Terje Sjöström

Fabrication of pillar-like titania nanostructures on titanium and their interactions with human skeletal stem cells.

Surface nanotopography is known to influence the interaction of human skeletal (mesenchymal) stem cells (hMSC) with a material surface. While most surface nanopatterning has been performed on polymer-based surfaces there is a need for techniques to produce well-defined topography features with tuneable sizes on relevant load-bearing implant materials such as titanium (Ti). In this study titania nanopillar structures with heights of either 15, 55 or 100 nm were produced on Ti surfaces using anodization through a porous alumina mask. The influence of the surface structure heights on hMSC adhesion, spreading, cytoskeletal formation and differentiation was examined. The 15 nm high topography features resulted in the greatest cell response with bone matrix nodule forming on the Ti surface after 21 days.

Cicada-inspired cell-instructive nanopatterned arrays

Biocompatible surfaces hold key to a variety of biomedical problems that are directly related to the competition between host-tissue cell integration and bacterial colonisation. A saving solution to this is seen in the ability of cells to uniquely respond to physical cues on such surfaces thus prompting the search for cell-instructive nanoscale patterns. Here we introduce a generic rationale engineered into biocompatible, titanium, substrates to differentiate cell responses. The rationale is inspired by cicada wing surfaces that display bactericidal nanopillar patterns. The surfaces engineered in this study are titania (TiO2) nanowire arrays that are selectively bactericidal against motile bacteria, while capable of guiding mammalian cell proliferation according to the type of the array. The concept holds promise for clinically relevant materials capable of differential physico-mechanical responses to cellular adhesion.

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.

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.

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 patients 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.

Osteogenic and bactericidal surfaces from hydrothermal titania nanowires on titanium substrates

Nanotopographical cues on Ti have been shown to elicit different cell responses such as cell differentiation and selective growth. Bone remodelling is a constant process requiring specific cues for optimal bone growth and implant fixation. Moreover, biofilm formation and the resulting infection on surgical implants is a major issue. Our aim is to identify nanopatterns on Ti surfaces that would be optimal for both bone remodelling and for reducing risk of bacterial infection. Primary human osteoblast/osteoclast co-cultures were seeded onto Ti substrates with TiO2 nanowires grown under alkaline conditions at 240 °C for different times (2, 2.5 or 3 h). Cell growth and behaviour was assessed by scanning electron microscopy (SEM), immunofluorescence microscopy, histochemistry and quantitative RT-PCR methods. Bacterial colonisation of the nanowire surfaces was also assessed by confocal microscopy and SEM. From the three surfaces tested the 2 h nanowire surface supported osteoblast and to a lesser extent osteoclast growth and differentiation. At the same time bacterial viability was reduced. Hence the 2 h surface provided optimal bone remodeling in vitro conditions while reducing infection risk, making it a favourable candidate for future implant surfaces.

Through-mask anodization of titania dot-?and pillar-like nanostructures on bulk Ti substrates using a nanoporous anodic alumina mask

Nanosized surface topography on an implant material has the capability of stimulating the acceptance of the material in its host surrounding. Fine-tuning of nanotopography feature size has been shown to trigger differentiation of mesenchymal stem cells into bone cells in vitro. For this purpose we have created well defined nanosized titania dot- and pillar-like structures on mechanically polished Ti substrates using a through-mask anodization technique with an anodic porous alumina template. The anodization technique allowed the titania structure dimensions to be precisely tuned in the range 15-140 nm in a single electrolyte system. The fabricated surfaces serve as good model surfaces for precise studies of in vitro cell behaviour. The through-mask anodization technique was used directly on bulk Ti surfaces, thus demonstrating a potential application for patterning of actual 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.

Analysis of Osteoclastogenesis/Osteoblastogenesis on Nanotopographical Titania Surfaces

A focus of orthopedic research is to improve osteointegration and outcomes of joint replacement. Material surface topography has been shown to alter cell adhesion, proliferation, and growth. The use of nanotopographical features to promote cell adhesion and bone formation is hoped to improve osteointegration and clinical outcomes. Use of block-copolymer self-assembled nanopatterns allows nanopillars to form via templated anodization with control over height and order, which has been shown to be of cellular importance. This project assesses the outcome of a human bone marrow-derived co-culture of adherent osteoprogenitors and osteoclast progenitors on polished titania and titania patterned with 15 nm nanopillars, fabricated by a block-copolymer templated anodization technique. Substrate implantation in rabbit femurs is performed to confirm the in vivo bone/implant integration. Quantitative and qualitative results demonstrate increased osteogenesis on the nanopillar substrate with scanning electron microscopy, histochemical staining, and real-time quantitative reverse-transcription polymerase chain reaction analysis performed. Osteoblast/osteoclast co-culture analysis shows an increase in osteoblastogenesis-related gene expression and reduction in osteoclastogenesis. Supporting this in vitro finding, in vivo implantation of substrates in rabbit femora indicates increased implant/bone contact by ≈20%. These favorable osteogenic characteristics demonstrate the potential of 15 nm titania nanopillars fabricated by the block-copolymer templated anodization technique.

The synergistic effects of lysophosphatidic acid receptor agonists and calcitriol on MG63 osteoblast maturation at titanium and hydroxyapatite surfaces

Successful osseointegration stems from the provision of a mechanically competent mineralised matrix at the implant site. Mature osteoblasts are the cells responsible for achieving this and a key factor for ensuring healthy bone tissue is associated with prosthetic materials will be 1 alpha,25 dihydroxy vitamin D3 (calcitriol). However it is known that calcitriol per se does not promote osteoblast maturation, rather the osteoblasts need to be in receipt of calcitriol in combination with selected growth factors in order to undergo a robust maturation response. Herein we report how agonists of the lysophosphatidic acid (LPA) receptor, LPA and (2S)-OMPT, synergistically co-operate with calcitriol to secure osteoblast maturation for cells grown upon two widely used bone biomaterials, titanium and hydroxyapatite. Efforts could now be focussed on functionalizing these materials with LPA receptor agonists to support in vivo calcitriol-induced osseointegration via heightened osteoblast maturation responses.