Design, Production and Characterisation of Anisotropic Collagen Scaffolds for Cardiac Tissue Repair

The helical arrangement of cardiomyofibres in the heart muscle wall contributes significantly to cardiac function. Recapitulating this complex structure *in vitro* is challenging and there has been limited success achieving this in tissue engineering scaffolds. Unidirectional ice-templating has been established in prior work and collagen structures with anisotropic porosity can be created via control of ice nucleation and growth. This thesis aims to advance knowledge using finite element simulations to design complex prototype architectures, and to produce and characterise the physical, mechanical, and biological properties of experimental scaffolds. Multi-directional freezing was undertaken for collagen slurries, each contained in a polycarbonate mould with a thermally conductive copper base plate. It was found that by controlling base topography, a Bouligand-like pore structure could be created. Scaffold architectures with a gradual orientational change (106±10)° were prepared which closely matches with the native 90° to 180° cardiomyofibre orientation change across the ventricle wall. External thermal influx was also investigated as a method to control the orientation of ice growth. The stability of the collagen at elevated temperatures was assessed and no denaturation was observed up to 80° C for up to two hours. Heat patches were placed in a spiral array around the circumference of the hollow mould. A sinusoidal helical orientation, of angles between −10° and 16°, was created around the circumference of the resulting tubular scaffold. The pore orientation in the radial direction was further adjusted using a double spiral arrangement of heat patches, and in the axial direction using a multi-turn spiral arrangement. Stiffness plays a crucial role in determining cell behaviour. Protocols were established to characterise chemical crosslinking, degradation and fatigue resistance. Chemical crosslinking increased compressive Young’s modulus (*E*) from (1.69±0.60) kPa to (6.42±1.54) kPa and critical stress (σ_crit) from (0.22±0.08) kPa to (0.71±0.11) kPa. Passive degradation tests resulted in a 30% reduction in *E* and σ_crit after 30 days in deionised water. Cyclic compressive and tensile loading regimes were designed to reflect the cardiac contraction and relaxation. *E* degraded rapidly in the first 100 cycles under cyclic compressive loading, but only decreased slightly under cyclic tensile loading. Cellular response to aligned collagen scaffolds was investigated using human dermal fibroblasts (HDFs) and human embryonic stem cell derived cardiomyocytes (hESC-CMs). Over 14 days in culture, HDFs migrated along the pore struts at an average speed of (0.34±0.02) μm min⁻¹. There was also parallel organisation of hESC-CMs with pore alignment, but penetration into the scaffold thickness was relatively low (20% of the thickness). Spontaneous beating was observed from Day 7. An out-of-plane bending motion was observed due to non-uniform cell distribution within the scaffold. However, the strain within the plane that cell seeding took place on resembled the native cardiac cycle, showing a maximum contraction strain of 3.2% with an inotropic strain rate of 0.28 s⁻¹ and a lusitropic strain rate of 0.16 s⁻¹. This thesis has established a design pipeline for the production of type I collagen scaffolds, from computational modelling, to fabrication and characterisation. Three-dimensional structural anisotropy, resembling the native myocardium, was created and the resulting mechanical and biological behaviour offer potential for cardiac tissue regeneration.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Zhang, Huijie

Advisor dc:contributor.advisor

• Best, Serena

• Cardiac tissue engineering
• Collagen
• Freeze-drying
• Ice-templating

Licence

• https://www.repository.cam.ac.uk/bitstreams/021d993a-6162-477f-a55a-e59f0c00c5b1/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0009-0005-4577-934X

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/370188