Abstract
Joint disorders have become a global health issue with the growth of
the aging population. Screening small active molecules targeting
chondrogenic differentiation of bone marrow-derived stem cells (BMSCs)
is of urgency. In this study, microfracture was employed to create a
regenerative niche in rabbits (n = 9). Cartilage samples were collected
four weeks post-surgery. Microfracture-caused morphological (n = 3) and
metabolic (n = 6) changes were detected. Non-targeted metabolomic
analysis revealed that there were 96 differentially expressed
metabolites (DEMs) enriched in 70 pathways involved in
anti-inflammation, lipid metabolism, signaling transduction, etc. Among
the metabolites, docosapentaenoic acid 22n-3 (DPA) and ursodeoxycholic
acid (UDCA) functionally facilitated cartilage defect healing, i.e.,
increasing the vitality and adaptation of the BMSCs, chondrogenic
differentiation, and chondrocyte functionality. Our findings firstly
reveal the differences in metabolomic activities between the normal and
regenerated cartilages and provide a list of endogenous biomolecules
potentially involved in the biochemical-niche fate control for
chondrogenic differentiation of BMSCs. Ultimately, the biomolecules may
serve as anti-aging supplements for chondrocyte renewal or as drug
candidates for cartilage regenerative medicine.
Keywords: metabolomics, endogenous biomolecules, bone marrow-derived
stem cells, chondrogenic differentiation, regeneration, cartilage
1. Introduction
Age-related changes in the chondrocyte renewal in the articular
cartilage predispose individuals to osteoarthritis [[38]1,[39]2].
Regenerative medicine aims to restore tissue functionality by
harnessing the differentiation potential of stem cells in tissue
replacement therapies [[40]3]. Microfracture (MF) is a commonly used
surgical method to stimulate cartilage regeneration, involving drilling
on the subchondral bone to access the resident bone marrow-derived stem
cells (BMSCs) [[41]4] and creating a biochemical niche to induce
chondrogenesis and sustain chondrocyte functionality [[42]5]. However,
the outcome of MF is correlated with age; i.e., greater improvement is
only shown in young patients [[43]6]. This may be due to BMSC aging,
failure in chondrogenic differentiation, and dysfunction of the
resident chondrocytes in old people [[44]7,[45]8]. Thus, the fate of
the activated BMSCs plays a determinative role in the modeling and
remodeling of cartilage, which are affected by both intrinsic and
extrinsic factors.
The biochemical niche which is created by a variety of small molecules
(metabolites) filling in the defect site in MF controls the fate of the
activated resident BMSCs [[46]9,[47]10]. For example, omega-3
polyunsaturated fatty acids (ω-3 PUFAs), including docosapentaenoic
acid 22n-3 (DPA) [[48]11], maintain the self-renewal of embryonic stem
cells [[49]12]. The amino acids, such as tryptophan, modulate the
senescence of mesenchymal stromal/stem cells (MSCs) by regulating
mitochondrial integrity and function [[50]13]. Yet, the excess reactive
oxygen species (ROS), oxidative markers, impair stem cell self-renewal
capacity [[51]1,[52]14,[53]15]; subsequentially, the ROS-induced DNA
damage causes replicative senescence in MSCs [[54]16]. The excess ROS
and related oxidative stress are correlated with the progression of
osteoarthritis, characterized by the changes in the hyaline cartilage
markers, i.e., an increased catabolism of proteoglycan aggrecan (ACAN)
and a loss of type II collagen (COL2A1) [[55]17]. Moreover, dietary
intake of ω-3 PUFAs attenuates osteoarthritis-associated cartilage
degradation [[56]18]. Hence, we hypothesize that the metabolic cues in
the regenerative niche contribute to BMSC activity and rebalance a
hostile joint environment, which is of paramount importance for the
fate control of BMSCs in chondrogenesis.
The aim of this study was to discover biomolecules with the potential
to serve as anti-aging supplements for chondrocyte renewal or as drug
candidates for cartilage regenerative medicine. In this study, MF was
employed in healthy rabbits to create a regenerative niche for BMSCs,
which maximizes the detection of the key components in cartilage
regeneration and excludes the background noise in deteriorative joints.
Non-targeted metabolomics was performed to determine the metabolic
differences between the normal (NOR) and regenerated (REG) cartilages.
A list of cartilage regeneration-related endogenous small molecules,
i.e., the differentially expressed metabolites (DEMs), were identified.
Two of the metabolites, DPA and ursodeoxycholic acid (UDCA), were
picked to determine their specific effects on cartilage regeneration,
i.e., BMSC vitality, chondrogenic differentiation, and chondrocyte
functionality, based on the chondrocyte-protective role of ω-3 PUFAs
[[57]19] and the chondrocyte-differentiation-facilitating role of
cholesterol (the substrate of UDCA) [[58]20].
2. Materials and Methods
2.1. Rabbit Microfracture Model
The animal study was approved by the Ethics Committee of Experimental
Animals of the Affiliated Hospital of Qingdao University (No.
AHQU-MAL20210419). A self-controlled design was used in this study.
Male New Zealand white rabbits (10–12 weeks in age, 2.0–2.5 kg in
weight, n = 9) were anesthetized through ear vein administration of 3%
w/v pentobarbital sodium at a dosage of 1.0 mg/kg. After sterilization,
a 2–3 cm anteromedial parapatellar incision was made on the left knee,
and the patella was everted. An articular cartilage defect (Ø ~ 4 × 6
mm, 2 mm in depth) was created on the trochlear groove of the left
distal femur with a sterilized cranial drill bit (ØO = 2.1 mm, RWD,
Shenzhen, China) ([59]Figure 1A). Afterward, the debris was removed and
hemostasis was achieved. All rabbits were treated with gentamycin for
three days after the surgery.
Figure 1.
[60]Figure 1
[61]Open in a new tab
The histological profiles of normal and regenerated cartilages in
rabbits. (A). The microfracture (MF) surgery is conducted on the
trochlear groove of the left distal femur. An articular cartilage
defect (Ø ~ 4 × 6 mm, 2 mm in depth) has been created on the trochlear
groove of the left distal femur with a sterilized cranial drill bit (ØO
= 2.1 mm). (B). The photographs of the trochlear grooves. Compared to
the normal (NOR) cartilage, the regenerated (REG) cartilage loses its
transparent color and smooth and glistening appearance, replaced with a
rough surface with fissures. (C,D) Examples of the H&E staining.
Compared to the NOR cartilage, the REG cartilage loses the highly
organized structure composed of four zones, i.e., the superficial,
middle, deep, and calcified zones. (E,F) Example of the Safranin-O/Fast
Green staining. Compared to the NOR cartilage, the reddish-stained ACAN
content in the REG cartilage was lower. The scale bars represent 40 μm
and 10 μm.
2.2. Tissue Collection
The rabbits were sacrificed by CO[2] asphyxiation, and the paired
cartilage samples from both the left and right distal femurs (n = 9)
were collected based on the reported protocol [[62]19]. For
histochemical analysis (n = 3), three pairs of trochlear grooves were
kept in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h
and then decalcified in EDTA (14% w/v, pH 7.4) for 21 days at 4 °C
until measurement. For metabolomic analysis (n = 6), six pairs of
cartilage tissues were washed with PBS and then stored at −80 °C until
analysis.
2.3. Histochemistry
The decalcified tissues were embedded in paraffin, and then sagittal
sections were cut at 3 μm. Hematoxylin–eosin (H&E) staining was
conducted to determine the cellular regularity of cartilage using a
commercial kit (G1120, Solarbio, Beijing, China) according to the
published procedure [[63]20]. A commercial kit (G1371, Solarbio) was
used for the Safranin-O/Fast Green staining, in which the depth of the
reddish color is correlated with the content of ACAN [[64]21], by
following the company’s instructions. All the slices were dehydrated,
transparentized, and then mounted with neutral resin for microscopic
observation (Leica, Herlev, Denmark). Images were representative
results of three biological repeats.
2.4. Metabolomics
2.4.1. Sample Preparation for Liquid Chromatography–Mass Spectrometry
L-2-Chlorophenylalanine (20 µL, 0.3 mg/mL in methanol) was used as the
internal standard. The cartilage sample (30 mg) was mixed with
ice-cooled methanol (400 µL, 80%), precooled for 2 min at −20 °C, and
then ground at 60 Hz for 2 min. After ultrasonic extraction, the
solution was centrifuged at 13,000 rpm for 10 min at 4 °C. Then, 300 µL
of the supernatant was evaporated and re-dissolved with 200 µL methanol
(20%). The mixture was incubated for 2 h at −20 °C and then centrifuged
at 11,400× g (TGL-16MS, Shanghai Lu Xiangyi Centrifuge Instrument Co.,
Ltd., Shanghai, China) for 10 min at 4 °C. Afterward, 150 µL of the
liquid supernatant from each tube was collected, filtered by an organic
phase pinhole filter (0.22 μm), and then stored at −80 °C until
analysis.
2.4.2. Liquid Chromatography–Mass Spectrometry
Liquid chromatography–mass spectrometry (LC-MS) analysis was conducted
by following a published protocol [[65]22]. Briefly, the LC was
performed using an ACQUITY UPLC HSS T3 1.8-micron column at 45 °C. The
mobile phase contained water and acetonitrile with 0.1% formic acid;
gradient elution was conducted at a flow rate of 0.35 mL/min. All
samples were kept at 4 °C during the analysis. The injection volume was
2 μL. The MS was performed on an AB TripleTOF 6600 plus system, with
electrospray ionization using both positive and negative ion modes. The
full mass scan range was set at 100–1000 mass to charge ratio (m/z).
2.4.3. Bioinformatics Data Processing
The raw data obtained from LC-MS were analyzed using Progenesis QI v2.3
(Nonlinear Dynamics, Newcastle, UK) with main parameters of 5 ppm/10
ppm precursor tolerance, 10 ppm/20 ppm product tolerance, and 5%
product ion threshold. The compounds were identified based on the
RT-m/z pairs. The supervised orthogonal partial least squares
discriminant analysis (OPLS-DA) analysis was employed to distinguish
the metabolic profiles of the NOR and REG groups, and a volcano plot
was employed to visualize the alterations of the metabolite
concentrations.
2.4.4. Identification of the Differentially Expressed Metabolites
The metabolites were identified using Progenesis QI v2.3, according to
the Human Metabolome database ([66]http://www.hmdb.ca/, assessed on 20
June 2007), Lipid Maps database (V2.3, [67]http://www.lipidmaps.org/,
assessed 15 July on 2003), the Metlin database, and the self-built
database of Shanghai Lu-Ming Biotech Co. Ltd. (Shanghai, China). To
identify the DEMs in the NOR and REG groups, the thresholds were set
based on the variable importance in projection (VIP) >1.0 and p < 0.05
from the paired Student’s t-test. Moreover, the DEMs were further
searched using online databases, including the Aging Atlas
([68]https://ngdc.cncb.ac.cn/aging/metabolomics, assessed on 29 October
2020) and Regeneration Roadmap
([69]https://ngdc.cncb.ac.cn/regeneration/metabolomics, assessed on 30
September 2021).
2.4.5. Pathway Enrichment Analysis
The pathway enrichment analysis for the DEMs was performed using the
Kyoto Encyclopedia of Genes and Genomes database (KEGG,
[70]https://www.kegg.jp/kegg/pathway.html, assessed 22 March on 1995)
through matching IDs (the KEGG ID of the corresponding DEM). A pathway
with a p-value < 0.05 was considered a significant one.
2.5. Cell Culture and Differentiation Induction
2.5.1. Bone Marrow-Derived Stem Cell Culture
Passage (P) 2 of human BMSCs was purchased from Haixing Biosciences
(BMHX-C106, Suzhou, China). The BMSCs were cultured in the Human BMSCs
Growth Medium (HyCyte, BMHX-G101, Haixing Biosciences) supplemented
with 1% penicillin/streptomycin, 1% glutamine, and 10% fetal bovine
serum under the condition of 5% CO[2] in the air at 37 °C.
2.5.2. Chondrogenic Differentiation
The chondrogenic differentiation in monolayer culture was induced
according to the previous protocol with minor modifications [[71]23].
Briefly, P4 BMSCs were cultured using a Human BMSCs Chondrogenic
Differentiation Kit (BMHX-D203R, Haixing Biosciences) for three weeks.
The differentiated chondrocytes were maintained in the chondrocyte
growth medium, i.e., the DMEM/F12 medium with 10% FBS and 1%
penicillin/streptomycin.
2.6. Stimuli and Chemicals Administrated
The oxidative stress and DNA damage to BMSCs were created by employing
hydrogen peroxide (H[2]O[2], 3%, LIRCON, Dezhou, China) and mitomycin C
(MC, GC12353, GLPBIO, Montclair, CA, USA), respectively
[[72]24,[73]25]. The dosages of H[2]O[2] (250 μM) and MC (1 μM) were
selected based on a previous study [[74]24] and our pilot experiments
([75]Supplementary Figure S1). The effects of UDCA (HY-13771, MCE,
Monmouth Junction, NJ, USA) and DPA (GC31637, GLPBIO) on the vitality
of BMSCs were examined under both control and challenge states. Various
concentrations of the biomolecules (0, 5, 10, 50, and 100 µM) were
added to the culture medium to determine the DEM’s contribution to the
vitality of BMSCs, while the biomolecules were added with the stimulus
to determine the DEM’s role under the H[2]O[2] and MC challenged
conditions. Thereafter, an optional dosage, 50 μM for both UDCA and
DPA, was used in this study for identifying the effects of small
molecules on chondrogenic differentiation and chondrocyte
functionality.
2.7. Cell Vitality
The cell vitality was detected using the Cell Counting Kit-8 (CCK-8,
C6005, NCM Biotech, Suzhou, China) after 24 h of incubation with and
without small molecules. The optical density was read at 450 nm using a
multimode plate reader (PerkinElmer, Waltham, MA, USA).
The data of the CCK-8 assay were presented as mean ± standard error
(SEM). Statistical analyses and graphics were conducted using GraphPad
Prism version 7.0 (San Diego, CA, USA). The effects of the small
molecules under control and challenge conditions were revealed by a
one-way ANOVA analysis. The Dunnett test was used to partition
differences among the dosages. A value of p < 0.05 was considered to be
statistically significant.
2.8. Immunofluorescence
The cells were grown on glass coverslips (ØO = 14 mm). The
immunofluorescence was determined according to the method previously
described with some modifications [[76]26]. Briefly, the cells were
incubated with anti-ACAN (1:500, bs-1223R, Boiss, Beijing, China)
overnight at 4 °C after fixation and permeabilization. The goat
anti-rabbit 594 secondary antibody (1:500, bs-0295G-AF594, Boiss) was
applied for 2 h at RT. The cells were counterstained with phalloidin
(C1033, Beyotime, Shanghai, China) to determine the cytoskeletal
arrangement [[77]27], and the nuclei were identified after staining
with DAPI (S2110, Solarbio) for 30 min at RT. The images were taken
under a Leica DM 6000B microscope (Leica, Herlev, Denmark) and were
representatives of three repeats.
2.9. Real-Time PCR
Total RNA was extracted using NcmZol Reagent (M5100, NCM Biotech). The
concentration and quality of RNA were measured using a
spectrophotometer (Nanodrop 2000c, Thermo Fisher, Waltham, MA, USA).
Reverse transcription was conducted using the Reverse Transcription
Reagent Pack (Applied Biosystems, Thermo Fisher). Real-time PCR
analysis was carried out using Quantstudio 5 (Applied Biosystems) with
the SYBR Green Master Mix (Applied Vazyme, Nanjing, China) and
gene-specific primers ([78]Table 1) of the key transcriptional factor
for chondrogenic differentiation (SRY-related high mobility group-box
gene 9, SOX9) and the chondrogenic indicators (ACAN and COL2A1)
[[79]21,[80]28]. The Ct-value of β-actin mRNA was used as the internal
control. The relative mRNA expression levels were analyzed using the
2−ΔΔCt method [[81]29]. The unpaired Students’ t-test was applied to
determine the difference between DPA or UDCA treatment with the CON
group. A value of p < 0.05 was considered to be statistically
significant.
Table 1.
Gene-specific primers for real-time PCR.
Gene Primer
SOX9 F: TAAGCTAAAGGCAACTCGTACC
R: TAGAGAATATTCCTCACAGAGGACT
ACAN F: TGAGCGGCAGCACTTTGAC
R: TGAGTACAGGAGGCTTGAGG
COL2A1 F: TCCAGATGACCTTCCTACGC
R: GGTATGTTTCGTGCAGCCAT
β-actin F: CCCTGGAGAAGAGCTACGAG
R: CGTACAGGTCTTTGCGGATG
[82]Open in a new tab
3. Results
3.1. The Histomorphologic Changes in the Regenerated Cartilage
Compared to the NOR cartilage, the REG cartilage lost its transparent
color and smooth and glistening appearance, replaced with a rough
surface with fissures ([83]Figure 1B). The REG cartilage also lost the
regularity seen in the H&E-stained NOR cartilage, i.e., a highly
organized structure composed of four zones ([84]Figure 1C,D).
Specifically, the thickness of the REG cartilage layer at the defect
site was lower than that of the NOR group, while the chondrocytes in
the REG group were more compactly organized than those in the NOR group
([85]Figure 1C–F). Furthermore, the shape of the chondrocytes in the
NOR group was plumper and more round ([86]Figure 1C,E) than that of the
chondrocytes in the REG group ([87]Figure 1D,F). Moreover, the
reddish-stained ACAN content in the REG cartilage ([88]Figure 1E) was
lower than that in the NOR cartilage ([89]Figure 1F).
3.2. The Metabolomic Changes in the Regenerated Cartilage
The OPLS-DA plot revealed the differences in metabolomic activities
between the NOR and REG groups ([90]Figure 2A). A total of 96 DEMs were
identified ([91]Table 2), with 31 decreased and 65 increased
metabolites induced by MF ([92]Figure 2B). Of these, 11 DEMs were
matched with the records in the Regeneration Roadmap and Age Atlas
([93]Figure 2C, [94]Supplementary Table S1). Moreover, the DEMs can be
divided into six categories, including lipids and lipid-like molecules
(47), organic oxygen compounds (16), organic acids and derivatives
(12), heterocyclic compounds (15), organosulfur compounds (2), and
hydrocarbons (4). Notably, the lipids and lipid-like molecules were
accountable for half (48.96%) of the entire metabolic alterations in
cartilage regeneration. Of the lipids and lipid-like molecules, the
majority were increased in the REG cartilages (85.11%), including DPA
([95]Figure 2D, p = 0.0087) and UDCA ([96]Figure 2E, p = 0.005). The
organic oxygen compounds, accounting for 16.67% of the DEMs, were
mainly second messengers and oligosaccharides. Among the organic oxygen
compounds, most of the glycolytic substances were decreased. The
heterocyclic compounds, organosulfur compounds, and others accounted
for the rest (21.88%) of the DEMs.
Figure 2.
[97]Figure 2
[98]Open in a new tab
The global metabolomic profiles of the normal and regenerated
cartilages. (A) The supervised orthogonal partial least squares
discriminant analysis (OPLS-DA) model reveals the metabolic differences
between the NOR and REG groups. (B) The volcano plot indicates the
upregulated differential metabolites (DEMs) in the REG group (red), the
downregulated DEMs (blue), and non-significantly altered metabolites
(gray). (C) Venn diagram shows the DEMs matched with the records in the
Regeneration Roadmap and Aging Atlas databases. (D,E) The different
expression levels of docosapentaenoic acid 22n-3 (DPA, (D)) and
ursodeoxycholic acid (UDCA, (E)) between the NOR and REG groups are
revealed. (F) The bubble diagram shows the top 20 differential
metabolic pathways enriched by the DEMs. X-axis represents rich
factors, and Y-axis represents pathway terms. The numbers of the
involved metabolites and p-value are listed on the right side. The
detailed information is presented in [99]Supplementary Tables S1 and
S2.
Table 2.
The list of DEMs identified in the NOR and REG groups.
Category Metabolite m/z ^a RT ^b (min) Error ^c (ppm) VIP ^d p-Value ^e
FC ^f Trend Formula
Lipids and lipid-like molecules (47) (7Z,10Z,13Z,16Z)-docosatetraenoate
333.2783 13.8609 −2.0938 2.0825 0.0247 2.0327 up C22H36O2
Arachidonic acid 305.2472 13.0585 −1.1653 5.4988 0.0064 1.8881 up
C20H32O2
Fludrocortisone acetate 405.2081 1.5828 2.2102 1.1184 0.0280 3.0242 up
C23H31FO6
L-Palmitoylcarnitine 400.3419 10.9240 −0.6476 2.5445 0.0051 2.4512 up
C23H45NO4
LysoPC(18:1(11Z)) 522.3545 11.1461 −1.7806 2.8324 0.0022 1.6622 up
C26H52NO7P
LysoPC(20:4(8Z,11Z,14Z,17Z)) 544.3386 10.7015 −2.1933 4.1157 0.0028
2.7755 up C28H50NO7P
LysoPC(22:5(7Z,10Z,13Z,16Z,19Z)) 570.3543 10.8646 −1.8896 1.1338 0.0039
5.7136 up C30H52NO7P
Prostaglandin E2 351.2173 8.2029 −1.5090 1.6011 0.0441 6.5508 up
C20H32O5
SM(d18:1/24:1(15Z)) 813.6829 13.1903 −1.8179 9.4552 0.0485 9.0986 up
C47H93N2O6P
Sphingosine 282.2784 10.4050 −2.4257 1.4741 0.0028 1.8594 up C18H37NO2
DPA 331.2630 13.2346 −0.4486 1.2545 0.0349 5.5002 up C22H34O2
Oleamide 304.2607 13.1326 −1.4413 2.3222 0.0257 1.6991 up C18H35NO
Ursodeoxycholic acid 391.2857 10.7032 0.8484 1.0112 0.0472 12.4921 up
C24H40O4
PI(O-18:0/0:0) 604.3833 14.9395 −1.7930 3.2441 0.0000 0.6783 down
C34H50O8
LysoPE(0:0/20:4(8Z,11Z,14Z,17Z)) 502.2918 10.6718 −2.0758 7.3932 0.0004
1.7392 up C25H44NO7P
6-[3]-ladderane-1-hexanol 280.2635 12.3763 −1.9348 6.3252 0.0251 1.7063
up C18H30O
PS(14:0/24:1(15Z)) 840.5728 11.4733 0.3863 1.6386 0.0196 14.4043 up
C44H84NO10P
LysoPE(22:4(7Z,10Z,13Z,16Z)/0:0) 530.3229 11.2206 −2.3436 4.2432 0.0010
1.7410 up C27H48NO7P
PS(14:1(9Z)/24:0) 840.5734 12.8225 1.0620 3.3834 0.0114 53.3843 up
C44H84NO10P
Linoelaidic Acid 263.2365 13.2051 −1.6593 3.3825 0.0354 1.6614 up
C18H32O2
Pelargonidin 3-(6″-p-coumarylglucoside)-5-(6‴-acetylglucoside) 763.1887
0.9317 0.9657 1.7822 0.0417 0.4288 down C38H38O18
PC(O-16:0/0:0) 482.3591 11.3248 −2.8591 2.1609 0.0008 2.6328 up
C24H52NO6P
LysoPC(18:2(9Z,12Z)) 520.3395 10.6718 −0.4349 2.1558 0.0049 2.6192 up
C26H50NO7P
LysoPE(0:0/18:2(9Z,12Z)) 476.2778 10.6394 −1.0077 1.3499 0.0031 3.4070
up C23H44NO7P
LysoPE(22:5(7Z,10Z,13Z,16Z,19Z)/0:0) 528.3073 10.8350 −2.1896 2.5262
0.0015 4.1787 up C27H46NO7P
1-O-(2R-hydroxy-hexadecyl)-sn-glycerol 355.2815 12.7332 −2.7314 2.4189
0.0170 0.8881 down C19H40O4
2,3,4,5,2′,3′,4′,6′-Octamethoxychalcone 895.3420 0.7166 2.9042 1.6332
0.0189 0.7754 down C23H28O9
15-hydroxy-tetracosa-6,9,12,16,18-pentaenoic acid 357.2786 10.7015
−0.4361 1.5497 0.0168 4.0203 up C24H38O3
Xestoaminol C 230.2473 9.4959 −2.5076 1.9590 0.0078 1.4550 up C14H31NO
LysoPE(18:2(9Z,12Z)/0:0) 478.2919 10.6421 −1.8674 1.8550 0.0005 2.6353
up C23H44NO7P
1,2-(8R,9R-epoxy-17E-octadecen-4,6-diynoyl)-3-(hexadecanoyl)sn-glycerol
839.5820 11.9032 −0.0844 1.4486 0.0101 3.2667 up C54H80O8
1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphate 507.2730 11.5926 0.2704
1.2619 0.0014 4.1891 up C23H43O7P
Stearoylcarnitine 428.3724 11.4139 −2.4569 1.5537 0.0045 4.1705 up
C25H49NO4
1-O-(2R-hydroxy-tetradecyl)-sn-glycerol 327.2499 11.9180 −3.8832 1.1413
0.0050 0.8497 down C17H36O4
LysoPE(0:0/22:5(7Z,10Z,13Z,16Z,19Z)) 528.3073 11.0426 −2.2949 1.3834
0.0059 2.6454 up C27H46NO7P
1-(2-methoxy-eicosanyl)-sn-glycero-3-phosphoethanolamine 508.3751
11.5454 −2.0199 1.5825 0.0022 2.4697 up C26H56NO7P
PC(0:0/18:1(9Z)) 566.3471 11.1411 1.5304 1.5653 0.0258 2.0328 up
C26H52NO7P
N-(3-oxo-butanoyl)-homoserine lactone 186.0753 0.9084 −4.0254 1.1941
0.0065 1.7216 up C8H11NO4
PS(17:0/0:0) 512.2973 11.6489 −1.9114 1.0334 0.0236 0.2002 down
C23H46NO9P
Oleoylcarnitine 426.3569 11.0426 −2.1469 1.1955 0.0417 1.4961 up
C25H47NO4
Linoleamide 302.2448 12.3763 −2.2846 1.0171 0.0368 1.6485 up C18H33NO
1-Arachidonoylglycerophosphoinositol 603.2915 10.9093 −2.2292 1.1105
0.0037 3.4729 up C29H49O12P
Dodecanoylcarnitine 344.2790 9.7305 −1.6266 1.3234 0.0324 0.2012 down
C19H37NO4
1-(2-methoxy-13-methyl-tetradecanyl)-sn-glycero-3-phosphoserine
522.2814 10.7905 2.3016 1.1583 0.0050 1.7855 up C22H46NO9P
Europinidin 330.0742 4.4330 −1.1294 1.1275 0.0143 1.8673 up C16H13O5+
LysoPC(P-16:0) 480.3432 11.3099 −3.5157 1.5174 0.0234 1.8926 up
C24H50NO6P
(E)-2-Penten-1-ol 104.1067 0.7885 −3.0443 2.9246 0.0097 1.3086 up
C5H10O
Organic oxygen compounds (16) D-Myoinositol 4-phosphate 259.0220 0.7671
−1.6433 2.6807 0.0147 0.1456 down C6H13O9P
N-Acetylgalactosamine 244.0790 0.8485 −0.5407 1.0594 0.0236 1.7493 up
C8H15NO6
D-glycero-L-galacto-Octulose 279.0470 0.7739 −2.7275 1.2166 0.0385
0.6264 down C8H16O8
Pelargonidin 272.0673 4.4259 −2.2420 2.8195 0.0058 0.4624 down
C15H11O5+
Malvidin 332.0888 4.4543 1.6129 3.5305 0.0099 2.1895 up C17H15O7+
Cellotetraose 684.2545 0.9383 −1.7043 3.2822 0.0375 0.4180 down
C24H42O21
N-Acetylgalactosaminyl lactose 546.2017 0.9232 −2.0868 2.2153 0.0058
0.4044 down C20H35NO16
Isopropyl β-D-ThiogalactoPyranoside 221.0837 2.0908 −1.9212 4.9497
0.0033 0.8273 down C9H18O5S
3,5-dihydroxy-4-(sulfooxy)benzoic acid 250.9848 0.8485 −3.1660 1.6067
0.0008 1.7730 up C7H6O8S
1,2,3,4-Tetramethoxy-5-(2-propenyl)benzene 261.1095 8.1917 −0.7636
2.3229 0.0175 0.8684 down C13H18O4
Vicianose 295.1024 2.0365 0.1620 1.6166 0.0289 0.8576 down C11H20O10
2-Deoxy-D-ribose 1,5-bisphosphate 292.9836 8.1902 1.0583 1.3599 0.0082
0.7965 down C5H12O10P2
Lacto-N-triaose 590.1927 0.9317 −2.5947 1.0686 0.0309 0.3734 down
C20H35NO16
2-(5,8-Tetradecadienyl)cyclobutanone 245.2258 13.2051 −2.3519 1.1487
0.0368 1.7212 up C18H30O
4-Acetylzearalenone 361.1658 13.0585 3.5409 1.1157 0.0164 1.8800 up
C20H24O6
Myrigalone H 304.1544 11.4883 0.2525 1.0970 0.0436 1.7111 up C17H18O4
Organic acids and derivatives (12) L-Arginine 175.1181 0.7447 −4.9509
1.1077 0.0232 1.4089 up C6H14N4O2
L-Phenylalanine 166.0856 3.5249 −4.1785 2.1999 0.0140 1.7636 up
C9H11NO2
L-Tryptophan 203.0824 4.3576 −1.1300 1.1625 0.0066 1.8097 up C11H12N2O2
L-Tyrosine 180.0665 2.2998 −0.8408 1.9941 0.0065 1.7151 up C9H11NO3
7,8-diaminononanoic acid 171.1484 6.5341 −4.3633 1.8274 0.0198 0.7210
down C9H20N2O2
Dopaxanthin 779.2082 0.9317 3.6779 1.0294 0.0464 0.4276 down C18H18N2O8
6-(5-carboxy-2-hydroxy-3-methoxyphenoxy)-3,4,5-trihydroxyoxane-2-carbox
ylic acid 361.0779 0.9383 3.8961 1.3373 0.0374 0.5079 down C14H16O11
S-Glutathionyl-L-cysteine 427.0942 0.9232 −2.2858 1.7851 0.0390 4.6303
up C13H22N4O8S2
Cysteineglutathione disulfide 425.0798 0.9189 −2.0096 1.7524 0.0369
4.9326 up C13H22N4O8S2
Propanoyl phosphate 306.9978 9.7584 −3.7713 1.3013 0.0154 0.7428 down
C3H7O5P
2-Hydroxycinnamic acid 182.0804 2.3357 −4.7335 2.6129 0.0184 1.9098 up
C9H8O3
Hydroxymethylphosphonate 110.9848 0.8047 −3.7569 1.1112 0.0311 1.5147
up CH5O4P
Heterocyclic Compounds (15) Uracil 113.0342 1.2618 −3.2771 1.4253
0.0280 1.7119 up C4H4N2O2
Deethylatrazine 188.0701 4.3813 −0.5980 2.6772 0.0062 2.5574 up
C6H10ClN5
dirithromycin 563.5503 13.1326 −4.2918 3.0670 0.0497 2.1749 up C39H72
2′-O-Methyladenosine 282.1185 3.3656 −4.2975 1.1541 0.0233 0.3554 down
C11H15N5O4
FAPy-adenine 171.0985 14.2547 −2.7181 10.0091 0.0045 0.7720 down
C5H7N5O
Pectachol 460.2687 10.2578 −1.3892 3.0608 0.0237 0.8238 down C26H34O6
1-(1,2,3,4,5-Pentahydroxypent-1-yl)-1,2,3,4-tetrahydro-beta-carboline-3
-carboxylate 367.1491 3.3656 −2.4233 1.6882 0.0314 0.4717 down
C17H22N2O7
Nocodazole 300.0449 0.8047 0.1909 1.7345 0.0100 0.4874 down C14H11N3O3S
Dipyridamole 527.3043 12.0652 1.5729 1.2210 0.0388 0.8693 down
C20H39N5O10
6-Methyltetrahydropterin 204.0861 0.9084 2.7681 1.2488 0.0079 1.8114 up
C7H11N5O
Gravacridonetriol 396.0855 0.0425 3.1419 1.5097 0.0248 0.8872 down
C19H19NO6
Nicorandil 256.0578 0.9062 1.3308 1.0936 0.0068 2.2259 up C8H9N3O4
Ethosuximide M5 200.0562 2.2873 −1.8422 1.1403 0.0168 1.9016 up C7H9NO3
Dihydrofolic acid 424.1355 2.2873 −4.4941 1.2621 0.0016 3.0187 up
C19H21N7O6
AFN911 550.2322 0.9232 −1.0333 1.0043 0.0310 0.5018 down C29H33N7O2
Organosulfur compounds (2) Ethyl isopropyl disulfide 137.0456 1.4293
1.9879 5.5947 0.0027 1.5142 up C5H12S2
Ethyl propyl disulfide 135.0312 1.4392 3.3806 2.2132 0.0018 1.3007 up
C5H12S2
Hydrocarbons and derivatives (4)
2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A) 358.9943
8.0025 −1.1090 2.5442 0.0177 0.7840 down C4H2F6O
(+/−)-N,N-Dimethyl menthyl succinamide 186.2209 14.2250 −4.3869 2.2373
0.0327 0.8893 down C12H24
2-Hexylidenecyclopentanone 331.2642 13.8606 −0.0544 1.2698 0.0331
2.6671 up C11H18O
Aluminium dodecanoate 663.4544 14.9840 0.4274 1.1638 0.0042 1.1576 up
C36H69AlO6
[100]Open in a new tab
^a mass to charge ratio of the features; ^b retention time of the
features; ^c mass error is obtained by using the experimental mass
minus the theoretical mass; ^d variable importance in projection; ^e
p-value is obtained from the two-tailed Student’s t-test; and ^f fold
change.
The enrichment analysis yielded 70 pathways related to cartilage
regeneration ([101]Supplementary Table S2), with the top 20 listed in
the bubble diagram ([102]Figure 2F), which revealed the key information
associated with the biochemical-niche control for chondrogenesis, such
as the rapamycin (mTOR) signaling pathway. Moreover, the detailed
information on the pathways with hits ≥ 2 and p-value < 0.05 is
presented in [103]Table 3.
Table 3.
The list of the potential pathways involved in cartilage regeneration.
NO. Annotation p-Value ^a Match Status ^b Rich Factor ^c Matching IDs
DEMs
1 Amoebiasis 0.0000 4/13 0.3077 [104]C00062 [105]C00219 [106]C00584
[107]C01074 L-Arginine, Arachidonic acid, Prostaglandin E2,
N-Acetylgalactosamine
2 Necroptosis 0.0000 3/9 0.3333 [108]C00219 [109]C00319 [110]C00550
Arachidonic acid, Sphingosine, SM(d18:1/24:1(15Z))
3 Aminoacyl-tRNA biosynthesis 0.0001 4/52 0.0769 [111]C00062
[112]C00078 [113]C00079 [114]C00082 L-Arginine, L-Tryptophan,
L-Phenylalanine, L-Tyrosine
4 Leishmaniasis 0.0004 2/6 0.3333 [115]C00219 [116]C00584 Arachidonic
acid, Prostaglandin E2
5 Phenylalanine, tyrosine, and tryptophan biosynthesis 0.0006 3/34
0.0882 [117]C00078 [118]C00079 [119]C00082 L-Tryptophan,
L-Phenylalanine, L-Tyrosine
6 African trypanosomiasis 0.0007 2/8 0.2500 [120]C00078 [121]C00584
L-Tryptophan, Prostaglandin E2
7 Oxytocin signaling pathway 0.0017 2/12 0.1667 [122]C00219 [123]C00584
Arachidonic acid, Prostaglandin E2
8 Regulation of lipolysis in adipocytes 0.0023 2/14 0.1429 [124]C00219
[125]C00584 Arachidonic acid, Prostaglandin E2
9 Sphingolipid signaling pathway 0.0026 2/15 0.1333 [126]C00319
[127]C00550 Sphingosine, SM(d18:1/24:1(15Z))
10 Serotonergic synapse 0.0034 2/17 0.1176 [128]C00078 [129]C00219
L-Tryptophan, Arachidonic acid
11 Phenylalanine metabolism 0.0034 3/60 0.0500 [130]C00079 [131]C00082
[132]C01772 L-Phenylalanine, L-Tyrosine, 2-Hydroxycinnamic acid
12 Sphingolipid metabolism 0.0072 2/25 0.0800 [133]C00319 [134]C00550
Sphingosine, SM(d18:1/24:1(15Z))
13 Inflammatory mediator regulation of TRP channels 0.0090 2/28 0.0714
[135]C00219 [136]C00584 Arachidonic acid, Prostaglandin E2
14 Protein digestion and absorption 0.0097 2/29 0.0690 [137]C00062
[138]C00079 L-Arginine, L-Phenylalanine
15 Biosynthesis of unsaturated fatty acids 0.0494 2/69 0.0290
[139]C00219 [140]C16527 Arachidonic acid
[141]Open in a new tab
The pathways listed in [142]Table 3 have hits ≥ 2 and p-value < 0.05.
^a p-value is obtained by
[MATH: 1−∑i=0<
/mn>m−1MiN−Mn−iNn
:MATH]
, where N represents the number of the total metabolites, n represents
the number of the DEMs, M represents the number of the total
metabolites in a certain pathway, and m represents the number of DEMs
in a certain pathway; ^b match status is indicated as
[MATH: mM
:MATH]
; ^c rich factor is identified as
[MATH: mM
:MATH]
.
3.3. Differentially Expressed Metabolites Involved in Promoting the Vitality
and Adaptation of BMSCs
The facilitating effect of both DPA (p < 0.0001, [143]Figure 3A) and
UDCA (p = 0.004, [144]Figure 3B) on the vitality of BMSCs was revealed.
Specifically, 50 and 100 μM of both DPA and UDCA significantly
increased the optical densities compared to the control group (0 μM).
Under oxidative challenge, adding DPA and UDCA rescued the damage
effect of 250 μM H[2]O[2] (p < 0.0001, [145]Figure 3C; p = 0.002,
[146]Figure 3D). Similarly, the administration of DPA and UDCA rescued
the injury effect of DNA damage caused by 1 μM MC (p < 0.0001,
[147]Figure 3E; p < 0.0001, [148]Figure 3F).
Figure 3.
[149]Figure 3
[150]Open in a new tab
The effects of DAP and UDCA on the vitality and adaptation of the
BMSCs. (A,B). The dosage effects of DPA and UDCA on the vitality of
BMSCs. (C,D). The dosage effects of DPA and UDCA on the vitality of
BMSCs in response to 250 μM hydrogen peroxide (H[2]O[2])-caused
oxidative stress damage. (E,F). The dosage effects of DPA and UDCA on
the vitality of BMSCs in response to 1 μM mitomycin C (MC)-caused DNA
damage. The data are presented as mean ± SEM (n = 7). * p < 0.05; ** p
< 0.01; *** p < 0.0001.
3.4. Differentially Expressed Metabolites Involved in Promoting Chondrogenic
Differentiation
After induced chondrogenic differentiation ([151]Figure 4A), compared
to the CON group ([152]Figure 4B), the intensity of ACAN was increased
in both the DPA ([153]Figure 4C) and UDCA ([154]Figure 4D) groups. The
phalloidin staining was of high intensity, with the microfilaments
easily distinguishable in the CON group ([155]Figure 4E), while the
staining in the DPA ([156]Figure 4F) and UDCA ([157]Figure 4G) groups
was almost faded; in particular, the microfilaments in the UDCA group
mostly disappeared ([158]Figure 4G). Moreover, the ACAN signal was
co-located with the nucleus in the CON group ([159]Figure 4B,H,K) and
exhibited cytoplasmic shuttling in the DPA ([160]Figure 4C,I,L) and
UDCA ([161]Figure 4D,J,M) groups. Furthermore, the quantitative
analysis indicated that the administration of DPA during chondrogenic
differentiation significantly elevated the mRNA expression of SOX9
([162]Figure 4N), ACAN ([163]Figure 4O), and COL2A1 ([164]Figure 4P)
compared to the CON group, while UDCA significantly increased the SOX9
([165]Figure 4N) and ACAN ([166]Figure 4O) mRNA expression levels.
Figure 4.
[167]Figure 4
[168]Open in a new tab
The effects of DPA and UDCA on chondrogenic differentiation. DPA and
UDCA were separately administrated during the entire process of
chondrogenic differentiation as indicated in (A). The distribution of
proteoglycan aggrecan (ACAN, red) is revealed with immunofluorescence;
F-actin (green) and nuclei (blue) are counterstained with phalloidin
and DAPI, respectively. The immuno-positive signal of ACAN (arrows) is
weak and restricted within the nucleus in the control (CON) group (B,H)
and is strong and located in the cytoplasm and nucleus in both the DPA
group (C,I) and UDCA group (D,J). F-actin is rearranged during the
chondrogenic differentiation; i.e., it displays a sharp image after
phalloidin staining in the CON group (E) and fades in both the DPA (F)
and UDCA (G) groups. (K,L,M) show the merge images. The scale bar
represents 50 μm. The mRNA expression levels of the SRY-related high
mobility group-box gene 9 (SOX9, (N)), ACAN (O), and type II collagen
(COL2A1, (P)) in the CON, DPA, and UDCA groups are quantified. The data
are presented as mean ± SEM (n = 3). ns indicates no significance (p >
0.05); * p < 0.05, ** p < 0.01, *** p < 0.001.
3.5. Differentially Expressed Metabolites Involved in Promoting Chondrocyte
Functionality
The ACAN signal was detected in the nucleus and cytoplasm in the CON
group ([169]Figure 5B,H,K), while it was restricted in the cytoplasm
and exhibited exocytosis (arrows) in both the DPA ([170]Figure 5C,I,L)
and UDCA ([171]Figure 5D,J,M) groups. In addition, the chondrocytes in
the CON group ([172]Figure 5E) tended to have a fibroblast-like
appearance, while those in the DPA ([173]Figure 5F) and UDCA
([174]Figure 5G) groups were more likely to exhibit a polygon shape.
Furthermore, the quantitative analysis indicated that the ACAN mRNA
expression in the chondrocytes was increased in both DPA and UDCA
groups ([175]Figure 5N), while the mRNA expression levels of COL2A1
([176]Figure 5O) were increased in the UDCA group only.
Figure 5.
[177]Figure 5
[178]Open in a new tab
The effects of DPA and UDCA on chondrocyte functionality. DPA or UDCA
was added to the chondrocytes for five days before sampling (A). The
distribution of ACAN (red); the distribution of F-actin (green) and
nuclei (blue), counterstained with phalloidin and DAPI, respectively.
The immuno-positive signal of ACAN (arrows) is relatively weak and
located in the nucleus and cytoplasm in the CON group (B,H,K), while it
becomes strong, moves to the cytoplasm, and exhibits external secretion
after DPA (C,I,L) or UDCA (D,J,M) administration. (E,F,G) indicate the
shape of chondrocyte. The scale bar represents 50 μm. The mRNA
expression levels of the ACAN (N) and COL2A1 (O) in the CON, DPA, and
UDCA groups are quantified. The data are presented as mean ± SEM (n =
3). ns indicates no significance (p > 0.05); * p < 0.05.
4. Discussion
Currently, the common osteoarthritis therapy with nonsteroidal
anti-inflammatory drugs or chondroprotective hyaluronic acid (HA) is
ineffective for cartilage regeneration [[179]30]. The pharmaceutical
industry is calling for novel biomolecules covering both safety and
effectiveness as anti-aging supplements or drug candidates for joint
health. The metabolomics modules in the Aging Atlas and Regeneration
Roadmap offer the molecular substances potentially related to the
degenerative and regenerative events in the blastema of axolotls, deer
antler stem cells, etc. However, orthopedic metabolic cues are lacking.
In this study, the minority of identified DEMs (11/96) were matched
with the records in the Regeneration Roadmap and Aging Atlas, which
stresses the uniqueness and significance of the cartilage
regeneration-specific metabolites. This study firstly reveals the
biomolecules for cartilage regeneration, which serve each step during
MF, i.e., the vitality and adaptation of BMSCs as well as chondrogenic
differentiation and chondrocyte functionality.
The vitality of the activated BMSCs may be the main restriction for
cartilage regeneration, especially in old people with an inadequate
BMSC pool and senescent BMSCs [[180]5]. The vitality of BMSCs is
altered by the biophysical perturbation during their migration, e.g.,
switched from the hypoxic bone marrow niche to the oxygen-exposed
defect site in MF [[181]31]. Oxidative metabolism, e.g., the oxidative
stress and the subsequent DNA damage in the defect site, has been
revealed by the increase in FAPγ-adenine in the heterocyclic compounds
in the REG group, exhibiting the damage effects on the vitality of
BMSCs [[182]32]. Adding DPA or UDCA increased the vitality of the BMSCs
under both the control condition and oxidative challenge. Moreover, the
fluctuation in the cellular metabolism directly alters the
microenvironment for cartilage regeneration in situ [[183]33]. In this
study, increases in phenylalanine, tyrosine, tryptophan,
2′-O-methyladenosine (an analog of adenosine), sphingosine, and
SM(d18:1/24:1(15Z)) have been identified in the REG group. Acetyl-CoA
is obtained from amino acids such as phenylalanine, tyrosine, and
tryptophan [[184]34], and oxidation of acetyl-CoA accounts for ATP
production [[185]34]. It has been reported that adenosine elevates the
ATP supply in MSCs [[186]35,[187]36]. Moreover, the sphingolipid
metabolism and sphingolipid signaling pathways are associated with stem
cell migration and signaling transduction [[188]37,[189]38]. Hence, the
DEMs may regulate the vitality of the activated BMSCs via mediating
acetyl-CoA synthesis, ATP supply, and signaling transduction.
The foreign supply in the defect site is critical for the renewal and
functionality of chondrocytes because cartilage is an avascular tissue
[[190]39]. The elevated second messengers in the REG group may reveal
the boosting of signaling transduction in the differentiating and
newborn chondrocytes. The activated mTOR signaling pathway is critical
for lipid biosynthesis [[191]40] and chondrogenic differentiation
[[192]41]. Blocking mTOR signaling inhibits chondrogenic
differentiation [[193]41]. Hence, the DEMs may participate in
regulating chondrogenic differentiation and chondrocyte functionality,
and they may potentially prevent cartilage loss in musculoskeletal
diseases via mediating mTOR signaling transduction. In addition, both
DPA and UDCA promoted chondrogenic differentiation by stimulating the
mRNA expression of SOX9 and ACAN and facilitating cytoskeleton
rearrangement during chondrogenic differentiation via regulating
F-actin depolymerization. The administration of DPA and UDCA enhanced
chondrocyte functionality by promoting the extracellular processing of
ACAN. The HA-bound extracellular ACAN regulates HA endocytosis
[[194]42], which may determine the chondroprotective effect of HA.
Compared to DPA, UDCA exhibited higher potential in facilitating
chondrogenic differentiation and chondrocyte functionality potentially
due to cholesterol’s facilitating role in chondrocyte differentiation
[[195]43]. The inconsistency between DPA and UDCA may be associated
with the dosage effect or functioning pathway, and the related
mechanism will be explored in further study.
5. Conclusions
Generally, the findings of this study (1) provided a list of
biomolecules potentially involved in the fate control of the activated
resident BMSCs in cartilage regeneration and (2) verified the functions
of UDCA and DPA in promoting BMSC vitality, chondrogenic
differentiation, and chondrocyte functionality. Ultimately, the small
molecules may work as anti-aging supplements for joint rejuvenation.
Clinical supply with the biomolecules in MF may confer a better outcome
and promote the surgery to a broad base of patients, especially the
elderly, by rebalancing and conquering the hostile joint environment.
Additionally, combining the biomolecules with BMSC therapy may be a
safe and effective strategy in stem cell-treated diseases, not limited
to orthopedic disorders.
Supplementary Materials
The following supporting information can be downloaded at:
[196]https://www.mdpi.com/article/10.3390/cells11192951/s1, Figure S1:
The dosages of the stimuli. Various concentrations of H[2]O[2] (A) and
MC (B) were added in the culture medium and co-incubated with the BMSCs
for 24 h, and the cell viability was decided by CCK8 assay. The data of
cell viability assay were presented as MEAN ± standard error (SEM). A
one-way ANOVA analysis was employed to determine the damage effect of
H[2]O[2] and MC. The Dunnett test was used to partition differences of
each stimulus-treated group with the control (0 μM); Table S1: The DEMs
matched with the records in Regeneration Roadmap and Aging Atlas; Table
S2: The list of the potential pathways induced by MF.
[197]Click here for additional data file.^ (232.8KB, zip)
Author Contributions
Conceptualization, H.P., X.H. and T.Y.; methodology, H.P. and Z.R.;
data curation, Z.W. and R.C.; writing—original draft preparation, H.P.
and X.H.; writing—review and editing, X.H., T.Y. and Y.Z. (Yingze
Zhang); supervision, T.Y. and Y.Z. (Yingze Zhang); funding acquisition,
T.Y. and Y.Z. (Yi Zhang). All authors have read and agreed to the
published version of the manuscript.
Institutional Review Board Statement
The animal study was approved by the Ethics Committee of Experimental
Animals of the Affiliated Hospital of Qingdao University (No.
AHQU-MAL20210419).
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are available from the corresponding author under reasonable
request.
Conflicts of Interest
The authors declare no conflict of interest.
Funding Statement
This research was funded by grants from the National Natural Science
Foundation of China (No. 31872310 to T.Y., No. 31802022 to Y.Z.).
Footnotes
Publisher’s Note: MDPI stays neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
References