Multiscale Characterisation of Ice and Collagen: Physicochemical Development of Lyophilised Scaffolds for Tissue Engineering

The structural cues that determine cellular response to ice-templated collagen-based constructs can be fine-tuned by modulating the scaffold fabrication process. To produce lyophilised scaffolds, ice crystals are grown in an aqueous collagen suspension then sublimed to create a highly porous and interconnected structure. The collagen scaffold can be subsequently chemically crosslinked to provide the desired rigidity and stability. Although previous work has demonstrated the effects of a number of processing parameters on the resulting structures and cellular behaviour, several questions remain unanswered. The aim of this thesis is to improve understanding of fundamental physicochemical phenomena which determine the structure of the collagen constructs, across several length scales. The main areas considered include: exploring mechanisms of ice morphology development during lyophilisation, improving characterisation methods for the architectures fabricated, isolating the effect of additives on scaffold microstructure and finally, characterising the effect of distinct crosslinking chemistries. The work reported in this thesis brings together a combination of computational models and experiments to explore these areas. Firstly, the ice crystal growth during freezing was investigated using a novel phase-field model and the freezing and sublimation processes were characterised through environmental scanning electron microscopy (ESEM) experiments. The dendrite growth rates predicted by the model were most significantly affected by temperature and thermal flux, whereas morphology was shown both computationally and experimentally to be influenced by the surface energy of the aqueous suspension. Furthermore, ice crystals imaged during sublimation revealed periodic mesoscopic facetting characterised by a vapour pressure-specific wavelength. Collagen scaffolds dried at different temperatures (273 and 293 K) and pressures (10, 100, 333 Pa) possessed this mesoscopic roughening as well as reduced scaffold connectivity at 293 K and 10 Pa. Consequently, these results suggest the ability to fine-tune the topography and morphology of a collagen scaffold as required for a particular cell type through the choice of freezing and drying parameters. Secondly, the ability to exploit and characterise microstructural connectivity was investigated. A standardised protocol for X-ray micro-computed tomography (MicroCT) data acquisition was devised, giving rise to a novel segmented percolation method to characterise interconnectivity. Analysis of artificial and real MicroCT datasets revealed that 3 μm was an ideal pixel size to image porous structures for tissue engineering without loss of resolution or computational feasibility. Scaffolds were then fabricated using 33 different conditions as modifications to the standard production route. The modifications encompassed changes to concentrations, solvents and solutes, the effects of which were evaluated using circular dichroism (CD), scanning electron microscopy (SEM) and MicroCT. The incorporation of sodium chloride was found to have the largest impact on scaffold connectivity and pore wall morphology. These results suggest that the morphology of the pore space can be controlled by the ice crystal growth, as predicted by the phase-field model, whereas the pore wall can be modified through collagen-solute interactions. Finally, the role of biochemical and electromechanical modifications on collagen through crosslinking was investigated with three different crosslinking chemistries. Characterisation of crosslinked collagen substrates using quantitative nanomechanical mapping (QNM) and piezoresponse force microscopy (PFM) at the nanoscale revealed that 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)- N-hydroxysuccinimide(NHS) induced alignment and self-assembly of collagen fibres that were 300 nm in width, and which exhibited localised piezoelectric response. The response of human dermal fibroblasts (HDFs) to films crosslinked with EDC-NHS, genipin and tissue transglutaminase 2 (TG2) revealed that genipin- and TG2-treated samples did not lose their integrin specificity unlike EDC-NHS crosslinked samples. Although both EDC-NHS- and genipin-treated films possessed a high tensile modulus, only genipin-treated films demonstrated high HDF proliferation. Therefore, this observation reinforces the conclusion that successful cell adhesion to biochemical ligands is required as a precursor to proliferation. In summary, this body of work identifies fundamental mechanisms that can modify structural elements in a collagen scaffold from the biomolecular to the micro-scale. The results from this thesis have critical implications since the prediction, modification and characterisation of the ice and collagen at various length scales can provide a comprehensive framework to tailor ice-templated collagen scaffolds for tissue engineering applications.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Nair, Malavika

Advisors dc:contributor.advisor

• Best, Serena
• Cameron, Ruth

• collagen
• ice
• characterisation
• tissue engineering

Statement dc:rights

• Figure 2.3, Image adapted from original by Dan Cojocari, distributed under a CC-BY-SA 3.0 license. Figure 2.5 Image by Sébastien Meghezi, Frédéric Couet, Pascale Chevallier, and Diego Mantovan International Journal of Biomaterials, Hindawi, distributed under a CC-BY 3.0 license. Figure 2.8 Copyright permission obtained to reuse 2 figures by Sylvain Deville, Eduardo Saiz and Antoni P.Tomsia in Biomaterials, Elsevier, in print and electronic forms of a thesis/dissertation. License No.: 4772820800701 Figure 2.9 Copyright permission obtained to reuse 1 figure by Kendell Pawelec, Anke Husmann, Serena M. Best, Ruth E. Cameron in Journal of the Royal Society: Interface, The Royal Society in print and electronic forms of a thesis/dissertation. License No.: 1018973-1 Figure 2.10 Copyright permission obtained to reuse 1 figure by Kendell Pawelec, Anke Husmann, Serena M. Best, Ruth E. Cameron in Journal of the Royal Society: Interface, The Royal Society in print and electronic forms of a thesis/dissertation. License No.: 1018973-1 Figure 2.17 Copyright permission obtained to reuse 2 figures by Matthew G. Haugh, Ciara M. Murphy, Ross C. McKiernan, Cornelia Altenbuchner and Fergal J. O'Brien in Tissue Engineering Part A, Mary Ann Liebert Inc, in print and electronic forms of a thesis/dissertation. Figure 2.18 Copyright permission obtained to reuse 1 figure by Barry M. Gumbiner in Cell, Elsevier, in print and electronic forms of a thesis/dissertation. License No.: 4772821061662 Figure 2.19 Copyright permission obtained to reuse 1 figure by A. A. Shibkov, Yu. I. Golovin, M. A. Zheltov, A. A. Korolev, and A. A. Vlasov in Crystallography Reports, Springer, in print and electronic forms of a thesis/dissertation. License No.: 4772821221937 Figure 2.20 Figure by N. B. Magee, A. Miller, M. Amaral, and A. Cumiskey in Atmospheric Chemistry and Physics, Copernicus Publications, distributed under a CC-BY 3.0 license. Figure 2.21 Copyright permission obtained to reuse 1 figure by W.J. Boettinger, J.A. Warren, C. Beckermann, A. Karma in Annual Review of Materials Research, in print and electronic forms of a thesis/dissertation. License No.: 1018982-1 Figure 2.22 Figure by Gilles Demange, Helena Zapolsky, Renaud Patte & Marc Brunel in npj Computational Materials, Springer Nature, distributed under a CC-BY 4.0 license. Figure 2.24 Copyright permission obtained to reuse 1 figure by Manuela Venturoni, Thomas Gutsmann, Georg E. Fantner, Johannes H. Kindt, Paul K. Hansma in Biochemical and Biophysical Research Communications, Elsevier, in print and electronic forms of a thesis/dissertation. License No.: 4772830426944 Figure 2.25 Copyright permission obtained to reuse figures by Jennifer C. Ashworth in print and electronic forms of a thesis/dissertation. Request granted via email. Figure 2.26 Copyright permission obtained to reuse figures by Jennifer C. Ashworth in print and electronic forms of a thesis/dissertation. Request granted via email.

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/9a11a802-aab0-46bc-b380-bf78b4653e8e/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

en

Author Identifier

0000-0002-5229-8991
0000-0001-7866-8607
0000-0003-1573-4923

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/303389