Summary of Study ST004276

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002702. The data can be accessed directly via it's Project DOI: 10.21228/M8D27Z This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST004276
Study Title(NMR data) Global metabolomics identifies new extracellular biomarkers of nanovibration-driven mesenchymal stem cells osteodifferentiation
Study SummaryThis study characterizes the metabolic adaptations of mesenchymal stem cells (MSCs) to chemical-free nanovibration (nanokicking, NK)-induced osteodifferentiation. Upon reaching the cell numbers required for metabolomic analysis (1 × 10⁶ cells per sample for NMR), day 0 samples were collected in triplicate prior to stimulation. The remaining T150 flasks were divided into unstimulated controls (CTR) and NK-stimulated groups. The NK bioreactor employs the reverse piezoelectric effect to generate 30 nm vertical displacements at a frequency of 1000 Hz, providing a purely mechanical stimulus without chemical induction. For intracellular metabolomics, CTR and NK cells were collected in triplicate on days 0, 7, and 21. For extracellular metabolomics, culture media samples were collected in triplicate on days 3, 6, 7, 10, 13, 17, 20, and 21, along with blank media controls. Through the integration of conventional osteogenic gene markers with global metabolomics and lipidomics analyses, our findings reveal that NK stimulation promotes a slow-paced osteodifferentiation process characterized by subtle, partially reversible intracellular changes and pronounced, largely irreversible extracellular alterations, highlighting the metabolic reprogramming underlying mechanically induced osteogenesis.
Institute
University of Aveiro
DepartmentChemistry
LaboratoryCICECO - Aveiro Institute of Materials
Last NameGil
First NameAna M.
AddressCICECO, Departamento de química, Campus de Santiago, Aveiro, Portugal
Emailagil@ua.pt
Phone+351234370707
Submit Date2025-09-11
Raw Data AvailableYes
Raw Data File Type(s)fid
Analysis Type DetailNMR
Release Date2025-10-18
Release Version1
Ana M. Gil Ana M. Gil
https://dx.doi.org/10.21228/M8D27Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002702
Project DOI:doi: 10.21228/M8D27Z
Project Title:Global metabolomics identifies new extracellular biomarkers of nanovibration-driven mesenchymal stem cells osteodifferentiation
Project Summary:Bone-related conditions are a leading cause of disability and rising healthcare costs, prompting interest in tissue engineering solutions using mesenchymal stem cells (MSC). As part of an effort to eliminate synthetic osteogenic compounds, this study characterizes the metabolic adaptations of MSC to chemical-free nanovibration (or nanokicking, NK)-induced osteodifferentiation. Through articulation of conventional gene markers and a global metabolomics/lipidomics strategy, our findings indicate successful slow-paced osteodifferentiation, expressed by subtle and partially reversible intracellular changes, and pronounced, largely irreversible, extracellular alterations.
Institute:University of Aveiro
Department:Chemistry
Laboratory:CICECO - Aveiro Institute of Materials
Last Name:Gil
First Name:Ana M.
Address:CICECO, Departamento de química, Campus de Santiago, Aveiro, Portugal
Email:agil@ua.pt
Phone:+351 234 370 707
Funding Source:This work was developed within the scope of the CICECO-Aveiro Institute of Materials, UIDB/50011/2020 project (doi: 10.54499/UIDB/50011/2020), UIDP/50011/2020 (doi: 10.54499/UIDP/ 50011/2020) and LA/P/0006/2020 (doi:10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC). We acknowledge funds from the Foundation for Science and Technology through the BetterBone project (2022.04286.PTDC, doi: 10.54499/2022.04286.PTDC), the Portuguese National NMR Network (RNRMN), supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC); and FCT/SPQ PhD grant (DSCB) (SFRH/BD/150655/2020, doi: 10.54499/SFRH/BD/150655/2020). EPSRC grant EP/P001114/1.

Subject:

Subject ID:SU004429
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:29
Weight Or Weight Range:Healthy
Gender:Female
Cell Strain Details:Human adipose tissue-derived stem cells
Cell Passage Number:3

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment
SA497998NK_D13_S1hASCs Osteogenic conditions
SA497999NK_D3_S3hASCs Osteogenic conditions
SA498000NK_D6_S1hASCs Osteogenic conditions
SA498001NK_D6_S2hASCs Osteogenic conditions
SA498002NK_D6_S3hASCs Osteogenic conditions
SA498003NK_D7_S1hASCs Osteogenic conditions
SA498004NK_D7_S2hASCs Osteogenic conditions
SA498005NK_D7_S3hASCs Osteogenic conditions
SA498006NK_D10_S1hASCs Osteogenic conditions
SA498007NK_D10_S2hASCs Osteogenic conditions
SA498008NK_D10_S3hASCs Osteogenic conditions
SA498009NK_D13_S2hASCs Osteogenic conditions
SA498010NK_D3_S1hASCs Osteogenic conditions
SA498011NK_D13_S3hASCs Osteogenic conditions
SA498012NK_D17_S1hASCs Osteogenic conditions
SA498013NK_D17_S2hASCs Osteogenic conditions
SA498014NK_D17_S3hASCs Osteogenic conditions
SA498015NK_D20_S1hASCs Osteogenic conditions
SA498016NK_D20_S2hASCs Osteogenic conditions
SA498017NK_D20_S3hASCs Osteogenic conditions
SA498018NK_D21_S1hASCs Osteogenic conditions
SA498019NK_D21_S2hASCs Osteogenic conditions
SA498020NK_D21_S3hASCs Osteogenic conditions
SA498021NK_D3_S2hASCs Osteogenic conditions
SA498022CTR_D0_S1hASCs Proliferation conditions
SA498023CTR_D0_S2hASCs Proliferation conditions
SA498024CTR_D10_S1hASCs Proliferation conditions
SA498025CTR_D0_S3hASCs Proliferation conditions
SA498026CTR_D3_S1hASCs Proliferation conditions
SA498027CTR_D3_S2hASCs Proliferation conditions
SA498028CTR_D3_S3hASCs Proliferation conditions
SA498029CTR_D6_S1hASCs Proliferation conditions
SA498030CTR_D6_S2hASCs Proliferation conditions
SA498031CTR_D6_S3hASCs Proliferation conditions
SA498032CTR_D7_S1hASCs Proliferation conditions
SA498033CTR_D7_S2hASCs Proliferation conditions
SA498034CTR_D7_S3hASCs Proliferation conditions
SA498035CTR_D10_S2hASCs Proliferation conditions
SA498036CTR_D21_S2hASCs Proliferation conditions
SA498037CTR_D10_S3hASCs Proliferation conditions
SA498038CTR_D13_S1hASCs Proliferation conditions
SA498039CTR_D13_S2hASCs Proliferation conditions
SA498040CTR_D13_S3hASCs Proliferation conditions
SA498041CTR_D17_S1hASCs Proliferation conditions
SA498042CTR_D17_S2hASCs Proliferation conditions
SA498043CTR_D17_S3hASCs Proliferation conditions
SA498044CTR_D20_S1hASCs Proliferation conditions
SA498045CTR_D20_S2hASCs Proliferation conditions
SA498046CTR_D20_S3hASCs Proliferation conditions
SA498047CTR_D21_S1hASCs Proliferation conditions
SA498048CTR_D21_S3hASCs Proliferation conditions
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Collection:

Collection ID:CO004422
Collection Summary:hAMSC were donated from Histocell (Bilbao, Spain), derived from a healthy 29-year-old female donor undergoing liposuction with written informed consent.
Collection Protocol ID:Protocol no. E08-30
Sample Type:Stem cells
Storage Conditions:-80℃
Tissue Cell Identification:Adipose Tissue

Treatment:

Treatment ID:TR004438
Treatment Summary:Upon reaching the cell numbers necessary for metabolomics (> 1 million cells/sample for NMR, and 500 k cells/sample for LC-MS), day 0 cell samples were collected in triplicate prior to stimulation (Figure S2). The remaining cell culture T150 flasks (for NMR metabolomics) and 24-well plates (for MS lipidomics, Alamar blue (AB) assay and qRT-PCR) were split into unstimulated (control, CTR) and NK-stimulated groups. As previously described,[30,32] the NK bioreactor employs the reverse piezoelectric effect to induce mechanical expansions from applied voltages, enabling 30 nm vertical displacements to cell cultures at a frequency of 1000 Hz (Figure 1). To ensure consistent amplitudes across the growth surfaces while allowing for easy removal and maintenance, culture flasks/plates were firmly attached to the bioreactor using magnetic sheets. For intracellular NMR metabolomics (endometabolomics) and MS lipidomics, CTR and NK cells were trypsinized and collected in triplicate on days 0, 7 and 21 (Figure S2). The resulting cell suspensions were filtered through 100 μm pore strainers, centrifuged (300 g, 5 min, 4 °C) and rinsed twice with phosphate-buffered saline (PBS) solution. For extracellular NMR metabolomics (exometabolomics), media samples were collected on days 3, 6, 7, 10, 13, 17, 20 and 21. Blank media (not cell-exposed) was also obtained. Media and cell samples were stored (− 80 °C) until analysis.
Cell Media:α-MEM (Gibco™ 12000063, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics
Cell Harvesting:Days 0, 7 and 21
Cell Pct Confluence:100%

Sample Preparation:

Sampleprep ID:SP004435
Sampleprep Summary:Endometabolites (intracellular metabolites) were extracted using a methanol-chloroform-water extraction method. In brief, cell pellets were re-suspended in 800 μL of cold methanol (ref. M-4000-PC17, Thermo Fisher Scientific, Loughborough, UK) and Milli-Q water solution (4:1 ratio), transferred into glass tubes containing 150 mg of glass beads (ref. G8772, Sigma-Aldrich, Dorset, UK) and vortexed (2 min, RT ~ 25 °C). Subsequently, 320 μL of cold chloroform (C-4960-17, Thermo Fisher Scientific, Loughborough, UK) was added and the mixture vortexed again (2 min, RT). Further 320 μL of cold chloroform were added, followed by 290 μL of cold Milli-Q water, with further vortexing (2 min, RT). After incubating at − 20 °C for 10 minutes, the samples were centrifuged (10,000 g, 15 min, 4 °C), and the upper (polar) and lower (lipophilic) phases were collected, dried, and stored at − 80 °C until analysis (NMR and LC-MS). For NMR exometabolomics, 500 µL of culture medium from each sample were centrifuged (1,000 g, 5 min, 4 °C) to eliminate cellular debris. Subsequently, protein precipitation was performed by adding 600 µL of 100% methanol at − 80 °C to microcentrifuge tubes containing 300 µL of the medium sample. Following 30 min incubation at − 20 °C, samples were centrifuged (13,000 g, 20 min, 4 °C). The supernatant containing the exometabolites (extracellular metabolites) was collected, dried under vacuum and stored at − 80 °C.
Processing Storage Conditions:On ice
Extraction Method:methanol-chloroform-water extraction method
Extract Storage:-80℃
Sample Resuspension:Prior to NMR analysis, cellular polar extracts were re-suspended in 625 μL of a 100 mM phosphate buffer (pH 7.4) in D2O (99.9% deuterium, Eurisotop D216), with 0.1 mM of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, in D2O, Sigma-Aldrich 293040) for chemical shift referencing. Dried media samples were resuspended in 700 μL of the same phosphate buffer, centrifuged (13,000 g, 5 min, RT) and the supernatant collected. Cellular lipidic extracts were re-suspended in 650 μL of deuterated chloroform (99.8% deuterium, Eurisotop D307F) containing 0.03% tetramethylsilane (TMS) for chemical shift referencing. Finally, 550 μL from each sample was transferred into a 5 mm NMR tube.

Analysis:

Analysis ID:AN007115
Laboratory Name:Metabolomics Group
Analysis Type:NMR
Software Version:Amix 3.9.15 and TopSpin3.2
Operator Name:Daniela Bispo
Num Factors:2
Num Metabolites:86
Units:Peak areas

NMR:

NMR ID:NM000318
Analysis ID:AN007115
Instrument Name:NMR 500 Bruker Avance III
Instrument Type:FT-NMR
NMR Experiment Type:1D-1H
NMR Comments:The following letters were added to each metabolite to specify the type of sample in which they were identified - M: MEDIA_corrected_integral; P: CELL_POLAR_normalized_integral; L: CELL_LIPID_normalized_integral
Spectrometer Frequency:500.13 Hz
NMR Probe:TXI probe
NMR Tube Size:5 mm
Pulse Sequence:POLAR: water presaturation (noesypr1d pulse sequence, Bruker library) & LIPID: standard 90° pulse sequence (zg pulse sequence, Bruker library).
Temperature:298 K
Number Of Scans:256 scans (exometabolome) and 512 (endometabolome)
Dummy Scans:8
Acquisition Time:2.3 s
Relaxation Delay:4 s
Spectral Width:7002.801 Hz
Num Data Points Acquired:32 k points
Line Broadening:0.3 Hz
Zero Filling:64 k points
Apodization:Exponential
Baseline Correction Method:Manual
Chemical Shift Ref Std:POLAR: 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP, in D2O, Sigma-Aldrich 293040) & LIPID: deuterated chloroform (99.8% deuterium, Eurisotop D307F) containing 0.03% tetramethylsilane (TMS)
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