Charras, Guillaume T; (2002) Cellular Mechanotransduction in Bone. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bone is a structure finely tuned to its mechanical environment. Strain detection in the skeleton is thought to be effected by bone lining cells and osteocytes embedded within the bone matrix. However, because bone is a composite material with multiple enclosed cavities and a complex architecture, the strain to which cells are subjected to in vivo and to which they react remains unknown. This thesis examined the sensitivity of cells of the osteoblastic lineage to mechanical strain using three different methods. First, Atomic Force Microscopy (AFM) was used to mechanically stimulate the cells and measure their elasticity, whilst changes in intracellular calcium concentrations were monitored with a confocal microscope. Post-hoc, the strain applied was calculated. The proportion of cells reacting by increasing their intracellular calcium concentrations followed a dose response curve with 50% of the cells reacting for 2.5% strain. Second, patch-clamp electrophysiology and simultaneous video-microscopy were combined to provide an experimental curve relating the length of the membrane extension into the micropipette to the aspiration pressure and the opening of mechanosensitive channels in response to pressure. A finite element simulation of the membrane aspiration into the micropipette revealed that strains superior to 20% were needed to open mechano-sensitive channels. Third, digital strain estimation was applied to Green Fluorescent Protein-actin or tubulin transfected melanoma cells to provide an estimate of the strain magnitude needed to elicit a rise in intracellular calcium concentration in response to micropipette poking. Intercellular communication was also examined. Many studies in vitro have studied the mechanisms of cellular adaptation to mechanical strains; however, the applied strains remain unknown and results from different techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by AFM with finite element modelling, we calculated the cellular strains exerted by common whole cell straining techniques and micromanipulation techniques.

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