Evaluation of early stage human bone marrow stromal proliferation, cell migration and osteogenic differentiation on µ-MIM structured stainless steel surfaces.

It is well established that surface topography greatly affect cell-surface interactions. In a recent study we showed that microstructured stainless steel surfaces characterized by the presence of defined hexagonally arranged hemisphere-like structures significantly affected cell architecture (shape and focal adhesion size) of primary human bone mesenchymal stromal cells. This study aimed at further investigating the influence these microstructures (microcline protruding hemispheres) on critical aspects of cell behaviour namely; proliferation, migration and osteogenic differentiation. As with previously reported data, we used primary human bone mesenchymal stromal cells to investigate such effects at an early stage in vitro. Cells of different patients were utilised for cell migration studies. Our data showed that an increase in cell proliferation was exhibited as a function of surface topography (hemispheres). Cell migration velocity also varied as a function of surface topography on patient specific basis and seems to relate to the differentiated state of the seeded cell population (as demonstrated by bALP positivity). Osteogenic differentiation, however, did not exhibit significant variations (both up and down-regulation) as a function of both surface topography and time in culture.

In vitro bioactivity of micro metal injection moulded stainless steel with defined surface features.

Micrometre- and nanometre-scale surface structuring with ordered topography features may dramatically enhance orthopaedic implant integration. In this study we utilised a previously optimised micron metal injection moulding (µ-MIM) process to produce medical grade stainless steel surfaces bearing micrometre scale, protruding, hemispheres of controlled dimensions and spatial distribution. Additionally, the structured surfaces were characterised by the presence of submicrometre surface roughness resulting from metal grain boundary formation. Following cytocompatibility (cytotoxicity) evaluation using 3T3 mouse fibroblast cell line, the effect on primary human cell functionality was assessed focusing on cell attachment, shape and cytoskeleton conformation. In this respect, and by day 7 in culture, significant increase in focal adhesion size was associated with the microstructured surfaces compared to the planar control. The morphological conformation of the seeded cells, as revealed by fluorescence cytoskeleton labelling, also appeared to be guided in the vertical dimension between the hemisphere bodies. Quantitative evaluation of this guidance took place using live cytoplasm fluorescence labelling and image morphometry analysis utilising both, compactness and elongation shape descriptors. Significant increase in cell compactness was associated with the hemisphere arrays indicating collective increase in focused cell attachment to the hemisphere bodies across the entire cell population. Micrometre-scale hemisphere array patterns have therefore influenced cell attachment and conformation. Such influence may potentially aid in enhancing key cellular events such as, for example, neo-osteogenesis on implanted orthopaedic surfaces.

Directed differentiation of pancreatic stem cells by soluble and immobilised signalling factors.

Recent findings demonstrate that adult stem cells exhibit a much higher differentiation potential than previously expected and that the sources within the adult organism seem to be surprisingly manifold. In this study, we investigated adult stem cells isolated from mouse and rat pancreas and showed for the first time their potential to differentiate towards osteoblasts, chondrocytes and adipocytes. These pancreatic stem cells (PSCs) have previously been described to differentiate spontaneously into cell types of all three germ layers. We could now demonstrate that a directed differentiation is also possible and compared a variety of cytokines as inducers. In addition to today's practice of applying only soluble differentiation factors we investigated the use of immobilised cytokines. For protein immobilization different strategies were evaluated such as covalent linkage of insulin-like growth factor-1 (IGF-1) to amino- or epoxy-silanised glass or affinity binding of the biotinylated cytokine to neutravidin-coated surfaces. Our results show that cytokines could be immobilised functionally, retaining their biological activity to effectively induce stem cell differentiation in long-term cell culture experiments. Our results emphasised the high differentiation potential of mouse and rat PSCs, showing that a directed cytokine-induced differentiation of both rodent PSC types is possible and demonstrating that differentiation factors, immobilised to different surfaces, can successfully trigger a stem cell's fate.

Immobilized cytokines as biomaterials for manufacturing immune cell based vaccines.

Manufacturing of bioactive cell culture substrates represents a major challenge for the development of cell therapy for tissue repair and immune treatment of cancers, infectious diseases, or immunodeficiencies. In this context, we evaluated the capacity of several differentiation factors, including Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF), to drive differentiation of primary cell cultures, once immobilized on surfaces. We show that covalently immobilized signal factors fully retain their biological properties and efficiently promote differentiation of mouse and/or human precursor cells leading to the production of dendritic cells and macrophages. For GM-CSF, we also show that the efficiency of receptor signaling is comparable using either soluble or tethered molecules. Such artificial bioactive interfaces are suitable for the development and automated production of cell-based vaccines and therapies.

Neural cell adhesion molecule polysialylation enhances the sensitivity of embryonic stem cell-derived neural precursors to migration guidance cues.

The development of stem cell-based neural repair strategies requires detailed knowledge on the interaction of migrating donor cells with the host brain environment. Here we report that overexpression of polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), in embryonic stem (ES) cell-derived glial precursors (ESGPs) strikingly modifies their migration behavior in response to guidance cues. ESGPs transduced with a retrovirus encoding the polysialyltransferase STX exhibit enhanced migration in monolayer cultures and an increased penetration of organotypic slice cultures. Chemotaxis assays show that overexpression of PSA results in an enhanced chemotactic migration toward gradients of a variety of chemoattractants, including fibroblast growth factor 2 (FGF2), platelet-derived growth factor, and brain-derived neurotrophic factor (BDNF), and that this effect is mediated via the phosphatidylinositol 3'-kinase (PI3K) pathway. Moreover, PSA-overexpressing ESGPs also exhibit an enhanced chemotactic response to tissue explants derived from different brain regions. The effect of polysialylation on directional migration is preserved in vivo. Upon transplantation into the adult striatum, PSA-overexpressing but not control cells display a targeted migration toward the subventricular zone. On the basis of these data, we propose that PSA plays a crucial role in modulating the ability of migrating precursor cells to respond to regional guidance cues within the brain tissue. Disclosure of potential conflicts of interest is found at the end of this article.