The role of microtopography in cellular mechanotransduction.

Mechanotransduction is crucial for cellular processes including cell survival, growth and differentiation. Topographically patterned surfaces offer an invaluable non-invasive means of investigating the cell response to such cues, and greater understanding of mechanotransduction at the cell-material interface has the potential to advance development of tailored topographical substrates and new generation implantable devices. This study focuses on the effects of topographical modulation of cell morphology on chromosomal positioning and gene regulation, using a microgrooved substrate as a non-invasive mechanostimulus. Intra-nuclear reorganisation of the nuclear lamina was noted, and the lamina was required for chromosomal repositioning. It appears that larger chromosomes could be predisposed to such repositioning. Microarrays and a high sensitivity proteomic approach (saturation DiGE) were utilised to identify transcripts and proteins that were subject to mechanoregulated changes in abundance, including mediators of chromatin remodelling and DNA synthesis linked to the changes in nucleolar morphology and the nucleoskeleton.

A biodegradable and biocompatible regular nanopattern for large-scale selective cell growth.

A biodegradable substrate with a regular array of nanopillars fabricated by electron-beam lithography and hot embossing is used to address the mechanisms of nanotopographical control of cell behavior. Two different cell lines cultured on the nanopillars show striking differences in cell coverage. These changes are topography- and cell-dependent, and are not mediated by air bubbles trapped on the nanopattern. For the first time, a strong cell-selective effect of the same nanotopography has been clearly demonstrated on a large area; while fibroblast proliferation is inhibited, endothelial cell spreading is visibly enhanced. The reduced fibroblast proliferation indicates that a reduction of available surface area induced by nanotopography might be the main factor affecting cell growth on nanopatterns. The results presented herein pave the way towards the development of permanent vascular replacements, where non-adhesive, inert, surfaces will induce rapid in situ endothelialization to reduce thrombosis and occlusion.

The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signalling in STRO-1+ enriched skeletal stem cells.

The physiochemical characteristics of a material with in vivo applications are critical for the clinical success of the implant and regulate both cellular adhesion and differentiated cellular function. Topographical modification of an orthopaedic implant may be a viable method to guide tissue integration and has been shown in vitro to dramatically influence osteogenesis, inhibit bone resorption and regulate integrin mediated cell adhesion. Integrins function as force dependant mechanotransducers, acting via the actin cytoskeleton to translate tension applied at the tissue level to changes in cellular function via intricate signalling pathways. In particular the ERK/MAPK signalling cascade is a known regulator of osteospecific differentiation and function. Here we investigate the effects of nanoscale pits and grooves on focal adhesion formation in human osteoblasts (HOBs) and the ERK/MAPK signalling pathway in mesenchymal populations. Nanopit arrays disrupted adhesion formation and cellular spreading in HOBs and impaired osteospecific differentiation in skeletal stem cells. HOBs cultured on 10 microm wide groove/ridge arrays formed significantly less focal adhesions than cells cultured on planar substrates and displayed negligible differentiation along the osteospecific lineage, undergoing up-regulations in the expression of adipospecific genes. Conversely, osteospecific function was correlated to increased integrin mediated adhesion formation and cellular spreading as noted in HOBS cultured on 100 microm wide groove arrays. Here osteospecific differentiation and function was linked to focal adhesion growth and FAK mediated activation of the ERK/MAPK signalling pathway in mesenchymal populations.

A hierarchical response of cells to perpendicular micro- and nanometric textural cues.

In this paper, we report on the influence of shallow micro- and nanopatterned substrata on the attachment and behavior of a human fibroblast [human telomerase transfected immortalized (hTERT)] cells. We identify a hierarchy of textural guidance cues with respect to cell alignment on these substrates. Cells were seeded and cultured for 48 h on silicon substrates patterned with two linear textures overlaid at 90 degrees, both with 24 microm pitch: a micrograting and a nanopattern of rows of 140- nm-diameter pits arranged in a rectangular array with 300 nm centre-to-centre spacing. The cell response to these textures was shown to be highly dependent on textural feature dimensions. We show that cells align to the stripes of nanopits. Stripes of 30-nm deep nanopits were also shown to elicit a stronger response from cells than 160-nm deep nanopits.

Differential in-gel electrophoresis (DIGE) analysis of human bone marrow osteoprogenitor cell contact guidance.

We have used a recent comparative proteomics technique, differential in-gel electrophoresis (DIGE), to study osteoprogenitor cell response to contact guidance in grooves. In order to increase protein output from small sample sizes, we used bioreactor culture before protein extraction and gel electrophoresis. Mass spectroscopy was used for protein identification. A number of distinct proteins were observed to exhibit significant changes in expression. These changes in protein expression suggest that the cells respond to tailored grooved topographies, with alterations in their proteome concurrent with changes in osteoprogenitor phenotype.

Interactions with nanoscale topography: adhesion quantification and signal transduction in cells of osteogenic and multipotent lineage.

Polymeric medical devices widely used in orthopedic surgery play key roles in fracture fixation and orthopedic implant design. Topographical modification and surface micro-roughness of these devices regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved the field of surface modification; in particular, nanotechnology has allowed the development of nanoscale substrates for the investigation into cell-nanofeature interactions. In this study human osteoblasts (HOBs) were cultured on ordered nanoscale pits and random nano "craters" and "islands". Adhesion subtypes were quantified by immunofluorescent microscopy and cell-substrate interactions investigated via immuno-scanning electron microscopy. To investigate the effects of these substrates on cellular function 1.7 k microarray analysis was used to establish gene profiles of enriched STRO-1+ progenitor cell populations cultured on these nanotopographies. Nanotopographies affected the formation of adhesions on experimental substrates. Adhesion formation was prominent on planar control substrates and reduced on nanocrater and nanoisland topographies; nanopits, however, were shown to inhibit directly the formation of large adhesions. STRO-1+ progenitor cells cultured on experimental substrates revealed significant changes in genetic expression. This study implicates nanotopographical modification as a significant modulator of osteoblast adhesion and cellular function in mesenchymal populations.

The response of fibroblasts to hexagonal nanotopography fabricated by electron beam lithography.

It has been known for many years that cells will react to the shape of their microenvironment. It is more recently becoming clear that cells can alter their morphology, adhesions, and cytoskeleton in response to their nanoenvironment. A few studies have gone further and measured cellular response to high-adhesion nanomaterials. There have, however, been practical difficulties associated with genomic studies focusing on low-adhesion nanotopographies. Because of advancement in fabrication techniques allowing the production of large area of structure and the ability to amplify mRNA prior to microarray hybridization, these difficulties can be overcome. Here, electron beam lithography has been used to fabricate arrays of pits with 120 nm diameters, 100 nm depth and 300 nm center to center spacing in hexagonal arrangement. Electron and fluorescent microscopies have been used to observe morphological changes in fibroblasts cultured on the pits. 1.7k gene microarray was used to gauge genomic response to the pits. The results show reduction in cellular adhesion, decrease in spreading, and a broad genomic down-regulation. Also noted was an increase in endocytotic activity in cells on the pits.

The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.

A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.

Nanomechanotransduction and interphase nuclear organization influence on genomic control.

The ability of cells to alter their genomic regulation in response to mechanical conditioning or through changes in morphology and the organization of the interphase nuclei are key questions in cell biology. Here, two nanotopographies have been used as a model surfaces to change cell morphology in order to investigate spatial genomic changes within the nuclei of fibroblasts. Initially, centromeres for chromosome pairs were labeled and the average distance on different substrates calculated. Further to this, Affymetrix whole genome GeneChips were used to rank genomic changes in response to topography and plot the whereabouts on the chromosomes these changes were occurring. It was seen that as cell spreading was changed, so were the positions along the chromosomes that gene regulations were being observed. We hypothesize that as changes in cell and thus nuclear morphology occur, that this may alter the probability of transcription through opening or closing areas of the chromosomes to transcription factors.

Osteoprogenitor response to low-adhesion nanotopographies originally fabricated by electron beam lithography.

It is considered that cells can use filopodia, or microspikes, to locate sites suitable for adhesion. This has been investigated using a number of mature cell types, but, to our knowledge, not progenitor cells. Chemical and topographical cues on the underlying substrate are a useful tool for producing defined features for cells to respond to. In this study, arrays of nanopits with different symmetries (square or hexagonal arrays with 120 nm diameters, 300 nm center-centre spacings) and osteoprogenitor cells were considered. The pits were fabricated by ultra-high precision electron-beam lithography and then reproduced in polycarbonate by injection moulding with a nickel stamp. Using scanning electron and fluorescence microscopies, the initial interactions of the cells via filopodia have been observed, as have subsequent adhesion and cytoskeletal formation. The results showed increased filopodia interaction with the surrounding nanoarchitecture leading to a decrease in cell spreading, focal adhesion formation and cytoskeletal organisation.

Nanotopographical stimulation of mechanotransduction and changes in interphase centromere positioning.

We apply a recently developed method for controlling the spreading of cultured cells using electron beam lithography (EBL) to create polymethylmethacrylate (PMMA) substrata with repeating nanostructures. There are indications that the reduced cell spreading on these substrata, compared with planar PMMA, results from a reduced adhesivity since there are fewer adhesive structures and fewer of their associated stress fibres. The reduced cell spreading also results in a reduced nuclear area and a closer spacing of centrosomes within the nucleus, suggesting that the tension applied to the nucleus is reduced as would be expected from the reduction in stress fibres. In order to obtain further evidence for this, we have used specific inhibitors of components of the cytoskeleton and have found effects comparable with those induced by the new substrata. We have also obtained evidence that these subtrata result in downregulation of gene expression which suggests that this may be due to the changed tension on the nucleus: an intriguing possibility that merits further investigation.

Group analysis of regulation of fibroblast genome on low-adhesion nanostructures.

Nanotopographical material modification represents a possible way of producing surfaces that influence cellular adhesion for biomaterials purposes. Here, two low-adhesion surfaces are studied with human genome microarrays (120nm diameter pits produced by electron beam lithography and 11nm high columns produced by colloidal lithography). In order to present the large numbers of results produced in a succinct and easy to understand manner, two types of recent bioinformatics methods were used; iterative group analysis and Ingenuity pathway analysis. These methods allowed the easy comparison of the nanomaterials and showed large-scale changes in areas of extracellular matrix, cell signalling and inflammation. The results demonstrate that whilst modes of cellular response to low-adhesion materials are similar, the more adhesion is reduced, the further 'shut-down' of critical cellular activities is observed. We also feel that the analysis used could be of interest to biomaterials scientists looking for easy ways to display microarray data efficiently.

Air-trapping on biocompatible nanopatterns.

The occurrence of air-trapping inside poly-eta-caprolactone nanopits was investigated by measuring the contact angles of water droplets on a set of defined nanotopographies. It is shown that the advancing angles follow the Cassie-Baxter theory, thus revealing the presence of air bubbles inside the biodegradable nanopatterns. The importance of these observations for the definition of hydrophilicity/hydrophobicity and in the context of in vitro cell behavior is discussed.

The effects of colloidal nanotopography on initial fibroblast adhesion and morphology.

Colloidal lithography offers a simple, inexpensive method of producing irregular nanotopographies, a pattern not easily attainable utilizing conventional serial writing processes. Colloids with 20- or 50-nm diameter were utilized to produce such an irregular topography and were characterized by calculating the percentage area coverage of particles. Interparticle and nearest neighbor spacing were also assessed for the individual colloids in the pattern. Two-way analysis of variance (ANOVA) indicated significant differences between the number of fibroblasts adhering to planar, 20-, and 50-nm-diameter colloidal topographies, the number of fibroblasts adhering to the substrates at the time intervals studied, namely 20 min, 1 h, and 3 h and significant interaction between time and topography on fibroblast adhesion (P < 0.01). Tukey tests were utilized for sensitive identification of the differences between the sample means and compounded ANOVA results. Cytoskeletal and general cell morphology were investigated on planar and colloidal substrates, and indicated cells in contact with irregular nanotopographies exhibit many peripheral protrusions while such protrusions are absent in cells on planar control surfaces. These protrusions are rich in microtubules on 20-nm-diameter colloidal surfaces while microfilaments are prevalent on 50-nm-diameter surfaces. Moreover, by 3 h, cells on the colloidal substrates initiate cell-cell adhesions, also absent in controls.

Osteoprogenitor response to defined topographies with nanoscale depths.

In the development of the next generation of orthopaedic implants for load-bearing applications, an ability to influence osteoprogenitor population activity and function will be highly desirable. This will allow the formation of hard-tissue directly onto the implant, i.e. osseointegration, rather than the formation of fibrous capsules which form around many of the current, non-bioactive, prosthesis. The formation of capsules leads to micromotion due to modulus mismatch and ultimately to fracture and the need for revision surgery. Microtopography and latterly nanotopography have been shown to elicit influence over adhesion, proliferation and gene expression of a wide number of cell types. This study has examined the possibility of modulating cell adhesion using a range of nanometric scale shallow pits and grooves. The topographies were manufactured using photolithography followed by the production of nickel shims and finally embossing into polymethylmethacrylate. Cell testing with human osteoprogenitor populations showed that the nanotopographies allowed control of cell adhesion, cytoskeleton, growth and production of the osteoblastic markers osteocalcin and osteopontin. It is concluded that the human bone marrow stromal cells are highly responsive to nanoscale features.

Superhydrophobicity and superhydrophilicity of regular nanopatterns.

The hydrophilicity, hydrophobicity, and sliding behavior of water droplets on nanoasperities of controlled dimensions were investigated experimentally. We show that the "hemi-wicking" theory for hydrophilic SiO(2) samples successfully predicts the experimental advancing angles and that the same patterns, after silanization, become superhydrophobic in agreement with the Cassie-Baxter and Wenzel theories. Our model topographies have the same dimensional scale of some naturally occurring structures that exhibit similar wetting properties. Our results confirm that a forest of hydrophilic/hydrophobic slender pillars is the most effective superwettable/water-repellent configuration. It is shown that the shape and curvature of the edges of the asperities play an important role in determining the advancing angles.

Investigating filopodia sensing using arrays of defined nano-pits down to 35 nm diameter in size.

In order for cells to react to topography, they must be able to sense shape. When considering nano-topography, these shapes are much smaller than the cell, but still strong responses to nano-topography have been seen. Filopodia, or microspikes, presented by cells at their leading edges are thought to be involved in gathering of special information. In order to investigate this, and to develop an understanding of what size of feature can be sensed by cells, morphological observation (electron and fluorescent microscopy) of fibroblasts reacting to nano-pits with 35, 75 and 120 nm diameters has been used in this study. The nano-pits are especially interesting because unlike many of the nanofeatures cited in the literature, they have no height for the cells to react to. The results showed that cell filopodia, and retraction fibres, interacted with all pit sizes, although direct interaction was hard to image on the 35 nm pits. This suggests that cells are extremely sensitive to their nanoevironment and that should be taken in to consideration when designing next-generation tissue engineering materials. We suggest that this may occur through nanocontact guidance as filopodia are moved over the pits.

Nucleus alignment and cell signaling in fibroblasts: response to a micro-grooved topography.

Cellular response to scaffold materials is of great importance in cellular and tissue engineering, and it is perhaps the initial cell contact with the scaffold that determines development of new tissue. Material surface morphology has strong effects on cell cytoskeleton and morphology, and it is thought that cells may react to the topography of collagen and surrounding cells during tissue embryology. A poorly understood area is, however, gene-level responses to topography. Thus, this paper used microarray to probe for consistent gene changes in response to lithographically produced topography (12.5 x 2-microm grooves) with time. The results showed many initial gene changes and also down-regulation of gene response with time. Cell and nucleus morphology were also considered, with nuclear deformation linked to cell signaling.