In vitro mechanotransduction studies, uncovering the basic science of the response of cells to mechanical forces, are essential for progress in tissue engineering and its clinical application. Many varying investigations have described a multitude of cell responses, however as the precise nature and magnitude of the stresses applied are infrequently reported and rarely validated, the experiments are often not comparable, limiting research progress. This thesis provides physical and biological validation of a widely available fluid stimulation device, a see-saw rocker, as an In vitro model for cyclic fluid shear stress mechanotransduction. This allows linkage between precisely characterised stimuli and cell monolayer response in a convenient six-well plate format. Computational fluid dynamic models of one well were analysed extensively to generate convergent, stable and consistent predictions of the cyclic fluid velocity vectors at a rocking frequency of 0.5 Hz, accounting for the free surface. Validation was provided by comparison with flow velocities measured experimentally using particle image velocimetry. Qualitative flow behaviour was matched and quantitative analysis showed good agreement at representative locations and time points. A maximum shear stress of 0.22Pa was estimated near the well edge, and time-average shear stress ranged between 0.029 and 0.068Pa, within the envelope of previous musculoskeletal In vitro fluid flow investigations. The CFD model was extended to explore changes in culture medium viscosity, rocking frequency and the robustness to position on the rocking platform. Shear stress magnitude was shown to increase almost linearly with an increase in the viscosity of culture medium. Compared with 0.5 Hz, models at 0.083 and 1:167 Hz, the operational limits of the see-saw rocker, indicated a change in shear stress patterns at the cell layer, and a reduction and increase in mean shear stress respectively. At the platform edge at 0.5 Hz, a 1.67-fold increase in time-average shear stress was identified. Extensive biological validations using human tenocytes underlined the versatility of the simple In vitro device. The application of fluid-induced shear stress at 0.5 Hz under varying regimes up to 0.714Pa caused a significant increase in secreted collagen (p < 0.05) compared to static controls. Tenocytes stimulated at a shear stress magnitude of 1.023Pa secreted significantly less collagen compared to static controls. The potential for a local maximum in the relationship between collagen secretion rate and shear stress was identified, indicating a change from anabolic to catabolic behaviour. Collagen biochemical assay results were echoed with antibody stains for proteins, where a co-localisation of connexin-32 with collagen type-I was also identified. A custom algorithm showed that four hours of fluid-induced shear stress of 0:033Pa intermittently applied to tenocytes encouraged alignment and elongation over an eight day period in comparison to static controls. Primary cilia were identified in human tenocyte cultures and bovine flexor tendon tissue; however primary cilium abrogation In vitro using chloral hydrate proved detrimental to cell viability. Collaborative investigations identified that ERK signalling and c-Fos transcription factor expression peaked after the application of 0.012Pa at 0.083 Hz for 20 minutes and anabolic collagen gene expression relative quantities increased after 48 hours of rocking at 0.083 Hz. In conclusion, validated shear stresses within a six-well plate, induced by cyclic flow from a see-saw rocker, provides an exceptional model for the In vitro study of dynamic fluid shear stress mechanobiology. Biological investigations have been linked to precise applied shear stress, creating a foundation for understanding the complex relationship between tenocytes and fluid-induced shear stress In vitro. Using this model, research is repeatable, comparable and accurately attributed to shear stress, accelerating the scientific advancement of musculoskeletal mechanobiology.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:618443 |
| Date | January 2013 |
| Creators | Tucker, Russell P. |
| Contributors | Thompson, Mark |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:0ea8b159-5cb6-4bf0-9a60-4c580824016a |
Page generated in 0.0025 seconds