Dynamic Surfaces for the Study of Mesenchymal Stem Cell Growth through Adhesion Regulation.

Out of their niche environment, adult stem cells, such as mesenchymal stem cells (MSCs), spontaneously differentiate. This makes both studying these important regenerative cells and growing large numbers of stem cells for clinical use challenging. Traditional cell culture techniques have fallen short of meeting this challenge, but materials science offers hope. In this study, we have used emerging rules of managing adhesion/cytoskeletal balance to prolong MSC cultures by fabricating controllable nanoscale cell interfaces using immobilized peptides that may be enzymatically activated to change their function. The surfaces can be altered (activated) at will to tip adhesion/cytoskeletal balance and initiate differentiation, hence better informing biological mechanisms of stem cell growth. Tools that are able to investigate the stem cell phenotype are important. While large phenotypical differences, such as the difference between an adipocyte and an osteoblast, are now better understood, the far more subtle differences between fibroblasts and MSCs are much harder to dissect. The development of technologies able to dynamically navigate small differences in adhesion are critical in the race to provide regenerative strategies using stem cells.

Systems Biology Approaches Applied to Regenerative Medicine.

Systems biology is the creation of theoretical and mathematical models for the study of biological systems, as an engine for hypothesis generation and to provide context to experimental data. It is underpinned by the collection and analysis of complex datasets from different biological systems, including global gene, RNA, protein and metabolite profiles. Regenerative medicine seeks to replace or repair tissues with compromised function (for example, through injury, deficiency or pathology), in order to improve their functionality. In this paper, we will address the application of systems biology approaches to the study of regenerative medicine, with a particular focus on approaches to study modifications to the genome, transcripts and small RNAs, proteins and metabolites.

Scanning electron microscopical observation of an osteoblast/osteoclast co-culture on micropatterned orthopaedic ceramics.

In biomaterial engineering, the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. On contact with an implant, bone marrow-derived mesenchymal stromal cells demonstrate differentiation towards bone forming osteoblasts, which can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of bone marrow-derived mesenchymal stromal cells with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina ceramics with 30 µm diameter pits. The aim was to establish whether the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropatterns towards osteogenesis, with more nodule formation and less osteoclastogenesis compared to planar controls. In addition, we found that that macrophage and osteoclast-like cells did not interact with the pits and formed fewer full-size osteoclast-like cells on the pitted surfaces. This may have a role when designing ceramic orthopaedic implants.

Nanotopographical induction of osteogenesis through adhesion, bone morphogenic protein cosignaling, and regulation of microRNAs.

It is emerging that nanotopographical information can be used to induce osteogenesis from mesenchymal stromal cells from the bone marrow, and it is hoped that this nanoscale bioactivity can be utilized to engineer next generation implants. However, the osteogenic mechanism of surfaces is currently poorly understood. In this report, we investigate mechanism and implicate bone morphogenic protein (BMP) in up-regulation of RUNX2 and show that RUNX2 and its regulatory miRNAs are BMP sensitive. Our data demonstrate that osteogenic nanotopography promotes colocalization of integrins and BMP2 receptors in order to enhance osteogenic activity and that vitronectin is important in this interface. This provides insight that topographical regulation of adhesion can have effects on signaling cascades outside of cytoskeletal signaling and that adhesions can have roles in augmenting BMP signaling.

Investigation of the limits of nanoscale filopodial interactions.

Mesenchymal stem cells are sensitive to changes in feature height, order and spacing. We had previously noted that there was an inverse relationship between osteoinductive potential and feature height on 15-, 55- and 90 nm-high titania nanopillars, with 15 nm-high pillars being the most effective substrate at inducing osteogenesis of human mesenchymal stem cells. The osteoinductive effect was somewhat diminished by decreasing the feature height to 8 nm, however, which suggested that there was a cut-off point, potentially associated with a change in cell-nanofeature interactions. To investigate this further, in this study, a scanning electron microscopy/three-dimensional scanning electron microscopy approach was used to examine the interactions between mesenchymal stem cells and the 8 and 15 nm nanopillared surfaces. As expected, the cells adopted a predominantly filopodial mode of interaction with the 15 nm-high pillars. Interestingly, fine nanoscale membrane projections, which we have termed 'nanopodia,' were also employed by the cells on the 8 nm pillars, and it seems that this is analogous to the cells 'clinging on with their fingertips' to this scale of features.

The use of microarrays and fluorescence in situ hybridization for the study of mechanotransduction from topography.

The combination of transcriptomic analysis and fluorescence in situ hybridization (FISH) provides a robust methodology to study genomic changes in different biological conditions. Microarrays allow a global study of gene expression in response to the conditions of interest, with comparison between control(s) and one or more test condition(s). The messenger RNA amplification step permits detection of even low abundance transcripts, a critical advantage for applications such as biomaterials research, where the starting material may be limited. Different types of microarrays are commercially available that allow the investigation of specific features, such as exon arrays, microRNA arrays, and gene arrays. Microarrays are available for different model organisms, but we use Affymetrix ® HuGene ® ST (Sense Target) arrays, a type of gene array for analysis of human samples. FISH involves fluorescent detection of probe DNA hybridized to an in situ chromosomal target that can be either whole chromosomes or chromosomal segments. The overall hybridization is similar to labeling with radioactive probes but the incorporation of fluorescent detection of the probe sequences allows for high sensitivity in a simple and quick assay. FISH can be applied to a variety of specimen types depending on the study of interest. In this chapter, we describe the methodologies of these two techniques and provide technical tips that should help overcome challenges in carrying them out.

Developments in stem cells: implications for future joint replacements.

Will stem cell research reverse the projected sevenfold increase in primary and revision knee replacements expected in the United States between 2005 and 2030? A focus on prevention and treatment of osteoarthritis may end the need for primary joint replacements. A more likely scenario can be described as slow and incremental changes in the prevention and treatment of osteoarthritis, accompanied by the continuing development of implant technology. Since the discovery of stem cells in the 1950s, research has increased exponentially. Expanded autologous chondrocytes, and more recently ex vivo expanded skeletal stem cells, are currently injected into osteochondral defects in the hope of regenerating cartilage and halting progression towards osteoarthritis. In addition, mesenchymal stem cells are being injected into human joints as a treatment for osteoarthritis despite a lack of quantitative research. Concurrently, stem cell research continues to contribute to chemical and topographical advancements in implant design. Advances in co-culture techniques mean it is possible that biologic articular replacements will develop prior to the cessation of the need for arthroplasty and radically change the nature of joint replacements. Whether it is through implant design or a potential cure for the pain attributable to osteoarthritis, as we hope to show in this 'forward look article', it is our opinion that stem cells will certainly impact future joint replacement.

2D and 3D nanopatterning of titanium for enhancing osteoinduction of stem cells at implant surfaces.

The potential for the use of well-defined nanopatterns to control stem cell behaviour on surfaces has been well documented on polymeric substrates. In terms of translation to orthopaedic applications, there is a need to develop nanopatterning techniques for clinically relevant surfaces, such as the load-bearing material titanium (Ti). In this work, a novel nanopatterning method for Ti surfaces is demonstrated, using anodisation in combination with PS-b-P4VP block copolymer templates. The block copolymer templates allows for fabrication of titania nanodot patterns with precisely controlled dimensions and positioning which means that this technique can be used as a lithography-like patterning method of bulk Ti surfaces on both flat 2D and complex shaped 3D surfaces. In vitro studies demonstrate that precise tuning of the height of titania nanodot patterns can modulate the osteogenic differentiation of mesenchymal stem cells. Cells on both the 8 nm and 15 nm patterned surfaces showed a trend towards a greater number of the large, super-mature osteogenic focal adhesions than on the control polished Ti surface, but the osteogenic effect was more pronounced on the 15 nm substrate. Cells on this surface had the longest adhesions of all and produced larger osteocalcin deposits. The results suggest that nanopatterning of Ti using the technique of anodisation through a block copolymer template could provide a novel way to enhance osteoinductivity on Ti surfaces.

Titanium nanofeaturing for enhanced bioactivity of implanted orthopedic and dental devices.

Titanium (Ti) is used as a load-bearing material in the production of orthopedic devices. The clinical efficacy of these implants could be greatly enhanced by the addition of nanofeatures that would improve the bioactivity of the implants, in order to promote in situ osteo-induction and -conduction of the patient's stem and osteoprogenitor cells, and to enhance osseointegration between the implant and the surrounding bone. Nanofeaturing of Ti is also currently being applied as a tool for the biofunctionalization of commercially available dental implants. In this review, we discuss the different nanofabrication strategies that are available to generate nanofeatures in Ti and the cellular response to the resulting nanofeatures. In vitro research, in vivo studies and clinical trials are considered, and we conclude with a perspective about the future potential for use of nanotopographical features in a therapeutic setting.

Novel anodization technique using a block copolymer template for nanopatterning of titanium implant surfaces.

Precise surface nanopatterning is a promising route for predictable control of cellular behavior on biomedical materials. There is currently a gap in taking such precision engineered surfaces from the laboratory to clinically relevant implant materials such as titanium (Ti). In this work, anodization of Ti surfaces was performed in combination with block copolymer templates to create highly ordered and tunable oxide nanopatterns. Secondary ion mass spectroscopy (SIMS) and X-ray photoelectron spectroscopy (XPS) analyses showed that the composition of the anodized structures was mainly titania with small amounts of nitrogen left from the block copolymer. It was further demonstrated that these nanopatterns can be superimposed on more complex shaped Ti surfaces such as microbeads, using the same technique. Human mesenchymal stem cells were cultured on Ti microbead surfaces, with and without nanopatterns, in vitro to study the effect of nanotopography on Ti surfaces. The results presented in this work demonstrate a promising method of producing highly defined and well-arranged surface nanopatterns on Ti implant surfaces.

Metabolomics: a valuable tool for stem cell monitoring in regenerative medicine.

Metabolomics is a method for investigation of changes in the global metabolite profile of cells. This paper discusses the technical application of the approach, considering metabolite extraction, separation, mass spectrometry and data interpretation. A particular focus is on the application of metabolomics to the study of stem cell physiology in the context of biomaterials and regenerative medicine. Case studies are used to illustrate key points, focusing on the use of metabolomics in the examination of mesenchymal stem cell responses to titania-nanopillared substrata designed for orthopaedic applications.

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.

Preventing and troubleshooting artefacts in saturation labelled fluorescence 2-D difference gel electrophoresis (saturation DiGE).

Saturation DiGE is a powerful but challenging technique for the characterisation of changes in protein expression between two or more scarce samples. In this paper, measures to prevent and troubleshoot artefacts in the saturation DiGE workflow are discussed, with illustration of some examples as they may be encountered in gel images or analysis.

Skeletal stem cell physiology on functionally distinct titania nanotopographies.

Functionalisation of the surface of orthopaedic implants with nanotopographies that could stimulate in situ osteogenic differentiation of the patient's stem or osteoprogenitor cells would have significant therapeutic potential. Mesenchymal stem cell (MSC) responses to titanium substrates patterned with nanopillar structures were investigated in this study. Focal adhesions were quantified in S-phase cells, the bone-related transcription factor Runx2 was examined, osteocalcin production was noted, and Haralick computational analysis was used to assess the relatedness of the cell responses to each of the titanium substrata based on cytoskeletal textural features. Metabolomics was used as a novel means of assessing cellular responses to the biomaterial substrates by analysing the global metabolite profile of the cells on the substrata, and shows promise as a technique with high data yield for evaluating cell interactions with materials of different surface chemistry or topography. The cell response to 15 nm high nanopillars was distinct, consistent with a transition from a more quiescent phenotype on the planar substrate, to an 'active' phenotype on the pillars. These studies illustrate the potential for clinically relevant titania nanopillared substrata to modulate MSCs, with implications for orthopaedic device design and application.