Integrins are a large family of membrane-spanning heterodimeric (αβ) receptors that bind to ligands on other cells or to extracellular matrix (ECM) proteins. These receptors mediate bidirectional signaling over the cell membrane to induce signaling cascades mediating functions as cell adhesion, spreading and migration. This signaling takes place at cell-matrix adhesions, which are sites where clustered and ligand-bound integrins connect to and mediate stabilization of the actin cytoskeleton, and induce signaling cascades. Integrins have a short cytoplasmic tail that is crucial for the bidirectional signaling, and the β1-integrin subunit exists in five splice variants only differing in the membrane-distal part of the cytoplasmic tail. This region of the almost ubiquitously expressed β1-integrin, β1A, contains two protein tyrosine motifs (NPXYs) interspaced with a threonine-rich region, while this region of the β1B splice variant is completely different and lacks known motifs. In contrast to the β1A-integrin, the β1B variant cannot mediate cell-matrix adhesion formation following binding to ECM ligands. The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1-integrins on the host cell with invasin, and this stimulates uptake of the bacterium. However, upon binding to the host cell, pathogenic Yersinia strains inject virulence effectors that block uptake. One effector responsible for the blocking is a tyrosine phosphatase, YopH. We identified the targets for this effector in the macrophage-like cell line J774A.1, which represent a professional phagocyte and thus is the likely target cell for the antiphagocytic effect of Yersinia. Two YopH target proteins were p130Cas and ADAP, of which the latter interestingly is an adapter protein specifically expressed in hematopoietic cells. ADAP has previously been implicated to participate in Fc-receptor-mediated phagocytosis and in communication between T-cell receptors and integrins. We also studied the importance of the cytoplasmic tail of β1-integrin for uptake of Yersinia. The GD25 cell line, which is a fibroblast-like cell line that lacks endogenous β1-integrins, was used together with GD25 cells transfected with β1B, β1Α or cytoplasmic tail mutants of β1A. These studies revealed that β1B-integrins could bind to invasin but not mediate uptake of Yersinia, while β1A both bound to invasin and mediated uptake. The first NPXY motif (unphosphorylated) and the double-threonines of the unique part of β1A were important for the ability of integrin to mediate uptake of Yersinia. These studies lead to the interesting finding that, when these cells were allowed to spread on invasin, those that expressed β1A spread as normal fibroblasts while for β1B-integrin-expressing cells, only finger-like protrusions of filopodia were formed. This provided us with a tool to study formation of filopodia without interference of the tightly linked process of lamellipodia formation. Initially, proteins that localized to the tip complex of these filopodia were identified. These were talin, VASP and interestingly the p130Cas-Crk-DOCK180 scaffold, while FAK, paxillin and vinculin were absent. In addition, VASP, p130Cas and Crk were shown to be important for the filopodia formation in GD25β1B. Further, the role of the actin motor myosin X, which previously has been implicated in formation of filopodia, was studied in the GD25Β1B cells and it was shown that myosin X not was important for filopodia formation, but that it recruited FAK and vinculin to the tip complexes of filopodia.

The interaction of materials with biological systems is of critical importance to a vast number of applications from medical implants, tissue engineering scaffolds, blood-contacting devices, cell-culture products, as well as many other products in industries as diverse as agriculture. This thesis describes a method for the modification of biomaterial surfaces and the generation of tissue engineering scaffolds that utilises the self assembly of poly (styrene)-block-poly (ethylene oxide) (PS-PEO) block copolymers. Block copolymers consist of alternating segments of two or more chemically distinct polymers. The salient feature of these materials is their ability to self organise into a wide range of micro-phase separated structures generating patterned surfaces that have domain sizes in the order of 10-100nm. Further, it is also possible to specifically functionalise only one segment of the block copolymer, providing a means to precisely locate specific biological signals within the 10-100nm domains of a nano-patterned surface, formed via the programmed micro-phase separation of the block copolymer system. The density and spatial location of signalling molecules can be controlled by altering several variables, such as block length, block asymmetry, as well as processing parameters, providing the potential to authentically emulate the cellular micro to nano-environment and thus greatly improving on existing biomaterial and tissue engineering technologies. This thesis achieved several aims as outlined below; Developed methods to control the self-assembly of PS-PEO block copolymers and generate nano-patterned surfaces and scaffolds with utility for biomaterials applications. PS-PEO diblock copolymers were blended with polystyrene (PS) homopolymer and spin cast, resulting in the rapid self-assembly of vertically oriented PEO cylinders in a matrix of PS. Due to the kinetically constrained phase-separation of the system, increasing addition of homopolymer is shown to reduce the diameter of the PEO domains. This outcome provides a simple method that requires the adjustment of a single variable to tune the size of vertically oriented PEO domains between 10-100nm. Polymeric scaffolds for tissue engineering were manufactured via a method that combines macro-scale temperature induced phase separation with micro-phase separation of block copolymers. The phase behaviour of these polymer-solvent systems is described, and potential mechanisms leading to this spectacular structure formation are presented. The result is highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available technologies. Characterised the properties of these unique nano-structured materials as well as their interaction with proteinaceous fluids and cells. Nano-patterned PS-PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control PS surfaces. The adhesion of NIH 3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-islands, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface. In order to understand the mechanism by which these surfaces resisted protein and cellular adsorption we utilised neutron reflection to study their solvation and swelling properties. The results indicate that the PEO domains are highly solvated in water; however, the PEO chains do not extend into the solvent but remain in their isolated domains. The data supports growing evidence that the key mechanism by which PEO prevents protein adsorption is the blocking of protein adsorption sites. Control the nano-scale presentation of cellular adhesion and other biological molecules via the self-assembly of functionalised PS-PEO block copolymers Precise control over the nano-scale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, including differentiation and apoptosis. We present the self-assembly of maleimide functionalised PS-PEO copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By controlling the phase separation of the functional PS-PEO block copolymer we alter the nano-scale (on PEO islands of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from ~ 450 to ~ 900 islands per um2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between islands of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces were utilised to immobilise poly-histidine tagged proteins and ECM fragments. The technologies developed in this thesis aim to improve on several weaknesses of existing biomaterials, in particular, directing cellular behaviour on surfaces, and within tissue engineering scaffolds, but also, on the prevention of fouling of biomaterials via non-specific protein adsorption. The application of block copolymer self-assembly for biomaterial and tissue engineering systems described in this thesis has great potential as a platform technology for the investigation of fundamental cell-surface and protein-surface interactions as well as for use in existing and emerging biomedical applications.

The interaction of materials with biological systems is of critical importance to a vast number of applications from medical implants, tissue engineering scaffolds, blood-contacting devices, cell-culture products, as well as many other products in industries as diverse as agriculture. This thesis describes a method for the modification of biomaterial surfaces and the generation of tissue engineering scaffolds that utilises the self assembly of poly (styrene)-block-poly (ethylene oxide) (PS-PEO) block copolymers. Block copolymers consist of alternating segments of two or more chemically distinct polymers. The salient feature of these materials is their ability to self organise into a wide range of micro-phase separated structures generating patterned surfaces that have domain sizes in the order of 10-100nm. Further, it is also possible to specifically functionalise only one segment of the block copolymer, providing a means to precisely locate specific biological signals within the 10-100nm domains of a nano-patterned surface, formed via the programmed micro-phase separation of the block copolymer system. The density and spatial location of signalling molecules can be controlled by altering several variables, such as block length, block asymmetry, as well as processing parameters, providing the potential to authentically emulate the cellular micro to nano-environment and thus greatly improving on existing biomaterial and tissue engineering technologies. This thesis achieved several aims as outlined below; Developed methods to control the self-assembly of PS-PEO block copolymers and generate nano-patterned surfaces and scaffolds with utility for biomaterials applications. PS-PEO diblock copolymers were blended with polystyrene (PS) homopolymer and spin cast, resulting in the rapid self-assembly of vertically oriented PEO cylinders in a matrix of PS. Due to the kinetically constrained phase-separation of the system, increasing addition of homopolymer is shown to reduce the diameter of the PEO domains. This outcome provides a simple method that requires the adjustment of a single variable to tune the size of vertically oriented PEO domains between 10-100nm. Polymeric scaffolds for tissue engineering were manufactured via a method that combines macro-scale temperature induced phase separation with micro-phase separation of block copolymers. The phase behaviour of these polymer-solvent systems is described, and potential mechanisms leading to this spectacular structure formation are presented. The result is highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available technologies. Characterised the properties of these unique nano-structured materials as well as their interaction with proteinaceous fluids and cells. Nano-patterned PS-PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control PS surfaces. The adhesion of NIH 3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-islands, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface. In order to understand the mechanism by which these surfaces resisted protein and cellular adsorption we utilised neutron reflection to study their solvation and swelling properties. The results indicate that the PEO domains are highly solvated in water; however, the PEO chains do not extend into the solvent but remain in their isolated domains. The data supports growing evidence that the key mechanism by which PEO prevents protein adsorption is the blocking of protein adsorption sites. Control the nano-scale presentation of cellular adhesion and other biological molecules via the self-assembly of functionalised PS-PEO block copolymers Precise control over the nano-scale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, including differentiation and apoptosis. We present the self-assembly of maleimide functionalised PS-PEO copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By controlling the phase separation of the functional PS-PEO block copolymer we alter the nano-scale (on PEO islands of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from ~ 450 to ~ 900 islands per um2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between islands of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces were utilised to immobilise poly-histidine tagged proteins and ECM fragments. The technologies developed in this thesis aim to improve on several weaknesses of existing biomaterials, in particular, directing cellular behaviour on surfaces, and within tissue engineering scaffolds, but also, on the prevention of fouling of biomaterials via non-specific protein adsorption. The application of block copolymer self-assembly for biomaterial and tissue engineering systems described in this thesis has great potential as a platform technology for the investigation of fundamental cell-surface and protein-surface interactions as well as for use in existing and emerging biomedical applications.

The interaction of materials with biological systems is of critical importance to a vast number of applications from medical implants, tissue engineering scaffolds, blood-contacting devices, cell-culture products, as well as many other products in industries as diverse as agriculture. This thesis describes a method for the modification of biomaterial surfaces and the generation of tissue engineering scaffolds that utilises the self assembly of poly (styrene)-block-poly (ethylene oxide) (PS-PEO) block copolymers. Block copolymers consist of alternating segments of two or more chemically distinct polymers. The salient feature of these materials is their ability to self organise into a wide range of micro-phase separated structures generating patterned surfaces that have domain sizes in the order of 10-100nm. Further, it is also possible to specifically functionalise only one segment of the block copolymer, providing a means to precisely locate specific biological signals within the 10-100nm domains of a nano-patterned surface, formed via the programmed micro-phase separation of the block copolymer system. The density and spatial location of signalling molecules can be controlled by altering several variables, such as block length, block asymmetry, as well as processing parameters, providing the potential to authentically emulate the cellular micro to nano-environment and thus greatly improving on existing biomaterial and tissue engineering technologies. This thesis achieved several aims as outlined below; Developed methods to control the self-assembly of PS-PEO block copolymers and generate nano-patterned surfaces and scaffolds with utility for biomaterials applications. PS-PEO diblock copolymers were blended with polystyrene (PS) homopolymer and spin cast, resulting in the rapid self-assembly of vertically oriented PEO cylinders in a matrix of PS. Due to the kinetically constrained phase-separation of the system, increasing addition of homopolymer is shown to reduce the diameter of the PEO domains. This outcome provides a simple method that requires the adjustment of a single variable to tune the size of vertically oriented PEO domains between 10-100nm. Polymeric scaffolds for tissue engineering were manufactured via a method that combines macro-scale temperature induced phase separation with micro-phase separation of block copolymers. The phase behaviour of these polymer-solvent systems is described, and potential mechanisms leading to this spectacular structure formation are presented. The result is highly porous scaffolds with surfaces comprised of nano-scale self-assembled block copolymer domains, representing a significant advance in currently available technologies. Characterised the properties of these unique nano-structured materials as well as their interaction with proteinaceous fluids and cells. Nano-patterned PS-PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control PS surfaces. The adhesion of NIH 3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-islands, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface. In order to understand the mechanism by which these surfaces resisted protein and cellular adsorption we utilised neutron reflection to study their solvation and swelling properties. The results indicate that the PEO domains are highly solvated in water; however, the PEO chains do not extend into the solvent but remain in their isolated domains. The data supports growing evidence that the key mechanism by which PEO prevents protein adsorption is the blocking of protein adsorption sites. Control the nano-scale presentation of cellular adhesion and other biological molecules via the self-assembly of functionalised PS-PEO block copolymers Precise control over the nano-scale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, including differentiation and apoptosis. We present the self-assembly of maleimide functionalised PS-PEO copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By controlling the phase separation of the functional PS-PEO block copolymer we alter the nano-scale (on PEO islands of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from ~ 450 to ~ 900 islands per um2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between islands of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces were utilised to immobilise poly-histidine tagged proteins and ECM fragments. The technologies developed in this thesis aim to improve on several weaknesses of existing biomaterials, in particular, directing cellular behaviour on surfaces, and within tissue engineering scaffolds, but also, on the prevention of fouling of biomaterials via non-specific protein adsorption. The application of block copolymer self-assembly for biomaterial and tissue engineering systems described in this thesis has great potential as a platform technology for the investigation of fundamental cell-surface and protein-surface interactions as well as for use in existing and emerging biomedical applications.

No description available.

Dans les tissus, les cellules génèrent et sont soumises en permanence à des forces mécaniques. Les perturbations biochimiques à l'intérieur des cellules, ainsi que les altérations de leur environnement mécanique peuvent modifier l'équilibre physiologique et mener à des pathologies, comme le cancer. Bien que les propriétés mécaniques puissent être modifiées à l'échelle du tissus, la compréhension de la mécanique au niveau de la cellule unique demeure importante. En particulier, la différenciation, la migration des cellules immunitaires et le caractère invasif d'un cancer dépendent fortement des propriétés mécaniques des cellules uniques. Les déformations mécaniques peuvent induire un changement de la surface et du volume cellulaires. Nous nous intéressons particulièrement à la régulation du volume cellulaire chez les cellules mammifères dans le contexte de déformations à différentes échelles de temps. Jusqu'à présent, la régulation du volume dans ce contexte n'a été que très peu étudiée, en raison de la difficulté d'obtention de mesures précises, et du fait que le volume de la cellule est généralement considéré comme constant. Nous avons développé une méthode de mesure du volume cellulaire reposant sur l'exclusion de fluorescence, qui nous permet d'effectuer des mesures de volume précise au niveau de la cellule unique. Dans cette étude, nous nous sommes concentrés sur la régulation du volume cellulaire au cours de l'étalement dynamique sur un substrat (échelle de temps : minutes). Nous avons démontré qu'il existe différents régimes de régulation du volume lors de l'étalement : les cellules réduisent, augmentent ou ne modifient pas leur volume, en fonction de l'état du cortex d'actomyosine et de la vitesse d'étalement. Nous avons constaté que les cellules s'étalant plus vite ont tendance à perdre davantage de volume. Notre hypothèse est que lors d'une extension rapide de lamellipode dépendante d'Arp2/3, l'actine tire sur la membrane et génère une tension et l'activation de transport ionique, s'accompagnant d'une perte de volume compensatoire. L'inhibition de la polymérisation de l'actine ou de sa ramification dépendante d'Arp2/3 réduit la vitesse d'étalement et ainsi la perte de volume. Nous avons ensuite montré que l'inhibition de la contractilité augmente la vitesse d'étalement et la perte de volume. Cependant, l'inhibition d'Arp2/3 dans des cellules à faible contractilité conduit à un étalement rapide sans perte de volume. En effet, l'inhibition d'Arp2/3 induit des bulles de membranes, une déformation rapide n'induirait donc pas de perte de volume car la cellule peut relâcher la tension en dépliant la membrane. Nous avons également montré que la régulation du volume en réponse à une compression mécanique rapide (échelle de temps : millisecondes) indépendante de l'adhérence dépend également de l'état du cortex d'actomyosine. Les cellules perdent jusqu'à 30% de leur volume lorsqu'elles sont confinées, car la membrane plasmique est attachée au cortex et ne peux pas être dépliée en réponse à l'augmentation de la tension. La perturbation du cortex d'actine induit le détachement de la membrane et limite la perte de volume. Enfin, nous avons montré que la réponse du volume à un choc osmotique (échelle de temps : secondes) est plus que complexe que décrite dans la littérature. Nos données indiquent qu'au niveau de la cellule unique, la réponse initiale du volume au changement de l'osmolarité extérieure n'est pas un processus passif uniforme. En utilisant la technique du choc osmotique, nous avons également confirmé que les cellules ont un large excès de membrane repliée dans des réservoirs. Nos résultats montrent que le volume et l'aire cellulaires sont couplés par l'homéostasie de la tension de surface, et, étant donné que les déformations induisent une augmentation de la tension de surface, elles conduisent à des modifications du volume et de l'aire de la cellule. / The field of biomechanics significantly progressed in the last two decades. The importance of the feedback between biochemical signaling and physical properties was revealed in many studies. Cells within tissues constantly generate and experience mechanical forces. Biochemical perturbations inside the cells as well as alterations in the mechanical environment can shift the tiny balance of normal physiological state and lead to pathologies, e.g. cancer. Although the mechanical properties of individual cells can alter when they are within the tissues, the understanding of single cell mechanics is still important. Differentiation, immune cell migration, and cancer invasion strongly depend on the mechanical properties of individual cells. Mechanical deformations can lead to a change in cell surface area and volume. We are particularly interested in single mammalian cell volume regulation in the context of deformations of different timescales. For the moment, volume regulation in this context was out from the research interest, probably due to the difficulties of accurate measurements, and cell volume often considered as a constant parameter. We developed a method for cell volume measurements based on a fluorescent exclusion that allowed us to perform precise volume measurements of individual live cells. In the present study, we mainly focused on cell volume regulation while dynamic spreading on a substrate (timescale – minutes). We demonstrated that there are different regimes for volume regulation while spreading: cells decrease, increase or do not change volume, and a type of the regime depends on the state of the actomyosin cortex and spreading speed. We obtained that faster-spreading cells tend to lose more volume. Our hypothesis is that during fast Arp2/3-driven lamellipodia extension actin pull on the membrane that generates tension and activation of ion transport and regulatory volume loss. Inhibition of actin polymerization or Arp2/3-dependent actin branching decreases spreading speed and volume loss. Next, we showed that inhibition of contractility increases spreading speed and volume loss. However, inhibition of Arp2/3 complex in cells with low contractility leads to fast spreading without volume loss. Our explanation is that inhibition of Arp2/3 induces cell blebbing and even fast deformation does not lead to volume loss as a cell can relax tension by membrane unfolding. We also showed that volume regulation in response to fast mechanical compression (timescale – milliseconds) independent of adhesion also depends on the actomyosin cortex state. Control cells lose up to 30% of volume under confinement, as the cell membrane is attached to the cortex and cannot be unfolded in response to the tension increase. Disruption of actin cortex leads to membrane detachment and prevents volume loss under confinement. Additionally, we showed that cell volume response to the osmotic shock (timescale – seconds) is more complex than it used to be known in the literature. For instance, our data indicate that at the level of individual cells initial volume response to the change of external osmolarity is not a uniform passive process. Using osmotic shock technique, we also confirmed that cells have a large excess of membrane folded in reservoirs. Taken together, our data show that cell volume and surface area are coupled through surface tension homeostasis and as deformations induce surface tension increase, they lead to change volume and surface area.

Motivé par l'étude de tissus biologiques, ce travail contribue aux développements d'outils numériques permettant de prédire la réponse mécanique de matériaux hétérogènes non linéaires dans lesquels les énergies d'interfaces deviennent prépondérantes. Ainsi, une méthode d'homogénéisation multi échelle combinée à une technique de réduction de modèle basée sur la décomposition orthogonale aux valeurs propres est proposée dans un cadre thermique et hyperélastique. Les énergies d'interfaces entre les différentes phases des composites sont décrites par un modèle d'interface cohérent et prises en compte numériquement par une approche liant la méthode des éléments finis étendus et la méthode level-set. Une étude de l'étalement d'une cellule vivante entre deux lamelles fixes est ensuite réalisée. Les deux modèles utilisés pour les simulations montrent que l'assemblage cortex d'actine-membrane plasmique ne joue qu'un rôle minime dans la réponse mécanique cellulaire / Motivated by the study of biological tissues, this work contributes to developing numerical tools to predict the mechanical response of nonlinear heterogeneous materials in which the energies of interfaces can no longer be ignored. First, a computational homogenization strategy combined with a model reduction technique based on the proper orthogonal decomposition is implemented in the cases of large elastic deformations and highly nonlinear conduction. The interfaces between the different phases of a composite are described by means of a coherent interface model and taken into account numerically by an extended finite element method in tandem with a level-set technique. Finally, experimental results of single cell spreading between two fixed parallel microplates are exploited through finite element modelling. Our two models show that the bilayer membrane and the actin cortex do not play a significant role in the cell mechanical response

Stable fixation of implants such as artificial teeth depends on the direct apposition of bone to the implanted material. While endosseous implants were traditionally allowed to "osseointegrate" over several months without carrying load, clinical and experimental data show that prostheses with roughened surfaces allow successful integration when subject to earlier loading and more challenging implant sites. However, to design implant surfaces for an optimal biological response requires an understanding of the mechanism by which roughened surfaces promote osseointegration. Research into this mechanism has, to date, focussed primarily on the response of osteoblastic cells to surface topography in vitro. While these have demonstrated some consistent trends in cell behaviour, the fundamental means by which cells sense and respond to roughness remain unclear. It has been suggested that cell responses to changes in topography may relate to differences in the proteins adsorbed from serum (in vitro). While experimental evidence indirectly suggests that physical features can affect protein adsorption, few studies have examined this with respect to surface roughness, particularly as a mediator of cell responses. To address this issue, cell culture and protein adsorption experiments were conducted on a limited range of surface textures. Titanium samples were ground to produce morphologically similar surfaces with three grades of roughness. A duplicate set of specimens were heated at 600°C for one hour, with the aim of masking potential variations in physicochemical properties with differing degrees of grinding. Osteoblast attachment and proliferation studies were conducted over a short time-frame of 48 hours or less, to highlight the effects of proteins adsorbed from serum rather than secreted by adherent cells. Gel electrophoresis provided a profile of the proteins adsorbed to each surface after 15 minutes, corresponding to the time by which the cells had settled onto the surface. Finally, confocal microscopy was used to examine cell morphology on each surface, and to visualize specific interactions between cellular structures and adsorbed adhesion-mediating proteins. Although the effects were inconsistent, attachment assays showed some indications that fewer cells attached in the first 90 minutes as roughness increased. This inverse cell number-roughness trend was significant at 48 hours; however, the variability in attachment assays prevented reliable separation of attachment and proliferation rate effects. While the reduction in cell number with increasing roughness is consistent with previous reports, it is typically observed at later time points, and thus may be increasingly confounded by contact inhibition and differentiation. Thermal oxidation of the titanium did not impact on osteoblast responses to roughness, although it significantly slowed cell proliferation. The latter result was unexpected on the basis of previous reports. One-dimensional gel electrophoresis revealed no significant differences in the composition of adsorbed layers with variations in roughness. However, as expected on account of wettability changes, the heat-treatment did correspond to significant changes in the adsorption profile. While this was not a highly sensitive analysis, it suggests that the cell responses to roughness changes were not governed by broadscale differences in the proteins initially available to adhering cells. In addition to the composition of the adsorbed layer, the distribution of proteins may also vary with topography. The immunofluorescence methods were not sufficiently sensitive to reveal the distribution of adsorbed adhesion proteins (vitronectin and fibronectin). However, the lack of clear labelling does suggest an absence of large accumulations due to specific topographic features. Further work is required to address this issue conclusively. Observations of cell morphology were consistent with widely-reported contact guidance phenomena on grooved surfaces, with elongation and alignment (with topography) increasing with groove depth. Cell elongation was also enhanced on the more hydrophilic, heat-treated titanium, but this effect diminished over time. Although increased elongation at 90 minutes corresponded to lower cell numbers at 48 hours, no causal relationship has yet been established.

Coupling of cells to biomaterials is a prerequisite for most biomedical applications; e.g., neuroelectrodes can only stimulate brain tissue in vivo if the electric signal is transferred to neurons attached to the electrodes’ surface. Besides, cell survival in vitro also depends on the interaction of cells with the underlying substrate materials; in vitro assays such as multielectrode arrays determine cellular behavior by electrical coupling to the adherent cells. In our study, we investigated the interaction of neurons and glial cells with different electrode materials such as TiN and nanocolumnar TiN surfaces in contrast to gold and ITO substrates. Employing single-cell force spectroscopy, we quantified short-term interaction forces between neuron-like cells (SH-SY5Y cells) and glial cells (U-87 MG cells) for the different materials and contact times. Additionally, results were compared to the spreading dynamics of cells for different culture times as a function of the underlying substrate. The adhesion behavior of glial cells was almost independent of the biomaterial and the maximum growth areas were already seen after one day; however, adhesion dynamics of neurons relied on culture material and time. Neurons spread much better on TiN and nanocolumnar TiN and also formed more neurites after three days in culture. Our designed nanocolumnar TiN offers the possibility for building miniaturized microelectrode arrays for impedance spectroscopy without losing detection sensitivity due to a lowered self-impedance of the electrode. Hence, our results show that this biomaterial promotes adhesion and spreading of neurons and glial cells, which are important for many biomedical applications in vitro and in vivo.

No description available.