This README.txt file was generated on 2022-10-20 by Maria Guillot-Ferriols ------------------- GENERAL INFORMATION ------------------- Title of Dataset: Piezoelectric 3D Platform Based on Poly(Vinylidene) Fluoride Microspheres for Osteogenic Differentiation of Mesenchymal Stem Cells_Data Author Information Principal Investigator: Maria Guillot-Ferriols; 1,2; maguifer@upv.es; https://orcid.org/0000-0003-1850-9094 Associate or Co-investigator: María Inmaculada García Briega; 1,2; inmagarciabriega@gmail.com; https://orcid.org/0000-0003-0811-2632 Laia Tolosa; 2,3; laiatolosa@hotmail.com; https://orcid.org/0000-0002-1740-0874 Carlos M. Costa; 4,5,6; cmscosta@fisica.uminho.pt; https://orcid.org/0000-0001-9266-3669 Senentxu Lanceros-Méndez; 4,5,7,8; senentxu.lanceros@bcmaterials.net; https://orcid.org/0000-0001-6791-7620 Gloria Gallego Ferrer; 1,2; ggallego@ter.upv.es; https://orcid.org/0000-0002-2428-0903 José Luís Gómez Ribelles; 1,2; jlgomez@ter.upv.es; https://orcid.org/0000-0001-9099-0885 Affiliations: 1- Centre for Biomaterials and Tissue Engineering (CBIT) Universitat Politècnica de València, 46022 Valencia, Spain 2- Biomedical Research Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 46022 Valencia, Spain 3- Experimental Hepatology Unit, Health Research Institute La Fe (IIS La Fe), Valencia, Spain 4- Centre of Physics, Universidade Do Minho, 4710-057 Braga, Portugal 5- Laboratory of Physics for Materials and Emergent Technologies, LapMET, University of Minho, 4710-057 Braga, Portugal 6- Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, 4710-057 Braga, Portugal 7- BCMaterials, Basque Centre for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain 8- IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain Date of data collection: From 2020-01-01 to 2021-09-30 Geographic location of data collection: Centro de Biomateriales e Ingeniería Tisular, Universitat Politècnica de València, Valencia, Spain (39.478006, -0.333463) Information about funding sources or sponsorship that supported the collection of the data: Spanish State Research Agency (AEI) through PID2019-106099RB-C41/AEI/10.13039/501100011033, PID2019-106099RB-C43/AEI/10.13039/501100011033 and PID2019-106000RB-C21 / AEI / 10.13039/501100011033 projects. Spanish Ministry of Science, Innovation and Universities through Grant BES-2017-080398 awarded to Maria Guillot-Ferriols. General description: The excel documents contain the crude numerical data of all the quantitative outputs of the article "Magnetically Activated Piezoelectric 3D Platform Based on Poly(Vinylidene) Fluoride Microspheres for Osteogenic Dif-ferentiation of Mesenchymal Stem Cells" published in October 2022 in the journal Gels (MDPI). The title of each file identifies which test the contained data corresponds to. The data is obtained in order to characterise the microspheres and cell behaviour in the 3D cell culture platform. Keywords: Mesenchymal stem cells; osteoblastogenesis; piezoelectricity; poly(vinylidene) fluoride; hydrogel. -------------------------- SHARING/ACCESS INFORMATION -------------------------- Open Access to data: Open Date end Embargo: non required Licenses/restrictions placed on the data, or limitations of reuse: Creative Commons (CC0, CC-BY, etc.) Citation for and links to publications that cite or use the data: not available yet Links/relationships to previous or related data sets: no other related data available Links to other publicly accessible locations of the data: do not exist -------------------- DATA & FILE OVERVIEW -------------------- File list: - Diameters_HistogramData: Obtained diameters of the microspheres. The diameters are expressed in microns after measuring them from electron microscopy images using ImageJ. - FTIR: It contains the results of the FTIR test conducted on both types of microspheres. These values are wavelengths and their corresponding transmittance. Calculations of b-phase percentage present in the samples are included. - DSC: Differential Scanning calorimetry data of PVDF and PVDF-CFO microspheres. Calculations of crystallinity degree. - VSM: Vibrational Sample Magnetometer measurements of PVDF-CFO microspheres. Calculations of CFO content in the samples from three different batches comparing it to pure CFO. - Live-Dead_INCELL: Results of the viability assessment of mesenchymal stem cells encapsulated in Gelatin, Gelatin-PVDF and Gelatin-PVDF-CFO hydrogels after 24h in culture. Results processed from images obtained after Hoechst-Propodium Iodide staining. - Proliferation_MTS: Results of the MTS proliferation test of mesenchymal stem cells after 2, 7, 14 and 21 days encapsulated in Gelatin, Gelatin-PVDF and Gelatin-PVDF-CFO hydrogels stimulated and non-stimulated. - RT-qPCR_GeneExpression: Gene expression of early and middle osteogenic markers in mesenchymal stem cells encapsulated in Gelatin-PVDF or Gelatin-PVDF-CFO hydrogels stimulated and non-stimulated after 7 and 14 days. DNA concentrations and Cp are reported for each gene and time point. Calculations of fold changes in mRNA expression are also reported. - ALP_Activity_Normalized_MTS: ALP activity determination for mesenchymal stem cells encapsulated in Gelatin-PVDF or Gelatin-PVDF-CFO hydrogels stimulated and non-stimulated after 7 and 14 days. Results of MTS proliferation tests for normalization at the same time points are also reported. Relationship between files: no needed Type of version of the dataset: Processed data in all files. Total size: 766 KB -------------------------- METHODOLOGICAL INFORMATION -------------------------- Description of methods used for collection/generation of data: - Diameters_HistogramData: Microspheres were morphologically characterised using a field emission scanning electron microscopy (FESEM) (AURIGA compact, Zeiss) with an accelerating voltage of 2 kV. Samples were coated with platinum following a standard sputtering protocol for 90 s (JFC 1100, JEOL). For observation of nanoparticle distribution, PVDF-CFO microspheres were cross-sectioned using a focused ion beam (FIB) device coupled to FESEM, and images were taken after sectioning. Microsphere diameter was assessed from FESEM images. At least 700 microspheres from three independent batches were measured using ImageJ software (National Institutes of Health, USA). - VSM Magnetic properties and nanoparticle content in the PVDF-CFO microspheres were determined using a Microsense 2 Tesla vibrational sample magnetometer (VSM). Magnetisation loops M(H) were evaluated up to ± 18 kOe, and pure CFO saturation magnetisation value (60 emu/g) was compared to the one obtained in the composite samples to obtain the effective filler content in the microspheres by means of Eq. 1: CFO wt %= (Saturation magnetization microspheres)/(Saturation magnetization pure CFO) x 100 (1) - FTIR Fourier transform infrared spectroscopy (FTIR) has proven to be an effective technique for determining the electroactive phase content of PVDF. Gregorio and Cestari described a method based on the identification of representative absorption bands at 840 cm−1 and 762 cm−1, which correspond to the β and α phase, respectively, and their quantification applying Eq. 2: F(β)= A_β/((K_β⁄(K_(α ) )) A_(α )+A_β ) (2) Assuming Lambert-Beer’s law Kα and Kβ are the characteristic absorption coefficients at the characteristic wavenumbers of the α and β-phases (762 and 840 cm−1, respectively). whose values are 6.1 × 104 and 7.7 × 104 cm2/mol, respectively. Aα and Aβ are the obtained absorbances at 762 and 840 cm−1, respectively, measured with an ALPHA FTIR spectrometer (Bruker) in ATR mode from 4000 to 400 cm−1 at a wavelength resolution of 4 cm−1. Measurements were taken from samples produced in three different batches. - DSC Samples were evaluated by differential scanning calorimetry (DSC) in a DSC 8000 (PerkinElmer). A mass of 2-4 mg of microspheres was encapsulated in aluminium pans and heated from 0 °C to 200 °C at a heating rate of 20 °C/min in a dry nitrogen atmosphere. The degree of crystallinity was calculated with the obtained data applying Eq. 3: Xc= 〖∆H〗_m/(w_(PVDF ) (x∆H_α+y∆H_β)) (3) where ∆Hm is the melting enthalpy of PVDF and PVDF-CFO microspheres measured by DSC and ΔHα and ΔHβ are the melting enthalpies of a 100 % crystalline sample in the α and β phases, whose values are 93.07 J/g and 103.4 J/g, respectively. wPVDF is the mass fraction of PVDF within the microspheres (provided by their magnetic properties), and x and y are the percentage of α and β phases present in the sample, obtained by FTIR measurements. - Live-Dead_INCELL After 24 hours, the viability of MSCs encapsulated with PVDF and PVDF-CFO microspheres were evaluated. Hoechst 3342 (1.5 ug/mL, Thermo Fisher) and propidium iodide (1.5 ug/mL, Sigma-Aldrich) were added to the cell culture medium and incubated for 20 minutes at 37 °C. After incubation with fluorescent probes, cells were imaged using the INCELL 6000 Analyzer system (GE Healthcare). Four randomised visual fields per well were analysed, and viability was determined using ImageJ software and applying Eq. 4: Viability (%)= (blue counts-red counts)/(blue counts) x 100 (4) -Proliferation_MTS The influence of piezoelectric stimulation on MSCs proliferation was assessed by analysing cell metabolic activity on days 2, 7, 14 and 21 under static (no applied stimuli) and dynamic (cell culture under magnetic stimulation) conditions. An alternating magnetic field (0-230 Oe) was provided using a homemade magnetic bioreactor placed inside the incubator, applying a 0.3 Hz frequency and a 10 mm magnet displacement under the 48-well plate. The stimulation program was divided into an active period of 16 h based on 5 minutes of magnetic stimulation and 25 minutes of resting time, followed by a non-active period of 8 h when no magnetic stimulation was applied. At different time points, hydrogels were transferred to a new culture plate. The basal medium was replaced for DMEM without phenol red (Sigma-Aldrich) containing the tetrazolium salt MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Biovision) at a working dilution of 1:10. Hydrogels were incubated for 1 hour at 37 °C. After that, the supernatant was transferred to a new plate and absorbance at 490 nm was read with a Victor3 microplate reader (PerkinElmer). Gelatin hydrogels without microspheres stimulated (S) and non-stimulated (NS) were used as controls. -RT-qPCR_GeneExpression Gel-PVDF and Gel-PVDF-CFO hydrogels were seeded, and after 24 h of culture in the basal medium, it was replaced by a commercial osteogenic differentiation medium (PromoCell). Stimulated samples were placed in the bioreactor. After 7 and 14 days of culture, hydrogels were digested with collagenase 993 U/mL (Collagenase from Clos-tridium Histolyticum, Sigma-Aldrich) in DPBS for 30 minutes at 37 °C. Qiazol lysis re-agent (Qiagen) and chloroform (Scharlab) were added with a ratio of 5:1 to purify nucleic acids. RNA was purified using an RNA extraction kit (RNeasy Micro Kit, Qiagen), and the obtained concentration was measured by spectrophotometer (Nanodrop ONE, Thermo Scientific). 300 ng of total RNA were reverse transcribed using the Superscript III reverse transcriptase (Invitrogen) and oligo dT primers (Invitrogen), following the manufacturer’s instructions. Real-time qPCR was performed using LightCycler 480 SYBR Green I Master (Roche) in a LightCycler 480 Instrument (Roche), and amplifications were performed for 40 cycles. For normalisation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used. Primer sequences were validated by dissociation curve/melt curve analysis. The relative changes in gene expression were calculated by E-method, applying Eq. 5: Fold difference = (Etarget) Ct(target) calibrator – Ct(target) sample / (Enormalizer) Ct(normalizer) calibrator – Ct(normalizer) sample (5) Where E is the efficiency of the target gene or the normaliser housekeeping gene GAPDH. Non-stimulated Gel-PVDF-CFO hydrogels were used as calibrators. The raw data were transferred using the LC480 conversion software (version 2014), and then PCR efficiency for each pair of primers was calculated by LineReg PCR (version 2021.1). -ALP_Activity_Normalized_MTS Hydrogels were kept in culture for 7 and 14 days, and alkaline phosphatase (ALP) activity was assessed using SensoLyte ® pNPP Alkaline Phosphatase Assay Kit (Anaspec). Briefly, hydrogels were digested by adding 200 uL of collagenase 993 U/mL in 1X Assay Buffer for 30 minutes at 37 °C. After digestion, 50 uL of Triton X-100 (Sigma-Aldrich) 1.2 % (v/v) in 1X Assay Buffer were added, and samples were incubated for 10 minutes at 4 °C in an orbital shaker. After that, samples were centrifuged at 4275 rpm for 10 minutes at 4 °C, and the supernatant was used to determine ALP activity following the kit’s manufacturer instructions. Acellular hydrogels were used as blanks. ALP activity was normalised against cell metabolic activity determined by MTS assay. Non-stimulated Gel-PVDF-CFO hydrogels were used as controls. Methods for processing the data: Cell culture experiments were performed, at least, in triplicates and a minimum of two replicates were used per technique. All results were expressed as mean ± standard deviation. Statistical analysis was performed on GraphPad Prism 9 (USA). Non-parametric Kruskal-Wallis and Dunn’s multiple comparison test were applied, and a 95 % confidence interval was set to accept significant inter-group differences (p-value < 0.05). Software- or Instrument-specific information needed to interpret the data, including software and hardware version numbers: - Graphpad 9 for statistical data analysis - ImageJ software for the analysis of microsphere diameter and viability assessment using INCELL images. - Excel software (version 16051.15629.20156.0 from Microsoft) for some data manipulation - Origin Pro 8 for FTIR, DSC and VSM data representation - LC480 conversion software (version 2014) for conversion of qPCR raw data and then PCR efficiency for each pair of primers was calculated by LineReg PCR (version 2021.1). - LightCycler 480 Instrument Software for qPCR analysis. Standards and calibration information, if appropriate: Environmental/experimental conditions: The temperature was always between 20-25ºC Describe any quality-assurance procedures performed on the data: -------------------------- DATA-SPECIFIC INFORMATION -------------------------- - Diameters_HistogramData Number of sheets and explanation: 1st sheet = Diameters of PVDF microspheres from 5 different batches 2nd sheet = Diameters of PVDF-CFO microspheres from 3 different batches Number of variables: 1 (Microsphere diameter) Number of cases/rows: Each column corresponds to the measured diameter of several microspheres (at least 100 rows per column) per batch. Variable list, defining any abbreviations, units of measure, codes or symbols used: Microsphere diameter (um) Specialized formats or other abbreviations used: um = micrometers PVDF = Poly(vinylidene) fluoride PVDF-CFO = Poly(vinylidene) fluoride - Cobalt Ferrite Oxide - FTIR Number of sheets and explanation: 1st sheet = PVDF microspheres FTIR measurement 2nd sheet = PVDF-CFO microspheres FTIR measurement Number of variables: 3 (wavenumber, absorbance and transmittance) Number of cases/rows: 1746/sample. We obtained the FTIR spectra for 3 different batches of microspheres. Variable list, defining any abbreviations, units of measure, codes or symbols used: Wavenumber (cm-1) Transmittance (a.u.) Absorbance (a.u.) Missing data codes: there is not missing data Specialized formats or other abbreviations used: a.u. = arbitrary units F(beta) = percentage of beta-phase - DSC Number of sheets and explanation: 1st sheet = % Xc (Calculations of crystalline content for PVDF and PVDF-CFO microspheres) 2nd sheet = raw data of DSC measurements and calculations of normalized heat flow Number of variables: 3 (temperature, heat flow, normalized heat flow) Number of cases/rows: 1200 rows per column (each column corresponds to temperature, heat flow and normalized heat flow for PVDF or PVDF-CFO microspheres) Variable list, defining any abbreviations, units of measure, codes or symbols used: Temperature (ºC) Heat Flow (mW) Normalized Heat Flow (J/gK) Missing data codes: there is not missing data Specialized formats or other abbreviations used: % Xc = percentage of crystalline content Tm = Melting temperature F(alfa) = alfa-phase percentage F (beta) = beta-phase percentage ∆Hm = melting enthalpy of PVDF and PVDF-CFO microspheres ΔHα = melting enthalpy of a 100 % alfa-phase sample ΔHβ = melting enthalpy of a 100 % beta-phase sample - VSM Number of sheets and explanation: 1st sheet = VSM measurements of PVDF-CFO microspheres 2nd sheet = CFO content calculations Number of variables: 2 (Magnetic field, magnetisation) Number of cases/rows: 617 rows per column. Each column corresponds to the measurement of the magnetic field, magnetisation or normalized magnetisation per sample mass (emu/g). The data is organized per batch. 3 different batches were measured. Variable list, defining any abbreviations, units of measure, codes or symbols used: Magnetic field (Oe) Magnetization (emu) Missing data codes:there is not missing data Specialized formats or other abbreviations used: Oe = oersted g = grams emu/g = emu/gram - Live_Dead_INCELL Number of sheets and explanation: 1st sheet = Numbers of live and dead cells and calculations of viability Number of variables: 2 (live and dead cells) Number of cases/rows: 4 rows per condition and biological replicate corresponding to 4 different analyzed zones of the hydrogel. Variable list, defining any abbreviations, units of measure, codes or symbols used: Alive cells Dead Cells Total number of cells Viability (%) Missing data codes: Specialized formats or other abbreviations used: R1, R2, R3 = Biological replicates 1, 2 and 3 for each condition Gel = Gelatin hydrogel Gel-PVDF = Gelatin hydrogel containing PVDF microspheres Gel-PVDF-CFO = Gelatin hydrogel containing PVDF-CFO microspheres - Proliferation_MTS Number of sheets and explanation: 1st sheet = Absorbance measurements and calculations of cell proliferation for different time points (2, 7, 14 and 21 days) for every condition (Gel, Gel-PVDF and Gel-PVDF-CFO) stimulated or non-stimulated Number of variables: 1 (Absorbance) Number of cases/rows: 3 rows per time point which correspond to Gel, Gel-PVDF and Gel-PVDF-CFO absorbance for technical and biological replicates. Variable list, defining any abbreviations, units of measure, codes or symbols used: Absorbance (a.u.) = arbitrary units Missing data codes: Specialized formats or other abbreviations used: R1, R2, R3 = biological replicates 1, 2 and 3 L1, L2, L3 = technical replicates for every biological replicate Gel = Gelatin hydrogel Gel-PVDF = Gelatin hydrogel containing PVDF microspheres Gel-PVDF-CFO = Gelatin hydrogel containing PVDF-CFO microspheres - RT-qPCR_GeneExpression Number of sheets and explanation: Sheets 1-10 = Raw data of qPCR analysis after processing in LightCycler 480 software for the different genes and time points analysed. Sheets 11 and 12 = Fold change in gene expression analysis at 7 and 14 days using the concentration data obtained in the previous sheets. Number of variables: 2 ( Cp and Concentration) Number of cases/rows: For every gene each row corresponds to a different biological and technical replicate for each sample tested. Variable list, defining any abbreviations, units of measure, codes or symbols used: Cp = threshold cycle Concetration Missing data codes: Specialized formats or other abbreviations used: ALP = Alkaline phosphatase COL I = Collagen type I RUNX = Runt-related transcription factor 2 GAPDH = Glyceraldehyde-3-phosphate dehydrogenase NS = Non-stimulated Gel-PVDF-CFO hydrogel PVDF-S = Stimulated Gel-PVDF hydrogel CFO-S = Stimulated Gel-PVDF-CFO hydrogel 7d = 7 days 14d = 14 days - ALP_Activity_NormalizedMTS Number of sheets and explanation: 1st sheet = Absorbance measurements at different time points for different conditions and calculations of ALP activity normalized by MTS. 2nd sheet = Absorbance measurements of MTS for normalization of different conditions at different time points. Number of variables: 1 (absorbance) Number of cases/rows: Rows and columns identified in the excel file. Variable list, defining any abbreviations, units of measure, codes or symbols used: Absorbance (a.u) = arbitrary units ALP mass / well (ng/well) ALP mass / unit of volume (ng/mL) Missing data codes: Specialized formats or other abbreviations used: ALP = Alkaline Phosphatase NS - CFO = Non-stimulated Gel-PVDF-CFO hydrogel S-PVDF = Stimulated Gel-PVDF hydrogel S-CFO = Stimulated Gel-PVDF-CFO hydrogel PVDF CTRL = acellular control of Gelatin-PVDF hydrogel CFO CTRL = acellular control of Gelatin-PVDF-CFO hydrogel 7d = 7 days 14d = 14 days R1, R2, R3 = biological replicates 1, 2 and 3 L1, L2, L3 = technical replicates for every biological replicate