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.

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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.

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.

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 marrowderived 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 confi rm 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 fi nding, 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.

Highlights Thermal oxidation was used to grow bioinspired nanospikes on Ti alloy. Nanospike dimension and arrangement could be controlled. The method worked on arbitrary shaped Ti alloy surfaces. A 40 % reduction of E. Coli viability was achieved on Ti alloy nanospike surfaces. AbstractWith the aim to fabricate bio-inspired antibacterial nanotopography surfaces, nanospikes with varying dimensions were grown on Ti alloy surfaces using a thermal oxidation method. By controlling the acetone vapour concentration inside the tube furnace, the resulting oxide surface changed from nanocolumn shapes to nanospikes with approximately 20 nm diameters. The nanospikee growth was demonstrated to work on 3D Ti alloy bead surfaces, which means translation of the method to implant surfaces would be possible. Microbiology studies using Escherichia coli. showed that the nanospikes on the Ti alloy surfaces has potential to reduce bacterial viability. More dead bacteria were present on the nanospike surfaces compared to a smooth control and a 40 % reduction of viability was noted in bacterial suspensions incubated with a nanospike surface. It was shown that by annealing the Ti alloy surfaces prior to thermal oxidation, it is possible to grow vertically aligned nanospikes. This could be highly valuable when designing implant surfaces with antimicrobial properties.

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.