京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12802号 / 論医博第2074号 / 新制||医||1001(附属図書館) / 80846 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 戸口田 淳也, 教授 松田 秀一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

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Objectives Intra-articular injections of local anaesthetics (LA), glucocorticoids (GC), or hyaluronic acid (HA) are used to treat osteoarthritis (OA). Contrast agents (CA) are needed to prove successful intra-articular injection or aspiration, or to visualize articular structures dynamically during fluoroscopy. Tranexamic acid (TA) is used to control haemostasis and prevent excessive intra-articular bleeding. Despite their common usage, little is known about the cytotoxicity of common drugs injected into joints. Thus, the aim of our study was to investigate the effects of LA, GC, HA, CA, and TA on the viability of primary human chondrocytes and tenocytes in vitro. Methods Human chondrocytes and tenocytes were cultured in a medium with three different drug dilutions (1:2; 1:10; 1:100). The following drugs were used to investigate cytotoxicity: lidocaine hydrochloride 1%; bupivacaine 0.5%; triamcinolone acetonide; dexamethasone 21-palmitate; TA; iodine contrast media; HA; and distilled water. Normal saline served as a control. After an incubation period of 24 hours, cell numbers and morphology were assessed. Results Using LA or GC, especially triamcinolone acetonide, a dilution of 1:100 resulted in only a moderate reduction of viability, while a dilution of 1:10 showed significantly fewer cell counts. TA and CA reduced viability significantly at a dilution of 1:2. Higher dilutions did not affect viability. Notably, HA showed no effects of cytotoxicity in all drug dilutions. Conclusion The toxicity of common intra-articular injectable drugs, assessed by cell viability, is mainly dependent on the dilution of the drug being tested. LA are particularly toxic, whereas HA did not affect cell viability.