1. Title:
Bone and cartilage differentiation of a single stem cell population driven by material interface.
2. Authors:
Hannah Donnelly1, Carol-Anne Smith1, Paula E Sweeten1, Nikolaj Gadegaard2, R.M. Dominic Meek3, Matteo D'Este4, Alvaro Mata5,6, David Eglin4, Matthew J Dalby1
Affiliations: 
1Centre for Cell Engineering, University of Glasgow, Glasgow, U.K.
2Division of Biomedical Engineering, University of Glasgow, Glasgow, U.K., 
3Department of Orthopaedics, Southern General Hospital, Glasgow, U.K.
4AO Research Institute Davos, Davos, Switzerland
5Institute of Bioengineering, Queen Mary University of London, London, U.K.
6School of Engineering and Materials Science, Queen Mary University of London, London, U.K.
3. Funder information:
This work was supported by 
AO Foundation grant S-12-01M. EPSRC project EP/K034898/1 
European Research Council Starting Grant STROFUNSCAFF
4. Dataset and readme information
Generation of FLAT and NSQ surfaces
Quartz slides of near-square (NSQ) topography (120nm pits in square arrangement, centre–centre spacing of 300 nm, with ±50 nm offset in pit placement in x and y axes 4,6) and glass coverslips of FLAT topography were used to create multiple polymer replicas by manual hot (80°C) embossing poly(ε-caprolactone) beads ((C6H10O2)n; PCL (Sigma Aldrich): typical disks 13 mm in diameter, suitable for cell culture in 24 well plates. PCL disks were treated for 30 seconds at MHz-range Radio Frequency (RF) in a plasma cleaner (PDC-002 Harrick Plasma) to remove organic contaminants and activate the surface to improve cell surface attachment, then sterilized in 70% ethanol for 30 minutes and two sequential 5 minute washes in cell culture media prior to cell seeding.

Synthesis of poly(N-isopropylacrylamide) hyaluronan derivative.
 The derivative was prepared as already reported.16 Briefly, amino-terminated poly(N-isopropylacrylamide) (PNIPAM-NH2) was synthesized by dissolving 10 g of N-isopropylacrylamide in 20 ml of dry N,N-dimethylformamide (DMF). Under nitrogen atmosphere, 15 mg of azobisisobutyronitrile and 30 mg of cysteamine hydrochloride were added, and the reaction was let to proceed for 6 hrs. The product was precipitated and washed with diethyl ether. The PNIPAM-NH2 Mw value was 40'300 gmol-1 as measured by gel permeation chromatography. HA sodium salt from Streptococcus equi (HANa) with Mw of 293'000 g.mol-1 and polydispersity PDI = 1.86 (Contipro Biotech.) was transformed in its tetrabutylammonium salt (HATBA) via cationic exchange. Then, 2.0 g of HATBA were dissolved in 200 ml of dry dimethyl sulfoxide at RT. Methanesulfonic acid and 1,1'-carbonyldiimidazole both equimolar to the repeating unit of HA were added and 3.7 g of PNIPAM-NH2 added and stirred at RT for 3 days. The solution was dialysed against demineralized water using regenerated cellulose dialysis tubes (MWCO 50 kDa) for 5 days and finally freeze dried. 1H NMR analysis was performed on a Bruker Avance AV-500 NMR spectrometer using deuterium oxide as solvent to assess the degree of grafting of pNIPAM-NH2 onto HA.

Reconstitution and rheology of thermoresponsive hyaluronan solution.
The hyaluronan derivative was reconstituted to 15% w:v in sterile phosphate buffered saline (PBS) and stored at 4oC for 24 hrs for complete dissolution. Rheological measurements were performed on an Anton Paar MCR-302 rheometer equipped with Peltier temperature control device and thermostatic hood. A 1° conical geometry of 25 mm diameter and 49 μm gap was used. For each sample an amplitude sweep was measured at 10 rad/s and 37.00 ± 0.03 °C. Storage moduli were measured as function of the temperature between 20 °C and 40 °C with a gradient of 1 °C/min at angular frequency of 10 rad/s and amplitude within the linear viscoelastic range. A thin layer of low-viscosity silicon oil was spread along the meniscus interface in order to avoid evaporation.

Cell isolation and culture.
MSCs were isolated from haematologically normal patients undergoing routine surgery as previously described.17 MSCs were cultured in growth media containing 86% DMEM (Sigma) supplemented with 10% Fetal Bovine Serum (FBS) (Sigma, UK), 2% penicillin streptomycin, 1% non-essential amino acids (Invitrogen, UK) and 1% 100mM sodium pyruvate (Life Technologies, UK) at 37°C with a 5% CO2 atmosphere. Media was changed every 3 days and cells passaged to passage 2 or 3. hMSCs were seeded on NQS topography PCL surfaces in 24 well plates at 1 x 104 cells per PCL disk (surface area of disk average 1.13 cm2), media as recipe above and incubated for 24 hours to allow for adherence. Then, 250 µL of 1 x 104 cells/ml of thermoresponsive hyaluronan composition at temperature around 10°C was added and allowed to flow on top of the PCL disk before incubation at 37°C for 10 – 15 minutes to allow gelation, resultant gel approximately 0.5 cm thickness. 13 mm glass coverslips sterilized in 70% ethanol were subsequently added on top to ensure that the hydrogel was always in contact with the underlying PCL disk. Note that MSCs from Promocell were used to generate Supplementary Figure 1.

Immunocytochemistry.
After 5 days of culture, cells were fixed (10 ml 37% formaldehyde, 2g sucrose in 90 ml PBS solution) for 15 minutes. Permeabilising buffer (10.3g sucrose, 0.292g NaCl, 0.06g MgCl2, 0.476g HEPES, 0.5 ml Triton X, in 100 ml of H2O, at pH 7.2) was then added for 15 minutes to control samples without hydrogel (-GEL), and for 2 hours to samples with hydrogel (+GEL). To block non-specific binding samples were incubated in 1% BSA/PBS for 15 min -GEL, and 1 hour +GEL. Primary antibodies (1:50 in 1% BSA/PBS) were added at 200 µL/well for 1 hour -GEL, and at 500 µl/well overnight (+GEL). Substrates were then washed three times in 0.5% Tween 20/PBS (5 minutes each –GEL, 20 minutes each +GEL). Corresponding secondary biotin-conjugated antibody (1:50 in 1% BSA/PBS) was added for 3 hours to –GEL and +GEL samples, followed by substrate washing as described above. FITC-conjugated streptavidin was added (1:50 in 1% BSA/PBS, Vector Laboratories) for 2 hours before samples were given a final wash. All immunostaining were carried out at 37°C with warmed solutions in order to maintain hydrogel integrity. Surfaces were mounted using mounting medium for fluorescence, with DAPI counterstain (Vector Laboratories, UK), and viewed by fluorescent microscopy (Zeiss Axiophot).  Digital images were captured in two fluorescent channels (x20 magnification) and saved for further processing. Primary antibodies presented in Table 1. Secondary antibodies: biotinylated monoclonal anti-mouse (IgG) raised in horse and fluorescein streptavidin (all Vector Laboratories, UK). 

Target Molecule	Figure	File Type	Location	
β3 Tubulin	1	.jpeg and .tiff	TUBULIN FIG 1>
FLAT – GEL FIG 1A
FLAT + GEL FIG 1B, 1C > GEL FIG 1C/PCL FIG 1B
NSQ – GEL FIG 1D
NSQ + GEL FIG 1E, 1F>GEL FIG 1F/PCL FIG 1E	
Phosphorylated RUNX2	5	.jpeg and .tiff	pRUNX2 FIG 5>
pRUNX2 FLAT – GEL FIG 5A
pRUNX2 FLAT + GEL FIG 5B, 5C > GEL FIG 5C/PCL FIG 5B
pRUNX2 NSQ – GEL FIG 5D
NSQ + GEL FIG 5E, 5F>GEL FIG 5F/PCL FIG 5E	
Vimentin	1	.jpeg and .tiff	VIMENTIN FIG 1>
FLAT – GEL FIG 1G
FLAT + GEL FIG 1H, 1I > GEL FIG 1I/PCL FIG 1H
NSQ – GEL FIG 1J
NSQ + GEL FIG 1K, 1L>GEL FIG 1L/PCL FIG 1K	
SOX9	5	.jpeg and .tiff	SOX9 FIG 5>
SOX9 FLAT – GEL FIG 5G
SOX9 FLAT + GEL FIG 5H, 5I > GEL FIG 5I/PCL FIG 5H
SOX9 NSQ – GEL FIG 5J
SOX9 NSQ + GEL FIG 5K, 5L>GEL FIG 5L/PCL FIG 5K	
Osteocalcin	6	.jpeg	OSTEOCALCIN FIG 6>
OCN FLAT – GEL FIG 6A
OCN FLAT + GEL FIG 6B
OCN NSQ – GEL FIG 6C
OSN NSQ + GEL FIG 6D	

Quantitative PCR.
Samples were harvested after 28 days of culture in triplicate by transferring hydrogels to a 2 ml Eppendorf tube and adding 1 ml of TRIZOL reagent and incubating at room temperature for 10 mins. Cells on PCL substrates were removed by trypsinization and cell pellet was added to corresponding hydrogel TRIZOL solution. Samples were stored at -80 °C until RNA isolation. RNA was isolated using RNAeasy micro kit according to manufacturer protocol (Qiagen, UK). RNA pellets were solubilized in RNase free water and assessed for concentration and purity with measured absorbance at 230 nm, 260 nm (nucleic acids) using a NanoDrop ND 100 spectrometer (Thermo Scientific). Reverse transcription was carried out on extracted RNA using an Omniscript Reverse Transcription kit (Qiagen, UK) according to manufacturer’s instructions, with Random Primers (Invitrogen, UK) and RNAsin (Promega, USA). Quantitative PCR was carried out using a qPCR detection system (model 7500, Applied Biosciences) by the SYBR green method. Expression of SOX9 (Table 2) were tested, and GAPDH expression was used as a reference gene to normalize all data. RQ (relative gene expression) values were automatically calculated by the delta delta CT method. Statistical analysis first determined that GAPDH did not vary under test conditions (one-way ANOVA;). Cycle threshold values were then converted from logarithmic to linear scale (2ΔΔ-CT) for further analysis.
Table 1. Primers used for qPCR
PRIMER	FORWARD	REVERSE	
GAPDH	TCAAGGCTGAGAACGGGAA	TGGGTGGCAGTGATGGCA	
SOX9	AGACAGCCCCCTATCGACTT	CGGCAGGTACTGGTCAAACT	

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: SOX9 PCR suppFIG3.xlsx. Data used to generate supplementary Figure 3. 

Von Kossa staining.
After 28 days of culture, cells were fixed for 15 minutes in 4% formaldehyde solution and stored in PBS overnight. 1 ml of 5% silver nitrate solution (5g silver nitrate, 100ml deionized H2O, kept in dark) was added to each sample well and exposed to U.V. light for 20 minutes. Samples were washed thrice in deionized water. 1 ml 5% sodium thiosulphate solution (5g sodium thiosulphate, 100ml deionized H2O, stored in dark) was added for 10 minutes, samples were washed as described above. 1 ml counterstain solution (0.1g nuclear fast red, 5g aluminium sulphate in 100ml deionized H2O, boiled for 5 minutes and filtered) was added for 3 minutes. Samples were washed as described above and finally rinsed in 70% ethanol. Digital images of PCL disks were captured and saved for further analysis. The whole procedure was carried out at 37°C to maintain gel integrity. The hydrogel was washed away during the staining protocol and therefore is not imaged.

Nuclear Area
Nuclear area was measured using imageJ software version J32. Scale was set and 20 nuclei per sample were measured and an average reading taken. Graph generated using Prism6 software, and stats generated via a one-way ANOVA with multiple comparisons.
Data file: N AREA fig 4.xlsx. Data was used to generate Figure 4. 

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