1. Title:
Stimulation of 3D osteogenesis by mesenchymal stem cells via a nanovibrational bioreactor.
2. Authors:
Penelope M. Tsimbouri1†, Peter G. Childs2,4†, Gabriel D. Pemberton1†, Jingli Yang1, Vineetha Jayawarna1, Wich Oripiriyakul1, Karl Burgess3, Cristina Gonzalez-Garcia4, Gavin Blackburn3, Dilip Thomas5, Catalina Vallejo-Giraldo5, Manus J.P. Biggs5, Adam S.G. Curtis1, Manuel Salmeron-Sanchez4, Stuart Reid2,6*, Matthew J. Dalby1* ‬‬‬‬
1Centre for Cell Engineering, Institute for Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
2SUPA, Institute of Thin Films, Sensors and Imaging, University of the West of Scotland, Paisley PA1 2BE, UK.
3Glasgow Polyomics facility, College of Medical, Veterinary and Life Sciences, University of Glasgow, Wolfson Wohl Cancer Research Centre, Garscube Campus, Bearsden, G61 1QH, UK.
4Microenvironments for Medicine, Division of Biomedical Engineering, School of Engineering, College of Science and Engineering, University of Glasgow, Glasgow, G12 8QQ, UK.
5Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Ireland.
6Now at SUPA, Department of Biomedical Engineering, Wolfson Centre, Strathclyde University, Glasgow, G4 0NW, UK.

†these authors contributed equally to this work
*corresponding authors Stuart.Reid@strath.ac.uk and matthew.dalby@glasgow.ac.uk
3. Funder information:
This work was supported by grants to Reid and Dalby from BB/N012690/1, BB/P00220X/1, EP/N013905/1, EP/N012631/1, EP/P001114/1 from BBSRC, BBSRC/SFI and EPSRC and a programme grant from Find a Better Way. Biggs is funded by SFI grant nos. 11/SIRG/B2135 and 13/RC/2073.

4. Dataset and readme information
Data folders
1. Finite element modelling (ANSYS) folder Fig 1 and 2. Simulated models of a Corning 6-well plate, the bioreactor top plate and the collagen gel were developed in ANSYS finite element software (ANSYS, Canonsburg, PA). Models were developed in ANSYS Workbench v14.0 with analysis involving use of the Harmonic Response package to provide values for deformation at user defined frequencies.  
The culture plate component was modelled with a polystyrene dish bonded to six ferrite magnets via a 0.2 mm layer of epoxy. A similar method was used to model the top plate with materials bonded with a 0.1 mm epoxy bond. Epoxy bonds were also added at the attachment points of the piezo actuators. For this analysis, a vibration frequency of 1000 Hz with 30 nm amplitude was applied to the models to compare amplitude consistency. Throughout all the analysis, mesh relevance was set at 0 giving a medium mesh density.
For studies of the collagen gel, the model incorporated the polystyrene culture ware (one well of a 6-well plate, and bonding to the vibration source through a 0.1 mm epoxy layer. Harmonic response analysis was used to study the frequency dependant effects of deformation of the gel construct including meniscus curvature at the edge (modelled using the linear shear moduli and assumption of incompressibility) along with estimates of the levels of shear stress within the construct. Vibration condition was set in the range of 500 to 5000 Hz at 20 nm to encompass the experimental value of 1000 Hz. Harmonic response analysis is strictly linear so accuracy would be limited to the linear range of the material however the calculated shear stress falls far below those measured through rheology. Meshing size was set to coarse to reduce computational time. Connections between the gel and the culture ware were set to bonded (assuming that the gel adheres to the culture ware) and constrained using multi point constraints (MPC) to ensure that the mesh was constrained to the container. The additional impact of gravity was incorporated through an inertial acceleration of 9.81 ms-1. Fig 1 and 2.

2. Data Folder: Interferometry 2.5 & 5 mL comparison. 3D MSC cell cultures in collagen gel. Assessment of vibration amplitude was carried out using laser interferometry. A USB interferometer (Model SP-S, SIOS Meßtechnik GmbH, Ilmenau, Germany) was used. Xls files of Interferometry results of 2.5 and 5ml collagen gels during nanokicking. Data was used to generate Figure 2h-I and Supplementary Figure 1. 

3. Data Folder: Rheology. Measurements were taken using a Malvern Instruments_Kinexus Pro - ID No 1001270 (University of Strathclyde Department of P&A Chemistry). Xls file of 3D collagen rheology measurements in the presence or absence of cells in the collagen gels. The data was used to generate Figure 2a-c and Supplementary Table 1.
Assessment of the physio‑elastic properties of the collagen matrix was performed by carrying out dynamic frequency sweep experimentation. These frequency sweeps were performed by using a strain-controlled rheometer (Kinexus rotational rheometer system- Malvern). The parallel-plate used to perform the frequency sweep had a 20 mm diameter with a 0.25 mm gap. An integrated thermostat mechanism was used to maintain the sample temperature at 25°C. In order to keep the sample hydrated and prevent drying out through evaporation the internal atmosphere was kept saturated through the use of a solvent trap.  An amplitude sweep was carried out and this confirmed no variation in elastic modulus (G′) and viscous modulus (G″). The amplitude sweep was performed up to a strain of 1% and these measurements were taken in a linear viscoelastic regime. The collagen matrix dynamic shear modulus was measured as a frequency function with a frequency range from 1 to 100 Hz. To ensure reproducibility all measurements per sample were performed in triplicates.

4. Data Folders ICW: Protein expression changes in nanokicked vs control samples or in the presence and absence of ion channel inhibitors were assessed by Incell western.
Subfolder: ICW Fig 1g. The quantitative spectroscopic analysis was carried out using the LI‑COR Odyssey Sa and all dyes and antibodies were sourced from LI‑COR corporate. Incell western data is in .txt  (intensity readings). The analysed data is provided in a .xls file which was used to generate Fig 1g.
Subfolder: 2DERK ICW data Fig 1h. The quantitative spectroscopic analysis was carried out using the LI‑COR Odyssey Sa and all dyes and antibodies were sourced from LI‑COR corporate. Incell western data images are in .png format. The analysed data is provided in  .xls files which were used to generate Fig 1h.
Subfolder: 21days OCN suplFig4. The quantitative spectroscopic analysis was carried out using the LI‑COR Odyssey Sa and all dyes and antibodies were sourced from LI‑COR corporate. Incell western data is in .txt  (intensity readings) and .tif file (scan images) formats. The analysed data is provided in a. .xls file which was used to generate Supplementary Fig 4, for OCN osteogenic marker expression.

5. Data Folder: 2D and 3D QPCR. Following different times points of stimulation, in the presence or absence of different inhibitors, in the bioreactor the MSCs were assessed by qRT‑PCR. PCR was quantified using the 2-∆∆Ct method and amplification was carried out using the 7500 Real Time PCR system. Unstimulated samples also cultured in a 3D collagen matrix (samples not nanokicked) were used as negative controls for comparison of osteogenic transcription expression.
-Sub Folder: 2D QPCR. MSCs were cultureD in 2D cultures with or without nanokicking. RNAs were extracted and reversed transcribed to cDNA. Osteogenic markers were tested by QPCR. Xls files of QPCR analysed data in excel used to generate Figure 1f.
- Sub Folder: 3D QPCRs. MSCs were cultures in 3D cultures with or without nanokicking in the presence or absence of cytoskeletal inhibitors for Rock and myosin (Blebbistatin) or TRPV1 and PKC inhibitors. RNAs were extracted and reversed transcribed to cDNA. Ion channel and Osteogenic markers were tested by QPCR. 
Folder:  day5 3d QPCRs fig 4b-h, contains .xls files for Fig 4b-h 
Folder: 3D QPCR Fig 3a and 4a, contains .xls files  for Fig 3a and 4a
.xls file: 3dgel qpcr nanokick day14 suppl Fig6
.xls file: 3dgelTRPPKCqpcrd5 Fig7

6. Data Folders: Von Kossa Stain. Von Kossa Stain_4weeks. Bone mineralisation in 3D through nanokicking. .Tif files of von Kossa staining performed after 28 days of nanokicking at 1000 Hz in a collagen matrix – gels used to generate Figure 3b. A .xls file contains the Von Kossa intensity data calculations used to generate Figure 3b graphs. Also .xls file 3DpaperVonkossa6week.

7. Data Folder: Raman Spectroscopy Raw Data Files. Files in .pptx format contain the Data that was used to generate Figure 3f and Suppl. Table 2. A Renishaw InVia Raman spectrometer with a 785 nm line focus laser was used to perform near IR spectroscopic scans. A light microscope (Leica) x 20 magnification was used to determine regions of interest and mineral deposition for assessment.  

8.  Data Folder: MicroCT: files in .bmp format used to generate Figure 3e and g. MSCs were nanokicked in 3D at 1000 Hz in a collagen gel extracellular matrix for 35 days the samples were fixed using 10% formaldehyde and transferred into PBS and subsequently imaged using µCT. The µCT system used was a Skyscan 1172 (Orthopaedic engineering department University of Edinburgh S/N=08F01110). The data was reconstructed using Skyscan reconstruction software and scanning was performed with a 360° rotation (Resolution /pixel size = 2.9278 µm, Source Voltage = 58 kV, Source Current = 171 µA, Exposure = 1178 ms).

9. Western blots. Following the required period of stimulation within the bioreactor (2 or 5 days experiments) MSC protein expression was assessed using western blotting. Total protein was measured using the Revert total protein stain (Li-COR) solution and used as loading control to normalize the samples where necessary. The quantitative spectroscopic analysis was carried out using the LI‑COR Odyssey Sa and all dyes and secondary antibodies were sourced from LI‑COR corporate. File .ppt 3DNanokick paper western images includes images used for Fig 5a and 5b, suppl. Fig 10 and 11 and Western blots data Fig5a and b.xls analysed data.

10. Metabolomics. MSCs were stimulated in 3D collagen gels (2mg/ml) at a density of 4×104 cells/ml of gel (2.5 ml) for 5 days or 2 days for the transient ion channel and PKC inhibition studies. Non-stimulated samples were used as controls. The metabolites were extracted using a 1:3:1 chloroform/methanol/water extraction buffer and placed on a shaking platform for 1 h at 4°C. The sample supernatant was transferred into 1.5 ml tubes and centrifuged for 3 min at 13,000g at 4°C. The samples were used for hydrophilic interaction liquid chromatography‐mass spectrometry analysis (UltiMate 3000 RSLC, Thermo Fisher, with a 150 x 4.6 mm ZIC-pHILIC column running at 300 uL/min and Orbitrap Exactive, Thermo Fisher, respectively). After Nanodrop (Thermo Fisher) measurement, total protein content was found identical in all samples, thus no further standardization was necessary. A standard pipeline, consisting of XCMS (peak picking), MzMatch (filtering and grouping), and IDEOM (further filtering, post‐processing and identification) was used to process the raw mass spectrometry data. Identified core metabolites were validated against a panel of unambiguous standards by mass and retention time. Further putative identifications were allotted by mass and predicted retention time. Means and standard errors of the mean were generated for every group of picked peaks and the resulting metabolomics data were uploaded to Ingenuity pathway analysis software for metabolite pathway analysis. 
Data folder: 3D Metabolomics day 5 Fig6, suppl. fig, contains MzXML files for Fig 6 and suppl Fig 12, 13 and 14.
Data Folder: QE_Matt_Dalby_pHilic_NKvsC MxXML files for suppl. Fig 7,8 and 9.

11. Alamar Blue. The Alamar blue assay was performed after 1 and 2 weeks of stimulation and compared to non-stimulated controls. 10% (v/v) of AlamarBlue® resazurin (Bio-Rad, Watford, UK) diluted in phenol-red free media (D5030, Sigma) was added to each gel. After further incubation for five hours at 37°C, 5% CO2, the culture supernatant was transferred into 96 well plates to measure the up-taken Alamar blue indicating cell metabolism. A microplate reader (Clariostar, BMG Labtech, Germany) was used to detect light absorbance at wavelengths of 570 nm and 600 nm. The percentage of Alamar blue reduction was calculated. Data provided in .xls files in folder Alamar Blue Raw data Suppl Fig 3e.

12. AFM. The fibrillar conformation of type I collagen gel matrices was assessed via AFM in AC mode (Nanowizard 3 Bioscience AFM, JPK Instruments AG, Berlin, Germany). Collagen gel (2 mg/ml) was gently dried with a nitrogen flow before evaluation in the AFM working in air. A pyramidal silicon nitride tip, with a cantilever spring constant of 3 N/m and a resonance frequency of 75 kHz (MPP-21120, Bruker), was used to scan the collagen matrix. Height and phase images were simultaneously recorded. The influence of cell seeding density in the local mechanical properties of the collagen gel matrices was evaluated via force spectroscopy. Silicon tipless cantilevers (Arrow TL1, Nanoworld) functionalised with 20 µm silicon beads were used to perform the measurements. Force-distance curves were obtained, after calibration of tip sensitivity and spring constant, with a set-point of 10 nN, a zeta length of 10 μm, a constant duration of 1 s, and at room temperature in liquid environment. Analysis was performed using the JPK processing software (v4.3.21), and force curves were fitted with a Hertz model. Images .tiff and data . xls measurements are provided in data folder:AFM suppl Fig1.

13. Livedead suppl Fig3a-d. 4×104 MCSs/ml of gel (2.5 ml) were seeded in each well of a 6 well plate for stimulated and control groups. MSCs were nanovibrated at 1,000 Hz for 2 weeks. Upon completion, the culture medium was removed and the samples were washed 3x in warm 1xPBS. 4 mM of calcein AM (Life Technologies, Carlsbad, USA) and 2mM of ethidium homodimer-1 (Life Technologies) diluted 1:1000 in phenol-red free media (D5030, Sigma) was added in each well. The samples were then re-incubated at 37°C, 5% CO2 for 2 hours. 10x magnification microscopic images were taken (Olympus, Pennsylvania, US) operated on Surveyor software version 9.0.1.4 (Objective Imaging, Cambridge, UK). FITC/TRITC channel images were stitched and processed using ImageJ (Version 1.50g, National Institutes of Health, USA) and Photoshop CS4 (version 11 extended, Adobe, Ireland). .jpg images.
5. The date the dataset can be made publicly would be once the paper is out on publication

6. No restrictions
7. No preference
8. DOI: 10.1038/s41551-017-0127-4