applications of micro- and nano-technology to study cell adhesion to material surfaces

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Applications of Micro- and Nano-technology to Study Cell Adhesionto Material SurfacesFranz Bruckert a & Marianne Weidenhaupt ba Laboratoire des Matériaux et du Génie Physique, UMRCNRS 5628, Grenoble Institute of Technology, Minatec,3 parvis Louis Néel, BP 257, 38016 Grenoble Cedex 1,France;, Email: [email protected] Laboratoire des Matériaux et du Génie Physique, UMRCNRS 5628, Grenoble Institute of Technology, Minatec,3 parvis Louis Néel, BP 257, 38016 Grenoble Cedex 1,FrancePublished online: 02 Apr 2012.

To cite this article: Franz Bruckert & Marianne Weidenhaupt (2010) Applications of Micro-and Nano-technology to Study Cell Adhesion to Material Surfaces, Journal of AdhesionScience and Technology, 24:13-14, 2127-2140, DOI: 10.1163/016942410X507957

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Journal of Adhesion Science and Technology 24 (2010) 2127–2140brill.nl/jast

Applications of Micro- and Nano-technology to Study CellAdhesion to Material Surfaces

Franz Bruckert ∗ and Marianne Weidenhaupt

Laboratoire des Matériaux et du Génie Physique, UMR CNRS 5628, Grenoble Institute ofTechnology, Minatec, 3 parvis Louis Néel, BP 257, 38016 Grenoble Cedex 1, France

AbstractMicro- and nano-technologies provide new tools to study and control cell adhesion. These technologiesare well suited to reconstitute the cell’s natural environment and mimic its biochemical, morphologicaland mechanical peculiarities. ‘Smart’ material surfaces and coatings, which allow controlling the bindingand release of specific macromolecules and/or cells, are being developed at a high pace. Moreover, theapplication of geometrical constraints at the micro- or nano-scale reveals some of the physico-chemicalprinciples underlying molecular and cellular organization.

These technologies, combined with the growing knowledge in molecular biology and the spatial and tem-poral resolutions given by the various microscopy techniques, are going to boost our understanding of cellphysiology. The availability of well-defined multicellular assemblies opens new ways to test and analyzecells, either in a cluster, or in assemblies mimicking tissue organization. These techniques help bridging thegap between molecular biology and tissue or organism physiology.

First, we list the main chemical and physical parameters in the cell micro-environment that influence itssurvival, proliferation, differentiation or migration. Then we review some examples where micro- and nano-technologies are used to control cell spreading and adhesion in different ways: (1) via the distance betweenadhesive molecules, (2) via the geometry of adhesive zones, (3) via surfaces with switchable adhesiveness,or (4) via three-dimensional coatings used as reservoir of active molecules.© Koninklijke Brill NV, Leiden, 2010

KeywordsLiving tissues, cell adhesion, cell differentiation, cell motility, mechanosensitivity, nanosciences, excitablesurfaces

1. Cell Microenvironment: an Overview

In the living tissues of pluricellular organisms, cells are attached to each other eitherdirectly by molecules embedded in the plasma membrane, or through the extracel-lular matrix, a mixture of polymers secreted by cells, onto which they adhere. Bloodvessels are a good example of such complex organized structures. They are made

* To whom correspondence should be addressed. Tel.: (33) 4 56 52 93 21; e-mail:[email protected]

© Koninklijke Brill NV, Leiden, 2010 DOI:10.1163/016942410X507957

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2128 F. Bruckert, M. Weidenhaupt

Journal of Adhesion Science and Technology 24 (2010) 2127–2140

of three layers. First, in contact with the blood flow, a monolayer of endothelialcells provides an impermeable barrier to most molecules and cells, but allows thetransport of specific molecules (e.g., glucose) by transcytosis and the transmigra-tion of specific cells (e.g., monocytes) between the lumen and the vessel wall. In thisvery thin layer (a few µm), cells are attached to each other by adherens junctions.Second, layers of smooth muscle cells, organized around the endothelial cell mono-layer, control the vessel diameter in response to mechanical stimuli (blood pressure,blood flow shear stress). These are relayed by chemical mediators secreted by theendothelial layer. For example vasodilation and vasoconstriction are regulated bynitric oxide and angiotensin, respectively. Depending on the vessel size, the smoothmuscle cell layer thickness varies from 50 µm to several mm. Third, an externallayer made of elastic fibers and fibroblast cells ensures mechanical stiffness. Inlarge vessels, this layer may be a few millimeters thick and also contains nutritivevessels and nerves that stimulate smooth muscle cells. A specific 80 nm thick ex-tracellular matrix layer, called the basal membrane, separates the endothelial cellsfrom smooth muscle cells [1]. Another specific extracellular matrix layer separatesthe smooth muscle cell layer from the external layer. This example shows that in liv-ing tissues, several differentiated cell types co-exist in oriented structures organizedat different scales, from about 10 nm to several mm.

Despite its apparent stability, the structure of living tissues is dynamic. Cellscontinuously synthesize and degrade the extracellular matrix [2], divide themselvesand die in response to precise molecular clues [3]. These mechanisms allow liv-ing tissue growth, physiological adaptation and repair. Cell signalling may occurdirectly, by cell–cell contact, by secretion of growth factors, differentiation fac-tors, chemo-attractants (molecules that influence cell proliferation, differentiationstate, or directed movement), or indirectly by the secretion of extracellular matrixand guidance molecules. Many proteins constituting the extracellular matrix indeedcontain molecular motives analogous to growth factors or differentiation factors(e.g., fibulins [4]). Therefore, a specific interaction between cells and the extracellu-lar matrix is necessary for cell survival, differentiation or proliferation. In addition,hydrated, charged polymers contained in the extracellular matrix bind many growthand differentiation factors with various specificities by hydrogen bonds and elec-trostatic interactions. These immobilized molecules may come in contact with cellexplorative structures such as filopodia, where they can stimulate, in an orientedmanner, intracellular signalling pathways. Conversely, cells produce enzymes thatdegrade specific molecules in the extracellular matrix (e.g., matrix metallopro-teinase [5]). The mixture of macromolecules and cells composing living tissuesreact therefore together in an interdependent manner. Taking again blood vessels asan example, cells may proliferate axially or radially, which either extends or widensthe vessel [6].

In living tissues, stem cells play a special role. Contrary to differentiated cellswhich undergo a limited number of symmetrical divisions, they may proliferate

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F. Bruckert, M. Weidenhaupt 2129Journal of Adhesion Science and Technology 24 (2010) 2127–2140

without limit, in specific conditions. They generate differentiated cells by asym-metric division: one daughter cell keeping stem cell properties while the otherdifferentiates. Stem cells maintain the steady-state number of differentiated cellsand are, therefore, essential for tissue development, renewal and repair. A well-known example of stem cells are blood stem cells, which are localized in the bonemarrow, generating erythrocytes, lymphocytes, monocytes and platelets in responseto specific growth and differentiation factors. For blood vessels, stem cells reside inthe wall around the endothelial cell layer (pericytes). During angiogenesis, endothe-lial cells are also able to degrade the basement membrane and together reorganizeinto new vessels. Recent progress has shown that stem cells exist in most tissues,and their stimulation could be a target for future regenerative medicine [7]. An al-ternative challenge is to integrate them in biomaterials to reconstitute tissues, ortissue components.

Thanks to the progress made in molecular and cellular biology, the use of mu-tants, the visualization of fluorescently labelled molecular structures and numer-ous molecular identification techniques (transcriptomics and proteomics), a goodknowledge of genes involved in cell differentiation and tissue biogenesis is nowa-days available. For instance, in blood vessels, VE-cadherin and the type 2 VEGFreceptor are specific for endothelial cells, caldesmon and β-actin for smooth mus-cle cells, and collagen I for fibroblasts. Similarly, signalling pathways triggeredby growth or differentiation factors, chemoattractants and adhesion molecules havebeen thoroughly studied. Generally speaking, the binding of extracellular moleculesto membrane-bound receptors triggers some conformational changes that allow thecooperative assembly of intracellular molecules at the plasma membrane, resultingin ion flow regulation, protein phosphorylation and second messenger biosynthesis(outside-in signalling). These transient molecular modifications assure the relay ofthe signal within the cell and they may amplify it. They have a direct, short-termeffect on macromolecules already present in the cell (primary response, ms to min)or an indirect, long-term effect by acting on transcription factors that translocateto the nucleus and control protein biosynthesis (delayed response, 20 min to days).Cells are also able to change the number and conformation of adhesion molecules(e.g., integrins) by membrane trafficking and protein phosphorylation (inside-outsignalling). In living tissues, many of these signals prevent programmed cell death(apoptosis) and control symmetrical or asymmetrical cell division (see below). It istherefore essential to understand and to be able to reconstitute the proper molecularenvironment of cells to ensure their survival, differentiation state and physiologi-cal function. Micro- and nano-technologies are well suited to conceive appropriateenvironments for different cell types in a very reproducible manner.

Although significant progress has been made in the identification of relevantgenes and macromolecules involved in cell physiology, an integrated picture of thecell molecular mechanisms is still lacking. Even for relatively simple cell struc-tures mediating attachment to the extracellular matrix such as focal adhesions, the

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hierarchical assembly and disassembly of these multi-molecular structures and thecross-talk between different signalling pathways are largely unknown [8]. More-over, focal adhesions, stable adhesion structures often observed in cells anchored inthe extracellular matrix, and podosomes, i.e., dynamic adhesion structures involvedin cell migration through tissues, share the same set of structural proteins: integrins,talin, paxillin, vinculin, actin, etc., but are controlled by different phosphorylationsignals (FAK versus Src, [9]). The assembly of adherens junctions is modulatedsimilarly. Cadherins connect cells together, and their assembly is stabilized by theirconnection to the actin cytoskeleton via catenins. All these various cell structuresconsist of the same basic elements that ensure plasma membrane binding to the out-side (e.g., integrins, cadherins), plasma membrane binding to the actin cytoskeleton(talin, paxillin), localized actin polymerization catalysis (ERM proteins) and me-chanical force application and mechanosensitivity (myosins).

Many studies have shown that mechanical forces can trigger biochemical sig-nalling at cell–cell or cell–extracellular matrix contacts. The effect of blood flowon endothelial cell morphology is one of the earliest documented examples [10]. Inadherens junctions, an increase of β-catenin tyrosine phosphorylation in responseto fluid shear stress regulates the linkage between VE-cadherin and the actin cy-toskeleton and is involved in their redistribution [11]. The molecular mechanismsof mechanotransduction are unknown. It is possible that specific proteins such asthose forming ion channels are sensitive to mechanical stimuli [12, 13] or thatlocal mechanical forces control the association and dissociation rates within co-operative molecular assemblies [14]. Recently, Whitehead et al. [15] reported thatheterozygous expression of a mutant form of APC (Adenomatous Polyposis Coli)protein renders colon tissue especially sensitive to mechanical stimuli. Compres-sion of the colon tissue increases the expression of c-myc and twist-1 transcriptionfactors by activating the Wnt/β-catenin pathway. Translocation of β-catenin fromadherens junctions to the nucleus in response to mechanical forces exerted duringembryogenesis has also been reported in drosophila and is dependent on a spe-cific Src activation [16]. Altogether, this shows that cell–cell adherens junctionsare mechanosensitive. Furthermore, focal adhesions are also sensitive to appliedmechanical forces [17, 18]. Since ICAPs (integrin cytoplasmic domain associatedproteins) shuttle between activated integrins and the nucleus, where they stimulatecell proliferation [19], long term effects of mechanical forces acting on cells em-bedded in the extracellular matrix can be anticipated [20]. These examples showthat for practical applications, the stiffness of materials should be taken into con-sideration, in addition to surface functionalization. This has nicely been illustratedby studies using thin elastic films of different stiffness [21–24]. From a theoreticalpoint of view, force application to deformable structures results in the localizationof mechanical stress in rather sharp zones. In the case of the interaction between aliving cell and a soft material, forces are concentrated at the border of cell–surface

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contact areas [25–27]. This explains why the distribution of mechanical forces pro-vides orienting clues for the cells.

In summary, cell adhesion to material surfaces depends on many parameters.First of all, the presence and the location of specific extracellular matrix mole-cules, chemoattractants and contact with other cells will be paramount. Adsorptionof macromolecules and surface functionalization are therefore essential. On a longterm, remodelling of the extracellular matrix, secretion or storage of growth and dif-ferentiation factors and proper material stiffness will be determinant. A completecharacterization of material properties is thus necessary. Mastering these processesis crucial for a good integration of substituting biomedical materials and for thecompatibility between medical implants and living tissues. It is also important forresearch in biology, since eukaryote cells are often grown on material surfaces.Furthermore, as the interaction between cells and materials extends over differentscales, from nm (typical size of macromolecules) to several µm (cell geometry),micro- and nano-technologies are, therefore, well suited to engineer material sur-faces for biological use, in order to provide cells with precise and well-characterizedconditions.

2. Examples of Applications of Nano-technologies to Cell Adhesion

In this section, we will review several advances in materials science and miniatur-ization technologies that open new ways to tackle biological or medical questions.

2.1. Surface Activation of Adhesion Proteins

The precise spatial arrangement of integrin molecules and actin microfilaments inadhesive zones is not known, but it is likely to be ordered, because of geometricalconstraints and the limited diffusion range of biochemical processes. An illustra-tion of precise integrin clustering during formation of cell adhesive structures isprovided by the effect of surface nanostructuring on cell spreading and motility.Using block copolymer nanolithography, Spatz and coworkers engineered materialsurfaces covered with microarrays of small gold particles exposing cyclic RGDfKmotives (an αVβ3 integrin ligand) separated by protein-repellent PEGylated sur-faces. The distance between adjacent gold particles was precisely defined by thesize of the polymer coating of the gold particles [28]. Their small size (8 nm) en-sured that only one integrin head domain (9 nm) could bind only a single goldparticle. Cells were strikingly sensitive to the distance between adjacent gold parti-cles. Spreading was similar on uniformly distributed RGDfK surfaces and on goldparticles exposing RGDfK motives separated by 25 or 58 nm, but they did notspread efficiently on nanoparticles separated by 73 nm or 108 nm [29]. This ef-fect was observed with REF-52 fibroblasts, 3T3 fibroblasts, MC3T3 osteoblasts orB16 melanocytes, all expressing αVβ3 integrins. The limitation in cell spreadingcould be due either to the increased separation distance between adjacent gold par-

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ticles or the global decreased RGDfK density. In order to address this issue, twoelegant experiments were done. First, combining block copolymer nanolithographyand electron-beam lithography, Arnold et al. [30] prepared squared surfaces withside lengths ranging from 100 nm up to 3 µm, separated by the same distance, andcontaining ordered gold particles separated by 58 nm. The distance between adja-cent squares was equal to their side length, and the global RGDfK density was thusequivalent to that of ordered gold particles separated by 116 nm, an experimentalcondition where cells did not spread. On all nano-patterned squares, cells were ableto spread showing that the local RGDfK density, but not the global one, drove ad-hesive structure formation. Second, using homopolymer polystyrene to prevent theordered hexagonal packing of gold particles, Huang et al. [31] prepared disorderedarrays of RGDfK-functionalized gold particles, keeping the average interparticledistance constant from 58 to 100 nm. In contrast to ordered arrays, MC3T3-E1 os-teoblasts were almost insensitive to RGDfK density when spreading on disorderedarrays. In such disordered arrays, small zones indeed exist, where a few RGDfKintegrin ligands are separated by less than 58 nm, the critical distance for the forma-tion of adhesive structures. Altogether, this shows that integrin clustering dependson the underlying order of RGDfK ligand arrangement on the surface (Fig. 1(a)).The formation of integrin clusters is evidenced by the increase of the force neces-sary to detach cells, which also critically depends on integrin binding site spacing[32]. At the molecular level, this critical distance corresponds to the cooperativeassembly of adjacent integrin molecules linked to the actin cytoskeleton. It should

Figure 1. Tailoring adhesive surfaces to control cell adhesion, morphology and physiology. (a) Thedistance between adhesive molecules controls the formation and mechanical properties of adhesivezones. (b) The geometry of adhesive zones controls cell polarization, the orientation of the cell divisionaxis and cell differentiation. (c) Array of adhesive zones can be used to direct cell movement and tosort cell types.

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be noted that this 50–70 nm distance, much larger than integrin or actin monomersizes (9 and 5 nm, respectively), is indicative of the formation of extensive molec-ular assemblies.

Besides cell adhesion studies, it should be noted that nanopatterning technolo-gies could have other applications in biology, especially because specific biomole-cules can be attached to gold particles [33]. For instance, this could be used toreproduce in vitro the geometry of large identical molecular assemblies: photosyn-thetic or respiratory complexes, vesicular coats, intermediate filaments. . . . Usingnanobeads of different sizes and different surface chemistries could even makemultiple-patterning possible in the near future. In this section, we have seen thatpatterning at the nanometer scale reveals interesting features of molecular adhesionmechanisms. In the next section, we will show that patterning at the micrometerscale is an important new tool to study cell internal organization.

2.2. Tailoring Adhesive Surfaces to Control Cell Geometry

Micropatterning technology has been used to study cell internal organization,which can be controlled by adhesion geometry at the microscale. Early studies byWhitesides, Ingber and Chen showed that cell fate (survival, differentiation or pro-grammed cell death) critically depends on the available adhesive area [34, 35]. Inaddition, the shape of the adhesive zone strongly influences the dynamics of theactin and microtubule cytoskeletons [36, 37] and the internal distribution of the cellorganelles [38, 39] (Fig. 1(b)). The rationale is that the adhesive zone geometry con-trols the distribution of mechanical forces and the release of signalling moleculesat adhesive contacts within the cell. Because of mechanical equilibrium, adhesionstructures localize in regions of maximal stress. The adhesive contact distributiondrives cell polarity, resulting in directional cell movement [40–42] and orientingthe cell division axis [43, 44] (Fig. 1(b) and 1(c)). Furthermore, adhesive patternscan be applied to multicellular assemblies, and therefore used to study intercellularinteractions [45, 46] or to separate cell types in a population [42, 47].

The key aspects of this technology that should attract the attention of biologistsare (i) the relative ease to prepare micropatterns using photolithography or imprit-ing techniques, (ii) the versatility of pattern design, and (iii) the fact that uniformcell size, shape and orientation greatly facilitate cell image analysis, in combina-tion with fluorescence microscopy [44]. In molecular cell biology, this techniqueopens the way to mutant library screens with much more complex — and bettercontrolled — environmental situations than ever envisioned. For instance, polar-ized cell arrays can be combined with parallel multiple optical trap technology tosimultaneously stimulate all cells at the same location [48]. In tissue engineering, itopens the way to reproduce the precise geometry of complex cell assemblies withinthe tissue, and to study the physiological relevance of geometrical parameters incell signalling [46].

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2.3. Smart Surfaces to Control Cell Adhesion

Material surfaces can be engineered not only to selectively control cell adhesion ina consistent manner, but also to switch from a non-adhesive to an adhesive state.A range of surfaces have been developed, whose hydrophobicity can be controlledeither electrically, electrochemically, thermally, or photoactively [49] (Fig. 2). Sur-face hydrophobicity is an interesting parameter to modulate cell adhesion becausemost proteins, including extracellular matrix ones, bind more strongly to hydropho-bic surfaces than to hydrophilic ones. However, large physico-chemical changes arenecessary to significantly modify protein adsorption and alter cell adhesion. In addi-tion, caution should be taken when using physical forces since living cells are verysensitive to their environment. Electrowetting, for instance, requires large electri-cal voltages to be effective in physiologically relevant solutions, which may triggerelectrophysiological responses. In the same way, strong UV illumination is neces-sary for photo-induced wetting, which is harmful to cells. As a consequence, thesetechniques have not yet been employed to control cell adhesion. Electrochemicaland thermal switchings are more cell-friendly techniques and several researchershave already demonstrated promising applications.

Electrochemical switching (Fig. 2(a) and 2(b)) can be achieved in different ways.One possibility is to change the redox state of a molecule grafted onto the materialsurface. The resulting surface voltage change exerts repulsive or attractive forceson adsorbed or covalently bound molecules, which drives a conformational change.Wang et al. [50] tethered bipyridinium molecules through an alkylated linker to anelectrode and showed that redox modification of the bipyrinidium group bent thelinker towards the surface, exposing the most hydrophobic part of the molecule.A reversible, but modest, surface energy change accompanies voltage application.Another approach is to release or bind biomolecules from or to the surface. Yeo etal. [51] prepared a self-assembled monolayer presenting an RGD peptide linked toan O-silyl hydroquinone group. Applying an electrical potential to the substrate oxi-dized the hydroquinone and released the RGD group, resulting in the detachment ofcells attached to the RGD moiety. Subsequent treatment of the surface with diene-tagged RGD peptides restored cell adhesion. They used this dynamic surface toconfine 3T3 fibroblasts to adhesive patterns and release them over the entire surface.Thiol chemistry on gold surfaces can also be used to drive reductive electrochem-ical desorption of self-assembled monolayers. Mali et al. [52] demonstrated thatproteins could be patterned on addressable gold electrodes and selectively releasedfrom them. Inversely, Tang et al. [53] coated an indium tin oxide (ITO) micro-electrode array with a protein-resistant poly(L-lysine)-g-poly(ethylene glycol) graftcopolymer. Application of a positive electrical potential resulted in localized poly-mer desorption, due to the positively charged poly-L-lysine moiety and freed theITO surface for subsequent protein binding. It should be noted that these techniquesare relatively slow, since tens of seconds are required to fully remove adsorbed

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Figure 2. Smart surfaces to control cell adhesion. (a, b): Electrochemical switching; (c, d): thermalswitching. In (a) and (c), a conformational change modifies the surface properties of the material,whereas in (b) and (d), this is achieved by desorption of a functional group.

molecules from the electrodes. State of the art electrochemical switching is, there-fore, well applicable to cells that spread or move rather slowly.

Thermal switching is based on hydrogels that are coated over the surface andexhibit a transition between a collapsed and a swollen structure at a lower criticalsolution temperature (LCST) (Fig. 2(c) and 2(d)). An example of such a thermo-

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responsive polymer is poly(N-isopropylacrylamide) or PNIPAAm, whose LCST isin the range of 32–35◦C. Cells adhere, spread and grow well on PNIPAAm hydro-gels at 37◦C, since the dehydrated polymer surface is hydrophobic which allowsstrong extracellular matrix protein binding. Reducing the temperature by about 3–5◦C makes the surface hydrophilic and swelling exerts large mechanical forces,which induce detachment of a cell layer including an intact extracellular matrix.This lift-off technique ensures only limited cellular damage and is now widely usedfor tissue engineering [54], and multiple cell layers can even be generated. As anexample of a medical application, corneal epithelial cell layers grown on PNIPAAmdishes can be directly implanted on the patient’s cornea [55], or co-cultures of he-patocytes and endothelial cells can be obtained that mimic liver tissue organization[56].

Recently, oligonucleotide–peptide and oligonucleotide–protein conjugates havebeen synthesized [57]. These molecules are interesting in the field of cell adhesionbecause their oligonucleotide moiety is able to reversibly bind a complementaryDNA sequence. Michael et al. [58] recently grafted oligonucleotides onto titaniumsurfaces and hybridized them to complementary oligonucleotide–GRGDSP con-jugates. This surface was then able to bind osteoblasts due to the RGD-containingmotif. Desorption of the cells should, in principle, be obtained by local denaturationabove the melting temperature of the oligonucleotide strands.

The examples given in this section show different ways to directly control cell–surface adhesion, using physico-chemical means. We will show in the next sectionhow to make use of more specific biochemical or cellular mechanisms to controlcell adhesion.

2.4. Polymers, Hydrogels and Multilayer Polyelectrolyte Film Coatings

In the presence of living cells, material surfaces are exposed to a changing envi-ronment and anti-biofouling coatings, currently available, do not last for more thana few days in cell culture or inside the human body. Active surfaces are there-fore needed that may release or capture molecules of interest. Polymers are ofspecial interest because their extended and flexible structure accommodates largeconformational changes, which allows large binding capacity compared to two-dimensional surface immobilization techniques. Furthermore, protein activity canoften be preserved in these hydrated molecules. Attaching polymers on materials,therefore, extends the volume in which cells interact with the underlying materialand allows a better control of bound molecules.

The surface modification of blood contacting materials provides a nice illustra-tion of the application of polymer versatility. An antithrombin–heparin complexcovalently linked to a protein-repellent poly(ethylene oxide) polymer efficientlycatalyzes the inhibition of either thrombin or factor Xa by blood antithrombin,preventing activation of the clotting cascade [59]. Similarly, a composite mole-cule made of a lysine aminoacid moiety exposing its ε-amino group attached to

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a poly(ethylene glycol) polymer both prevents fibrinogen binding and adsorbs plas-minogen and tissue plasminogen activator, resulting in fibrin clot lysis [60]. Thesecoatings strongly reduce platelet adhesion and may greatly improve the hemocom-patibility of cardiovascular devices. Active surfaces are, therefore, an efficient wayto control cell adhesion.

Polyelectrolyte Multilayer Films are assembled by the alternate adsorption ofpolyanions and polycations [61]. They can be prepared from a large variety ofnatural or synthetic polymers. Linearly growing films maintain a stratified struc-ture, but not exponentially growing ones in which polyelectrolytes can diffuse [62].These coatings are interesting to control cell adhesion because in addition to surfacefunctionalization with adhesion receptor ligands, their mechanical stiffness can bemodulated by crosslinking [63, 64]. As previously explained, adherent cells exertlarge forces on material surfaces and soft materials thus prevent cell spreading. Celladhesion patterns could, therefore, be engineered using photo-crosslinking to ad-just the coating stiffness over the surface [65]. The development of thermosensitivefilms or their electrochemical destabilization may also provide temporal control ofcell adhesion. Another interest of polyelectrolyte multilayer films is the possibilityto fill them with bioactive molecules, such as growth factors [66] or differentiationfactors [67] and thus create a material-associated reservoir for cell stimulation.

The increasing complexity of polyelectrolyte multilayer film technology is sim-ilar to actual trends in hydrogel technology [68]. Tissue engineering scaffolds alsorelease bioactive molecules such as growth factors and cytokines [69–71]. The maindifference between hydrogels and polyelectrolyte multilayer films is that hydrogelsadditionally provide multiple scale porosity, allowing therefore cells to grow withinthe material and even angiogenesis to occur [72, 73].

3. Future Directions in Nanobiosciences to Study Cell–Material Adhesion

The examples given show that nanosciences can be used both to study and to in-fluence cell–surface adhesion. The temporal and spatial control of cell adhesionis the focus of many current studies. In addition, there is a trend to extend thecell–material interaction zone towards the third dimension. Simple surface func-tionalization is progressively replaced by multi-layers of molecules, each fulfillinga specific function.

This increased complexity requires the development of new experimental toolsand conceptual models to monitor protein conformational changes and homol-ogous and heterologous interactions. Similarly, studying cellular organization inthree dimensions requires powerful microscopy techniques and analytical methods.The presence of macromolecules in a reduced effective volume should enhancecooperative effects in their interaction, which needs to be adequately monitored.Theoretically, the increased complexity should drive more studies to model theconsequences of 3D confinement on macromolecule interactions: diffusion in a

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crowded environment, electrostatic and electrochemical effects under the Debyelength and kinetic and thermodynamic control of reaction specificity.

4. Conclusion

In conclusion, these emerging technologies open the way to new biological discov-eries at different scales, from internal molecular assemblies to cell–cell interactionsand tissue morphogenesis. By using materials to reduce the variations associatedwith cell shape or concentrations, modelling signalling pathways becomes easierand can better explain experimental results.

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applications of micro- and nano-technology to study cell adhesion to material surfaces

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