1. Title:
Osteogenic and bactericidal surfaces from hydrothermal titania nanowires on titanium substrates
2. Authors:
P.M. Tsimbouri1*, N. Holloway2, L. Fisher3, T. Sjostrom3. A.H. Nobbs3, R.M.D Meek4, B. Su3, M.J. Dalby1 
1 Centre for Cell Engineering, University of Glasgow, Glasgow, Scotland, UK
2 Golden Jubilee National Hospital, Clydebank
3 School of Oral and Dental Sciences, University of Bristol, Bristol, UK
4 Department of Orthopaedics, Southern General Hospital, Glasgow, Scotland, UK
3. Funder information:
This work was supported by 
EPSRC grant EP/K034898/1 and EP/K035142/1.
4. Dataset and readme information
Generating titania nanowires on Ti substrates
Use commercial grade 1 Titanium discs.
Polishing titanium samples to a mirror finish by mechanical/chemical (OP-S solution - colloidal silica suspension with 10% H2O2) means 
Use a circular glass disk, glued to a stub which fits into the automatic rotating wheel on the polishing machine. Cover the glass with double sided tape and attach a layer of parafilm, this makes removing the disks easier. Glue the Ti disks onto the parafilm with superglue, aim to get all the disks level (approx. 19 Ti disks per glass disk).
Polishing machine: Main menu: preparation button to set settings. Water on, turn on tap slightly and press water button on polisher machine. Press start (green button).
1.	SiC grinding paper #80 (only use 1 sheet per set of samples).
Settings:
•	300 rpm
•	25 N
•	Water on
•	4min

2.	SiC grinding paper #500 (only use 1 sheet per set of samples).
Settings:
•	300 rpm
•	25 N
•	Water on
•	4min

3.	Polishing on MD-Largo disk (smooth diamond pattern disc). Make sure the bottle dispenser on the side says Largo and that there is diamond solution in the bottle. Dispense some of the diamond lubricant on the disk before starting. Press spray button on polishing machine

Settings:
•	150 rpm
•	Time 4 min
•	30 N
•	Water off

Wash the samples off with water before the next step.

4.	Polishing on MD-Chem disk (felt like disc). Use OPS suspension with 10 % H2O2. Shake well. Wet the polishing disk with some water before starting (from bottle). Add OPS/H2O2 mixture with disposable pipette. Add pipette load by load.

The disks can easily become scratched in this step, avoid this by ensuring the MD-Chem disk is as clean as possible.
Settings:
•	150 rpm
•	Time as needed (5 – 30 min) (remove and view to see how polished)
•	35 N
•	Water on. Try to adjust the water so that a small drop is applied every other second

Cleaning the samples
Remove the samples from the stubs using a knife and put them in a beaker of dH2O. Try to scrape off all of the glue from the samples using a scalpel/razor or similar.  Wash the samples in the ultrasonic bath for 20 min, hot water aids the washing. After ultrasonication pick each Ti disk up with tweezers and rinse with distilled water. Soak the samples in Isopropanol for at least 5 mins. Remove the samples from the Isopropanol and dry them with the air gun.

HYDROTHERMAL PROCESS - For preparation of TiO2 nanowires on Ti substrate

Never fill the hydrothermal vessel above the “critical volume” for the temperature you are using at. As the liquid expands to gas state this may cause damage to the vessel and ultimately destroy it.

1.	Place the Ti disks in the ptfe sample holders (will hold 20 disks) and subsequently place the holder in the Teflon cup inside the stainless steel vessel (125mL capacity).
2.	For 20 samples, add 52ml of 1M NaOH to the cup and seal the vessel.
3.	The vessel should be tightened by hand first and then use the allen key to tighten the bolts.
4.	Place the vessel in the oven which should be pre-heated to 240oC.
5.	After completed processing time, remove the vessel from the oven, using oven gloves, and allow to cool. To ensure the cooling rate is always the same it is wise to place the vessel in an oven kept at for instance 25oC. 
6.	Once at room temperature, remove the samples and soak/wash them in dH2O.
7.	Soak the samples in ethanol and then allow to dry.
8.	Heat treat the Ti samples at 300oC for 1h. (this is needed to fix the nanowires to the substrate or they tend to peel during ion exchange)
9.	Immerse the Ti disks in 0.6M HCl for 1h. (exchanges the Na+ ions for H+ ions to create TiO2)
10.	Soak/wash in dH2O.
11.	Soak in ethanol and then allow to dry.
12.	Anneal the samples at 600oC for 2h, in the small muffle furnace. (10 oC/min to 600oC).
13.	When the furnace is cool remove the samples.

Human BMSC and human BMHC isolation 
BM was obtained from hip and knee arthroplasty surgeries from three healthy patients. The BM aspirate was split into two vials and washed twice in mDMEM (DMEM (Sigma) supplemented with 10% foetal bovine serum, 200 mM L-glutamine (Invitrogen), 100 mM sodium pyruvate, 1% MEM NEAA (Gibco) and antibiotics (6.74 U/ml Penicillin-Streptomycin, 0.2 µg/ml Fungizone)) and each time centrifuged at 376 g for 10 min. The cell pellets were resuspended in basal medium and overlaid on a Ficoll layer. This was centrifuged at 445 g for 45 min. The mononuclear interface layer was transferred in a new vial and further washed twice in basal media. The cells were transferred to an appropriately sized cell culture flask and incubated at 37  ͦC with 5% humidified CO2. At day 3, post selection, the non-adherent cells were transferred to a new flask and cultured separately as bone marrow hematopoeitc cells (BMHC). The remaining adherent cells were cultured for a further 7-10 days until a confluent bone marrow stromal cells (BMSC) layer was identified. 

Co-cultures on titania nanowire surfaces
After 7 days of culture, the BMSCs were trypsinised and resuspended in mDMEM at a density of 1 x 104 cells/ml/Ti substrate. Five days later, 1 ml of BMHC suspension was added at a concentration of 1.5 x 105 cells/ml to form the co-culture. 
Histochemistry: TRAP staining
At day 35 of co-culture, histochemical analysis was performed on duplicate 2 h, 2.5 h and 3 h nanowire or polished control substrates to assess osteoclastogenesis. TRAP staining allows assessment of osteoclastogenesis, as TRAP is expressed by osteoclast progenitors and osteoclasts. Samples were fixed in 4% formaldehyde fixative for 30 s, stained with TRAP kit (Acid Phosphatase Leukocyte No.387, Sigma-Aldrich) according to manufacturer’s protocol and counterstained for 10 min in haematoxylin solution followed by a wash in H2O. Imaging was performed using the Leica DM750M microscope, with a ThorLabs DCC1645C colour camera running uc480 viewer to capture pictures. Files generated in JPEG or TIFF format.
Immunofluorescence
At day 35, 2 discs of each substrate were analysed by immunofluorescence staining as described previously in 8. Briefly, cells were fixed in 4% formaldehyde fixative at 37 °C for 15 min and permeabilized at 4 °C for 5 min. The samples were blocked with 1% BSA/PBS at 37  ͦC for 5 min and stained with the appropriate primary antibodies for osteopontin (OPN) and OCN (1:50 in 1% BSA/PBS, Autogen Bioclear). Actin fillaments were stained with phalloidin (1:100, Invitrogen). Two washes in 1 x PBS/0.5% Tween-20 (3 x 5 min at room temperature) followed and a secondary, biotin-conjugated antibody (1:50, horse monoclonal anti-mouse (IgG), Vector Laboratories) was added for 1 h at 37 °C. The samples were washed and streptavidin was added (1:50, Vector Laboratories) at 4 °C for 30 min, before the samples were given a final wash and mounted in Vectashield mounting medium (Vector Laboratories) containing DAPI to stain the nucleus. Visualisation was via a fluorescence microscope (Zeiss Axiovert 200M, 10x magnification, NA 0.5). For cell count osteoclasts were defined as cells greater than 30µm diameter, with greater than 3 nuclei and presence of actin ring. Files generated in JPEG or TIFF format.
Image analysis
For TRAP stain and immunofluorescence, comparisons of staining intensity between substrates was analysed by Image J software version 1.42q using the TRAP macro_intensity_sequence.txt file provided.
After analysis by Image J software, statistical analysis of TRAP histochemical staining and immunostaining for osteopontin and osteocalcin intensity was performed by the student’s t-test. 
Scanning Electron Microscopy (SEM) for eukaryotic cells
SEM was performed at days 3, 14 and 35 of co-culture on duplicate 2 h, 2.5 h and 3 h nanowire or polished control Ti substrates. The samples were fixed in 4% gluteraldehyde for 1 h at 4 oC and then washed in 0.2 M sodium cacodylate (pH 7.4). They were post fixed in 1% osmium tetroxide and washed once in sodium cacodylate. The samples were then immersed in a 1% (v/v) tannic acid/0.1 M sodium cacodylate solution for 1 h and then washed in 0.2 M sodium cacodylate before the dehydration steps through an incremental (70-100% v/v) alcohol series. A hexamethyldisiloxane step was conducted prior to sputter coating (20 nm gold/palladium) A Carl Zeiss Sigma Variable Pressure Analytical SEM with Oxford Microanalysis at the GU (ISAAC) Imaging Spectroscopy and Analysis Centre was used for sample visualisation.  Imaging conditions on data bar on images, KV and working distance. Also 30um aperture, 450pA (probe Current). Files generated in JPEG or TIFF format.

qRT-PCR
Total RNA was extracted from day 14 and 35 time point cultures using a Qiagen RNeasy Micro kit and protocol. To standardise for quantitative analysis, equal amounts of RNA from each sample were used for cDNA synthesis using the Qiagen QuantiTect RT-PCR kit and protocol. qRT-PCR was carried out using the Qiagen Quantifast SYBR Green kit and the reactions run in the 7500 Real Time PCR cycler from Applied Biosystems. Three biological (and 2 technical replicates from each) replicates were tested at each time point. Expression of test genes (OPN, OCN, SOX9, OPG, OSCAR, RANKL, interleukin 6 (IL-6), TRAP, tumour necrosis factor  (TNF) and cathepsin-K) was normalised against house keeping gene GapDH. The primer sequences (Table 1) for the genes were validated by dissociation curve/melt curve analysis. 
Table 1: PCR primer sequences
Target Gene	Forward Sequence	Reverse Sequence	
OSCAR	CCAGCTCTAGCGGGTATCTG	GACGGAGTGATGTCTGTGTGAC	
OPG	GAAGGGCGCTACCTTGAGAT	GCAAACTGTATTTCGCTCTGG	
RANKL	TGATTCATGTAGGAGAATTAAACAGG	GATGTGCTGTGATCCAACGA	
IL-6	GATGAGTACAAAAGTCCTGATCCA	CTGCAGCCACTGGTTCTGT	
TRAP	GGACTGAAGGGACTCCTGAAT	GGTCCCTGAGCCTTTATTCC	
TNF	CAGCCTCTTCTCCTTCCTGAT	GCCAGAGGGCTGATTAGAGA	
Cathepsin-K	GCCAGACAACAGATTTCCATC	CAGAGCAAAGCTCACCACAG	
GapDH	GTCAGTGGTGGACCTGACCT	ACCTGGTGCTCAGTGTAGCC	
OPN	AGCTGGATGACCAGAGTGCT	TGAAATTCATGGCTGTGGAA	
OCN	CAGCGAGGTAGTGAAGAGACC	TCTGGAGTTTATTTGGGAGCAG	
QPCR data analysis was performed using the 2(–DDCT) Method (doi:10.1006/meth.2001.1262, available online at http://www.idealibrary.com). 

1)	Using the Ct column data from the original data file, the average of the technical replicates from both house keeping and test gene were calculated.
2)	The house keeping ct average was subtracted from the test gene equivalent (In our case normalize to GapDH).
3)	The control group was selected and the average the biological replicates calculated.
4)	The control average was subtracted from each one of the test gene equivalent replicates (in our case normalized to Flat).
5)	The resulting values were converted using the 2(–DDCT) formula.

Data file of calculations provided in Excel format.
Statistical analysis of qPCR was performed using the Graphpad Prizm software version 4c by the non-parametric Kruskal-Wallis test and Dunn's Multiple Comparison Test.

Bacterial culture preparation 
P. aeruginosa PAO1 was grown aerobically for 16 h in 10 mL Luria-Bertani broth (LBB) in a 37 ºC shaker incubator set at 220 rpm. The bacterial suspension was then diluted in LBB to OD600 0.1 and further incubated until mid-exponential phase was reached. At this time bacterial cells were harvested by centrifugation (7 min,  5000 g), washed twice in 10 mM Tris-HCl buffer adjusted to pH 7 with hydrochloric acid, and suspended in Tris-HCl buffer to OD600 0.3 (approx. 107 cfu ml-1). 

Bacterial adhesion
All test and control surfaces were rinsed with absolute ethanol prior to bacterial adhesion experiments. The test surfaces and controls were then placed into a 12-well microtitre plate and submerged in 2 mL of bacterial suspension. Plates were incubated for 1 h at 37 ºC under static conditions. After incubation, surfaces were rinsed to remove non-adherent bacteria by passing back and forth five times in a Universal container containing Tris-HCl buffer, repeated three times in total. 

Live/Dead staining and fluorescence microscopy
Following rinsing, 1 ml of Live/Dead® BacLight™ bacterial viability stain (Invitrogen) was applied to the surfaces (as per manufacturers’ instructions) and incubated in the dark for 15 min at room temperature. Surfaces were then rinsed in Tris-HCl buffer as above to remove excess stain. Surfaces were maintained in 1 ml of Tris-HCl buffer, and bacterial adhesion and viability was visualised by confocal microscopy. Imaris software was used to calculate the number of cells with intact membranes (SYTO 9, green) and the number of cells with damaged membranes (propidium iodide, red) based on 5 images per surface. The average % damaged cells was determined by (no. of damaged cells / total no. of cells) * 100. All tests were carried out in triplicate. A 2 tailed homoscedastic Student’s t-test was performed using Microsoft Excel 2013 to compare data sets. If the p value was less than 0.05 then results were statistically significant.

Scanning Electron Microscopy (SEM) of bacterial cultures
Surfaces attached with P. aeruginosa were fixed by immersion in a 2.5% gluteraldehyde solution (Sigma Aldrich) dissolved in 0.1 M potassium phosphate buffer (Sigma Aldrich) for 2 h at room temperature. An alcohol dehydration stage was then performed by immersing the surfaces in 20%, 40%, 60%, 80% and 100% ethanol (Sigma Aldrich) for 10 mins each prior to 10 mins in hexamethyldisilazane (Sigma Aldrich). Surfaces were then air dried, mounted onto carbon stubs and sputtered with platinum for 30 seconds.  A Quanta 400FEI Scanning Electron Microscope (SEM) was used to visualise the surfaces.

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