W. Richard Bowen and Nidal Hilal 4

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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
7.2 ENgINEERINg THE ECM FOR PRObINg CELL SENSINg 209 Ordered pattern (a) (d) (b) 200 μm (e) (c) 1 μm 200 μm in signalling and genetic analysis. With ever closer collaboration between biologists and engineers and the availability of advanced tools for nanoscale measurements, such as AFM, a greater understanding of the underlying mechanisms of cellular interactions will be possible. This will lead to the development of more effective methods for regenerative medicine. 7.2.3 nanoscale Measurement: Challenges and opportunities for AFM It might have not been possible to discover many of the findings related to nanotopography-induced cellular reactions without the AFM. AFM imaging permits reliable and routine characterisation of nanotopographic features with high resolution particularly in the z-direction, (f) Disordered pattern FIgurE 7.6 The effect of nanotopography on cell differentiation. SEM images of nanotopographies fabricated by EBL (a and d). The nanopits (120 nm diameter, 100 nm deep) arranged in a highly ordered square (a) and with each pit randomly displaced from the square pattern (�50 nm from the true centre) (d). The disordered nanostructures stimulate the human MSCs to express the bone-specific ECM protein osteopontin, as shown in (e and f) (arrows) in contrast to no effect seen with the highly ordered pits (b and c). Figure reprinted from Dalby et al. [61] with permission.
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Butterworth-Heinemann is an imprint
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x Preface in their work. Hence, it
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xii Preface them from hostile condi
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xiv About thE Editors Professor Nid
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xvi List of Contributors Dr Daniel
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2 1. BAsIC PRINCIPLEs OF ATOMIC FOR
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20 1. BAsIC PRINCIPLEs OF ATOMIC FO
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32 2. MEASUREMENT OF PARTICLE ANd S
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82 3. QUANTIFICATION OF PARTICLE-BU
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100 3. QUANTIFICATION OF PARTICLE-B
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C H A P T E R 4 Investigating Membr
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4.2 THE RANgE OF POssIbILITIEs FOR
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4.2 THE RANgE OF POssIbILITIEs FOR
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4.3 CORREsPONdENCE bETwEEN sURFACE
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4.3 CORREsPONdENCE bETwEEN sURFACE
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4.4 IMAgINg IN LIqUId ANd THE dETER
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4.4 IMAgINg IN LIqUId ANd THE dETER
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4.5 EFFECTs OF sURFACE ROUgHNEss ON
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4.6 ‘vIsUALIsATION’ OF THE REjE
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4.7 THE UsE OF AFM IN MEMbRANE dEvE
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Dfractional 0.18 0.16 0.14 0.12 0.1
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4.8 CHARACTERIsATION OF METAL sURFA
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At pH 5.5, dissolution began to occ
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REFERENCEs 137 [4] W.R. Bowen, N. H
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C H A P T E R 5 AFM and Development
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5.2 MEAsUREMENT OF ADHEsION OF COLL
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5.2 MEAsUREMENT OF ADHEsION OF COLL
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The antibacterial activity of initi
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5.3 MODIFICATION OF MEMBRANEs 147 t
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5.3 MODIFICATION OF MEMBRANEs 149 B
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Force (nN m -1 ) Loading force (mN
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5.3 MODIFICATION OF MEMBRANEs 153 p
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Adhesion force (mN m -1 ) 10 8 6 4
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258 9. APPLICATION OF ATOMIC FORCE
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276 10. FuTuRE PRosPECTs most AFM s
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278 10. FuTuRE PRosPECTs targeted d
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A Actin stress fiber, 197 Adhesion,
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Natural organic matter, 140 Negativ
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W. Richard Bowen and Nidal Hilal 4

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