Stem Cell Differentiation & Genomic Processes in Response to Bioactive Nanotopography

With an increasing ageing population the clinical requirement to replace degenerated tissues, such as musculoskeletal tissue, is a major socio-economic requirement. A key issue is an understanding of stem cell activity on different materials, specifically a need to understand how stem cells behave on a material surface. We have generated novel data that shows small changes in the shape of a material can relate to large changes in cell behaviour when they are grown on the material surface. These changes in material shape can be at the nanoscale (1 x 10-9 meters); for examples pits, pillars and grooves with widths and heights of under 100 nm can cause cell alignment, increases in adhesion and even cause total non-adhesion (non-contact) through adjustments of spacing and aspect ratio. Other effects nanoscale designs can have on cells are changes in cytoskeleton (proteins involved in cell adhesion, spreading, metabolism and signalling), cell growth and the function of the cell (differentiation). Stem cells are immature cells that have the ability to differentiate into a number of mature cell types. For example, stem cells from bone can differentiate into cells for bone formation and maintenance (osteoblasts and osteocytes) or cells for cartilage formation and maintenance (chondroblasts and chondrocytes), ligament and tendon formation (fibroblasts) and a number of other cell types (fat, endothelial, epithelial). The understanding of the environmental cues allowing cells to chose one type (bone or fat - referred to as lineage) over another would be of great advantage for stem cell biologists and subsequently for materials researchers and tissue engineers could then optimise material design for e.g. hip and knee replacements. In the replacements of load bearing implants for bone (such as the knee and hip), once the material is implanted, bone stem cells in the bone marrow (called mesenchymal or skeletal stem cells) differentiate to become fibroblasts due to lack of appropriate cues from the material. Thus, the material is surrounded by soft tissue rather than hard bone. Over time this causes implant failure leading to older patients undergoing complicated secondary (revision) surgery. Here, we plan to investigate how materials can pass nanoscale mechanical signals to the cell nucleus and how this leads to changes in DNA organisation and subsequent cell differentiation - a process known as direct mechanotransduction. We would view changes in structural proteins of the nucleus (nucleoskeleton) with changes in cell spreading on nanomaterials. These changes could then be related to changes in DNA positioning and gene regulation alongside studies of differentiation. Very little is know about what in their environment triggers stem cell differentiation, we believe that surface shape, also known as topography (like a mountain surface can be flat, rugged, smooth and bumpy), is important. If we can understand these processes we can produce better materials (informed design) that will encourage direct bone bonding (apposition) to an implant, thus removing the need for revision surgery. This would save patient worry, surgical time and the NHS millions of pounds.

Topographical changes as small as 20 nm can be the difference between bone formation or lack of differentiation in stem cells. We intend to exploit this exquisite control to study the skeletal stem cell / material interface. It has been shown that the interphase nucleus has relative organisation, with chromosomes being arranged into discrete territories. Whereupon, it has then been speculated that by changing interphase chromosome positioning, the probability of transcription is altered. While research in this area is in its infancy, we have spent the last five years collecting data towards a grant application and have a unique system with which to study this without the use of invasive methodology. It is known that the cytoskeleton is central to mechanotransduction, both indirect (signalling cascades) and direct (force transduction). Our previous studies with skeletal stem cells and nanotopography have shown that topography confers changes in cell spreading and cytoskeletal organisation upon the cells. Furthermore, our previous studies with fibroblasts have shown changes in nuclear organisation and gene regulation with changes in cytoskeletal organisation. Here, it is intended than nanotopography will be used to cause changes in nuclear organisation (lamin nucleoskeleton, chromosome territories and methylation patterns) and differentiation will be monitored. Thus, the topography would be used to induce mechanotransductive effects in mesenchymal stem cells. Liking these results to microarray data and using resources such as the Stanford Source, we can plot where in the genome changes due to direct mechanotransduction are occurring. This will allow us to observe if large changes in interphase chromosome positioning due to direct mechanotransduction lead to large changes in gene regulation. Potentially, this could be a major route of cell differentiation and help us to develop materials, through rational design, to elicit directed differentiation.