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Title: The use of a novel miniature strain device to investigate the response of tendon cells to In vitro tensile strain
Author: Way, Louise
ISNI:       0000 0004 2742 4161
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2012
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The aim of this work was to study the cell biology of tendons and their response to in vitro tensile strain with a view to elucidating how cells sense mechanical load and what changes occur to the cells in response. This work was done in order to provide some scientific background to aid the improvement of strategies for the treatment and prevention of tendon damage and develop appropriate methodologies related to the mechanical stimulation of tissue engineered tendons. A miniature cyclic straining device was designed (in collaboration with Electrical Engineering) which utilised a 50ml centrifuge tube containing two clamps, controlled via a stepper motor. This was programmed using Visual Basic enabling a defined magnitude and duration of strain to be applied to the tissue or monolayer of cells within the incubator. The design offered numerous benefits over the more commonly-used commercially available bioreactors; in that the system was relatively inexpensive to make, multiple repeats could be carried out simultaneously, only comparatively small amounts of media were required and, as the device was small, it could be kept in the incubator for the duration of the experiment. This design was developed further by re-orientating the clamps and stepper motor to create a horizontal device which could be placed in a Petri-dish and used with a dipping lens con focal microscope to image the response of cells to strain in real-time. Due to the variability in primary cell attachment to the PLA surfaces, alternative methods of cell seeding were investigated. Cytocentrifugation provided a reliable method of attaching a pre-determined number of cells to the surface; cells had an improved plating efficiency and less variation in distribution, whilst remaining viable. This method was also tested on Azowipes tm, which were used as a model 3D scaffold in order to see whether this method had potential for use in tissue engineering. Cells penetrated to the centre of the scaffold, in the order of mm's, an improved penetration compared to the gravity-seeded controls. Cells continued to produce collagen and synthesise DNA after cytocentrifugation onto the 3D scaffold. Monolayer cultures of cells on collagen-coated PLA were exposed to cyclic mechanical strain in the custom-made mechanical straining devices. The effect of specific durations and magnitude of strains on cell number was compared to mono layers of cells which were either maintained in an unloaded state or experienced static tensile strain. However, monolayer cultures of cells do not accurately represent in vivo cell behaviour, as proliferation is much increased in 20 cultures as opposed to what is seen physiologically in tendon tissue. For this reason, intact rat tail tendon fascicles were investigated as they have a regular and relatively uniform composition and the cells remain in their native extracellular matrix. In situ 3D information regarding the cells residing in intact tail tendon fascicles under tension was obtained using multiphoton microscopy. Mechanical stress was applied to the fascicles and a wavelength of 900nm was used to image tendon cells stained with Acridine Orange whilst simultaneously imaging the collagen extracellular matrix using Second Harmonic Generation. The data was then used to inform 'The Virtual Tendon Project' about local variations in strain and demonstrated the requirement for 3D image processing to reliably describe local strains in the matrix. Cell proliferation in response to mechanical stimulation was studied by the uptake of EdU, a relatively new method which was optimised for tendon tissue (Way et al., 20 I 0). The EdU technology, together with histological sectioning, was used to study the effects of obesity and age on tendons in vivo. Rats were fed a high fat diet and tendons from the tail, Achilles and patellar were dissected. Fewer collagen fibres with greater inter-fibrillar spaces were seen in the histological sections taken from the Achilles of the rats fed a high fat diet and the cells had a greater proliferative index than those of the control. In the ageing studies a decrease in cell area and an increase in proliferative index were apparent in the Achilles of the mature rat compared to the 2 month old rat. The methodologies developed in this project predominantly use the simple tendons of the tail to help describe how cells residing in tendon respond to mechanical strain and together with 'The Virtual Tendon Project' go some way towards describing the complex 3D local strain environment present in this tissue.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available