1	Title 

Nanovibrational Stimulation of Mesenchymal Stem Cells Induces Therapeutic Reactive Oxygen Species and Inflammation for Three- Dimensional Bone Tissue Engineering
2	Authors: 

Wich Orapiriyakul1,2, Penelope M. Tsimbouri1, Peter G. Childs3, Paul Campsie4, Julia Wells5, Marc Fernandez-Yague6, Karl Burgess7, K. Elizabeth Tanner3,8, Manlio Tassieri3, R.M. Dominic Meek9, Massimo Vassali3, Manus JP Biggs6, Manuel Salmerón-Sánchez3, Richard O.C. Oreffo5, Stuart Reid4, Matthew J Dalby1*.

1Centre for the Cellular Microenvironment, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
2Department of Orthopedics, Faculty of Medicine, Prince of Songkla University, Songkhla, 90110, Thailand.
3Centre for the Cellular Microenvironment, Division of Biomedical Engineering, School of Engineering, University of Glasgow, Glasgow G12 8LT, UK.
4SUPA Department of Biomedical Engineering, University of Strathclyde, Glasgow G1 1QE, UK.
5Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton SO16 6YD, UK.
6Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Galway, Ireland. 
7Glasgow Polyomics, College of Medical, Veterinary and Life Sciences, University of Glasgow, Switchback Rd, Bearsden, Glasgow G61 1BD, UK.
8School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London E1 4NS, UK.
9Department of Orthopedics, Queen Elizabeth II University Hospital, Glasgow G51 4TF, UK.

KEYWORDS: Mesenchymal stem cells, nanovibration, mechanotransduction, bioreactor, bone tissue engineering 

*corresponding author matthew.dalby@glasgow.ac.uk

3	
Funder information:
W.O. was supported by a scholarship from the Royal Thai Government and Faculty of medicine, Prince of Songkla University. The work was supported by grants to M.J.D. from BBSRC (BB/P00220X/1, BB/S018808/1) and EPSRC (EP/ N013905/1, EP/P001114/1) and and the Sir Bobby Charlton Foundation and to M.J.P.B from Science Foundation Ireland (16/BBSRC/3317).

4	Abstract:
ABSTRACT: There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs’  responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level infl ammation as well as to ROS- and infl ammation-balancing pathways.These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells’  mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaff old design are underpinned by a process that is free of inductive chemicals.

5	Dataset and readme information:
5.1	Scanning electron microscopy (SEM). 
Data Folder: 		Figure6 (6A)

The samples were prepared for SEM using the sputter coating technique. The samples were mounted onto SEM stubs using double sided conductive tape and silver paint. They were then coated with Gold/ Palladium (approximately 10-20 nm) using a SEM coating system (Q150T ES, Quorum, UK). The samples were viewed on a JEOL6400. SEM running at 10 kV and Tif image captured using Olympus Scandium Software. Porosity diameter was analysed by ImageJ software.

5.2	Hydrogels-CHSCs contraction measurement and time lapse microscopy.
Data Folder: 		Figure6 (6C, 6F, 6G)
FigureS2 (S2B)
 
In order to monitor the hydrogel or CHSCs contraction without NS, the percentage of the hydrogel contraction comparing to initial hydrogel or CHSCs surface area in top view. The hydrogel or CHSCs images were taken in top view every 3-4 days. The hydrogel or CHSCs surface areas in top view were measured and analysed by ImageJ software. To evaluate cell migration, CHSCs were cultured for 6 (pre-CHSCs contraction) and 10 days (post-CHSCs contraction). CHSCs were imaged using a 10x objective lens at 120 sec intervals for 24 hours at 37°C. Cell velocity and migration directions were analysed using imageJ plugin (manual tracking) and Chemotaxis and Migration tool (Ibidi). Data and calculation were provided in .xlsx files (Microsoft Excel) and .pzfx file (GraphPad Prism software).

5.3	Cryosection and immunostaining.
Data Folder:	 	Figure6 (6H)

After CHSCs were stimulated with NS, the samples were fixed in 4% formadehyde and infused in 30% sucrose in PBS for cryoprotection, overnight at 4 C. The samples were then transferred to embedding molds and O.C.T. (Tissue-Tek, Sakura) was aliquoted to cover the samples. The samples were then frozen in liquid nitrogen and kept at -80 C. The frozen samples were sliced into 60 mm thickness and attached onto adhesive slides (9597, Tissue-Tek, Sakura, Netherlands). Immunofluorescent staining was then carried out. Samples were rinsed with 1xPBS and fixed with fixative at 37°C for 15 minutes. After that, permeabilisation buffer was added and incubated at 4°C for 5 minutes. Samples were then blocked with 1% BSA in 1xPBS at 37°C for 5 minutes. Primary antibody (P-myosin light chain 2, cell signaling, 3671S, rabbit, 1:50) and rhodamine-phalloidin (Invitrogen, Thermo fisher, 1:500) diluted in 1% BSA in 1xPBS were added and incubated at 37°C for 1 hour. Samples were then washed with 1xPBS/0.5% Tween-20 for 3 times (5 minutes each). Biotinylated secondary antibody (anti-rabbit; Vector Laboratories, USA, 1:50) diluted in 1% BSA in 1xPBS was added and incubated at 37°C for 1 hour. Samples were washed again with 1xPBS/0.5% Tween for 3 times. Streptavidin-FITC (Vector Laboratories, USA, 1:50) diluted in 1% BSA in 1xPBS was incubated at 4°C for 30 minutes. Samples were washed for 3 times. A small drop of DAPI (4’,6-diamidino-2 phenylindole, Vectashield) was placed onto the samples and covered with coverslips. FITC/TRITC channel images were taken (Olympus, US) operated on Surveyor software version 9.0.1.4 (Objective Imaging, UK). Images were processed using ImageJ (Version 1.50g, USA) and Photoshop CS4 (Adobe, version11 extended, Ireland). 

5.4	Interferometric measurement. 
Data Folders: 	Figure1 (1B, 1C, 1D, 1E and 1F)
Figure6 (6D, 6E)

0.8 mg/ml or 1.8 mg/ml collagen hydrogels were prepared. The hydrogel nanovibration was measured by laser interferometric vibrometer (wavelength = 632.8 nm, CW power; 5mW; SIOS, Meβtechnik GmbH, Germany). The vibration distance was analysed using INFAS Vibro 1.8.4 software (SIOS, Meβtechnik GmbH, Germany) and recorded in .xls file. Data and calculation were provided in .xlsx files (Microsoft Excel) and .pzfx file (GraphPad Prism software).

5.5	Rheology. 
Data Folder:	 	FigureS2 (S2A)

To investigate viscoelastic property of the hydrogels, a modular compact rheometer (MCR 302, Anton Paar, Austria) with parallel plate method (25 mm of Parallel plate) was used. The strain sweep test was measured to evaluate the breaking point of the hydrogels. Following that, hydrogel viscoelasticity was assessed by frequency sweep test. Hydrogels were prepared 24 hours prior to study and transferred on the rheometer plate. At 23°C of controlled temperature, paralell plate was applied with a 0.01 N of normal force compression and 1.5 mm gap size. The strain sweep was measured from 0.01 to 1% (of 0.8 mg/ml hydrogel) and 0.1 to 10% (of 1.8 mg/ml hydrogel) at 10 rad/sec in order to select the strain which was in linear-viscoelastic regime. Consequently, the frequency sweep test was measured at 1% strain starting from 100-0.1 rad/sec. Data and calculation were provided in .xlsx files (Microsoft Excel) and .pzfx file (GraphPad Prism software)

5.6	AlamarBlue assay.
Data Folders: 	FigureS2 (S2C)

4x104 cells/ml of Stro1 selected MSCs in CHSCs and hydrogels were prepared and stimulated for 1 and 2 week. The previous culture media were removed. Samples were washed with warm 1xPBS. 10% (v/v) of AlamarBlue resazurin (Bio-Rad, Watford, UK) diluted in phenol-red free media (D5030, Sigma) was prepared and added into each sample. After incubated at 37°C and 5% CO2 for 5 hours, the supernatant containing the up-taken AlamarBlue was pipetted and transferred into 96 well plates. A microplate reader (Clariostar, BMG Labtech, Germany) was used to detect light absorbance at wavelengths of 570 nm and 600 nm. The percentage of AlamarBlue reduction was calculated as Tsimbouri et al, 2017 (1). Data and calculation were provided in .xlsx files (Microsoft Excel) and .pzfx file (GraphPad Prism software). 

5.7	Quantitative polymerase chain reaction with reverse transcription (qRT-PCR) 
Data Folders: 	Folder: Figure1 (1G, 1H)
Figure2 (2B, 2C)
Figure4 (4E)
Figure5 (5A, 5B, 5C)
Figure7 (7A, 7B, 7E)
FigureS5

To assess gene expression changes, hydrogels or CHSCs were prepared for quantitative RT-PCR (qRT-PCR). Real time PCR was carried out (7500 Real Time PCR system, Applied Biosystem, USA) and quantified by 2-∆∆CT method (2). Data and calculation were provided in .xlsx files (Microsoft Excel) and .pzfx file (GraphPad Prism software).

Folder: Figure1 (1G)
4x104 cells/ml of MSCs (2 donors) were cultured in hydrogels. qRT-PCR at day 9 compared between with (N30 and N90) and without NS were carried out. Osteogenic markers were tested.

Folder: Figure1 (1H), Figure5 (5C)
4x104 cells/ml of MSCs (3 donors) were cultured in hydrogels. Temporal study of qRT-PCR at day 7, 14 and 21 were performed to compare N90 with non-stimulated group (control). Osteogenic (1H) and inflammatory (5C) markers were tested.

Folder: Figure2 (2B, 2C)
4x104 cells/ml of MSCs (2 donors) were cultured in hydrogels. qRT-PCR at day 9 compared between with (N30 and N90) and without NS were carried out. Osteogenic-related pathways markers were tested. 

Folder: Figure2 (2D) and Figure 4 (4E)
4x104 cells/ml of MSCs in hydrogels were cultured with ROCK (2D) and ROS (4E) inhibitor. qRT-PCR at day 9 was performed to compare NS (N30 and N90) with non-stimulated group (control). RUNX2 and OSX were studied.

Folder: Figure5 (5A)
4x104 cells/ml of MSCs were cultured in hydrogels. qRT-PCR at day 9 compared between with N90 and non-stimulated group was carried out. Inflammatory markers were investigated.

Folder: Figure5 (5B), Figure S5
4x104 cells/ml of MSCs in hydrogels were cultured with NFκB inhibitors. qRT-PCR at day 9 was carried out to compare NS (N30 and N90) with non-stimulated group (control). RUNX2, OSX and OPN were studied. Effect of dependent pathways were calculated.

Folder: Figure7 (7A, 7B)
4x104 cells/ml of MSCs (3 donors) were cultured in CHSCs. Temporal study of qRT-PCR at day 7, 14 and 21 were performed to compare NS (N30 and N90) with non-stimulated group (control). Osteogenic (7A) and inflammatory (7B) markers were tested.

Folder: Figure7 (7E)
4x, 10x and 20x 104 cells/ml of MSCs in CHSCs were prepared and stimulated with N30 and N90 for 1 and 2 weeks. qRT-PCR was studied at week 2 and 4. Osteogenic markers were tested.

5.8	Protein antibody microarrays. 
Data Folder:	 	Figure2 (2A)

4x104 cells/ml of Stro1 selected MSCs in 1.8 mg/ml collagen hydrogels were stimulated with N30 and N90. After stimulation for 1 and 2 weeks, samples were processed. Data was analysed by Microsoft Excels. Heatmaps were generated by Hierarchical Clustering Explorer v3.0 (http://www.cs.umd.edu/hcil/hce/hce3.html). Data and calculation were provided in .xlsx files (Microsoft Excel).

5.9	Metabolomics. 
Data Folders: 	Figure3 (3A, 3B, 3C, 3D)
Figure4 (4A, 4B, 4C, 4F)
FigureS3, S4

Stro1-selected MSCs seeded with 4 x104 cells/ml density in 1.8 mg/ml collagen hydrogels were stimulated with N30 and N90. After 1 and 2 weeks NS the gels were homogenized on ice and metabolites were then extracted using a Chloroform/Methanol/ water (1:3:1 ratio) extraction buffer. Samples were agitated on a shaker at 4C for 1 hour and in turn centrifuged at 13,000xg at 4 C for 5 minutes. Supernatants were transferred to and analysed by the Glasgow Polyomics team. Hydrophilic interaction liquid chromatography-mass spectrometry was performed (Dionex, UltiMate 3000 RSCL system, Thermo Fisher Scientific, Hemel Hempstead, UK) using a ZIC-pHILIC column (150 mm x 4.6 mm, 5 m. column, Merck Sequant). The datasets were processed using XCMS (peak picking), MzMatch (filter and grouping), IDEOM (post processing filtering and identification). Metaboanalyst was used to generate heatmaps and PCA analysis. KEGG database and Ingenuity Pathway Analysis (IPA) software were used for metabolomic pathway analysis. 

5.10	Reactive oxygen species measurement. 
Data Folder:	 	Figure4 (4D)

Stro1-selected MSCs seeded with 4 x104 cells/ml density in hydrogels (3 donors) were stimulated with N30 and N90 for 7 days. The samples were incubated in 2.5 mg/ml collagenase for an hour and were then centrifuged at 200xg for 4 minutes. Following that, the cell pellets were incubated for an hour in 2 uM 2’,7’- dichlorodihydrogen-fluorescein diacetate (H2DCF-DA, Invitrogen) in phenol red free media (Sigma, D5030). In the positive control group, 500 uM hydrogen peroxide was added. After incubation, the samples were then centrifuged and resuspended in 250 ul of flow cytometry buffer (2% FBS, 2 mM EDTA in 1xPBS) and transferred to 96 well plates. Resuspended MSCs in 96 well plates were incubated for 30 minutes. Signal of H2DCF-DA fluorescein was detected by using flow cytometry at 492-295 nm of excitation and 517-527 nm of emission. Data and calculation were provided in .xlsx files (Microsoft Excel) and .pzfx file (GraphPad Prism software)

5.11	ELISA of interleukin-1β. 
Data Folder:	 	Figure5 (5D)

Stro1-selected MSCs seeded with 4 x104 cells/ml density in 1.8 mg/ml collagen hydrogels were nanostimulated for 7 days. Hydrogels were digested with collagenase (Sigma-Aldrich, UK). Protein was extracted using RIPA lysis buffer containing phosphatase and protease inhibitors. Total protein concentration was quantified using BCA kits (Pierce, ThermoFisher). Human IL-1β kits (DY201-05, R&D systems) was used. Working concentration of reagents (Human IL-1 capture antibody; 840168, Human IL-1 detection Antibody; 840169, Human IL-1 standard; 840170, Streptavidin-HRP; 893975) was prepared as Certificate of Analysis (COA) sheet. To coat the captured antibody onto ELISA plate, 100 μl captured antibody was added into 96-well plate and incubated overnight at 4°C. A captured antibodies coated plate was then washed with washing buffer (0.05% Tween 20 in 1xPBS (pH 7.4) for 3 times using multichannel pipettes. 300 μl of reagent diluent (Reagent diluent concentrate 2, DY995, R&D systems) was added and incubated for an hour to block the coated plate. 100 l samples and standard in reagent diluent (Human IL-1, 840170) were added and incubated for 2 hours at room temperature. Samples were washed 3 times with washing buffer. 100 l of streptavidin-HRP was added and incubated for 30 minutes at room temperature. Samples were washed 3 times. 100 l substrate solution (1:1 of colour reagent A; H2O2 and colour reagent B; Tetramethylbenzidine) was added and incubated for 20 minutes. 50 l stop solution (2NH2SO4, DY994, R&D systems) was added. Microplate reader was used to determine the optical density at 450 nm and 570 nm. Data and calculation were provided in .xlsx files (Microsoft Excel) and .pzfx file (GraphPad Prism software)

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. paper link https://pubs.acs.org/doi/10.1021/acsnano.0c03130 .

References

1.	Tsimbouri P.M. CPG, Pemberton G.D., Yang J, Jayawarna V., Orapiriyakul W., Burgess K., Gonzalez-Garcia C., Thomas D., Giraldo C.V., Biggs M.P., Curtis A.G., Salmeron-Sanchez M., Reid S., Dalby M.J. 3D osteogenesis of mesenchymal stem cells stimulated by nanovibrational bioreactor in vitro. Nature Biomedical engineering. 2017;1:758-70.
2.	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.