2.1

Production of Ti surfaces with different micrometer-scale roughness values

2.1.1

Micrometer-featured roughness masters

Micrometer-featured masters with different roughness values were prepared in a similar way as described previously . A two-step process was used to create micrometer-scale-featured roughness masters. Rolled aluminum sheets (purity 99.5%, dimensions: 20 mm × 40 mm × 2 mm (Metall Service Menzikon AG, Switzerland)) were sand-blasted with corundum particles (81500-826-074, Sablux, Switzerland) to achieve a uniformly rough surface morphology. In a second step, the roughened substrate was fully immersed into a chemical polishing solution (77.5% (v/v) phosphoric acid, 16.5% (v/v) sulfuric acid and 6% (v/v) nitric acid), which preferentially removes small and sharp features as a function of time. The substrates were exposed to the chemical polishing solution for 33, 12 or 2 min. The different polishing times result in homogenous roughness masters with a Ra value of approximately 1, 2 or 4 μm, respectively (referred as surfaces 1 , 2 and 4 ). A flat silicon wafer (〈1 0 0〉 orientation, Si-mat, Germany) was also used as a master with an Ra value of approximately 0 μm (referred as surface 0 ).

2.1.2

Replication

In order to produce multiple identical samples for reproducibility reasons, the masters were replicated, as described by Wieland et al. and Schuler et al. . Impressions of the different roughness masters were prepared in polyvinylsiloxane (PROVIL novo light, Heraeus-Kulzer, Switzerland). A 24-well plate (TPP, Switzerland) was used as a mold to achieve cylindrical negatives with a diameter of 16.2 mm. Negative replicas were inserted into a polytetrafluoroethylene (PTFE) template with holes of a diameter of 15 mm, leaving a rim of 1 mm height. Epoxy resin (EPO-TEK 302-3M, Polyscience AG, Switzerland) was cast onto the polyvinylsiloxane negatives and cured at 65 °C for at least 24 h. The epoxy samples were cleaned for 10 min in an ultrasonic bath containing a 2 vol% Hellmanex solution (HELLMA GmbH & Co, Müllheim, Germany) and extensively rinsed in ultra-pure water 10 times. Samples were pre-dried with a jet of nitrogen and air dried overnight. Clean epoxy replicas were sputter-coated with 40 nm of metallic titanium (Reactive magnetron sputtering, Paul Scherrer Institute, Villigen, Switzerland). SEM investigation of freeze-fractured cross-sections of epoxy samples with highest roughness (Ra ∼ 4 μm) showed that the epoxy surface is entirely covered with a 30–50 nm thick layer of TiO ₂ . No tendency of a thinner coating on peaks and thicker coating in valleys could be observed (see also exemplary images in Supplementary material). The replicas were cleaned in a plasma cleaner (Harrick, PDC-32G, USA) for 2 min in an oxygen atmosphere of roughly 2 × 10 ⁻³ mbar at high RF level. The replicas were sterile packed under laminar flow in containers containing ultra-pure water and stored at room temperature until further use.

2.1.3

Characterization

The micrometer-scale roughness of the titanium-coated epoxy replicas was analyzed by an optical profilometer (Sensofar Plu Neox, Sensofar, Spain) in confocal mode. 2.75 mm long strips were stitched together by using the 20× objective. The evaluation was carried out with the SensoMap software (v.6.0, Sensofar, Spain) according to the DIN EN ISO 4287 standard.

The contribution of different wavelength windows to the overall surface roughness was determined using a fast-Fourier-transformation (FFT)-based analysis method . With this method, contributions in different wavelength windows can be individually evaluated. Three different windows with wavelengths between 3–10 μm, 10–50 μm and 50–250 μm were used to calculate the arithmetic roughness value Ra.

2.2

Cell culture

Osteoblast-like MG-63 cells (American Type Culture Collection, Rockville, USA) were used in the present study. MG-63 is a cell line that exhibits osteoblastic features and has constant properties , whereas characteristics of primary osteoblasts may differ depending on the source, donor, and isolation method . The cells were cultured in modified Eagle’s minimum essential medium (MEM, Gibco ^® , Carlsbad, USA) supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (50 μg/ml) at 37 °C in a humidified atmosphere containing 5% CO ₂ . Cells were grown in culture up to passage four, in order to obtain sufficient cell numbers for experiments. Cells between the fourth and the seventh passage were used in the experiments.

2.3

Measurements of cell proliferation and viability

Cell proliferation was measured by two methods: manual cell counting and cell counting kit 8 (CCK-8, Dojindo Laboratories, Japan). In proliferation experiments, 2 × 10 ⁴ cells were seeded on each Ti-coated (diameter 15 mm, cylinder height 1 mm) surface in 500 μl MEM. Cells seeded at similar density on tissue culture plastic (TCP) were used as control. Each group included three different wells. Cell proliferation was measured after 2, 5, and 9 days of culture. For manual cell counting, cells were collected by accutase and counted in blind fashion using a cell-counting chamber under a microscope. Cell viability/proliferation was determined using the cell counting kit-8 (CCK-8; Dojindo Laboratories, Japan) assay. After incubation, 50 μl of CCK-8 reagent were added into each well and culture plates were incubated in 5% CO ₂ at 37 °C for 4 h. Thereafter 100 μl of each well were transferred to a separate 96-well plate and the optical density (OD) was measured at 450 nm using a microplate reader (Spectramax Plus 384; Molecular Devices, USA).

2.4

Time-lapse microscopy

The behavior of MG-63 cells on different surfaces was observed using time-lapse microscopy, as described in our previous studies . Cells were stained with CellTracker™ Orange CMRA (Molecular Probes™, Invitrogen, UK) according to manufacturer’s instructions and 1 × 10 ⁵ stained cells, resuspended in 800 μl of MEM, were seeded on Ti discs with different surface treatments. Experiments for each surface were repeated at least three times. Cells were observed with an upright fluorescence microscope (Nikon Eclipse E 800 M microscope; Nikon Instruments Europe B.V, Badhoevedorp, Netherlands), and photographed with a digital imaging system (Photometrics1, Cascade 512F, Germany) every 30 min for over 120 h with the aid of NIS-elements software (Nikon Instruments). Cell motility was analysed using the tracking module in manual tracking mode. For each experiment, 10 randomly selected cells per well/group were tracked in the time period from 6 h until 12 h after seeding. Cell motility was described by average migration speed.

2.5

Alkaline phosphatase activity

MG-63 cells were seeded at an initial density of 5 × 10 ⁴ cells per well in 0.5 ml of MEM on Ti surfaces with different micrometer-scale roughness values, as well as on tissue-culture plastic. After 2, 5, or 9 days of culture, the activity of alkaline phosphatase was measured with a similar method to that described in our previous studies . In brief, culture cells were collected by accutase and lysed in 200 μl phosphate-buffered saline (PBS) containing 0.2% Triton X-100, which facilitates the destruction of the plasma membranes, and then homogenized by sonification. The total protein content in cell homogenates was determined using a commercially available kit (Micro/Macro BCA; Pierce Chemical Co., Rockford, IL, USA). The ALP activity was assayed using the conversion of a colorless p -nitrophenyl phosphate to a colored p -nitrophenol according to the manufacturer’s protocol (Sigma, St. Louis, MO, USA). The color changes were measured spectrophotometrically at 405 nm. The amounts of released phosphate were quantified by comparison with a standard curve and normalized to the total protein content in cell homogenates. ALP activity experiments were repeated three times.

2.6

Real-time PCR

The expression levels of different osteogenesis-related proteins in MG-63 cells were quantified by means of real time PCR, applying a similar method to those described earlier . MG-63 cells were seeded at a density of 5 × 10 ⁴ cells per well in 0.5 ml MEM and cultured for 2, 5, or 9 days. Isolation of mRNA and transcription into cDNA was performed using the TaqMan ^® Gene Expression Cells-to-CT™ kit (Ambion/Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. This kit provides high accuracy and superior sensitivity in gene-expression analysis . qPCR was performed on an ABI StepOnePlus device (Applied Biosystems) in paired reactions using TaqMan ^® gene expression assays with the following ID numbers (all from Applied Biosystems): alkaline phosphatase (ALP), Hs01029141_g1; osteocalcin (OC), Hs00609452_g1; VEGF, Hs00900055_m1; Hs; β-actin, Hs99999903_m1. β-actin was used as a house-keeping gene. The PCR reactions were performed in triplicate under the following thermocycling conditions: 95 °C for 10 min; 40 cycles, each for 15 s at 95 °C and at 60 °C for 1 min. The point at which the PCR product was first detected above a fixed threshold (cycle threshold, C _t ), was determined for each sample. Changes in the expression of target genes were calculated using the 2 ^−ΔΔCt method, where <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='ΔΔCt=(Cttarget−Ctβ-actin)sample−(Cttarget−Ctβ-actin)control’>ΔΔCt=(Cttarget−Ctβ-actin)sample−(Cttarget−Ctβ-actin)controlΔΔCt=(Cttarget−Ctβ-actin)sample−(Cttarget−Ctβ-actin)control
Δ Δ C t = ( C t t a r g e t − C t β -actin ) s a m p l e − ( C t t a r g e t − C t β -actin ) c o n t r o l
, taking MG-63 cells grown on plastic as a control.

2.7

Measurement of osteocalcin, and VEGF in conditioned medium

Commercially available ELISA kits were used for osteocalcin (Ebioscience, San Diego, USA) and VEGF (BosterBio, Pleasanton, USA) assays in the conditioned media after 5 days of culturing. Undiluted samples were used for measurements.

2.8

Statistical analysis

The normal distribution of all data was tested with the Kolmogorov–Smirnov test. For normally distributed data, the statistical differences between different groups were analysed by one-way analysis of variance (ANOVA) followed by a post-hoc LSD test. Data are expressed as mean ± S.E.M. Differences were considered to be statistically significant at p < 0.05.