Angiogenesis in congenital vascular malformations: a dynamic view on a static lesion

Aangeboren afwijkingen in de aanleg van bloedvaten zouden, omdat de vaten langzaam groeien, pas op latere leeftijd klachten geven. Dat blijkt niet altijd zo te zijn, want bij een deel van de afwijkingen vormen zich nieuwe, kleine bloedvaten, waardoor de afwijkende groei sneller gaat. Ludovica Jorna laat zien dat deze nieuwe vaten vaak worden gevormd in die afwijkingen waar het bloed snel stroomt door aanwezigheid van fistels (niet natuurlijke verbindingen) tussen slagaders en aders. Een toename van kleine bloedvaten was tot nu toe bekend bij zeer jonge patiënten met een goedaardige woekering van de vaten. Zij worden met succes behandeld met medicijnen. Mogelijk kunnen die middelen ook worden toegepast bij kinderen met snel groeiende aangeboren afwijkingen in de aanleg van de bloedvaten

Development of angiogenic models to investigate neovascularisation for tissue engineering applications

The aim of this project was to develop in vitro models for angiogenesis that have the capability of combining pro-angiogenic cells with an extracellular matrix (ECM) component that can be monitored under flow conditions to learn more about the ‘rules’ of angiogenesis. Significant advancement has been made in the field of tissue engineering in recent years, however one of the current obstacles limiting progression is the production of thick, complex tissues due to the lack of rapid neovascularisation of the constructs upon implantation. Blood vessel formation is tightly regulated and relies on the chronologically precise adjustment of vessel growth, maturation and suppression of endothelial cell growth - all of which are controlled by a large number of factors which influence each other. To induce vascularisation within tissue-engineered (TE) substitutes the same processes need to occur. A number of different vascularisation strategies have been investigated in an attempt to overcome this issue but as yet there is no unified solution to this problem. The most promising attempts have used scaffolds with vascular architectures, perfusion conditions and relevant cell types. Although it is recognised that perfusion conditions, the cell type and scaffold architecture are important with regards to vascularisation strategies many of the techniques fail to consider them in combination. It is therefore important to take a step back and understand how these factors work together to i promote angiogenesis in order to advance this crucial area. This lack of understanding is further compounded by the deficiencies of current angiogenesis models. Current in vitro models fail to combine the use of supporting cells, the extracellular matrix and fluid flow in 3D. Although this complexity exists within in vivo models such assays are primarily limited by the species used, organ sites available and complicated analysis techniques. In this project two in vitro angiogenesis models were developed. The first was derived from the decellularisation of a rat jejunum. Characterisation showed the retention of key extracellular matrix (ECM) components and the removal of almost all cellular material. Re-endothelialisation with human dermal endothelial cells (HDMECs) of the patent vascular network showed enhanced results when co-cultured with human dermal fibroblasts (HDFs). In an attempt to induce angiogenesis, vascular endothelial growth factor (VEGF) loaded gels were placed on top of the scaffold whilst being continuously perfused with media. Placing VEGF loaded gels onto the recellularised jejunum led to the expression of the Notch ligand Delta- like-4 (DLL4) by HDMECs indicating their transformation into tip cells which are synonymous with sprouting angiogenesis. The second was produced through the combination of robocasting and electrospinning. Nanofibrous poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) scaffolds with hollow channels capable of perfusion were produced that could be re-endothlialised with HDMECs. Again the addition of HDFs enhanced cellular distribution in the channels. Placing VEGF loaded gels onto the surface of the scaffolds led to the outgrowth of HDMECs into the gel, forming perfusable tubules. Overall these two models overcome limitations of current in vitro models since they offer the capability of combining pro-angiogenic cells with ECM components that can be monitored under flow conditions. With further development they could provide more sophisticated platforms upon which to investigate the angiogenic process

Development of a synthetic small calibre vascular bypass graft

Polyurethanes are an attractive class of material for bioprosthesis development due to the ability to manipulate their elasticity and strength. However, their use as long term biological implants is hampered by biodegradation. A novel polyurethane has been developed which incorporates nano-engineered polyhedral oligomeric silsesquioxane within poly(carbonate-urea) urethane to improve the biostability of the latter. Previous investigators have found this material to be cytocompatible and to have low thrombogenicity. The medium and long term clinical results of currently available prosthetic small calibre vascular bypass grafts are poor, due to neo-intimal hyperplasia associated with their non-compliant properties. The investigation reported here commences with the benchtop manufacture of compliant small calibre grafts using an original extrusion- phase inversion technique. The reproducibility of the technique as well as the effect on the pore structure of different coagulation conditions is demonstrated. Fundamental mechanical characterisation of the grafts produced is then presented, by way of tensillometry to demonstrate the viscous and elastic properties of the material. These are made more relevant to the clinical setting with functional mechanical characterisation of the grafts, showing graft compliance in a biomimetic flow circuit along with viscoelastic hysteresis, along with burst pressure testing. An examination of burst pressure testing methodology is also shown, in the light of the various non-standardised strategies reported in the graft-testing literature. Mechanical characterisation shows the short-term safety for use, but durability studies in the biological haemodynamic environment serve to assess longer term fatigability as well as confirming biostability. This has been reported using a stringent ovine carotid interposition model which remained patent over the full investigation period representing at least 45 million pulsatile cycles. Physico-chemical analysis; integrity of the structure, microstructure and ultrastructure; preservation of mechanical properties and immunohistological analysis were used to examine the grafts after implantation to show their healing properties and biostability

Development and Modeling of a Polymer Construct for Perfusion Imaging and Tissue Engineering.

The physical and computational modeling of distributed fluid flow to vascular beds remains a challenging issue. The computational resources required, and the complexity of capillary networks makes modeling infeasible. The resolution limits of manufacturing techniques make physical models difficult to fabricate and manipulate under experimental conditions. As such, an in vitro polymer construct was developed with structural properties of small arteries and the bulk flow characteristics of capillary beds. Rapid prototyping and scaffolding techniques were used to fabricate vascular trees amendable to scaffold compartments. Several scaffold architectures were evaluated to achieve target fluid flow characteristics for implementation in a dynamic contrast-enhanced computed tomography (DCE-CT) imaging phantom and endothelial cell bioreactor, respectively. Experimental flow measurements were compared to measurements from computational simulations. In addition, the flow-induced shear stress across the construct was modeled to identify the optimal settings within the bioreactor. In addition, the cytocompatibility of the polymer construct was optimized. Vascular trees were reliably fabricated to achieve arteriole-like flow. Rapid prototyped polycaprolactone (PCL) scaffolds produced distinct differential flow ranges, marked by a decrease in flow rate across the network. The construct served as a viable dynamic flow phantom capable of generating signals typical of organs imaged with DCE-CT. Furthermore, simulations of the construct as a bioreactor provided guidance on the boundary conditions required for stimulatory shear stress within the scaffolds. Under static conditions, endothelial cells were cultured on PCL scaffolds modified with extra-cellular matrix mimicking biological and chemical agents. All surface modifications exhibited similar cell proliferation and function. However, the Arg-Gly-Asp (RGD) surface-modified constructs exhibited an optimal spatial distribution for future endothelial cell bioreactor investigations. This work demonstrates a method for modeling and physically simulating a bifurcating vascular tree adjoined to scaffold compartments with tunable flow, for application to perfusion imaging and in vitro tissue engineering (tissue and tumors).PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107136/1/auresa_1.pd