The influence of substrate creep on mesenchymal stem cell behaviour and phenotype

Andrew Cameron (2011). The influence of substrate creep on mesenchymal stem cell behaviour and phenotype PhD Thesis, Aust Institute for Bioengineering & Nanotechnology, The University of Queensland.

       
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Author Andrew Cameron
Thesis Title The influence of substrate creep on mesenchymal stem cell behaviour and phenotype
School, Centre or Institute Aust Institute for Bioengineering & Nanotechnology
Institution The University of Queensland
Publication date 2011-10
Thesis type PhD Thesis
Supervisor Professor Justin Cooper-White
Dr Jessica Frith
Associate Professor Darren Martin
Dr Barbara Rolfe
Total pages 207
Total colour pages 17
Total black and white pages 190
Language eng
Subjects 060106 Cellular Interactions (incl. Adhesion, Matrix, Cell Wall)
090301 Biomaterials
100404 Regenerative Medicine (incl. Stem Cells and Tissue Engineering)
Abstract/Summary In Tissue Engineering (TE) applications, it is important to consider the mechanical properties that are being presented to the cells. Previous studies have shown that variations in substrate compressive modulus (rigidity) can affect a range of cell behaviours including cell spreading, motility, proliferation and differentiation. While these studies focused primarily on the dominant elastic component of a substrate, most matrices surrounding cells in vivo are viscoelastic in nature – that is, they have separable elastic (storage, G`) and viscous (loss, G``) modulus components. This thesis aims to isolate and analyse the effect of the viscous component (i.e. loss modulus) of a substrate on cell behaviour. It was hypothesised that a time dependent dissipation of energy after an initial matrix deformation (or dynamic creep) imposed by cells would influence cell behaviour. Human mesenchymal stem cells (hMSCs) were utilised for the analysis as they have great potential as a cell source in regenerative medicine due to their capacity to self-renew and differentiate towards multiple lineages. Polyacrylamide (PAM), due to its easily tunable properties, was used to develop a platform with which to study the effect of substrate loss modulus on hMSC behaviour. By varying the initial monomer concentrations of bis and acrylamide, a series of gels were produced with a constant storage modulus (G` ~ 4.7 kPa, representing a constant compressive modulus of ~13.5 kPa) but varying loss moduli over two orders of magnitude (1-130 Pa). This platform was then used to test the effect of loss modulus on hMSC behaviours. As substrate loss modulus increased, cell spread area, rate of membrane protrusion, motility and proliferation all increased but the size of focal adhesions decreased. Consistent with the cellular tensegrity model, it was hypothesised that much of the cell behaviour displayed on the high loss modulus (HLM) substrates related to the loss of cytoskeletal tension caused by the inherent creep of the substrates. Inhibitors were used to selectively impair isometric tension (using the non-muscle myosin II inhibitor, Blebbistatin) or both isometric and isotonic tension (using the Rho-Kinase inhibitor, Y-27632). Blebbistatin treated hMSCs on low loss modulus (LLM) substrates exhibited behaviour similar to untreated hMSCs on HLM substrates. Furthermore, Blebbistatin treatment did not affect the behaviour of hMCSs on the HLM substrates while Y-27632 had a substantiative effect. This indicated that isotonic tension plays an important role in directing the behaviour of hMSCs on HLM substrates and suggests that any further reduction in isometric tension on these substrates is limited. The effect of dynamic substrate creep on hMSC differentiation was also analysed. While variations in substrate loss modulus did not cause spontaneous differentiation towards osteogenic or adipogenic lineages, hMSCs on HLM substrates showed significantly enhanced differentiation towards both lineages in the presences of soluble induction cues. Furthermore, hMSCs on HLM substrates showed no bias towards either adipogenic or osteogenic lineages when cultured in medium with mixed inductive cues. This demonstrates that rather than directing hMSC differentiation, increasing substrate loss modulus acts to enhance the differentiation induced by soluble cues. The adipogenic differentiation of hMSCs exposed to Blebbistatin and Y-27632 on the different gels once again indicated that the effect of substrate loss modulus on hMSC behaviour was due to a loss in cytoskeletal tension. Previous studies have shown that the differentiation of hMSCs towards smooth muscle cells (SMCs) is enhanced by increased cell spread area. Given the increased cell spread area on HLM substrates, the effect of substrate loss modulus on SMC differentiation of hMSCs was analysed. Increasing substrate loss modulus was shown to enhance the expression of SMC specific markers within hMSCs in both basal and myogenic media. It was suggested that the increased potential for SMC differentiation on the HLM substrates was due, in part, to the observed increase in expressions of proteins known to promote SMC differentiation. N-Cadherin (N-Cad) expression (which is known to mediate SMC differentiation) was also increased in hMSCs on HLM substrates. It was hypothesised that this would facilitate enhanced SMC differentiation on HLM substrates, in addition to a probable increase in Rac1 activity (which is known to mediate SMC differentiation and N-Cad expression). In basal medium, inhibition of Rac1 activity (via the Rac1 inhibitor, NSC23766) reduced N-Cad and SMC marker expressions by hMSCs on HLM substrates to levels equivalent to those of untreated hMSCs on LLM substrates. This indicated that increased N-Cad on HLM substrates was due to increased Rac1 activity, which was supported by the observed translocation of Tiam1 (a guanine exchange factor responsible for Rac1 activation) from the nucleus to the periphery of hMSCs on these substrates. In contrast to basal medium, inhibition of Rac1 activity in hMSCs on HLM substrates in myogenic medium did not result in a decrease of N-Cad expression (unlike those on LLM substrates) suggesting a complex interplay between soluble and mechanical directive cues. Finally, as the majority of previous studies into the effect of substrate mechanical properties on cell behaviour have focused upon substrate rigidity, the effect of substrate rigidity was compared to the effect of substrate loss modulus on SMC differentiation of hMSCs. The enhanced differentiation caused by increasing substrate loss modulus on enhancing SMC differentiation was maintained when the compressive moduli of gels were varied and furthermore, substrate loss modulus was shown to have a greater effect on SMC differentiation than substrate compressive modulus. In these studies we have developed a platform for testing the effect of substrate loss modulus on cell behaviour and shown that it has a significant impact on the behaviour and phenotype of hMSCs. While previous studies into the effect of substrate mechanical properties on cell behaviour have focused primarily on substrate compressive modulus, this thesis highlights the importance of considering the effect of other mechanical properties, namely loss modulus and creep. It is hoped that the information presented here can be used in the future optimisation of biomaterial scaffolds for TE applications, such as those involving blood vessel regeneration.
Keyword Biomaterial
Viscoelastic
Mechanical Properties
Creep
Loss Modulus
Mesenchymal Stem Cells
Differentiation
Cytoskeletal Tension
Mechanotransduction
Tensegrity
Additional Notes 29, 33, 35, 43, 47, 53, 84, 93, 103, 114, 116, 118, 123, 124, 149, 181, 184

 
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