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.

Genomic expression of mesenchymal stem cells to altered nanoscale topographies.

The understanding of cellular response to the shape of their environment would be of benefit in the development of artificial extracellular environments for potential use in the production of biomimetic surfaces. Specifically, the understanding of how cues from the extracellular environment can be used to understand stem cell differentiation would be of special interest in regenerative medicine. In this paper, the genetic profile of mesenchymal stem cells cultured on two osteogenic nanoscale topographies (pitted surface versus raised islands) are compared with cells treated with dexamethasone, a corticosteroid routinely used to stimulate bone formation in culture from mesenchymal stem cells, using 19k gene microarrays as well as 101 gene arrays specific for osteoblast and endothelial biology. The current studies show that by altering the shape of the matrix a cell response (genomic profile) similar to that achieved with chemical stimulation can be elicited. Here, we show that bone formation can be achieved with efficiency similar to that of dexamethasone with the added benefit that endothelial cell development is not inhibited. We further show that the mechanism of action of the topographies and dexamethasone differs. This could have an implication for tissue engineering in which a simultaneous, targeted, development of a tissue, such as bone, without the suppression of angiogenesis to supply nutrients to the new tissue is required. The results further demonstrate that perhaps the shape of the extracellular matrix is critical to tissue development.

Osteoprogenitor response to semi-ordered and random nanotopographies.

In bone tissue engineering, it is desirable to use materials to control the differentiation of mesenchymal stem cell populations in order to gain direct bone apposition to implant materials. It has been known for a number of years that microtopography can alter cell adhesion, proliferation and gene expression. More recently, the literature reveals that nanotopography is also of importance. Here, the reaction of primary human osteoprogenitor cell populations to nanotopographies down to 10 nm in size is considered. The topographies were originally produced by colloidal lithography and polymer demixing on silicon and then embossed (through an intermediate nickel shim) into polymethylmethacrylate. The biological testing considered cell morphology (image analysis of cell spreading and scanning electron microscopy), cell cytoskleton and adhesion formation (fluorescent staining of actin, tubulin, vimentin and vinculin) and then subsequent cell growth and differentiation (fluorescent staining of osteocalcin and osteopontin). The results demonstrated that the nanotopographies stimulated the osteoprogenitor cell differentiation towards an osteoblastic phenotype.

The fibroblast response to tubes exhibiting internal nanotopography.

The use of three-dimensional scaffolds in cell and tissue engineering is widespread; however, the use of such scaffolds, which bear additional cellular cues such as nanotopography, is as yet in its infancy. This paper details the novel fabrication of nylon tubes bearing nanotopography via polymer demixing, and reports that the topography greatly influenced fibroblast adhesion, spreading, morphology and cytoskeletal organisation. The use of such frameworks that convey both the correct mechanical support for tissue formation and stimulate cells through topographical cues may pave the way for future production of intelligent materials and scaffolds.

Increasing fibroblast response to materials using nanotopography: morphological and genetic measurements of cell response to 13-nm-high polymer demixed islands.

It is becoming clear that cells can respond not only to micometric scale topography, but may also to nanometric scale topography. The production of reproducibly sized nanometric features has relied heavily on expensive and time-consuming methods of manufacture, such as electron beam lithography. Polymer demixing of polystyrene and polybromostyrene has been found to produce nanoscale islands of reproducible height, and the islands have been previously shown to effect cell spreading compared to planar surfaces. This study observes morphological, cytoskeletal, and molecular changes in fibroblast reaction to 13-nm-high islands. The methods employed include scanning electron microscopy, fluorescent microscopy, and 1718 gene microarray. The results show that the cells respond to the islands by broad gene up-regulation, notably in the areas of cell signaling, proliferation, cytoskeleton, and production of extracellular matrix proteins. Microscopical results provide confirmation of the microarray findings.

Fibroblast signaling events in response to nanotopography: a gene array study.

When considering the complicated nature of cell/tissue interactions with biomaterials, especially materials with nanometric surface features, observation of changes in one or two selected genes or proteins may not be sufficient. To get a fuller understanding of the scope of responses effected by nanotopography on cells, many genes need to be surveyed. Recent developments in molecular biology have lead to the commercial production of microarrays. Microarray presents a powerful tool by which many genes (up to many thousands) can be probed simultaneously. In this study, 1718 gene arrays have been used to measure human fibroblast response to 13-nm-high polymer demixed islands. The results have shown many changes in genes involved in signaling, cytoskeleton, extracellular matrix, gene transcription, and protein translation; these results have been used to build a more complete overview of fibroblast response to the islands. The use of microarray has expanded the range of observations possible using established microscopical and biochemical techniques.

Interactions of human blood and tissue cell types with 95-nm-high nanotopography.

Two of the major concerns for tissue engineering materials are inflammatory responses from blood cells and fibrous encapsulation by the body in order to shield the implant from blood reaction. A further hurdle is that of vascularization. In order to develop new tissues, or to repair parts of the vascular system, nutrients need to be carried to the basal cell layers. If a material promotes tissue formation, but not vascularization, necrosis will be observed as multilayered cells develop. In this paper, polymer demixed island topography with a 95-nm Z axis was tested using human mononuclear blood cells, platelets, fibroblasts, and endothelial cells. The results showed no difference in blood response between the islands and the flat controls, suggesting that in vivo there would be negligible immunological difference. Fibroblasts reacted by changing morphology into a rounded shape with thick processes and poorly developed cytoskeleton. Retardation of fibroblast growth may be an advantageous, as it is this cell type that forms the fibrous capsule, preventing growth of the required tissue type. Finally, endothelial cells were seen to form arcuate, or curved, morphologies in response to the islands. This is the normal, in vivo, morphology for vascular endothelium. This result suggests that the nano-features are promoting a more phenotypically correct morphology.