despite the advantages of autografts, the limited availability of autologous vessels has led to the development of synthetic prostheses. However, their high thrombogenic surface limits their use as small calibre vascular substitutes (< 6 mm). To prevent narrowing of small diameter vascular grafts, the reconstruction of a luminal surface close to the native endothelium is essential. However, the retention of the neo-endothelium subjected to shear stress is poor on the coatings currently available. In this work, we developed a small calibre endothelialized vascular substitute thanks to three elements: 1) a natural matrix prepared from umbilical artery, 2) an innovative coating based on the polyelectrolytes multilayer film (PEM) (PAH-PSS)3-PAH, and 3) used for cell culture of mature or progenitor endothelial cells. In vitro studies have shown that the formation, the retention under shear stress and the endothelial function of the neoendothelium on the luminal surface were improved by PEM film. The anastomosis of this substitute on rabbits has shown that the specifications essential to small calibre vascular grafts were reached, mainly in terms of permeability and diameter but also of resistance to suture and infections. In conclusion, PEM films helped us to develop a small diameter vascular substitute with long term patency

An injured vascular system has a substantial impact on an individuals overall health, and an understanding of the mechanisms that underlie blood vessel pathophysiology is required for the development of rational and effective treatment strategies. The phenotypic modulation of smooth muscle cells (SMC) during vascular injury, characterized by altered adhesion, migration and synthetic behavior, plays an important role in the eventual outcome. Specifically, the ability of SMCs to adhere to and remodel their extracellular environment via regulation of the syndecan class of cell adhesion molecules dictates the response of the vascular wall to local injury. The effect of in vitro syndecan-4 regulation on SMC adhesion was investigated through the use of a glass microsphere centrifugation assay, and an antisense-mediated reduction in gene expression was found to correlate with decreased adhesive strength. Regulation of syndecan-1, syndecan-2, and syndecan-4 gene expression was observed experimentally by mechanical strain of SMCs. Using real-time polymerase chain reaction (PCR), the kinetics of both static and cyclic mechanical strain were found to modify the gene expression in a time and strain magnitude-dependent manner unique to each syndecan. In particular, the responses of syndecan-4 were acute, but transient, while the evolution of syndecan-1 and syndecan-2 regulation was delayed by comparison. Mechanical strain also modulated syndecan-4 protein expression and ectodomain shedding, as measured by Western immunoblotting, and this effect was found, through selective inhibition, to be at least in part dependent on mitogen-activated protein (MAP) kinase signaling. In particular, intact extracellular signal-regulated MAP kinase (ERK) 1/2 and c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK/SAPK) signaling pathways were found to be required for the observed strain-induced shedding. These findings offer a better understanding of syndecan function in response to mechanical strain and suggest potential new mechanisms by which physical forces may modulate vascular SMC behavior and regulation during normal physiology, pathologic conditions, and engineered arterial substitute development.

Mesenchymal stem cells (MSCs) may benefit vascular cell-based therapies as smooth muscle or endothelial cell substitutes or through paracrine actions to repair, replace, or regenerate vascular tissue. Previous studies have demonstrated that MSCs can adopt traits of smooth muscle cells (SMCs) or endothelial cells (ECs), as well as secrete specific factors that tune signaling and material properties in the local environment. Few studies have investigated the cell signaling response of MSCs to mechanical forces present in the vasculature: specifically, shear stress due to blood flow and cyclic strain due to pulsatile blood flow. Thus, the central objective of this dissertation was to determine the signaling responses of MSCs to vascular-relevant applied physical forces, in comparison with that of differentiated vascular cells. Vascular-relevant mechanosignaling of MSCs was assessed through two comparisons: (1) MSC and SMC responses to applied cyclic strain and (2) MSC and EC responses to applied fluid shear stress. MSCs and SMCs were seeded on fibronectin-coated silicone and subjected in vitro to cyclic strain (10%, 1 Hz) or parallel static culture using a custom-built equibiaxial cyclic strain device. Gene expression analysis of 84 signal transduction molecules demonstrated both cell types respond with significant (p<0.05, n=3) fold-changes (|FC|≥ 1.5) within 24 hours of applied equibiaxial strain. Most strain-responsive genes identified were significantly strain-responsive in only one cell type. A signaling trio of Interleukin 8, Vascular cell adhesion molecule 1, and Heme oxygenase 1 was significantly altered in both MSCs and SMCs, suggesting cyclic strain regulates immune and inflammatory functions in both cell types. The response to shear stress of MSCs and ECs was compared using cells seeded on type I collagen or fibronectin and exposed to steady laminar shear stress (5 or 15 dyn/sq-cm) using a parallel plate shear chamber system. Gene expression was compared in MSCs and ECs for a panel of immune and inflammation-related markers. Expression of Cox-2 and Hmox-1 increased significantly (p<0.05, n≥3; |FC|≥1:5) in both cell types. Reduced shear stress-responses of Mcp-1, Pecam-1, and VE-Cad in MSCs relative to ECs suggests that MSCs promote less inflammation and immune activation in response to shear stress than ECs. Mechanosensitivity profiles for MSCs and differentiated vascular cells were broadened using whole genome microarrays. These high-throughput studies confirmed that (1) signaling profiles between sample groups vary significantly more (p<0.05, n=3) with cell type than applied force condition and (2) a subset of conserved mechanosensitive genes alter expression levels significantly and in the same direction fold-change in multiple cell types. Bioinformatics analysis of these conserved mechanoresponsive genes highlighted oxidative stress, cell cycle, and DNA replication as functions regulated by vascular-relevant mechanical cues. These studies demonstrate that MSCs partially reproduce differentiated vascular cell mechanosignaling, while simultaneously altering expression of genes not typically force-responsive in vascular cells. This work defines a role for conserved mechanosignals, based on genes whose expression in response to applied force alters significantly (p<0.05, n≥3) and by at least 1.5-fold change in multiple cell types and/or force types. Comparisons completed for this dissertation motivate future studies to track the functional impact of specific similar or unique MSC mechanoresponses. This work contributes to design of MSC-based vascular therapies and an understanding of stem and differentiated cell mechanobiology.

Introduction: Cardiovascular surgery is a relatively young but progressively evolving field in medicine. More specifically, in the past decades, cardiac surgery achieved significant advances in understanding the causes, progression and treatments of ischemic heart disease (IHD). The IHD is the most common coronary disease, and it ranks first in morbidity and mortality in the developed world. It justifies the need for significant fundamental research as well as its study in clinical practice. It now includes specialized cardiovascular centres with the complex specialized treatments. A group of interventional cardiologists capable of performing routine examinations of the coronary veins using selective angiography has been established. They can eventually also perform percutaneous coronary interventions with direct stent implants. Thus the advances have been made in comprehensive indication of the patients towards their optimal treatments under the regime of a cardio-team. Such team comprises of a cardio-surgeon, interventional cardiologist, echocardiography specialist and the attending physician who is usually the cardiologist. The IHD treatments involve preventive cardiology with the regime measures and checks, pharmacotherapy, interventional cardiology and cardiac surgery to spa treatment and...

The development of vascular replacement for low flow rates is a topical issue. The model for developing the development of replacement properties, which are based on the idea of assuming the characteristics of the biological model - vena saphena and programming these properties into a model of constructed replacement is one of the possible directions of development. The presented replacement, which is the result of the work of the author's team, consists of three parts - a non-absorbable scaffold representing the media, and two absorbable collagen layers - pseudointima and pseudoadventice. Target parameters of the prosthesis were determined by test results of the basic physical testing method - uniaxial tensile test and inflation-extension test, as well as other procedures in human saphenous specimens. The key issue is the technology of producing the collagen layer of the prosthesis. However, other manufacturing processes can also have a significant impact on vascular prosthesis properties, such as collagen hardening, antithrombogenic treatment of the inner surface of the vascular replacement, and the use of a sterilization method. Furthermore, the author deals with the development of a new female component of the three- layer vascular prosthesis of the Czech carp, which is characterized by lower...

Introduction: Cardiovascular surgery is a relatively young but progressively evolving field in medicine. More specifically, in the past decades, cardiac surgery achieved significant advances in understanding the causes, progression and treatments of ischemic heart disease (IHD). The IHD is the most common coronary disease, and it ranks first in morbidity and mortality in the developed world. It justifies the need for significant fundamental research as well as its study in clinical practice. It now includes specialized cardiovascular centres with the complex specialized treatments. A group of interventional cardiologists capable of performing routine examinations of the coronary veins using selective angiography has been established. They can eventually also perform percutaneous coronary interventions with direct stent implants. Thus the advances have been made in comprehensive indication of the patients towards their optimal treatments under the regime of a cardio-team. Such team comprises of a cardio-surgeon, interventional cardiologist, echocardiography specialist and the attending physician who is usually the cardiologist. The IHD treatments involve preventive cardiology with the regime measures and checks, pharmacotherapy, interventional cardiology and cardiac surgery to spa treatment and...