Experimental and computational investigation into mechanobiology of osteocytes

Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which comprise over 90% of the cells in mature bone tissue and are known to be highly sensitive to extracellular mechanical cues. The effect of extracellular mechanical stimuli on osteoblast differentiation in particular has been well studied, with both mesenchymal and embryonic stem cells shown to differentiate into the osteoblast phenotype when cultured on substrates that mimic the mechanical properties of developing bone tissue, osteoid. However, relatively little attention has been paid thus far to the effects of extracellular mechanics on the differentiation of osteocytes, despite their clear importance to the function of bone tissue as a whole. The aim of this thesis is to uncover the effects of extracellular mechanical stimuli on osteocyte differentiation, and thus inform future bone tissue engineering strategies. The first study of this thesis examined the effects of passive substrate stiffness and intercellular separation on osteocyte differentiation of the pre-osteoblast MC3T3-E1 cell line. Cells were cultured on Type 1 collagen based substrates of different stiffnesses while seeding density was used to control intercellular separation. It was found that osteocyte differentiation, as measured by morphological analysis, alkaline phosphatase (ALP) activity, substrate mineralisation and gene expression profile, occurred on substrates of approximately 300 Pa at an initial seeding density of 103 cells/cm2. Interestingly, this stiffness was much lower than those previously found to induce osteoblast differentiation. The second study of this thesis used finite element modelling to examine the isometric stress generated in the cell body when cultured on the substrates investigated in the first study. Cell and substrate mechanical properties were measured through atomic force microscopy, while cell morphologies and the locations of focal adhesion complexes were determined through confocal microscopy. The results demonstrated that intracellular tension was influenced by cell morphology, focal adhesion location and density as well as substrate stiffness, suggesting a role for isometric tension in osteocyte mechanobiology. The third study presented here investigated the combined effects of substrate stiffness, thickness, fibrosity and crosslinking density on cell spread area, a known indicator of osteogenic differentiation potential. MC3T3-E1 cells were cultured on flat and wedge shaped collagen and polyacrylamide (PA) gels, with stiffness being controlled through the polymerisation process of PA gels or 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking of collagen. It was found that cells on the fibrous collagen substrates adopt the same spread area as those cultured on much stiffer non-fibrous PA substrates, while a reduction in substrate thickness caused an increase in cell area in both collagen and PA substrates. The increase in cell area due to thickness was reduced as crosslinking density of collagen substrates was increased. Finite element simulations demonstrated that the effective modulus of a substrate increased when discrete fibres were included. The effective modulus was further increased as crosslinking density was increased and as substrate thickness was reduced. Interestingly, the effect of thickness on the effective modulus was lessened at higher crosslinking density, offering an explanation for the reduced effect of substrate thickness in highly crosslinked collagen. These results suggest that cell behaviour is regulated by the modulus of a substrate, as experienced at a local cell level, rather than bulk material properties. The results of this study may explain, in part, seemingly contradictory results as to the optimal stiffness for the promotion of osteogenic differentiation. The final study of this thesis investigated the combined effects of fluid flow and substrate stiffness on osteocyte differentiation of MC3T3-E1 cells. Cells were cultured on collagen substrates previously shown to induce osteocyte differentiation (Study 1) and subjected to physiologically relevant levels of pulsatile fluid flow after 7 days of static culture. It was found that the application of flow at day 7 of culture followed by a further 7 days of static culture caused an increase in the percentage of cells undergoing osteocyte differentiation, as measured through morphological analysis and ALP activity. Together these studies provide new evidence that osteocyte differentiation can be induced by culture of pre-osteoblast like cells on soft collagen substrates, provided substrate thickness and heterogeneity are also controlled, and that this differentiation may be initiated through the generation of isometric stress within the cell body. The importance of intercellular separation in osteocyte differentiation was also demonstrated for the first time, while the number of cells undergoing the differentiation process can be further enhanced through the application of fluid flow induced stress. Through the research studies of this PhD Thesis, fundamental information has been uncovered about the differentiation behaviour of osteogenic cells in response to extracellular mechanical cues, and these results can be used to inform future bone tissue engineering strategies