#METABOLOMICS WORKBENCH dbispo_20220406_041746 DATATRACK_ID:3173 STUDY_ID:ST002142 ANALYSIS_ID:AN003507 PROJECT_ID:PR001357
VERSION             	1
CREATED_ON             	April 7, 2022, 3:30 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 (Lipid 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           	-	OI1_D0_S1_LE	Experiment day:D0	Sample type=Lipidic extract; Replica number=1; RAW_FILE_NAME=OI1_D0_S1_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D0_S2_LE	Experiment day:D0	Sample type=Lipidic extract; Replica number=2; RAW_FILE_NAME=OI1_D0_S2_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D0_S3_LE	Experiment day:D0	Sample type=Lipidic extract; Replica number=3; RAW_FILE_NAME=OI1_D0_S3_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D1_S1_LE	Experiment day:D1	Sample type=Lipidic extract; Replica number=1; RAW_FILE_NAME=OI1_D1_S1_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D1_S2_LE	Experiment day:D1	Sample type=Lipidic extract; Replica number=2; RAW_FILE_NAME=OI1_D1_S2_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D1_S3_LE	Experiment day:D1	Sample type=Lipidic extract; Replica number=3; RAW_FILE_NAME=OI1_D1_S3_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D7_S1_LE	Experiment day:D7	Sample type=Lipidic extract; Replica number=1; RAW_FILE_NAME=OI1_D7_S1_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D7_S2_LE	Experiment day:D7	Sample type=Lipidic extract; Replica number=2; RAW_FILE_NAME=OI1_D7_S2_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D7_S3_LE	Experiment day:D7	Sample type=Lipidic extract; Replica number=3; RAW_FILE_NAME=OI1_D7_S3_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D14_S1_LE	Experiment day:D14	Sample type=Lipidic extract; Replica number=1; RAW_FILE_NAME=OI1_D14_S1_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D14_S2_LE	Experiment day:D14	Sample type=Lipidic extract; Replica number=2; RAW_FILE_NAME=OI1_D14_S2_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D14_S3_LE	Experiment day:D14	Sample type=Lipidic extract; Replica number=3; RAW_FILE_NAME=OI1_D14_S3_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D21_S1_LE	Experiment day:D21	Sample type=Lipidic extract; Replica number=1; RAW_FILE_NAME=OI1_D21_S1_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D21_S2_LE	Experiment day:D21	Sample type=Lipidic extract; Replica number=2; RAW_FILE_NAME=OI1_D21_S2_LE
SUBJECT_SAMPLE_FACTORS           	-	OI1_D21_S3_LE	Experiment day:D21	Sample type=Lipidic extract; Replica number=3; RAW_FILE_NAME=OI1_D21_S3_LE
#COLLECTION
CO:COLLECTION_SUMMARY            	At days 0, 1, 7, 14, and 21 cells were trypsinized, filtered through 100 μm
CO:COLLECTION_SUMMARY            	pore strainers, and rinsed twice with PBS, and at least 1 × 10 6 (range 1–4.5
CO:COLLECTION_SUMMARY            	× 10 6) cells per pellet were collected for lipidomics.
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            	Cellular metabolites were extracted using the methanol-chloroform-water method
SP:SAMPLEPREP_SUMMARY            	and the resulting lipophilic extracts were dried under a nitrogen flow. Prior to
SP:SAMPLEPREP_SUMMARY            	NMR analysis, dried lipidic extracts were re-suspended in deuterated chloroform
SP:SAMPLEPREP_SUMMARY            	(99.8% deuterium, Eurisotop D307F) containing 0.03% tetramethylsilane (TMS),
SP:SAMPLEPREP_SUMMARY            	homogenised and transferred to 5 mm NMR tubes.
SP:EXTRACTION_METHOD             	Methanol-chloroform-water method
SP:EXTRACT_STORAGE               	-80℃
SP:SAMPLE_RESUSPENSION           	Deuterated chloroform (99.8% deuterium, Eurisotop D307F) containing 0.03%
SP:SAMPLE_RESUSPENSION           	tetramethylsilane (TMS)
#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          	CDCl3
NM:SPECTROMETER_FREQUENCY        	500 MHz
NM:NMR_PROBE                     	TXI
NM:NMR_SOLVENT                   	CDCl3
NM:NMR_TUBE_SIZE                 	5 mm
NM:SHIMMING_METHOD               	Topshim
NM:RECEIVER_GAIN                 	203
NM:TEMPERATURE                   	298 K
NM:NUMBER_OF_SCANS               	512
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                  	65k
NM:BASELINE_CORRECTION_METHOD    	Manual
NM:CHEMICAL_SHIFT_REF_STD        	TMS
NM:NMR_RESULTS_FILE               	ST002142_AN003507_Results.txt	UNITS:ppm
#END