Hindawi
Stem Cells International
Volume 2022, Article ID 3674931, 20 pages
https://doi.org/10.1155/2022/3674931

Research Article
Energy Metabolism and Lipidome Are Highly Regulated during
Osteogenic Differentiation of Dental Follicle Cells
Oliver Pieles,1 Marcus Höring,2 Sadiyeh Adel,1 Torsten E. Reichert,1 Gerhard Liebisch,2and Christian Morsczeck 1
1

Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11,93053 Regensburg, Germany
2
Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11,93053 Regensburg, Germany
Correspondence should be addressed to Christian Morsczeck; christian.morsczeck@ukr.deReceived 21 January 2022; Revised 14 June 2022; Accepted 30 June 2022; Published 16 July 2022
Academic Editor: Toru Ogasawara
Copyright © 2022 Oliver Pieles et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Dental follicle cells (DFCs) are stem/progenitor cells of the periodontium and give rise to alveolar osteoblasts. However,understanding of the molecular mechanisms of osteogenic diﬀerentiation, which is required for cell-based therapies, isdelimited. This study is aimed at analyzing the energy metabolism during the osteogenic diﬀerentiation of DFCs. Human
DFCs were cultured, and osteogenic diﬀerentiation was induced by either dexamethasone or bone morphogenetic protein 2(BMP2). Previous microarray data were reanalyzed to examine pathways that are regulated after osteogenic induction.
Expression and activity of metabolic markers were evaluated by western blot analysis and speciﬁc assays, relative amount of
mitochondrial DNA was measured by real-time quantitative polymerase chain reaction, the oxidative state of cells wasdetermined by a glutathione assay, and the lipidome of cells was analyzed via mass spectrometry (MS). Moreover, osteogenic
markers were analyzed after the inhibition of fatty acid synthesis by 5-(tetradecyloxy)-2-furoic acid or C75. Pathway
enrichment analysis of microarray data revealed that carbon metabolism was amongst the top regulated pathways after
osteogenic induction in DFCs. Further analysis showed that enzymes involved in glycolysis, citric acid cycle, mitochondrial
activity, and lipid metabolism are diﬀerentially expressed during diﬀerentiation, with most markers upregulated and moremarkedly after induction with dexamethasone compared to BMP2. Moreover, the cellular state was more oxidized, andmitochondrial DNA was distinctly upregulated during the second half of diﬀerentiation. Besides, MS of the lipidome revealed
higher lipid concentrations after osteogenic induction, with a preference for species with lower numbers of C-atoms anddouble bonds, which indicates a de novo synthesis of lipids. Concordantly, inhibition of fatty acid synthesis impeded theosteogenic diﬀerentiation of DFCs. This study demonstrates that energy metabolism is highly regulated during osteogenic
diﬀerentiation of DFCs including changes in the lipidome suggesting enhanced de novo synthesis of lipids, which are required
for the diﬀerentiation process.

1. Introduction
Periodontal diseases are a common health issue that can lead tosevere tissue damage and tooth loss. Tissue engineering basedon biomaterials and stem cells is a promising approach forregeneration of functional periodontal tissue [1–3]. Dental follicle cells (DFCs) are stem/progenitor cells of the periodontium
and give rise to the alveolar bone, cementum, and periodontal
ligament during tooth development. Thus, DFCs are suitable

for stem cell-based therapies of periodontal diseases [4, 5]. Furthermore, they can be easily isolated from extracted wisdom
teeth [6]. However, besides much progress in the recent years,
the molecular mechanisms of diﬀerentiation processes intofunctional tissue cells are still insuﬃciently understood, which
impedes their optimal use for targeted therapies [5, 7].
The osteogenic diﬀerentiation of DFCs can be inducedin vitro by stimulating the cells with dexamethasone or bonemorphogenetic protein 2 (BMP2) amongst others [8, 9].

2
Several studies indicate that the two inducers target diﬀerent
molecular signaling pathways. For example, BMP2 drives thediﬀerentiation via the expression of runt-related transcription
factor 2 (RUNX2) and distal-less homeobox 3 (DLX3), while
dexamethasone-induced osteogenesis requires other transcription factors like zinc ﬁnger and BTB domain-containing
16 (ZBTB16) [10–12]. However, the two diﬀerentiation procedures also share similarities like the fact that classical isoforms
of protein kinase C (PKC) disturb osteogenesis with both
inducers [13]. Besides particular molecular pathways, only little attention has been spent to the role of general cellular
metabolism for DFC diﬀerentiation. A previous study showedthat osteogenesis of DFCs is sensitive to changes in AMPactivated protein kinase (AMPK) activity [14], which is a key
molecular sensor for cellular energy [15]. It is presumable thatenergy metabolism might play a crucial role for osteogenic
diﬀerentiation of precursor cells. Bone marrow-derived mesenchymal stem cells (MSCs) were reported to activate oxidative phosphorylation, but not glycolysis, after osteogenic
induction [16, 17], and hypoxia inhibits the diﬀerentiation[18]. Reactive oxygen species (ROS), which are mainly generated by mitochondrial metabolism, can regulate the osteogenic
diﬀerentiation [19]. Mitochondrial activity was further shownto stimulate the osteogenic diﬀerentiation via acetylation of
β-catenin [20]. Beyond, also metabolism of cellular lipids
participates in energy homeostasis and is implicated in several molecular pathways like activation of PKC [21–23].
In order to further unravel the molecular mechanisms
behind the osteogenic diﬀerentiation of DFCs, this study is
aimed at analyzing the energy metabolism including markers
for glycolysis, citric acid cycle, oxidative phosphorylation,and lipid metabolism after induction of diﬀerentiation withdexamethasone and BMP2. Moreover, the lipidome of diﬀerentiating DFCs and the role of fatty acid synthesis wereanalyzed in this study.

2. Materials and Methods
2.1. Cell Culture. Human dental follicle cells were purchasedfrom AllCells (Alameda, CA, USA) and cultured in Dulbecco’s
Modiﬁed Eagle’s Medium (DMEM) containing 10% fetal
bovine serum (FBS) and 1% penicillin (100 U/ml)/streptomycin (100 μg/ml) solution (all Sigma-Aldrich, St. Louis, MO,
USA) with 37°C and 5% CO2 in humidiﬁed atmosphere.
Growth medium was changed three times in a week. Cells
until passage 8 were used for the experiments.
2.2. Osteogenic Diﬀerentiation. For induction of osteogenic
diﬀerentiation, subconﬂuent DFCs were treated with osteogenic diﬀerentiation medium consisting of DMEM supplemented with 2% FBS, antibiotics as above, 20 mM HEPES,
10 mM β-glycerophosphate, 100 μM phospho-ascorbic acid,and 100 nM dexamethasone (all Sigma-Aldrich), or with
BMP2 diﬀerentiation medium which contains 50 ng/ml
BMP2 (Biomol, Hamburg, Germany) in place of dexamethasone. As control, cells were simultaneously cultured in
DMEM with 2% FBS and antibiotics as above. The diﬀerentiation media were changed twice in a week.

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2.3. Microarray Analysis of Transcriptome. Publicly available
microarray data (GSE20963) were used for analysis. The data
include the gene expression proﬁle of DFCs that were treatedwith diﬀerent osteogenic inducers including dexamethasone
and BMP2 for seven days compared to control medium in biological triplicates for each group. Data were processed as
described earlier [24]. Processed gene expression data wereused to search for regulated genes after osteogenic induction:
The genes selected were up- or downregulated with at least
factor 2 and with a p value below 0.05 in Student’s t-test byboth dexamethasone and BMP2. A list of the selected genes
is available in Supplementary Table 1. Afterwards, a pathway
enrichment analysis was performed with the selected genes
with DAVID bioinformatics resources [25, 26] using the
KEGG (Kyoto Encyclopedia of Genes and Genomes)
pathway database [27–29]. A p value was determined with
the EASE score, and pathways with a p value below 0.05
were considered signiﬁcantly addressed.
2.4. Western Blot Analysis. DFCs were lysed and scraped in a
buﬀer consisting of 20 mM trisHCl (pH 8.0), 137 mM NaCl,
48 mM NaF, 1% (v/v) NP-40, 10% (v/v) glycerol, 2 mM
Na3VO4, phosphatase inhibitor cocktail 3 (Sigma-Aldrich),and cOmplete™ mini protease inhibitor cocktail (1 tablet in
10 ml, Sigma-Aldrich) in H2O. Supernatants were collected
after centrifugation of the lysates at 14,000 rpm for 5 min at
4°C, and the protein concentration was determined with the
Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientiﬁc,Waltham, MA, USA). Subsequently, lysates were mixed with
the Laemmli sample buﬀer (BIO-RAD, Hercules, CA, USA)
before boiling for 5 min at 95°C. Samples were loaded onto a
4–15% Mini-PROTEAN® TXG StainFree™ Protein Gel (BIORAD), and proteins were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). After activation by UV light, proteins were transferred onto Amersham
Protran® 0.2 μm nitrocellulose membranes (Sigma-Aldrich)using either a Trans-Blot® SD Semi-Dry Transfer Cell or aTrans-Blot® Turbo™ Transfer System (both BIO-RAD). Photographs of total lane protein were taken with a ChemiDoc™
Touch Imaging System (BIO-RAD). Membranes were thenwashed in Tris-buﬀered saline (TBS) for 5 min before blocking
in 5% bovine serum albumin (BSA) in Tween-containing TBS(TBST) for 1 hour at room temperature. Subsequently, themembranes were incubated overnight at 4°C in a primary antibody against hypoxia-inducible factor 1-alpha (HIF-1α), cytochrome c, prohibitin 1, cytochrome c oxidase subunit 4(COX IV), pyruvate dehydrogenase, succinate dehydrogenase
complex subunit A (SDHA), heat shock protein 60 (HSP60),
voltage-dependent anion channel (VDAC), hexokinase I, hexokinase II, phosphofructokinase, glycerinaldehyd-3-phosphatdehydrogenase (GAPDH), pyruvate kinase M1/2, pyruvatekinase M2, lactate dehydrogenase A, aconitase 2, isocitrate
dehydrogenase 1, isocitrate dehydrogenase 2, dihydrolipoamide succinyltransferase (DLST), fumarase, citrase synthase,mitochondrial pyruvate carrier 1, mitochondrial pyruvate carrier 2, acetyl-CoA carboxylase, phospho-acetyl-CoA carboxylase (Ser79), ATP-citrate lyase, phospho-ATP-citrate lyase(Ser455), acetyl-CoA synthetase, acyl-CoA synthetase, fatty
acid synthase (all Cell Signaling Technology, Danvers, MA,

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USA), or elongation of very long chain fatty acids protein 6(ELOVL6) (Abcam, Cambridge, UK). The primary antibodies
were all diluted 1 : 1000 in TBST with 5% BSA. Afterwards,
membranes were washed three times in TBST for 10 min each,before incubation in the secondary antibody horseradish peroxidase- (HRP-) linked anti-rabbit IgG (Cell Signaling Technology), which was diluted 1 : 1000 in TBST with 5%
skimmed milk powder for 1 hour at room temperature. Thereupon, membranes were washed twice in TBST for 10 min each,once in phosphate-buﬀered saline (PBS) for at least 5 min andonce in TBS for 5 min, before a chemiluminescent signal wasdeveloped by using the Clarity™ western ECL substrate (BIORAD) and recorded with a ChemiDoc™ Touch Imaging System (BIO-RAD). Densitometric quantiﬁcation was performed
with the software Image Lab version 6.0.1 (BIO-RAD) withnormalization of protein bands to total lane protein and thecontrol group.
2.5. L-Lactate Assay. The Glycolysis Cell-Based Assay Kit(Cayman Chemical, Ann Arbor, MI, USA) was used according
to the manufacturer’s handbook. Brieﬂy, DFCs in a 96-well
cell culture plate with 200 μl medium per well were centrifuged, and 20 μl of supernatant was used to determine theamount of l-lactate in a colorimetric reaction, which was measured spectrophotometrically at OD = 490 nm.
2.6. Hexokinase Activity Assay. The Hexokinase Colorimetric
Assay Kit (Sigma-Aldrich) was used according to manufacturer’s instructions. Brieﬂy, DFCs were washed with PBS, scrapedin assay buﬀer from the kit, and centrifuged. The supernatant
was further used to determine hexokinase activity in a colorimetric reaction, which was measured spectrophotometricallyat OD = 450 nm in kinetic mode.
2.7. Malate Dehydrogenase Activity Assay. The Malate Dehydrogenase Activity Assay Kit (Abcam) was used according to
the manufacturer’s protocol booklet. Brieﬂy, cells were
washed with PBS and scraped in assay buﬀer from the kit.
After centrifugation, supernatants were used to determine
malate dehydrogenase activity in a colorimetric reaction,
which was measured spectrophotometrically at OD = 450
nm in kinetic mode.
2.8. Complex I Activity Assay. Mitochondria were isolated from
DFCs with the Mitochondria Isolation Kit for Cultured Cells(Abcam) according to manufacturer’s instructions. Afterwards,
the Complex I Enzyme Activity Microplate Assay Kit (Abcam)
was used to determine complex I activity following the manufacturer’s protocol booklet. Brieﬂy, proteins from isolated mitochondria were extracted using the detergent solution from the
kit and loaded onto a microplate precoated with complex I
antibodies. After 3 hours incubation, the microplate wells were
washed, before the assay solution from the kit was added inorder to initiate a colorimetric reaction, which was measured
spectrophotometrically at OD = 450 nm in kinetic mode.
2.9. Real-Time Quantitative Polymerase Chain Reaction(qPCR). The QIAamp® DNA Mini Kit (Qiagen, Hilden,
Germany) was used to isolate DNA from DFCs. For this, the“Protocol for Cultured Cells” in the manufacturer’s handbook

3
was used. Both trypsinization and scraping were applied todetach the adherent cells from the culture plates. The qPCR
analysis was performed with the SsoAdvanced™ Universal
SYBR Green Supermix (BIO-RAD) and primers against mitochondrial DNA (forward primer: 5′-GCCTTCCCCCGTAAATGATA-3′, reverse primer: 5′-TTATGCGATTACCGGG
CTCT-3′) or B2M (forward primer: 5′-TGCTGTCTCCA
TGTTTGATGTATCT-3′, reverse primer: 5′-TCTCTGCTCCCCACCTCTAAGT-3′) as reference for genomic DNA.
The qPCR was run on a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientiﬁc) with the following protocol:
polymerase activation for 2 min at 95°C, then 40 cycles eachconsisting of a denaturation step for 15 sec at 95°C, and an
annealing/elongation step for 30 sec at 62°C, then melt curve
analysis (for amplicon control) starting at 95°C for 15 sec, then
1 min at 60°C following temperature increase in 0.3°C steps foreach 15 sec until 95°C. The amount of mitochondrial DNA
was normalized to the amount of B2M genomic DNA andto the control group at day 1 by using the ΔΔCt method[30]. The qPCR protocol including primer sequences was
adopted from Venegas and Halberg [31].
2.10. Glutathione Assay. The GSH/GSSG-Glo™ Assay kit(Promega, Madison, WI, USA) was used according to manufacturer’s instructions to determine relative amounts ofoxidized and reduced glutathione in DFCs. Brieﬂy, cells were
washed with PBS and lysed in a Glutathione Lysis Reagent
from the kit for analysis of either oxidized or total glutathione. The amount of glutathione was then quantiﬁed using
a luciferase reporter system. For each experimental group,
three biological replicates were used for determination ofoxidized and total glutathione, respectively. The amount ofoxidized from total glutathione, which is used as indicatorof oxidative stress, was normalized to the control group.
2.11. Mass Spectrometry Analysis of Lipidome (MS). DFCs
were washed with PBS and scraped in 0.2% sodium dodecyl
sulfate (SDS) and cOmplete™ mini protease inhibitor cocktail(1 tablet in 5 ml, Sigma-Aldrich) in H2O. The protein concentration in the lysates was measured with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientiﬁc). The samples werestored at -80°C until analysis. Cell homogenates were subjected to lipid extraction according to the method of Blighand Dyer [32] in the presence of not naturally occurring lipid
species as internal standards. The following lipid species wereadded as internal standards: PC 14 : 0/14 : 0, PC 22 : 0/22 : 0, PE
14 : 0/14 : 0, PE 20 : 0/20 : 0 (diphytanoyl), PS 14 : 0/14 : 0, PS
20 : 0/20 : 0 (diphytanoyl), PI 17 : 0/17 : 0, LPC 13 : 0, LPC
19 : 0, LPE 13 : 0, Cer d18:1/14 : 0, Cer d18 : 1/17 : 0, D7-FC,
CE 17 : 0, CE 22 : 0, TG 51 : 0, TG 57 : 0, DG 28 : 0, and DG
40 : 0. The chloroform phase was recovered by a pipetting
robot (Tecan Genesis RSP 150) and vacuum dried. The residues were dissolved in either 10 mM ammonium acetate inmethanol/chloroform (3 : 1, v/v) (for low mass resolution tandem mass spectrometry) or chloroform/methanol/2-propanol(1 : 2 : 4, v/v/v) with 7.5 mM ammonium formate (for highresolution mass spectrometry).

4
Mass spectrometry analysis of lipids was performed bydirect ﬂow injection analysis (FIA) using a triple quadrupole
mass spectrometer (FIA-MS/MS; QQQ triple quadrupole)
and a hybrid quadrupole-Orbitrap mass spectrometer(FIA-FTMS; high mass resolution).
FIA-MS/MS (QQQ) was performed in positive ion modeusing the analytical setup and strategy described previously[33]. A fragment ion of m/z 184 was used for lysophosphatidylcholine (LPC) [34]. The following neutral losses wereapplied: phosphatidylethanolamine (PE) 141, phosphatidylserine (PS) 185, phosphatidylglycerol (PG) 189, and phosphatidylinositol (PI) 277 [35]. PE-based plasmalogens (PEP) were analyzed according to the principles described by
Zemski Berry and Murphy [36]. Sphingosine-based ceramides (Cer) and hexosylceramides (HexCer) were analyzed
using a fragment ion of m/z 264 [37]. Quantiﬁcation wasachieved by calibration lines generated by addition ofnaturally occurring lipid species to the respective sample
matrix. Calibration lines were generated for the following
naturally occurring species: PC 34 : 1, 36 : 2, 38 : 4, 40 : 0,
and PC O-16 : 0/20 : 4; SM 18 : 1; O2/16 : 0, 18 : 1, and 18 : 0;
LPC 16 : 0, 18 : 1, and 18 : 0; PE 34 : 1, 36 : 2, 38 : 4, and40 : 6; PE P-16 : 0/20 : 4; PS 34 : 1, 36 : 2, 38 : 4, and 40 : 6; Cer
18 : 1; O2/16 : 0, 18 : 0, 20 : 0, 24 : 1, 24 : 0; and FC, CE 16 : 0,
18 : 2, 18 : 1, and 18 : 0.
The Fourier transform mass spectrometry (FIA-FTMS)
setup is described in detail in Höring et al. [38]. Triglycerides(TG), diglycerides (DG), and cholesteryl ester (CE) wererecorded in positive ion mode FTMS in range m/z 500-1000
for 1 min with a maximum injection time (IT) of 200 ms, anautomated gain control (AGC) of 1 × 106 , three microscans,and a target resolution of 140,000 (at m/z 200). Phosphatidylcholine (PC) and sphingomyelin (SM) were measured in anegative ion mode with a mass range m/z 520-960. Multiplexed acquisition (MSX) was used for the [M+NH4]+ of freecholesterol (FC) (m/z 404.39) and D7-cholesterol (m/z411.43) 0.5 min acquisition time, with a normalized collisionenergy of 10%, an IT of 100 ms, AGC of 1 × 105 , isolation window of 1 Da, and a target resolution of 140,000 [39]. Data processing details were described in Höring et al. [38] using the
ALEX software [40] which includes peak assignment andintensity picking. The extracted data were exported to Microsoft Excel 2016 and further processed by self-programmed
Macros. FIA-FTMS quantiﬁcation was performed by multiplication of the spiked IS amount with the analyte-to-IS ratio.
The lipid species were annotated according to the latest
proposal for shorthand notation of lipid structures that arederived from mass spectrometry [41]. For QQQ glycerophospholipid species, annotation was based on the assumption of even numbered carbon chains only. Concentration
data are available in the supplementary ﬁle.
2.12. Inhibition of Fatty Acid Synthesis. In order to analyze
the role of fatty acid synthesis on osteogenic diﬀerentiation,
DFCs were treated with 5 μg/ml fatty acid synthesis inhibitors C75 or 5-(tetradecyloxy)-2-furoic acid (TOFA, bothSigma-Aldrich) in diﬀerentiation or control medium. The
corresponding medium with 0.05% (v/v) DMSO was usedas vehicle control.

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2.13. Reverse Transcription—Quantitative Polymerase Chain
Reaction (RT-qPCR). The RNeasy® Plus Mini Kit (Qiagen)was used to isolate RNA from DFCs. Concentration andpurity of RNA were analyzed using a NanoDrop™ 2000
spectrophotometer (Thermo Fisher Scientiﬁc). Next, theiScript™ cDNA Synthesis Kit (BIO-RAD) was used to convert mRNA into cDNA (reverse transcription). The qPCR
analysis was performed with the SsoAdvanced™ Universal
Probes Supermix (BIO-RAD) and probe-containing PrimePCR™ primers (BIO-RAD) against GAPDH, RUNX2,
and COL1A2 (Collagen type 1 alpha 2 chain) on a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientiﬁc)with the following protocol: 2 min polymerase activation at
95°C, then 40 cycles of 5 sec denaturation at 95°C, and
30 sec annealing/elongation at 60°C. Target gene expression
was normalized to the expression of GAPDH and to the
DMEM control group by use of the ΔΔCt method [30].
2.14. Alkaline Phosphatase (ALP) Activity Assay. For examining
the ALP activity in DFCs, the cells were washed with PBS andlysed with Triton-X (0.1% in PBS). A portion of the lysate waspipetted in a fresh 96-well plate in order to measure the protein
concentration using the Pierce™ BCA Protein Assay Kit(Thermo Fisher Scientiﬁc). Besides, p-nitrophenylphosphate(Sigma-Aldrich) was added to the remaining lysates, whichthen incubated at 37°C for 60 min. Immediately thereafter, theconversion into yellow p-nitrophenol was quantiﬁed spectrophotometrically at OD = 415 nm. The ALP activity values
were normalized to the total protein of each sample and to
the DMEM control group.
2.15. Alizarin Red Staining. Mineralization of extracellularmatrix was analyzed by Alizarin Red staining. Cells were
washed with PBS and ﬁxed with 4% formalin. Subsequently,
cells were washed with H2O three times, stained with Alizarin Red Solution (Merck Millipore, Billerica, MA, USA) for
20 min, and washed again with H2O three times. Microscopic photographs were then taken and staining was quantiﬁed by dissolving Alizarin Red crystals in cetylpyridinium
chloride (10% in PBS) and measuring spectrophotometrically at OD = 595 nm. The obtained values were normalized
to the DMEM control group.
2.16. Statistical Analysis. Experiments were performed inbiological triplicates unless otherwise stated. Student’s
t-test and one-way analysis of variance (ANOVA) were performed with the software SPSS version 25 (IBM, Armonk,
NY, USA) as described in the ﬁgure legends. p values < 0:05
were considered statistically signiﬁcant and labelled as indicated in the ﬁgure legends.

3. Results
3.1. Carbon Metabolism Plays a Major Role during Osteogenic
Diﬀerentiation of DFCs. Aiming at further unraveling themolecular mechanisms behind osteogenesis of DFCs, former
microarray data were reanalyzed. The KEGG pathway analysis
was performed to determine pathways that are signiﬁcantly
regulated after both osteogenic induction with dexamethasone
and induction with BMP2. Amongst pathways related toStem Cells International
biosynthesis and metabolism of proteins, carbon metabolism
was signiﬁcantly addressed (Table 1), which conﬁrms thatenergy metabolism plays a major role during osteogenesis.

5
Table 1: Pathway enrichment analysis of genes signiﬁcantly
regulated by dexamethasone and BMP2.
% of regulated
genes

p value

Biosynthesis of amino acids

6.8

3:43E − 07

Glycine, serine, and threonine
metabolism

4.5

3:12E − 05

Biosynthesis of antibiotics

7.5

1:70E − 04

Aminoacyl-tRNa biosynthesis

3.8

3:57E − 03

Carbon metabolism

4.5

4:41E − 03

Protein digestion and absorption

3.8

9:86E − 03

Arginine biosynthesis

2.3

1:54E − 02

Alanine, aspartate, and glutamate
metabolism

2.3

4:40E − 02

KEGG pathway

3.2. Glycolysis and Citric Acid Cycle Are Regulated during
Osteogenic Diﬀerentiation of DFCs. Hence, we investigated
the signaling pathways involved in carbon metabolism,
which is highly correlated with the energy metabolism ofcells, more precisely. Starting with the glycolysis, weanalyzed the expression of the most important enzymes by
western blot analysis (Figure 1(a), upper part, and Supplementary Table 2). Protein expression of hexokinase I, hexokinase II,
phosphofructokinase, and lactate dehydrogenase A was
upregulated after osteogenic induction and more distinctly
with dexamethasone, except for lactate dehydrogenase A,
which was more upregulated by BMP2. In contrast, GAPDH
and pyruvate kinase were expressed rather consistently.
Additionally, the amount of produced L-lactate (Figure 1(b))
and hexokinase activity (Figure 1(c)) were signiﬁcantly
enhanced during osteogenic diﬀerentiation, which was again
more pronounced with dexamethasone. Moreover, enzymesof the subsequent citric acid cycle were examined (Figure 1(a)lower part and Supplementary Table 2). The western blot
analysis showed that expression of aconitase 2, isocitrate
dehydrogenase 2, DLST, fumarase, and citrate synthase wasupregulated at later stages of osteogenesis. In contrast, theenzymes isocitrate dehydrogenase 1, mitochondrial pyruvate
carrier 1, and mitochondrial pyruvate carrier 2 were expressedrather consistently. Besides the protein expression of citricacid cycle markers, the activity of malate dehydrogenase wassigniﬁcantly upregulated during osteogenic diﬀerentiation,with a higher increase after induction with dexamethasone(Figure 1(d)).
3.3. Mitochondrial Metabolism Is Highly Regulated duringDiﬀerentiation. Subsequently, we investigated the regulation of
mitochondrial markers during osteogenic diﬀerentiation of
DFCs (Figure 2). We found that protein expression of cytochrome c, pyruvate dehydrogenase, and SDHA was distinctly
upregulated, while the expression of HSP60 was slightly andnot signiﬁcantly upregulated, and the expression of prohibitin
1 and VDAC was downregulated after induction withdexamethasone. In contrast, these markers were less regulated
or even downregulated after BMP2 induction until day 14,
while the expression of cytochrome c, prohibitin 1, and SDHA
was signiﬁcantly upregulated at day 28. Besides, protein expression of COX IV was downregulated after seven days and upregulated at the later time points with both osteogenic inducers(Figure 2(a) and Supplementary Table 2). Additionally,
complex I activity in DFCs was enhanced after seven days
osteogenic diﬀerentiation, but only signiﬁcantly withdexamethasone treatment (Figure 2(b)). The relative amountof mitochondrial DNA in DFCs was regulated similarly to
COX IV expression: At days 1 and 7 during diﬀerentiation,
relative amount of mitochondrial DNA was signiﬁcantly
reduced, while signiﬁcantly upregulated at days 14 and 28
with both inducers (Figure 2(c)). Besides, protein expressionof HIF-1α was inhibited at all investigated time points(Figure 2(d)), which suggests that more oxygen species arepresent in the cells. Concordantly, a higher proportion ofoxidized glutathione was found in DFCs seven days after
osteogenic induction (Figure 2(e)), which indicates higher
oxidative stress.
3.4. Lipid Metabolism Is Regulated during Osteogenic
Diﬀerentiation of DFCs. Next, we explored the metabolismof lipids as another major type of carbon-based moleculesin cells. Initially, the expression of relevant enzymes involved
in lipid metabolism was analyzed by western blot analysis(Figure 3 and Supplementary Table 2). The results show
weak—and not signiﬁcant—upregulation of ATP-citratelyase and fatty acid synthase and signiﬁcant upregulationof acetyl-CoA-synthetase by both osteogenic inducers.
However, expression of phosphorylated ATP-citrate lyase
was only upregulated by dexamethasone and tendentiallydownregulated by BMP2, which indicates a higher enzyme
activation state in dexamethasone-treated DFCs [42]. Besides,
the expression of acetyl-CoA carboxylase is reduced at days
14 and 28, while no signiﬁcant upregulation of the Ser79
phosphorylation was observed at the later time points.
Instead, Ser79-phosphorylated acetyl-CoA carboxylase isupregulated by dexamethasone until day 14, however not
signiﬁcantly, which is an indicator for inhibition of thisenzyme [43]. At day 28, almost no phosphorylated acetylCoA carboxylase was detected in any sample. Additionally,
expression of ELOVL6 markedly increased in all culture media
until day 28, most distinctly in DMEM control medium, while
expression of acyl-CoA synthetase was unaltered during
diﬀerentiation. We next analyzed the lipidome of
diﬀerentiating DFCs by MS. In the overview, the sum of all
lipids was slightly enhanced—more pronounced withdexamethasone—during osteogenesis from day 7 (Figure 4(a)).
However, some particular lipid classes were more regulated
than others. Analyzing the proportions of the most abundantclasses of membrane lipids revealed that especially the
proportion of phosphatidylethanolamines (PE) varied during
diﬀerentiation (Figure 4(b)). Moreover, phosphatidylcholines(PC) showed a similar regulation pattern, but less distinctly(Figure 4(c)). Interestingly, the fraction of PE and PC wasmarkedly decreased in ODM-treated cells at day 14 compared6

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Day 1

Day 7

DMEM ODM BMP2DMEM ODM BMP2

Day 14

Day 28DMEM ODM BMP2 DMEM ODM BMP2

Hexokinase I
Hexokinase II
Phosphofructokinase
GAPDH

Glycolysis

Pyruvate kinase M1/2
Pyruvate kinase M2
Lactate dehydrogenase A
Aconitase 2
Isocitrate dehydrogenase 1
Isocitrate dehydrogenase 2
DLSTCitricacid cycle

Fumarase
Citrate synthase
Mitochondrialpyruvate carrier 1
Mitochondrialpyruvate carrier 2(a)L-lactate⁎⁎

1.5

Hexokinase activity
⁎⁎⁎

2.5

⁎⁎
Relative units

Relative amount

2

1
0.5

⁎⁎⁎

2
1.5
1
0.5
0

0
DMEM

ODM

BMP2

DMEM

(b)

ODM

BMP2

(c)

Malate dehydrogenase activity⁎⁎⁎⁎⁎

Relative units

2
1.5
1
0.5
0
DMEM

ODM

BMP2

(d)

Figure 1: Expression of glycolysis and citric acid cycle markers during osteogenic diﬀerentiation of DFCs. (a) DFCs were cultured in osteogenic
diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for one, seven, 14, and 28 days before protein
expression of the glycolysis markers hexokinase I, hexokinase II, phosphofructokinase, glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH),
pyruvate kinase M1/2, pyruvate kinase M2, and lactate dehydrogenase A, and protein expression of the citric acid cycle markers aconitase 2,
isocitrate dehydrogenase 1, isocitrate dehydrogenase 2, dihydrolipoamide succinyltransferase (DLST), fumarase, citrase synthase, mitochondrial
pyruvate carrier 1, and mitochondrial pyruvate carrier 2 were determined by western blot analysis. Quantiﬁcation results and statistical analysis
are shown in the Supplementary Table 2. (b–d) DFCs were cultured in osteogenic diﬀerentiation medium (ODM), BMP2 diﬀerentiation
medium, or control medium (DMEM) for seven days before the amount of produced L-lactate (b) and hexokinase activity (c) were determinedas markers of glycolysis and malate dehydrogenase activity was measured (d) as marker of citric acid cycle. Results are shown as means +standard deviation, and Student’s t-test was performed to compare diﬀerentiation medium with control medium. ∗∗ p < 0:01, ∗∗∗ p < 0:001.

Stem Cells International

7

Day 1

Day 7

Day 14

Day 28DMEM ODM BMP2DMEM ODM BMP2DMEM ODM BMP2DMEM ODM BMP2

Complex I activity
3

Cytochrome c⁎⁎⁎

COX IV
Pyruvate dehydrogenase
SDHA

Relative units

2.5

Prohibitin 1

2
1.5
1
0.5

HSP60

0
DMEM

VDAC

ODM

(a)

(b)

DMEM
mtDNA relative to 𝛽2 m (gDNA)

Mitochondrial DNA
100

⁎⁎
⁎⁎⁎

⁎⁎⁎

⁎

HIF-1α
ODM

BMP2

Day 1

Day 7

10
⁎

⁎
⁎

1

0.1

BMP2

DMEM

ODM

Day 1

BMP2 DMEM⁎⁎⁎
Day 14

ODM

BMP2 DMEM

Day 7

ODM

BMP2 DMEM

Day 14

ODM

BMP2

Day 28Day 28Relative units

(c)

(d)

Oxidative stress
⁎⁎

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
DMEM

ODM

BMP2

(e)

Figure 2: Evaluation of mitochondrial metabolism during osteogenic diﬀerentiation of DFCs. (a) DFCs were cultured in osteogenic
diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for one, seven, 14, and 28 days before
protein expression of cytochrome c, prohibitin 1, cytochrome c oxidase subunit 4 (COX IV), pyruvate dehydrogenase, succinate
dehydrogenase complex subunit A (SDHA), heat shock protein 60 (HSP60), and voltage-dependent anion channel (VDAC) was
determined by western blot analysis. Quantiﬁcation results and statistical analysis are shown in the Supplementary Table 2. (b) DFCs
were cultured in osteogenic diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for seven days
before complex I activity was determined in isolated mitochondria. (c, d) DFCs were cultured in osteogenic diﬀerentiation medium(ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for one, seven, 14, and 28 days before the amount of mitochondrial
DNA relative to genomic DNA was measured by qPCR analysis (c) and protein expression of HIF-1α was determined by western blot
analysis (n = 1, d). (e) DFCs were cultured in osteogenic diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control
medium (DMEM) for seven days. The amount of oxidized glutathione from total glutathione was measured as indicator for oxidative
stress. Results are shown as means + standard deviation, and Student’s t-test was performed to compare diﬀerentiation medium with
control medium at the same time point (b, c, e). ∗ p < 0:05, ∗∗ p < 0:01, ∗∗∗ p < 0:001.

to days 7 and 28. In addition, the proportion ofphosphatidylinositols (PI) was steadily upregulated duringosteogenic diﬀerentiation from day 7 (Figure 4(d)). Besides, thelipid classes phosphatidylserines (PS), PE-based plasmalogens(PE P), PC-ethers (PC O), sphingomyelins (SM), and freecholesterol (FC) were less regulated (Figures 4(e)–4(i)).

Regulation patterns of less abundant lipid classes are shownin Supplementary Figure S1.
3.5. The Composition of Lipid Species Is Altered during
Osteogenic Diﬀerentiation. In order to analyze the regulationof lipids in more detail, we investigated the composition of8

Stem Cells International
Day 1
DMEM ODM BMP2

Day 7

Day 14

Day 28DMEM ODM BMP2 DMEM ODM BMP2 DMEM ODM BMP2

Acetyl-CoA carboxylase
P-Acetyl-CoA carboxylase
ATP-citrate lyase
P-ATP-citrate lyase
Acetyl-CoA synthetase
Acyl-CoA synthetase
Fatty acid synthase
ELOVL6

Figure 3: Expression of lipid metabolism markers during osteogenic diﬀerentiation of DFCs. DFCs were cultured in osteogenic
diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for one, seven, 14, and 28 days before protein
expression of acetyl-CoA carboxylase, phospho-acetyl-CoA carboxylase, ATP-citrate lyase, phospho-ATP-citrate lyase, acetyl-CoA synthetase,acyl-CoA synthetase, fatty acid synthase, and elongation of very long chain fatty acids protein 6 (ELOVL6) was determined by western blot
analysis. Quantiﬁcation results and statistical analysis are shown in the Supplementary Table 2.

PEs—as highly regulated membrane lipid—during osteogenic diﬀerentiation and found a heterogeneous regulationof PEs with diﬀerent numbers of C-atoms and double bonds(Figure 5). While almost no changes were detected at day 1(Figures 5(a) and 5(e)), lipid species were highly regulated
at day 7: PEs with 32, 34, and 36 C-atoms and with one,
two, or three double bonds were distinctly upregulated,which was accompanied by a decrease of PEs with 40 Catoms and polyunsaturated PEs with four, ﬁve, or six double
bonds (Figures 5(b) and 5(f)). The same regulation pattern
was observable at day 14 (Figures 5(c) and 5(g)). More precisely, PE species 32 : 1, 34 : 1, 34 : 2, 36 : 2, 36 : 3, 38 : 2, and
38 : 3 were upregulated at days 7 and 14 and PE 40 : 3 only
at day 14, while the species 38 : 6, 40 : 5, and 40 : 6 were
downregulated at both days 7 and 14, PE 36 : 1, 38 : 1, and40 : 4 only at day 7, and PE 38 : 4 and 38 : 5 only at day 14(Supplementary Figure S2). It is worth noting that thechanges were usually more pronounced in dexamethasoneinduced DFCs compared to osteogenic induction with
BMP2. At day 28, the composition of PE species indexamethasone-induced cells was similar to days 7 and 14,
with the only diﬀerence that PEs with 38 C-atoms wereupregulated additionally. In BMP2-treated DFCs, PEs with
38 C-atoms were also elevated, as well as PEs with two,
three, or four double bonds, while species with 40 C-atoms
and with ﬁve or six double bonds were downregulated atday 28 (Figures 5(d) and 5(h)).
3.6. Storage Lipids and Diacylglycerol Are Highly Regulated
during the Osteogenic Diﬀerentiation of DFCs. Furthermore,
we investigated the regulation of storage lipids and diacylglycerols (DG), as DGs are precursors for triacylglycerols (TG)and glycerophospholipids, during the osteogenic diﬀerentiation of DFCs (Figure 6). The proportion of DGs was initiallydecreased compared to the control medium at day 1, while it
was steadily increasing at the later time points and more
distinctly in dexamethasone-treated cells (Figure 6(a)).
Regarding the regulation of triacylglycerols, a decrease in com-pare to control medium is observable later, namely, at day 7,
while upregulated at days 14 and 28, except for
dexamethasone-treated DFCs, where the proportion of TGs isdecreased again at day 28 (Figure 6(b)). By contrast, cholesterylesters (CE) are constantly downregulated compared to control
medium from day 7 after osteogenic induction (Figure 6(c)). As
the DG fraction is highly increased in dexamethasone-treated
DFCs, we investigated the DG species proﬁle at the diﬀerent
time points in detail (Figure 6(d)). The results show that monounsaturated DGs with 34 and 36 C-atoms are the most abundant species at days 1 and 7, while polyunsaturated DGs with
38 C-atoms are dominant at days 14 and 28. We also observed
the species proﬁle of TGs in dexamethasone-treated cells andconcordantly found more species with lower numbers of Catoms and double bonds at the earlier time points, while polyunsaturated species with a higher number of C-atoms weremore abundant at days 14 and 28 (Figure 6(e)).
3.7. Inhibition of Fatty Acid Synthesis Impedes the Osteogenic
Diﬀerentiation of DFCs. As lipid concentrations were elevatedafter osteogenic induction, further experiments were performed
investigating the role of fatty acid synthesis for the osteogenic
diﬀerentiation of DFCs. Cells were treated with either TOFAor C75, which act as inhibitors of fatty acid synthesis by inhibiting the enzymes acetyl-CoA carboxylase or fatty acid synthase,respectively [44–46]. Treating DFCs with TOFA resulted in ahigher phosphorylation of acetyl-CoA carboxylase, whichindicates inactivation of the enzyme [43], while C75 treatment
reduced the expression of fatty acid synthase (Supplementary
Figure S3). Subsequent analysis of osteogenic markers
showed that gene expression of COL1A2 as well as ALP
activity and Alizarin Red staining were reduced after
inhibitor treatment, more distinctly and throughout
signiﬁcantly with TOFA (Figures 7(a), 7(c), and 7(d)).
However, gene expression of RUNX2 was only reduced byfatty acid synthesis inhibition in DMEM control medium,
while unaltered or even elevated in the diﬀerentiation media(Figure 7(b)).

Stem Cells International

9
PE
12

⁎

Fraction of total
lipid sum (%)

Lipid concentration(nmol/mg protein)

Lipid sum
400
350
300
250
200
150
100
50
0

10

⁎
⁎

⁎⁎⁎
⁎⁎⁎

⁎⁎⁎
⁎⁎⁎

⁎

Day 1

Day 7

Day 14

Day 28

⁎⁎
⁎⁎

⁎⁎
⁎⁎

Day 14

Day 28⁎⁎⁎⁎⁎⁎Day 14

Day 28

8
6
4
2
0

Day 1

Day 7

Day 14

Day 28

DMEM
ODM
BMP2

DMEM
ODM
BMP2
(a)

Fraction of total
lipid sum (%)

25

PC

⁎⁎
⁎⁎⁎

⁎⁎
⁎

12
⁎⁎

20
15
10
5

Fraction of total
lipid sum (%)

⁎⁎
⁎⁎⁎

30

(b)

0

10
8
6
4
2
0

Day 1

Day 7

Day 14

Day 28

Day 1

DMEM
ODM
BMP2

(d)

⁎

⁎

⁎⁎
⁎

⁎⁎⁎
⁎⁎⁎

12
Fraction of total
lipid sum (%)

Fraction of total
lipid sum (%)

PS
10
9
8
7
6
5
4
3
2
1
0

Day 7

DMEM
ODM
BMP2
(c)

10
8

PE P

⁎

6
4
2
0

Day 1

Day 7

Day 14

Day 1

Day 28

DMEM
ODM
BMP2

Day 7

DMEM
ODM
BMP2
(e)

(f)

PC O
9
8
7
6
5
4
3
2
1
0

⁎⁎

Day 1

Day 7

SM
⁎⁎⁎

⁎⁎

Day 14

Day 28Fraction of total
lipid sum (%)

Fraction of total
lipid sum (%)

PI

10
9
8
7
6
5
4
3
2
1
0

⁎⁎

Day 1

⁎⁎⁎

⁎⁎

Day 7

Day 14

DMEM
ODM
BMP2

DMEM
ODM
BMP2
(g)

(h)

Figure 4: Continued.

⁎

Day 28

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Stem Cells International

Fraction of total
lipid sum (%)

FC
35
30
25
20
15
10
5
0

⁎⁎

Day 1

Day 7

Day 14⁎

Day 28(i)

Figure 4: Regulation of membrane lipids during osteogenic diﬀerentiation of DFCs. DFCs were cultured in osteogenic diﬀerentiation
medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for one, seven, 14 and 28 days before the lipidome wasanalyzed by MS. This ﬁgure shows the regulation of total lipids (a) and the fraction of phosphatidylethanolamines (PE, b),phosphatidylcholines (PC, c), phosphatidylinositols (PI, d), phosphatidylserines (PS, e), phosphatidylethanolamine-based plasmalogens(PE P, f), phosphatidylcholine-ethers (PC O, g), sphingomyelins (SM, h), and free cholesterol (FC, i). Data points show means ±standard deviation, and Student’s t-test was performed to compare diﬀerentiation medium with control medium at the same time point.
∗
p < 0:05, ∗∗ p < 0:01, ∗∗∗ p < 0:001. Asterisks relate to the diﬀerentiation medium with data points and line in same color.

4. Discussion
The results of this study showed that energy metabolism ishighly regulated after osteogenic induction in DFCs. Against
previous ﬁndings reporting that only oxidative phosphorylation and not glycolysis are upregulated [16], our experiments
showed induction of some glycolysis markers as well. Presumably, glycolytic energy production is not dispensable for osteogenesis. Concordantly, a previous study showed that miR-34a
suppresses the osteogenic diﬀerentiation of MSCs by the downregulation of lactate dehydrogenase A [47], which was upregulated during osteogenesis in DFCs. Nonetheless, mitochondrial
markers were also regulated in DFCs and analysis of mitochondrial DNA copy number revealed a high upregulation in thesecond half of the diﬀerentiation process. This is accompanied
by inhibition of HIF-1α expression and a more oxidized cellular state. Downregulation of HIF-1α was also reported duringosteogenic diﬀerentiation of bone marrow-derived MSCs,
while reactivation of HIF-1α inhibits oxidative phosphorylation [16]. Interestingly, inhibition of HIF-1α expression occurs
directly after osteogenic induction in DFCs, while mitochondrial DNA is even downregulated at days 1 and 7. Probably,other molecular mechanisms are important for the inductionof diﬀerentiation, while a suﬃcient energy supply and oxidative
phosphorylation activity are necessary in later stages when premature osteoblasts lead to biomineralization of extracellularmatrix. However, a higher energy demand is probably not the
only purpose of enhanced mitochondrial activity during osteogenesis, since a previous study reported that inhibition of oxidative phosphorylation can indeed be compensated byglycolysis, which maintains the ATP level, but still impedes
osteogenic diﬀerentiation [20]. Against this ﬁnding, inductionof pseudohypoxia enhanced the proliferation and also the osteogenic diﬀerentiation of PDL cells while concurrently shiftingenergy metabolism from oxidative phosphorylation to glycolysis [48]. Hence, glycolytic metabolism is not necessarily related
to impeded diﬀerentiation. Upregulation of mitochondrial
activity is further reported to depend on Akt [49], which ishighly regulated during osteogenesis in DFCs [13]. The results
indicate that mitochondrial upregulation plays a pivotal anddiverse role in the molecular clockwork of osteogenic diﬀerentiation and that energy metabolism aﬀects osteogenesis in more
respects than only ATP supply.
The results further revealed diﬀerences between dexamethasone and BMP2 induction. With regard to mitochondrial
markers, it is noticeable that admittedly both inducers lead toupregulation of mitochondrial DNA copy number from day
14 but upregulate diﬀerent proteins as dexamethasone predominantly enhanced expression of cytochrome c and pyruvate
dehydrogenase, while BMP2 induced the expression of prohibitin 1. Diﬀerences in carbon metabolism comparing osteogenic
induction with dexamethasone or BMP2 were also detected in
MSCs [50]. The observations strengthen the hypothesis thatboth inducers lead to the same outcome—upregulation of mitochondrial activity, a more oxidative state, inhibition of HIF-1α,
and ﬁnally matrix mineralization—but function via regulating
diﬀerent proteins and signaling pathways, which are worth tobe further explored in future studies.
Analysis of enzymes concerning lipid metabolism also
showed high regulation after osteogenic induction with both
inducers including upregulation of some important markers
for fatty acid metabolism. This is in line with the MS analysisof the lipidome which concurrently showed higher lipid concentrations. Overall, regulation of lipidome was similar withboth osteogenic inducers, although more pronounced in
dexamethasone-treated DFCs as the expression of related
enzymes and the concentration of lipids were higher here.
This is very interesting with regard to the previous observation that dexamethasone-induced mineralization is more
intense compared to BMP2-treated DFCs [13]. The transcription factor ZBTB16, whose expression is highly induced
by dexamethasone [11], might play a role for this diﬀerence
as it is also related to energy metabolism [51]. A study on
rabbit MSCs could further show that the lipidome changes
during osteogenic diﬀerentiation of precursor cells and,moreover, that lipidomes are similar in cells from diﬀerent
origins [52]. This implies a fundamental role of lipid metabolism for osteogenesis. Detailed analysis with the example ofPE species revealed that especially species with a number of
C-atoms until 36—which mainly corresponds to PEs with

11

Fraction of total PE (%)

Stem Cells International
PE day 1

50
45
40
35
30
25
20
15
10
5
0

⁎

⁎⁎

⁎
⁎⁎⁎
⁎⁎
⁎⁎
C32

C34

C36

C38

C40

C42

DMEM
ODM
BMP2
(a)

PE day 7

45⁎⁎

Fraction of total PE (%)

40
35

⁎⁎⁎
⁎⁎⁎

⁎⁎⁎
⁎⁎

30
25
20

⁎⁎⁎
⁎⁎⁎

15
10

⁎⁎⁎
⁎⁎⁎

5

⁎⁎⁎
⁎

0
C32

C34

C36

C38

C40

C42

DMEM
ODM
BMP2
(b)

PE day 14

45
Fraction of total PE (%)

40

⁎⁎⁎
⁎⁎⁎

35
⁎⁎⁎
⁎⁎

30
25
20
15
10
5

⁎⁎⁎
⁎⁎⁎

⁎⁎⁎
⁎⁎⁎
⁎⁎

0
C32

C34

C36

C38

DMEM
ODM
BMP2
(c)

Figure 5: Continued.

C40

C42

Stem Cells International

Fraction of total PE (%)

12

50
45
40
35
30
25
20
15
10
5
0

PE day 28
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎
⁎⁎⁎
⁎⁎⁎

⁎⁎⁎
⁎⁎

C32

C34

C36

C38

C40

C42

DMEM
ODM
BMP2
(d)

PE day 1

Fraction of total PE (%)

30
25
⁎

20

⁎
⁎⁎⁎
⁎⁎

15

⁎⁎

10
5
0
DB0

DB1

DB2

DB3

DB4

DB5

DB6

DB7

DMEM
ODM
BMP2
(e)

PE day 7

Fraction of total PE (%)

30

⁎⁎⁎
⁎⁎⁎

25

⁎⁎⁎
⁎⁎⁎

20

⁎⁎⁎
⁎⁎⁎

⁎⁎⁎
⁎⁎⁎

⁎
⁎
⁎⁎⁎
⁎⁎

15
10
5

⁎⁎

0
DB0

DB1

DB2

DB3

DB4

DMEM
ODM
BMP2
(f)

Figure 5: Continued.

DB5

DB6

DB7

Stem Cells International13
PE day 14

Fraction of total PE (%)

30

⁎⁎⁎
⁎⁎⁎

⁎⁎⁎

25

⁎⁎⁎
⁎⁎⁎

⁎⁎⁎
⁎⁎⁎

⁎⁎⁎
⁎⁎⁎

20

⁎⁎⁎
⁎⁎⁎

15
10
5

⁎

0
DB0

DB1

DB2

DB3

DB4

DB5

DB6

DB7

DMEM
ODM
BMP2
(g)

PE day 28

Fraction of total PE (%)

30

⁎⁎⁎
⁎⁎⁎

25
⁎⁎

20

⁎⁎⁎
⁎⁎⁎

⁎⁎
⁎⁎

⁎⁎⁎
⁎⁎

⁎⁎⁎
⁎⁎⁎

15
10
5

⁎⁎
⁎

0
DB0

DB1

DB2

DB3

DB4

DB5

DB6

DB7

DMEM
ODM
BMP2
(h)

Figure 5: Regulation of phosphatidylethanolamines with diﬀerent numbers of C-atoms and double bonds during osteogenic diﬀerentiationof DFCs. DFCs were cultured in osteogenic diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for
one, seven, 14, and 28 days before the lipidome was analyzed by MS. This ﬁgure shows the regulation of phosphatidylethanolamines (PE)with diﬀerent C-atom numbers at day 1 (a), day 7 (b), day 14 (c), and day 28 (d), and PEs with diﬀerent numbers of double bonds (DB) at
day 1 (e), day 7 (f), day 14 (g), and day 28 (h). Results are shown as means + standard deviation, and Student’s t-test was performed tocompare diﬀerentiation medium with the control medium at the same time point. ∗ p < 0:05, ∗∗ p < 0:01, ∗∗∗ p < 0:001. Asterisks relate to
the diﬀerentiation medium with bars in same color.

two fatty acids with each 18 C-atoms—and a higher degreeof saturation was increased at day 7, which indicates thatde novo synthesis is the main source for elevated lipid levels.
Concordantly, PE species with C-atom numbers until 38 andmore double bonds were found until day 28, presuming
chain elongation and desaturation following synthesis. This
assumption is in accordance with the observation that
expression of ELOVL6, which is known to elongate fatty
acids with up to 16 C-atoms into species with up to 18 Catoms [53], was increased over time.
Eventually, inhibition of fatty acid synthesis resulted in
downregulation of most investigated osteogenic markers.
Especially COL1A2 gene expression, ALP activity, andAlizarin Red staining were impacted, which are important
markers for extracellular matrix production and mineralization. Besides, gene expression of RUNX2 was less aﬀected.

Interestingly, ALP activity and RUNX2 expression werebarely upregulated in diﬀerentiation media compared with
control medium. This implies that the DFCs used in ourstudy already exhibit a high basal expression of thesemarkers, which is probably associated with a high osteogenic
potential. Thus, further regulation of RUNX2 and ALP
might not be required to drive the diﬀerentiation process.
However, as the inhibition of fatty acid synthesis impedes
mineralization but not RUNX2 expression, the relevance oflipid metabolism for osteogenesis is likely not connected to
RUNX2. Instead, de novo synthesis of fatty acids and lipids
is presumably required for proper function of osteogenicallyinduced DFCs to produce hard tissue. Fatty acid synthesis in
eukaryotic cells occurs in both the cytoplasm and mitochondria [54]. Hence, enhanced mitochondrial activity might be
an important driver for lipogenesis during the diﬀerentiation.

Stem Cells International

Fraction of total lipid sum (%)

14
DG

1.6

⁎⁎⁎
⁎⁎⁎

1.4
1.2
1

⁎
⁎

⁎⁎⁎
⁎

0.8
0.6
0.4
0.2
0
Day 1

Day 7

Day 14

Day 28

DMEM
ODM
BMP2
(a)

TG

Fraction of total lipid sum (%)

8

⁎⁎⁎
⁎⁎

7

⁎
⁎⁎

6
⁎⁎
⁎⁎

5
4
3
2
1
0
Day 1

Day 7

Day 14

Day 28Day 14

Day 28

DMEM
ODM
BMP2

Fraction of total lipid sum (%)

(b)

CE

2.5
2
1.5
1
0.5
0
Day 1

Day 7

DMEM
ODM
BMP2
(c)

Figure 6: Continued.

Stem Cells International

15
DG species profile after ODM treatment

Fraction of total DG (%)

70
60

⁎⁎

50

⁎⁎⁎

40
30

⁎⁎⁎

20
⁎⁎⁎

10

⁎⁎

⁎⁎⁎

0
DG
32:0

DG
32:1

DG
34:1

DG
34:2

DG
36:1

DG
36:2

DG
36:4

DG
38:2

DG
38:3

DG
38:4

DG
38:5

Day 14
Day 28

Day 1
Day 7
(d)

TG species profile aft er ODM treatment

Fraction of total TG (%)

30

⁎⁎⁎

25
20

⁎⁎

15
10

⁎⁎⁎

⁎⁎⁎
⁎⁎⁎

5

⁎⁎⁎
⁎⁎

⁎⁎⁎
⁎⁎

⁎⁎⁎

⁎⁎⁎

⁎⁎⁎

⁎⁎

⁎⁎⁎

⁎⁎⁎
⁎⁎⁎

⁎⁎⁎

⁎⁎⁎ ⁎⁎⁎⁎⁎⁎⁎⁎⁎0
TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG TG
40:3 44:0 46:0 46:4 48:0 48:1 48:4 50:1 50:2 51:2 52:1 52:2 52:3 54:2 54:3 54:4 56:2 56:3 56:4 56:5 56:6 58:3 58:4
Day 1
Day 7

Day 14
Day 28(e)Figure 6: Regulation of storage lipids and diacylglycerol during osteogenic diﬀerentiation of DFCs. DFCs were cultured in osteogenic
diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for one, seven, 14, and 28 days before thelipidome was analyzed by MS. (a–c) The line charts show the fraction of diacylglycerols (DG, a), triacylglycerols (TG, b), and cholesterylesters (CE, c). Data points show means ± standard deviation, and Student’s t-test was performed to compare diﬀerentiation medium with
control medium at the same time point. ∗ p < 0:05, ∗∗ p < 0:01, ∗∗∗ p < 0:001. Asterisks relate to the diﬀerentiation medium with data
points and line in same color. (d, e) Furthermore, the composition of DG species (d) and TG species (e) in cells treated with ODM at
the diﬀerent time points is shown. Only species with an abundance of more than 2% for at least one time point are shown as means +standard deviation, and one-way ANOVA was performed to determine signiﬁcant diﬀerences in the fraction of a speciﬁc species at
diﬀerent time points. ∗∗ p < 0:01, ∗∗∗ p < 0:001.

The deﬁnite role of enhanced lipid production for biomineralization remains elusive and probably has diﬀerent
functions for the mineralization process. Predominantly,
cells might use fatty acids as energy source, which was also
reported by a previous study [55]. This is further supportedby the fact that the fraction of DGs was elevated from day 7,
as intermediate products, and eventually TGs at day 14,
whose role is predominantly deﬁned as energy storage[56]. Concordantly, DG species 32 : 0, 32 : 1, and 34 : 1—presumably due to de novo synthesis—showed peaks at day 7
during dexamethasone-induced diﬀerentiation. However,
DG species 38 : 3 and 38 : 4 and TG species 56 : 5 and 56 : 6
were highly induced at days 14 and 28. Presumably, upregulation of polyunsaturated species occurred due to uptake
from the culture medium in order to enhance the cellularenergy resources.

Interestingly, concentration of DGs at day 1 was lower inthe diﬀerentiation media compared to control medium. As
DGs are an important cofactor for activation of classical PKC
isoforms [57], which inhibit the osteogenic diﬀerentiation of
DFCs [13], downregulation of DGs right after osteogenic
induction might be important to impede PKC activation. Furthermore, it is worth noting that cholesteryl esters are the only
lipid class that is strictly downregulated during diﬀerentiationcompared to control medium. By contrast, the proportion offree cholesterol is constantly high. Remaining a stable level offree cholesterol might play another pivotal role during diﬀerentiation. Previous studies showed that oxysterols—oxidized
derivatives of cholesterol—support the osteogenic diﬀerentiation of stem cells and counteract negative impacts of oxidative
stress [58, 59]. Thus, the abundance of free cholesterol together
with enhanced mitochondrial activity might be an important

16

Stem Cells International
COL1A22.5

Relative gene expression

⁎⁎⁎
2
⁎⁎⁎
⁎

⁎⁎⁎

1.5
⁎⁎
1
0.5
0

CTRL

C75

TOFA

CTRL

DMEM

C75

TOFA

CTRL

C75

ODM

TOFA

BMP2

(a)

RUNX2
1.4

⁎⁎

1
0.8
0.6
0.4
0.2
0

CTRL

C75

TOFA

CTRL

C75

TOFA

ODM

DMEM

CTRL

C75

TOFA

BMP2

(b)

1.6
1.4
Relative ALP activity

Relative gene expression

⁎⁎

⁎⁎⁎

1.2

ALP activity
⁎

⁎⁎

⁎⁎⁎

1.2
1
0.8
0.6
0.4
0.2
0
CTRL C75 TOFA CTRL C75 TOFA CTRL C75 TOFA
ODM

DMEM
(c)

Figure 7: Continued.

BMP2

Stem Cells International

17
Alizarin red staining⁎⁎⁎⁎

Relative units

100

⁎⁎

10

⁎

⁎⁎⁎
⁎⁎
1

0.1

CTRL C75 TOFACTRL C75 TOFA

DMEM

ODM

CTRL

C75

TOFA

BMP2

(d)

Figure 7: Regulation of osteogenic diﬀerentiation in DFCs after inhibition of fatty acid synthesis. DFCs were cultured in osteogenic
diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) and simultaneously treated with 5 μg/ml
fatty acid synthesis inhibitors TOFA or C75 or vehicle control for diﬀerent time periods before evaluation of osteogenic diﬀerentiation
markers: (a, b) Gene expressions of COL1A2 (a) and RUNX2 (b) relative to gene expression of GAPDH were determined after threedays treatment by RT-qPCR. (c) Activity of alkaline phosphatase (ALP) relative to total protein was measured after seven days
treatment. (d) Mineralization of extracellular matrix was determined by Alizarin Red staining after 28 days treatment. Photographic
pictures of the staining are shown below the bar chart which shows the relative quantiﬁcation results as means + standard deviation.
Student’s t-tests were performed to compare inhibitor treatment with the vehicle control. ∗ p < 0:05, ∗∗ p < 0:01, ∗∗∗ p < 0:001.

mechanism to support the osteogenic diﬀerentiation of DFCs.
Mitochondrial activity, a more oxidative cellular state andupregulation of free cholesterol together could be anothermechanism that contributes to osteogenesis. Further research
will be required to evaluate the signiﬁcance of those pathways
for osteogenesis.

5. Conclusion
Energy metabolism is highly regulated during osteogenic differentiation of DFCs. Several enzymes involved in glycolysis,
citric acid cycle, mitochondrial respiratory chain, and fatty
acid metabolism are upregulated upon osteogenic induction,
more notably by dexamethasone, accompanied by a moreoxidative cellular state. Eventually, analysis of lipidome
showed higher lipid concentrations with essential changes
in the composition of lipid species, while inhibition of fatty
acid synthesis impeded osteogenesis. The results demonstrate that energy metabolism plays a vital role during theosteogenic diﬀerentiation of DFCs.

Abbreviations
AGC:
ALP:
AMPK:
ANOVA:
BMP2:
BSA:
CE:
Cer:
COL1A2:

Automated gain control
Alkaline phosphatase
AMP-activated protein kinase
Analysis of variance
Bone morphogenetic protein 2
Bovine serum albumin
Cholesteryl ester
Ceramides
Collagen type 1 alpha 2 chain

COX IV:
DB:
DFC:
DG:
DLST:
DLX3:
DMEM:
ELOVL6:
FBS:
FC:
FIA:
FTMS:
GAPDH:
HexCer:HIF-1α:HRP:
HSP60:
IT:
KEGG:
LPC:
LPE:
MS:
MSC:
MSX:
ODM:
PBS:
PC:
PC O:
PE:
PE P:
PG:

Cytochrome c oxidase subunit 4
Double bonds
Dental follicle cell
Diglycerides
Dihydrolipoamide succinyltransferase
Distal-less homeobox 3
Dulbecco’s modiﬁed Eagle’s medium
Elongation of very long chain fatty acids protein 6
Fetal bovine serum
Free cholesterol
Flow injection analysis
Fourier transform mass spectrometry
Glycerinaldehyd-3-phosphat-dehydrogenase
Hexosylceramides
Hypoxia-inducible factor 1-alphaHorseradish peroxidase
Heat shock protein 60
Injection time
Kyoto Encyclopedia of Genes and Genomes
Lysophosphatidylcholine
Lysophosphatidylethanolamine
Mass spectrometry
Mesenchymal stem cell
Multiplexed acquisition
Osteogenic diﬀerentiation medium
Phosphate-buﬀered saline
Phosphatidylcholine
Phosphatidylcholine-ether
Phosphatidylethanolamine
PE-based plasmalogens
Phosphatidylglycerol

18
PI:
Phosphatidylinositol
PKC:
Protein kinase C
PS:
PhosphatidylserineqPCR:
Real-time quantitative polymerase chain reaction
ROS:
Reactive oxygen species
RUNX2:
Runt-related transcription factor 2
SDHA:
Succinate dehydrogenase complex subunit A
SDS:
Sodium dodecyl sulfate
SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gelelectrophoresis
SM:
Sphingomyelin
TBS:
Tris-buﬀered saline
TBST:
Tris-buﬀered saline with Tween20
TG:
Triglycerides
TOFA:
5-(Tetradecyloxy)-2-furoic acid
VDAC:
Voltage-dependent anion channel
ZBTB16:
Zinc ﬁnger and BTB domain-containing 16

Data Availability
The lipidome concentration data are available as supplementary ﬁle. The other raw data are available from the authors
on reasonable request.

Ethical Approval
Human dental follicle cells for this study were purchasedcommercially.

Conflicts of Interest
The authors declare that there is no conﬂict of interest
regarding the publication of this article.

Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, project number 319390412).

Supplementary Materials
Supplementary 1. List of genes signiﬁcantly regulated bydexamethasone and BMP2. Genes involved in the pathway
carbon metabolism are in bold type.
Supplementary 2. Quantiﬁcation results of western blots in
Figures 1(a), 2(a), and 3 with means ± standard deviationand molecular weight of antigens. Statistically signiﬁcant(p < 0:05) values compared to control medium (DMEM) at
the same time point are in bold type.
Supplementary 3. Regulation of less abundant lipid classes during osteogenic diﬀerentiation of DFCs. DFCs were cultured inosteogenic diﬀerentiation medium (ODM), BMP2 diﬀerentiation medium, or control medium (DMEM) for one, seven, 14,
and 28 days before the lipidome was analyzed by MS. This ﬁgure shows the fraction of lysophosphatidylethanolamines (LPE,a), lysophosphatidylcholines (LPC, b), ceramides (Cer, c), hexosylceramides (HexCer, d), and phosphatidylglycerols (PG, e).
Data points show means ± standard deviation, and Student’s

Stem Cells Internationalt-test was performed to compare diﬀerentiation medium
with control medium at the same time point. ∗ p < 0:05,∗∗
p < 0:01, ∗∗∗ p < 0:001. Asterisks relate to the diﬀerentiation
medium with data points and line in same color.
Supplementary 4. Species proﬁles of phosphatidylethanolamines during osteogenic diﬀerentiation of DFCs. Thisﬁgure shows the composition of phosphatidylethanolamine(PE) species analyzed by MS in DFCs cultured in osteogenic
diﬀerentiation medium (ODM), BMP2 diﬀerentiation
medium, or control medium (DMEM) at days 7 (a) and 14(b). Results are shown as means + standard deviation, andStudent’s t-test was performed to compare diﬀerentiation
medium with control medium at the same time point.
∗
p < 0:05, ∗∗ p < 0:01, ∗∗∗ p < 0:001. Asterisks relate to thediﬀerentiation medium with bars in same color.
Supplementary 5. Inhibition of fatty acid synthesis by TOFA
and C75 in DFCs. DFCs were treated with 5 μg/ml fatty acidsynthesis inhibitors TOFA (a) or C75 (b) or vehicle control
for two hours (a) or one day (b) before protein expressionof phospho-acetyl-CoA carboxylase (a) or fatty acid synthase (b) was determined by western blot analysis. Results
are shown as means + standard deviation, and Student’s
t-test was performed to compare inhibitor treatment withthe vehicle control. ∗ p < 0:05.

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