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Title: Effect of calcium phosphate ceramic architectural features on the self-assembly of microvessels in vitro
Author: Gariboldi, Maria Isabella
ISNI:       0000 0004 7651 7380
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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One of the greatest obstacles to clinical translation of bone tissue engineering is the inability to effectively and efficiently vascularise scaffolds. This limits the size of defects that can be repaired, as blood perfusion is necessary to provide nutrient and waste exchange to tissue at the core of scaffolds. The goal of this work was to systematically explore whether architecture, at a scale of hundreds of microns, can be used to direct the growth of microvessels into the core of scaffolds. A pipeline was developed for the production of hydroxyapatite surfaces with controlled architecture. Three batches of hydroxyapatite were used with two different particle morphologies and size distributions. On sintering, one batch remained phase pure and the other two batches were biphasic mixtures of α-tricalcium phosphate (α-TCP) and hydroxyapatite. Sample production methods based on slip casting of a hydroxyapatite-gelatin slurry were explored. The most successful of these involved the use of curable silicone to produce moulds of high-resolution, three dimensional (3D) printed parts with the desired design. Parts were dried and sintered to produce patterned surfaces with higher resolution than obtainable through conventional 3D printing techniques. Given the difficulties associated with the structural reproducibility of concave pores architectures in 3D reported in the literature, in this work, a 2.5D model has been developed that varies architectural parameters in a controlled manner. Six contrasting architectures consisting of semi-circular ridges and grooves were produced. Grooves and ridges were designed to have widths of 330 μm and 660 μm, with periodicities, respectively, of 1240 μm and 630 μm. Groove depth was varied between 150 μm and 585 μm. Co-cultures of endothelial cells and osteoblasts were optimised and used to grow microcapillary-like structures (referred to as "microvessels") on substrates. Literature shows that these precursors to microcapillaries contain lumina and can produce functional vasculature, demonstrating their clinical promise. The effects of the composition and surface texture of grooved samples on microvessel formation were studied. It was found that surface microtopography and phase purity (α-TCP content) did not affect microvessel formation. However, hydroxyapatite architecture was found to significantly affect microvessel location and orientation. Microvessels were found to form predominantly in grooves or between convexities. Two metrics - the degree of alignment (DOA) and the degree of containment (DOC) - were developed to measure the alignment of endothelial cell structures and their localisation in grooves. For all patterned samples, the CD31 (an endothelial cell marker) signal was at least 2.5 times higher along grooves versus perpendicular to grooves. In addition, the average signal was at least two times higher within grooves than outside grooves for all samples. Small deep grooves had the highest DOA and DOC (6.13 and 4.05 respectively), and individual, highly aligned microvessels were formed. An image analysis method that compares sample X-ray microtomography sections to original designs to quantify architectural distortion was developed. This method will serve as a useful tool for improvements to architectural control for future studies. This body of work shows the crucial influence of architecture on microvessel self-assembly at the hundreds of micron scale. It also highlights that microvessel formation has a relatively low sensitivity to phase composition and microtopography. These findings have important implications for the design of porous scaffolds and the refinement of fabrication technologies. While important results were shown for six preliminary architectures, this work represents a toolkit that can be applied to screen any 2.5D architecture for its angiogenic potential. This work has laid the foundations that will allow elucidating the precise correspondence between architecture and microvessel organisation, ultimately enabling the "engineering" of microvasculature by tuning local scaffold design to achieve desirable microvessel properties.
Supervisor: Best, Serena M. Sponsor: Gates Cambridge Scholarship ; Geistlich Pharma AG
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Biomaterials ; Hydroxyapatite ; Biomaterial Architecture ; Bioceramics ; Architecture ; Co-cultures ; Endothelial Cells ; Vascularisation ; Angiogenesis ; Microvessels ; Osteoblasts ; Medical Materials ; Bone Substitution ; Bone Substitute Materials ; 3D Printing ; Additive Manufacturing ; Bioprinting ; Microcapillary-Like Structures