Influence of nanostructural environment and fluid flow on osteoblast-like cell behavior: a model for cell-mechanics studies.

Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering.

Dynamic cell adhesion and migration on nanoscale grooved substrates.

Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.

The influence of nanoscale topographical cues on initial osteoblast morphology and migration.

The natural environment of a living cell is not only organized on a micrometer, but also on a nanometer scale. Mimicking such a nanoscale topography in implantable biomaterials is critical to guide cellular behavior. Also, a correct positioning of cells on biomaterials is supposed to be very important for promoting wound healing and tissue regeneration. The exact mechanism by which nanotextures can control cellular behavior are thus far not well understood and it is thus far unknown how cells recognize and respond to certain surface patterns, whereas a directed response appears to be absent on other pattern types. Focal adhesions (FAs) are known to be involved in the process of specific pattern recognition and subsequent response by cells. In this study, we used a high throughput screening "Biochip" containing 40 different nanopatterns to evaluate the influence of several nanotopographical cues like depth, width, (an)isotropy and spacing (ridge-groove ratio) on osteoblast behavior. Microscopical analysis and time lapse imaging revealed that an isotropic topography did not alter cell morphology, but it highly induced cell motility. Cells cultured on anisotropic topographies on the other hand, were highly elongated and aligned. Time-lapse imaging revealed that cell motility is highly dependent on the ridge-groove ratio of anisotropic patterns. The highest motility was observed on grooves with a ratio of 1:3, whereas the lowest motility was observed on ratios of 1:1 and 3:1. FA measurements demonstrated that FA-length decreased with increasing motility. From the study it can be concluded that osteoblast behavior is tightly controlled by nanometer surface features.

The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion.

The differences in morphological behaviour between fibroblasts cultured on smooth and nanogrooved substrata (groove depth: 5-350 nm, width: 20-1000 nm) have been evaluated in vitro. The aim of the study was to clarify to what extent cell guidance occurs on increasingly smaller topographies. Pattern templates were made using electron beam lithography, and were subsequently replicated in polystyrene cell culture material using solvent casting. The replicates were investigated with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. AFM revealed that the nanogroove/ridge widths were replicated perfectly, although at deeper levels the grooves became more concave. The smooth substrata had no distinguishable pattern other than a roughness amplitude of 1 nm. Interestingly, microscopy and image analysis showed that fibroblast after 4 h had adjusted their shape according to nanotopographical features down to cut-off values of 100 nm width and 75 nm depth. After 24 h culturing time, fibroblasts would even align themselves on groove depths as shallow as 35 nm. It appears depth is the most essential parameter in cellular alignment on groove patterns with a pitch ratio of 1:1. On the smooth substrata, cells always spread out in a random fashion. Analysis of variance (ANOVA) demonstrated that both main parameters, topography and culturing time, were significant. We conclude that fibroblast cells cultured on nanotopography experience a threshold feature size of 35 nm, below this value contact guidance does no longer exist.