Ex Vivo Tissue Vascularisation

Lien Chau (2010). Ex Vivo Tissue Vascularisation PhD Thesis, Aust Institute for Bioengineering & Nanotechnology, The University of Queensland.

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Author Lien Chau
Thesis Title Ex Vivo Tissue Vascularisation
School, Centre or Institute Aust Institute for Bioengineering & Nanotechnology
Institution The University of Queensland
Publication date 2010-04
Thesis type PhD Thesis
Supervisor Professor Justin Cooper-White
Dr Barbara Rolfe
Total pages 210
Total colour pages 51
Total black and white pages 159
Subjects 11 Medical and Health Sciences
Abstract/Summary An overriding limiting factor in tissue engineering is the current inability to vascularise large three-dimensional scaffolds. In an attempt to alleviate this problem, this thesis is focused on generating a viable microvascular construct for tissue engineering purposes. Through a holistic approach, it was recognised that the construct will require an effective combination of cells, scaffold material, and microvascular network design. Since endothelial cells line all blood vessels, and are exposed and regulated by shear stress, understanding the effects of shear on endothelial cells was recognised as necessary to ensure cell survival in an artificial microvascular network. Furthermore, the network is required to provide uniform distribution of nutrients to all cells within the construct, which is made from a material that encourages relevant cell development and angiogenesis. Therefore, three key objectives were identified in this thesis: 1. To understand the effects of shear on endothelial cells and determine the optimal flow and shear stress ranges for culturing endothelial cells in microchannels of vasculature dimensions; 2. To design a vascular network structure that mimics relevant architectural features and dimensions of natural vasculature, and allows uniform distribution of nutrients to all cells in the network; and 3. To incorporate the vascular network design into a gelatin hydrogel system to study angiogenesis. To investigate the effects of shear on human umbilical vein endothelial cells (HUVECs), a multishear device was developed (Chapters 3 and 4). The device has one inlet, one outlet and 10 channels, all with dimensions within natural microvasculature (height = 100 µm and width = 200 µm), and allows the simultaneous evaluation of 10 different shear stresses, covering physiological shear stresses (1 - 130 dynes cm-2, 0.1 - 13 Pa). The multishear microdevice was fabricated using poly-dimethylsiloxane (PDMS), and HUVECs were seeded and perfused for 20 h in the device. To probe for the optimal flow and shear range, HUVECs in the microchannels were compared to the static control in terms of cell morphology (cell and nuclear sizes, perimeters and circularities) and expression level of von Willebrand Factor (vWF), a large multimeric plasma protein produced specifically by endothelial cells and involved in promoting platelet adhesion to damaged vessels walls. In brief, HUVECs under shear stresses range of 1 - 3 dynes cm-2 (0.1 - 0.3 Pa) showed similar vWF content, cell and nuclear size and perimeter to static cultures. This shear range was considered to be the most effective for HUVECs’ survival under perfusion in microchannels and networks, and it was implemented in the microvascular network device in Chapter 5 to create a fully artificial, endothelialised microvascular network. The microvascular network in this thesis was designed as a simple ladder structure with an inlet and outlet connected by five microchannels (Chapter 5). It was also designed to mimic natural vasculature dimensions and fluid dynamics, and have uniform velocity profiles in all its channels to allow uniform distribution of nutrients to all cells throughout the network. The device was then fabricated using PDMS, and the flow profiles in all five microchannels were assessed to be similar using micro particle image velocimetry (µPIV), validating the design concept. HUVECs were then seeded into the device and perfused for 24 h at shear stresses within the promising shear range (1 - 3 dynes cm-2, 0.1 - 0.3 Pa) found for HUVECs using the multishear microdevice. Immunostaining showed that HUVECs lined and formed a monolayer on all four sides of the microchannels, and bridged the corners. In addition, VE-cadherin was present throughout the vascular network, indicating an EC monolayer with stable cell-cell adhesions, and cells were similar in size throughout the network. Overall, the vascular network design was validated and it was used in a gelatin-based system to generate a microvascular construct (Chapter 6). A gelatin-based microvascular microdevice (Chapter 6), featuring the vascular network, was seeded with HUVECs and perfused at the same inlet flow rate as that used in PDMS microvascular device (Chapter 5) for 24 h and 5 d periods. After 24 h of perfusion, the cells were found to line all channels, but they did not form a monolayer like that observed in the PDMS microvascular device. Interestingly, the cells invaded and sprouted into the gelatin gel from the bottom and top of each channel and corners of the network after 5 d of perfusion. This observation validates the gelatin-based microvascular device as a promising microvasculature construct and platform for further investigations in angiogenesis. Overall, a microvascular construct with a 3D vascular network that reflects fluid flow equivalent to natural vasculature, has a single inlet and outlet, and allows angiogenesis has been developed in this thesis through a holistic and systematic approach, incorporating an effective combination of cells, microvascular network design and scaffold material.
Keyword vascularisation
vascular network
shear stress
endothelial cells
Additional Notes Pages to be printed in colour (NOTE these are page numbers that actually appear on the page): 4, 5, 7-10, 14, 18, 19, 21, 23-26, 38, 62, 74, 76, 83, 94-97, 105, 122, 123, 127-133, 143-145, 148, 150, 152, 155-157, 159, 164, 166-170, 178, 184

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Created: Fri, 29 Oct 2010, 17:53:34 EST by Miss Lien Chau on behalf of Library - Information Access Service