W. Richard Bowen and Nidal Hilal 4

208 7. MICRO/NANOENgINEERINg ANd AFM FOR CELLULAR SENSINg
possibly entrap significant amounts of ECM proteins from the culture
medium; and it is not clear as to what extent the chemistry and texture of
the nanofibre matrix (or the nanoislands) interact with cells. As a random
nanophase meshwork with poorly defined ‘nanotopography’ features, it is
difficult to isolate the many parameters which might affect cells.
Precisely Defined Nanostructures
E-beam lithography and associated techniques can produce precisely
defined nanostructures, so that individual variables can be investigated
thoroughly. Features as small as 3 nm can be reliably fabricated on a substrate
[59] and using modern high throughput machines, sufficiently large
areas can be written for cellular studies. Through combination of EBL
and NIL techniques, arbitrary nanopatterns can be replicated in thermoplastic
polymers. Using this method, a large number of replicates having
identical patterns of designed nanofeatures have been produced on polylactide,
polycarbonate, PMMA, and polycaprolactone [60–62]. This mass
production using different materials has enabled direct comparison of the
influence of substrates having different surface chemistries but the same
nanotopography on cell behaviour for a number of cell lines.
The use of arbitrary nanopatterns provides a very flexible and systematic
way to explore the interactions between cells and the nanoworld, e.g.
when highly regular arrays of nanopit structures with pit diameters of 35,
75 and 120 nm were used for fibroblast growth. Within this range of subtle
variation in pit size, it was found that cell spreading reduced and there
was less apparent stress fibre formation [60]. Following on from this study,
EBL was used to create different levels of disorders in the nanopatterns.
Using these levels it has been found that human mesenchymal stem cells
(MSCs) were prompted to produce more bone mineral when there was
a certain degree of disorder to the array patterns of nanopits (Figure 7.6;
[61]). However, highly ordered nanopits resulted in low to negligible cellular
adhesion and osteroblast differentiation. This discovery suggested
that nanotopography might be an efficient method to guide MSC cells to
be used in regenerative medicine and tissue engineering devices, since at
present, many examples of failed bone implants have been found to be
associated with encapsulation by soft tissue without direct bone bonding.
Clearly, substantial studies have advanced our understanding of the
influence of topography on cellular processes. However, it is still to be
resolved as to how cells detect and respond to these nanofeatures. Much
evidence has shown that nanotopography influences cellular cytoskeleton
formation, and thus is likely to modulate membrane receptor organisation,
integrin cluster formation, and intracellular signalling – all of which are
related to focal adhesion formation. It is not clear as to whether the mechanosensitivity
of cells to physical topography features employs the same
machinery that cells use to sense changes in surface chemistry (e.g. adhesive
patterns). New investigations have started to understand the changes