Cell response to microtopography

Topographical cues can influence cell behaviour independent of biochemistry. An overview of the field is described in Section 1.4.3, so here, the focus is on micrometre scale features. Many studies of topo- graphical effects on cell behaviour have revolved around microstructured surfaces due to the wide range of fabrication techniques and the relative simplicity of surface characterisation. The most common tech- nique is lithography, which can be used to create substrates with high intra- and inter-batch consistency and fine control over the features. A wide range of topographical features have therefore been studied.

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a b

Figure 2.3: SEM micrographs of two tissues, demonstrating the types of architectural features found in vivo. a) The corneal basement membrane has a complex architecture. Scale: 500 nm, adapted from Flemming et al.117b) Articular cartilage has a topography that varies throughout the depth of the tissue. Adapted from Jeffery et al.118

When cells come in contact with a substrate, they probe their surroundings with filopodia. The resulting feedback initiates a cascade of biochemical events that have been demonstrated to affect virtually all as- pects of cell behaviour. Cell response to topography is reasonable, considering the structural organisation present in extracellular matrix (Figure 2.3). The first observation of this response in vitro was in 1911, when frog embryo cells were seen to align along spider web fibres.119 _{The term “contact guidance”, now} in wide use was first used by Paul Weiss in 1939 when he observed that neurite outgrowth followed the topography of the underlying substrate.120 _{Since then, it has been repeatedly reported that microscale} topography can influence a wide range of cell responses, including cytoskeletal arrangement,121,122 os- teoblast mineralisation,123and stem cell differentiation. Mesenchymal stem cell differentiation into either adipocytes or osteoblasts can be controlled by microscale topography, and was found to be activated by the RhoA pathway.82_{Similarly, differentiation into either chondrocytes or myoblasts was found to be me-} diated by Rac1 and N-cadherin.124 _{These differentiation studies were particularly interesting examples} of the use of topography as the features were designed to put the cell into a specific orientation, which caused the observed effects. For a summary of many microstructured surfaces and the effects they had on a wide variety of cells, see the reviews by Flemming et al.117 _{and Ross et al.}125

2.1.3.1 Embryonic stem cell response to topographical cues

Embryonic stem cells (ESCs) have the potential to be a potent cell source for regenerative medicine ther- apies due to their ability to differentiate into all somatic cell types found in the adult organism, and their sensitivity to small changes in culture conditions.39In the presence of leukaemia inhibitory factor (LIF), a differentiation-inhibiting cytokine, murine ESCs can be maintained in culture in an undifferentiated state. Differentiation protocols in vitro typically involve the formation of embryoid bodies (EBs), which are aggregates of cells derived from ESCs that represent the earliest stages of differentiation. ESCs can also be differentiated on two-dimensional substrates in the presence of exogenous factors, such as soluble growth factors or substrate-adsorbed ECM molecules.126Compared to other adherent cell types, little is known about the effects of substrate properties on ESC behaviour, perhaps because ESCs are typically grown on a layer of feeder cells, which effectively obscures the substrate.

It follows that there is little known about the effects of topographical features on ESCs. Most of the work on topography affecting stem cell differentiation has pertained to mesenchymal stem cells (MSCs). However, one group recently used lithography to produce an array of microstructures, composed of square or round pillars in different lateral combinations and different pillar heights, for a total of 504 different topographies.127 _{They seeded two strains of ESCs and observed changes in morphology and phenotype.} Colony number was affected by both the vertical and lateral dimensions. It decreased as the vertical height of the pillars was decreased and as pillar size increased. Colony spreading was associated with diminished alkane phosphatase activity, a marker of stem cells, demonstrating an effect of topography on ESC differentiation.

Before that, it had been shown that human ESCs participate in contact guidance in a similar manner as other adherent cell types.128 _{PDMS (polydimethylsiloxane) substrates with line grating of 600 nm} ridges, with 600 nm spaces, and 600 nm height were coated with fibronectin to promote cell attachment. The ESCs aligned and elongated with the ridges, which led to cytoskeletal organisation and reduced proliferation. This effect was diminished when actin disrupting agents were used.

Topography has also been used to control the size of EBs. The protocol for differentiation of ESCs into all three germ layers begins with forming cell aggregates. These aggregates typically take the form of EBs, which are suspended, spherical cell aggregates. It is known that the size of the EBs affects the efficiency of the subsequent differentiation and so one group used photolithography to make a substrate that could control the size of ESC aggregates.129 Interestingly, their aggregates remained adhered to the substrate instead of becoming free-floating. However, they observed enhanced cardiac differentiation when they made aggregates 200 µm in diameter, an indication that topography can be used to control stem cell differentiation through the EB intermediary.