#METABOLOMICS WORKBENCH dbispo_20220406_062203_mwtab.txt DATATRACK_ID:3175 STUDY_ID:ST002143 ANALYSIS_ID:AN003508 PROJECT_ID:PR001357 VERSION 1 CREATED_ON April 7, 2022, 3:33 am #PROJECT PR:PROJECT_TITLE A Metabolomics-guided Bioreactor for Improved Engineered Bone Implants PR:PROJECT_TITLE (BioImplant) PR:PROJECT_TYPE NMR-based untargeted metabolomics PR:PROJECT_SUMMARY In an “omic” era, metabolomics offers exquisite insight into the complex PR:PROJECT_SUMMARY metabolic network of living organisms and their adaptation mechanisms towards PR:PROJECT_SUMMARY disease, therapy and environment. Metabolic markers (sets of metabolites) are PR:PROJECT_SUMMARY emerging as new means of diagnostics, personalized follow-up and prediction of PR:PROJECT_SUMMARY patient health status. Here, metabolomics is used for the first time to guide PR:PROJECT_SUMMARY the development of a stem cell (SC) bioreactor to produce improved quality bone PR:PROJECT_SUMMARY tissue for implantation. 3D porous scaffolds will be produced using PR:PROJECT_SUMMARY biodegradable polymer poly-L-lactic acid (PLLA), both bare and collagen-coated PR:PROJECT_SUMMARY to improve cell adhesion. These scaffolds will be 3D-printed with controlled PR:PROJECT_SUMMARY architectures (both internally and externally), namely with struts alignment of PR:PROJECT_SUMMARY 90o, with and without offset, expected to trigger distinct biological behaviors, PR:PROJECT_SUMMARY particularly under mechanical cues. Scaffolds will be tested in a bioreactor for PR:PROJECT_SUMMARY growth and differentiation of human mesenchymal SCs (hMSCs) into osteogenic PR:PROJECT_SUMMARY lineage. hMSCs obtained from adipose tissue or bone marrow will be compared, as PR:PROJECT_SUMMARY they have shown secretome differences and possible different potentials for PR:PROJECT_SUMMARY osteogenic differentiation. The bioreactor will allow the application of PR:PROJECT_SUMMARY controlled compression, to help mimic bone physiological conditions, and PR:PROJECT_SUMMARY scaffold piezoelectricity will be studied in the same context. Scaffolds and PR:PROJECT_SUMMARY physical cues will be tested in vitro (in osteogenic media) and monitored by PR:PROJECT_SUMMARY biological measurements (proliferation, viability, differentiation indicators) PR:PROJECT_SUMMARY and, for the first time, by cell metabolomics to identify the impact of each PR:PROJECT_SUMMARY variable (scaffold composition, morphology, piezoelectricity and compression) on PR:PROJECT_SUMMARY hMSC metabolism and define metabolic markers of hMSC function. Untargeted PR:PROJECT_SUMMARY Nuclear Magnetic Resonance (NMR) metabolomics of cell extracts will identify PR:PROJECT_SUMMARY dynamic metabolic cellular profiles associated to i) hMSC self-renewal and PR:PROJECT_SUMMARY differentiation mechanisms, and their adaptations to ii) scaffold PR:PROJECT_SUMMARY characteristics and iii) physical cues (compression and/or piezoelectricity). PR:PROJECT_SUMMARY Statistical correlation of metabolic profiles with scaffold/bioreactor features PR:PROJECT_SUMMARY and biological parameters will unveil metabolic markers of bioreactor PR:PROJECT_SUMMARY performance and novel knowledge on SC osteogenic metabolism. Key metabolites PR:PROJECT_SUMMARY will be identified as potentially osteogenesis-inducing, a role to be PR:PROJECT_SUMMARY demonstrated using metabolite-tailored cell media to potentially substitute PR:PROJECT_SUMMARY osteogenic growth factors and, thus, tackle related implantation challenges. PR:PROJECT_SUMMARY Putative hypotheses of responsive hMSCs metabolic pathways will be validated PR:PROJECT_SUMMARY through pathway network analysis, isotope-labeled tracers (NMR) and specific PR:PROJECT_SUMMARY protein and genetic measurements. To our knowledge, this project proposes the PR:PROJECT_SUMMARY first use of metabolomics to guide in vitro bone tissue engineering, building on PR:PROJECT_SUMMARY recent proposals to exploit “omics” to understand, monitor and guide SC PR:PROJECT_SUMMARY behavior for effective tissue engineering and implantation. PR:INSTITUTE University of Aveiro PR:DEPARTMENT Department of Chemistry PR:LABORATORY CICECO - Aveiro Institute of Materials PR:LAST_NAME Bispo PR:FIRST_NAME Daniela S.C. PR:ADDRESS Campus Universitário de Santiago, Aveiro, Aveiro, Aveiro, 3810-193, Portugal PR:EMAIL d.bispo@ua.pt PR:PHONE none PR:FUNDING_SOURCE The authors acknowledge the Portuguese Foundation for Science and Technology PR:FUNDING_SOURCE (FCT) for co-funding the BIOIMPLANT project (PTDC/BTM-ORG/28835/2017) through PR:FUNDING_SOURCE the COMPETE2020 program and European Union fund FEDER PR:FUNDING_SOURCE (POCI-01-0145-FEDER-028835). CSHJ is grateful to the same project for funding PR:FUNDING_SOURCE her contract with the University of Aveiro. DSB acknowledges the Sociedade PR:FUNDING_SOURCE Portuguesa de Química and FCT for her PhD grant SFRH/BD/150655/2020. This work PR:FUNDING_SOURCE was developed within the scope of the project CICECO-Aveiro Institute of PR:FUNDING_SOURCE Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by PR:FUNDING_SOURCE national funds through the FCT/MEC (PIDDAC). The NMR spectrometer used in this PR:FUNDING_SOURCE work is part of the National NMR Network (PTNMR) and, partially supported by PR:FUNDING_SOURCE Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, PR:FUNDING_SOURCE POCI and PORL and FCT through PIDDAC). #STUDY ST:STUDY_TITLE Endo- and Exometabolome Crosstalk in Mesenchymal Stem Cells Undergoing ST:STUDY_TITLE Osteogenic Differentiation (Media Samples) ST:STUDY_SUMMARY The holistic nature of NMR enabled the time-course evolution of cholesterol, ST:STUDY_SUMMARY mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), ST:STUDY_SUMMARY several phospholipids (phosphatidylcholine, phosphatidylethanolamine, ST:STUDY_SUMMARY sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. ST:STUDY_SUMMARY Lipid changes occurred almost exclusively between days 1 and 7, followed by a ST:STUDY_SUMMARY tendency for lipidome stabilization after day 7. On average, phospholipids and ST:STUDY_SUMMARY longer and more unsaturated fatty acids increased up to day 7, probably related ST:STUDY_SUMMARY to plasma membrane fluidity. Articulation of lipidome changes with previously ST:STUDY_SUMMARY reported polar endometabolome profiling and with exometabolome changes reported ST:STUDY_SUMMARY here in the same cells, enabled important correlations to be established during ST:STUDY_SUMMARY hAMSC osteogenic differentiation. Our results supported hypotheses related to ST:STUDY_SUMMARY the dynamics of membrane remodelling, anti-oxidative mechanisms, protein ST:STUDY_SUMMARY synthesis, and energy metabolism. Importantly, the observation of specific ST:STUDY_SUMMARY up-taken or excreted metabolites paves the way for the identification of ST:STUDY_SUMMARY potential osteoinductive metabolites useful for optimized osteogenic protocols. ST:INSTITUTE University of Aveiro ST:DEPARTMENT Department of Chemistry ST:LABORATORY CICECO - Aveiro Institute of Materials ST:LAST_NAME Bispo ST:FIRST_NAME Daniela S.C. ST:ADDRESS Campus Universitário de Santiago, Aveiro, Aveiro, Aveiro, 3810-193, Portugal ST:EMAIL d.bispo@ua.pt ST:PHONE none #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Medium_Blank_S1 Experiment day:Blank Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=Medium_Blank_S1 SUBJECT_SAMPLE_FACTORS - Medium_Blank_S2 Experiment day:Blank Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=Medium_Blank_S2 SUBJECT_SAMPLE_FACTORS - Medium_Blank_S3 Experiment day:Blank Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=Medium_Blank_S3 SUBJECT_SAMPLE_FACTORS - Medium_Blank_S4 Experiment day:Blank Sample type=Media Sample; Replica number=4; RAW_FILE_NAME=Medium_Blank_S4 SUBJECT_SAMPLE_FACTORS - OI1_D0_S1_Medium Experiment day:D0 Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=OI1_D0_S1_Medium SUBJECT_SAMPLE_FACTORS - OI1_D0_S2_Medium Experiment day:D0 Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=OI1_D0_S2_Medium SUBJECT_SAMPLE_FACTORS - OI1_D0_S3_Medium Experiment day:D0 Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=OI1_D0_S3_Medium SUBJECT_SAMPLE_FACTORS - OI1_D1_S1_Medium Experiment day:D1 Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=OI1_D1_S1_Medium SUBJECT_SAMPLE_FACTORS - OI1_D1_S2_Medium Experiment day:D1 Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=OI1_D1_S2_Medium SUBJECT_SAMPLE_FACTORS - OI1_D1_S3_Medium Experiment day:D1 Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=OI1_D1_S3_Medium SUBJECT_SAMPLE_FACTORS - OI1_D6_S1_Medium Experiment day:D6 Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=OI1_D6_S1_Medium SUBJECT_SAMPLE_FACTORS - OI1_D6_S2_Medium Experiment day:D6 Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=OI1_D6_S2_Medium SUBJECT_SAMPLE_FACTORS - OI1_D6_S3_Medium Experiment day:D6 Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=OI1_D6_S3_Medium SUBJECT_SAMPLE_FACTORS - OI1_D9_S1_Medium Experiment day:D9 Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=OI1_D9_S1_Medium SUBJECT_SAMPLE_FACTORS - OI1_D9_S2_Medium Experiment day:D9 Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=OI1_D9_S2_Medium SUBJECT_SAMPLE_FACTORS - OI1_D9_S3_Medium Experiment day:D9 Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=OI1_D9_S3_Medium SUBJECT_SAMPLE_FACTORS - OI1_D12_S1_Medium Experiment day:D12 Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=OI1_D12_S1_Medium SUBJECT_SAMPLE_FACTORS - OI1_D12_S2_Medium Experiment day:D12 Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=OI1_D12_S2_Medium SUBJECT_SAMPLE_FACTORS - OI1_D12_S3_Medium Experiment day:D12 Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=OI1_D12_S3_Medium SUBJECT_SAMPLE_FACTORS - OI1_D14_S1_Medium Experiment day:D14 Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=OI1_D14_S1_Medium SUBJECT_SAMPLE_FACTORS - OI1_D14_S2_Medium Experiment day:D14 Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=OI1_D14_S2_Medium SUBJECT_SAMPLE_FACTORS - OI1_D14_S3_Medium Experiment day:D14 Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=OI1_D14_S3_Medium SUBJECT_SAMPLE_FACTORS - OI1_D16_S1_Medium Experiment day:D16 Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=OI1_D16_S1_Medium SUBJECT_SAMPLE_FACTORS - OI1_D16_S2_Medium Experiment day:D16 Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=OI1_D16_S2_Medium SUBJECT_SAMPLE_FACTORS - OI1_D16_S3_Medium Experiment day:D16 Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=OI1_D16_S3_Medium SUBJECT_SAMPLE_FACTORS - OI1_D21_S1_Medium Experiment day:D21 Sample type=Media Sample; Replica number=1; RAW_FILE_NAME=OI1_D21_S1_Medium SUBJECT_SAMPLE_FACTORS - OI1_D21_S2_Medium Experiment day:D21 Sample type=Media Sample; Replica number=2; RAW_FILE_NAME=OI1_D21_S2_Medium SUBJECT_SAMPLE_FACTORS - OI1_D21_S3_Medium Experiment day:D21 Sample type=Media Sample; Replica number=3; RAW_FILE_NAME=OI1_D21_S3_Medium #COLLECTION CO:COLLECTION_SUMMARY hAMSCs conditioned media samples were collected in triplicate at days 1, 6, 9, CO:COLLECTION_SUMMARY 12, 14, 16, and 21 after filtration through 40 μm pore strainers. CO:SAMPLE_TYPE Stem cells #TREATMENT TR:TREATMENT_SUMMARY hAMSCs were detached, at passage 7, from the flasks by trypsinization, counted TR:TREATMENT_SUMMARY in a Neubauer chamber and seeded at a density of 0.5 × 10 6 cells/flask. Cells TR:TREATMENT_SUMMARY were maintained under basal conditions until reaching ~100% confluence, then the TR:TREATMENT_SUMMARY basal culture medium was exchanged and supplemented with osteoinductive factors, TR:TREATMENT_SUMMARY specifically 10 mM β-glycerophosphate (β-GP, Sigma-Aldrich G9422), 50 µg/mL TR:TREATMENT_SUMMARY L-ascorbic acid (Sigma A0278), and 10 nM Dexa (ACROS Organics™ 230300010). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Both blank and conditioned media samples were subjected to a SP:SAMPLEPREP_SUMMARY protein-precipitation procedure and dried under vacuum. Prior to NMR analysis, SP:SAMPLEPREP_SUMMARY dried media samples were resuspended in 100 mM phosphate buffer at pH 7.4 SP:SAMPLEPREP_SUMMARY (prepared in D2O) containing 0.1 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid SP:SAMPLEPREP_SUMMARY (TSP in D2O, Sigma-Aldrich 293040), homogenised and transferred to 5 mm NMR SP:SAMPLEPREP_SUMMARY tubes. SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION 100 mM phosphate buffer at pH 7.4 (prepared in D2O) containing 0.1 mM SP:SAMPLE_RESUSPENSION 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP in D2O, Sigma-Aldrich 293040) #ANALYSIS AN:ANALYSIS_TYPE NMR AN:OPERATOR_NAME Daniela Bispo AN:SOFTWARE_VERSION Topspin 4.0.8 #NMR NM:INSTRUMENT_NAME Bruker Avance III NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK D2O NM:SPECTROMETER_FREQUENCY 500 MHz NM:NMR_PROBE TXI NM:NMR_SOLVENT 100 mM phosphate buffer at pH 7.4 (prepared in D2O) containing 0.1 mM NM:NMR_SOLVENT 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid NM:NMR_TUBE_SIZE 5 mm NM:SHIMMING_METHOD Topshim NM:RECEIVER_GAIN 203 NM:TEMPERATURE 298 K NM:NUMBER_OF_SCANS 256 NM:DUMMY_SCANS 4 NM:ACQUISITION_TIME 2.3 s NM:RELAXATION_DELAY 4 s NM:SPECTRAL_WIDTH 7002.801 Hz NM:NUM_DATA_POINTS_ACQUIRED 32k NM:ZERO_FILLING 131k NM:BASELINE_CORRECTION_METHOD Manual NM:CHEMICAL_SHIFT_REF_STD TSP NM:NMR_RESULTS_FILE ST002143_AN003508_Results.txt UNITS:ppm #END