Melatonin rescued interleukin 1β-impaired chondrogenesis of human mesenchymal stem cells
Gao et al. Stem Cell Research & Therapy
Melatonin rescued interleukin 1β-impaired chondrogenesis of human mesenchymal stem cells
Bo Gao 2
Wenjie Gao 1 5 6
Zizhao Wu 2
Taifeng Zhou 1 6
Xianjian Qiu 2
Xudong Wang 2
Chengjie Lian 1 6
Yan Peng 2
Anjing Liang 2
Jincheng Qiu 2
Yuanxin Zhu 2
Caixia Xu 3
Yibing Li 5
Peiqiang Su 1 6
Dongsheng Huang 2
0 107 West Yan Jiang Road , Guangzhou 510120, Guangdong , China
1 Department of Orthopedics, The First Affiliated Hospital of Sun Yat-sen University , Guangzhou, Guangdong , China
2 Department of Orthopedics, Sun Yat-sen Memorial Hospital of Sun Yat-sen University
3 Research Centre for Translational Medicine, The First Affiliated Hospital of Sun Yat-sen University , Guangzhou, Guangdong , China
4 58 Zhongshan Road II , Guangzhou 510080, Guangdong , China
5 Department of Spine Surgery, Xi'an Honghui Hospital, Xi'an Jiaotong University , Xi'an , China
6 Guangdong Provincial Key Laboratory of Orthopedics and Traumatology, The First Affiliated Hospital of Sun Yat-sen University
Background: Osteoarthritis (OA) is a widespread arthritic disease and a primary cause of disability. Increasing evidence suggests that inflammation has a pivotal part in its pathogenesis. Interleukin-1β (IL-1β) is a primary mediator of local inflammatory processes in OA. Current therapies for OA mainly focus on the symptoms of the advanced stage of the disease. The possible utilization of bone marrow mesenchymal stem cells (BMSCs) to regenerate cartilage is an appealing method, but in the case of OA requires chondrogenesis to take place within an inflamed environment. Our previous study showed that melatonin (MLT) can promote chondrogenic differentiation of MSCs, but whether MLT can rescue IL-1β-impaired chondrogenesis in human BMSCs has not yet been established. MLT, which can have anti-inflammatory and prochondrogenic effects, has demonstrated potential in defeating IL-1β-induced inhibition of chondrogenesis and further study should be conducted. Methods: Human bone marrow-derived MSCs were separated and cultured based on our system that was already documented. A high-density micromass culture system was used for the chondrogenic differentiation of human BMSCs, which was also described previously. Human BMSCs were induced for chondrogenesis for 7, 14, and 21 days with the treatment of IL-1β and MLT. The cultured cartilage pellets were then evaluated by morphology, extracellular matrix accumulation, and chondrogenic, metabolic, and apoptotic marker expression. Furthermore, cell apoptosis was assessed by TUNEL assay. The phosphorylation level P65 and IκBα of the NF-κB pathway activity was explored on day 21 of chondrogenic differentiation of BMSCs. (Continued on next page)
(Continued from previous page)
Results: The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs in
different aspects. Firstly, MLT can restore the chondrogenic pellet size, and rescue matrix synthesis and
accumulation. Secondly, MLT can upregulate chondrogenic marker COL2A1 expression at both mRNA and protein
levels, and also regulate the expression levels of other chondrogenic markers like ACAN, SOX9, and COL10A1 in the
presence of IL-1β. Thirdly, MLT can maintain the metabolic balance of the chondrogenic process by suppressing
expression of catabolic genes, such as MMP, MMP13, and ADAMTS4. Furthermore, MLT can subdue IL-1β-induced
cell apoptosis of BMSCs throughout chondrogenesis. Meanwhile, MLT suppressed the phosphorylation level of P65
and IκBα, which were elevated by IL-1β treatment, indicating that MLT can attenuate the IL-1β-induced activation
of NF-κB signaling.
Conclusion: The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs by
restoring the pellet size and matrix accumulation, and maintaining the metabolic balance, reducing cell apoptosis.
Our study also showed that MLT can attenuate the IL-1β-induced activation of the NF-κB signaling pathway, which
is the most important pathway downstream of IL-1β, and plays a crucial role in inflammation, apoptosis, and
metabolism. Thus, MLT has prospects for treating OA due to its multifaceted functions, such as mitigating
inflammation, maintaining metabolic balance, and mitigating apoptosis.
Osteoarthritis (OA) is the most widespread degenerative
arthritic disease across the globe, and it is a primary
cause of disability, with radiographically determined OA
impacting about 37% of the US population older than
60 years of age [
]. Typical clinical characteristics
include pain, joint dysfunction, and deformity, which all
lower health-related quality of life. OA is anticipated to
be the fourth-leading reason for disability by the year
2020 because of the aging of the world’s population .
This motivates us to further explore more OA treatment
options, including stem-cell-based therapy.
OA has been thought of as a degenerative disease of
the cartilage for a long time; however, increasing
evidence suggests that inflammation plays a pivotal part in
its pathogenesis. Inflammation takes part in the early
course of OA, resulting in the metabolic dysfunction of
chondrocytes, advancing the malfunction of articular
cartilage, and eventually leads to the functional
breakdown of synovial joints. Interleukin-1β (IL-1β) is a
primary mediator of local inflammatory processes in OA
]. There are extensive studies that suggest elevated
levels of IL-1β during the cartilage destruction cascade
in the OA process [
4, 6, 7
]. IL-1β can change the
differentiation and function of chondrocytes, which can then
prompt the expression and activation of matrix
metalloproteinases (MMPs) and a disintegrin and
metalloproteinase with thrombospondin motifs (ADAMTS),
enzymes that break down the cartilage matrix,
encourage cell apoptosis, and are believed to be the
downstream effectors of OA pathogenesis [
Moreover, there is extensive literature that demonstrates
the effects of IL-1β on chondrogenic MSCs [
Wehling et al.  found that IL-1β inhibited
chondrogenesis of MSCs in a dose-dependent manner
and cell-based repair of lesions in articular cartilage will
be compromised in inflamed joints.
Contemporary treatments for OA mainly focused on
pain management, viscosupplementation, and joint
replacement, which all simply target the clinical symptoms
of the progressive stage of OA [
]. Drugs focused on fixing
the injured cartilage due to OA are desperately required.
Possible treatments, including anti-IL-1β, in OA animal
models revealed lowered infiltration of inflammatory cells
and cartilage injury [
]. Unfortunately, IL-1β blockade is
linked to liver toxicity [
]. Since articular cartilage
has a limited self-repair capacity, the use of BMSCs to
regenerate cartilage is an attractive approach due to the
multiple differentiation abilities and the extensive
resources of harvestable BMSCs available [
which inhabit bone marrow and numerous adult tissues,
are able to self-renew and differentiate into various cell
lineages, such as osteoblasts and chondrocytes. MSCs
have been established in healthy and damaged cartilage
and seem to keep at a minimum some promising ability to
regenerate cartilage [
]. Cartilage tissue-engineering
repair strategies that depend on the chondrogenesis of
MSCs are attractive, but in instances of OA they require
chondrogenesis to take place within an inflamed
environment. Moreover, MSCs interact with both the innate and
adaptive immune systems, generally leading to abatement
of ongoing inflammatory responses, which aggravates the
damage caused by inflammatory factors like IL-1β .
However, there is surprisingly little in the literature
concerning ways to stop or reverse IL-1β-induced impairment
of chondrogenesis, which could greatly improve the
clinical outcomes of cartilage tissue-engineering repair
strategies for OA treatment [
5, 11, 20
Melatonin (MLT), best known as a modulator of
circadian rhythms [
], is reported to have multiple
functions, including restriction of tumor development,
immunomodulation, and antioxidation [
and its metabolites modulate a variety of molecular
signaling pathways including proliferation, apoptosis,
metastasis, and inflammation, across a wide range of
pathophysiological situations [
]. Further, MLT
plays a pivotal part in managing skeleton establishment
and growth. Our prior evaluation revealed that MLT can
halt adipogenesis and encourage both osteogenic and
chondrogenic differentiation of MSCs [
]. IL-1β is
an important ligand of the NF-κB pathway, which is one
of the most important pathways involved in
inflammation and apoptosis. We believe that the NF-κB pathway
plays a crucial role in IL-1β’s inhibitory effects in the
process of chondrogenesis. With its powerful
anti-inflammatory and prochondrogenic effects, we
suggest that MLT could be a potential therapeutic
compound for IL-1β-inhibited chondrogenesis by
suppressing the activation of NF-κB signaling.
In the current evaluation, MLT was examined for its
potential to encourage chondrogenic differentiation, retain
metabolic balance, and lower cell apoptosis of human
MSCs with the inflammatory factor IL-1β. MLT’s influence
on the NF-κB pathway was also assessed. The objectives of
this evaluation are to additionally establish the main part
of MLT in the management of the differentiation of MSCs
in a pathological environment and its potential underlying
mechanism, offering additional evidence for the utilization
of MLT in stem-cell-based OA treatment.
Antibodies and reagents
Recombinant human IL-1β was purchased from R&D
(Minneapolis, MN, USA). MLT, Alcian blue solution,
hydrochloride, EDTA, 1,9-dimethylmethylene blue
(DMMB), and dye Hoechst 33,258 were purchased from
Sigma-Aldrich (St. Louis, MO, USA). COL2A1 antibody
was from Abcam (Cambridge, UK). The DAB Horseradish
Peroxidase Color Development Kit was from (Beyotime
Biotechnology, Beijing, China), and the MEBSTAIN
Apoptosis TUNEL Kit Direct was from MBL International Co.
(Woburn, MA, USA). The subsequent antibodies (Abs)
were bought from Cell Signaling Technology (CST,
Danvers, MA, USA): P65, phospho-P65, IκBα, phospho-IκBα,
GAPDH, goat anti-rabbit IgG H&L (HRP), and goat
anti-mouse IgG H&L (HRP).
obtained from healthy volunteer donors as described
]. In short, the bone marrow specimens
were diluted with PBS. Cells were then fractionated on a
lymphoprep density gradient by centrifugation at 500 × g
for 20 min. Interfacial mononuclear cells were gathered,
resuspended in low-glucose Dulbecco’s modified Eagle
medium (DMEM; Gibco, Waltham, MA, USA)
augmented with 10% FBS (Gibco), and then seeded and
incubated at 37 °C/5% CO2. After 48 h, nonadherent cells
were eliminated by replacing the medium with fresh
medium. The medium was then replaced every 3 days.
When the cells approached 80–90% confluence, they
were trypsinized, quantified, and plated again. Cells from
passages 3–6 were utilized for the experiments.
A high-density micromass culture system was used for
the chondrogenic differentiation of human MSCs as
described previously [
]. In short, MSCs were trypsinized,
washed, and then resuspended at 2 × 107 cells/ml in
OriCell™ Human Mesenchymal Stem Cell Chondrogenic
Differentiation Medium (Cyagen Biosciences Inc.).
Droplets (12.5 μl) were carefully placed in each interior well
of a 24-well plate. Cells were allowed to adhere at 37 °C
for 2 h, followed by addition of 500 μl chondrogenic
medium containing vehicle, 10 ng/ml IL-1β and vehicle
(PBS with 0.1% BSA), or 10 ng/ml IL-1β and 50 nM
MLT. The medium was replaced every 3 days and the
pellets were harvested on days 7, 14, and 21.
Real-time RT-PCR assay
Total RNA was removed with RNAiso Plus Reagent
(Roche, Basel, Switzerland) and then changed to cDNA
with PrimeScript™ RT Master Mix (Roche) based on the
manufacturer’s instructions. Real-time PCR was
conducted on a Light Cycler 480 Real-Time PCR Detection
System (Roche) with SYBR Green I Master Mix (Roche).
Expression levels were established for the following
genes: ACAN, COL2A1, COL10A1, SOX9, MMP9,
MMP13, and ADAMTS4. The expression level of the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
gene acted as a reference. Every PCR was processed in
triplicate. The Ct value of GAPDH was subtracted from
the Ct value of the target gene (ΔCt), and the average
ΔCt value of each replicate was documented. The
relative expression levels of every gene were established with
the 2–ΔΔCt method. Primer sequences utilized in this
evaluation are presented in Table 1.
Separation and culture of MSCs
The study was authorized by the Ethical Committee of
Sun Yat-sen University, and written informed consent
was gained from each of the participants enrolled in the
evaluation. MSCs were separated from bone marrow
Alcian blue staining
Micromasses were fixed in 4% paraformaldehyde for 3 h
and then dehydrated with ethanol, washed with xylene,
and embedded in paraffin. Sections with a thickness of
4 μm were cut and coated on the glass slides. We then
deparaffinized the slides and hydrated them three times
with distilled water, and Alcian blue solution (pH 2.5;
Sigma-Aldrich) was added and incubated for 1 h at
room temperature. After a removal of staining reagents,
the slides were washed in running tap water for 2 min.
Then mount with resinous mounting medium. Finally,
the sections were photographed with an Olympus BX51
microscope (Olympus, Tokyo, Japan).
Quantitative analysis of glycosaminoglycan
Pellets were cleaned and digested in PBS with 0.03%
papain (Merck, Darmstadt, Germany), 5 mM cysteine
hydrochloride, and 10 mM EDTA for 16 h at 65 °C. The
glycosaminoglycan (GAG) concentration was quantified
with a 1,9-dimethylmethylene blue dye binding assay. In
short, a portion of the lysate was reacted with DMMB
solution for 10 min, and the absorbance at 525 nm was
established with Varioskan Flash (Thermo Scientific,
Waltham, MA, USA). Pellet digests were taken through
three freeze–thaw cycles, and aliquots were added to
100 ng/ml of Hoechst Dye 33,258 (Sigma) in 10 mM Tris
(pH 7.4), 1 mM disodium EDTA, and 100 mM NaCl.
DNA concentration was determined by fluorescent dye
Hoechst 33,258 binding assay with a SpectraMax M5
microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Fluorescence was measured using excitation and emission
wavelengths of 485 nm and 528 nm, respectively, and
DNA concentrations were determined relative to a lambda
DNA standard curve. For GAG synthetic activity, the
resulting GAG amounts were normalized to the amount
of DNA for each sample.
We used 4% paraformaldehyde for fixation of the tissues
at room temperature for 1 h. Paraffin sections (4 μm
thick) were prepared and immunohistochemical (IHC)
analysis was performed using a Histostain-Plus Kit
(Thermo Fisher Scientific, Waltham, MA, USA). We
used 5% bovine serum albumin as the blocking reagent.
The specimen was treated for 30 min at room
temperature, and the tissues were incubated with the
anti-COL2A1 antibody (1:500) at 4 °C overnight.
Detection was performed with a DAB Horseradish Peroxidase
Color Development Kit (Origin Technologies, Inc.).
Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay
A terminal deoxynucleotidyl-transferase-mediated dUTP
nick end labeling (TUNEL) assay was conducted with a
MEBSTAIN Apoptosis TUNEL kit direct (MBL
International Co.) based on the manufacturer’s instructions.
The percentage of TUNEL-positive cells relative to
propidium iodide (PI)-stained cells was calculated. Three
independent experiments were conducted and quantified
for each experimental group.
Pellets were cleaned three times with cold PBS and
gathered in RIPA (Beyotime, Shanghai, China), adding 1%
protease inhibitor and phosphatase inhibitor. Pellets were
exposed to the liquid nitrogen for 15 min and pellet lysates
were obtained using a TissueLyser (QIAGEN, Germany).
Identical portions of each specimen were subjected to
SDS-polyacrylamide gel electrophoresis (PAGE) and moved
to PVDF transfer membranes (Millipore). Membranes were
halted with 5% nonfat milk for 1 h at room temperature
and then incubated with the anti-P65, anti-phospho-P65,
anti-IκBα, and anti-phospho-IκBα specified antibodies
(1:1000) at 4 °C overnight. Antibody-specific labeling was
noted by incubation with secondary antibodies (1:2000) for
1 h at room temperature and observed with an ECL kit
(Millipore). The band was established with ImageJ software
and normalized to GAPDH (1:2000) as the loading control.
Comparisons of perimeters, GAG content, gene expressions,
and quantitation of protein expressions were performed
using a two-tailed independent Student’s t test. Statistical
analyses for comparisons of apoptosis rate and relative
protein expressions were performed using chi-square or Fisher’s
exact tests when appropriate. All statistical analyses were
conducted with SPSS 20.0 statistical software (SPSS,
Chicago, IL, USA) and GraphPad Prism 5.01. The level of
statistical significance was established at P < 0.05.
To examine the impacts of MLT on chondrogenic
differentiation of MSCs with IL-1β, chondrogenic
differentiation was prompted in human MSCs in chondrogenic
medium with vehicle, 10 ng/ml IL-1β and vehicle, or
10 ng/ml IL-1β and 50 nM MLT. No difference was
discovered between groups in pellet size on day 7 of
chondrogenesis (Fig. 1a); quantitative analysis of the
perimeters of the pellets also confirmed that there was
no difference in chondrogenesis on day 7 between
different groups (Fig. 1b). However, the cartilage pellets
treated with IL-1β were smaller than those of the
controls in the 14-day and 21-day groups and additional
MLT treatment partially restored the size of the pellets
(Fig. 1cf–f ).
Alcian blue staining was used for evaluation of
cartilage matrix synthesis and accumulation. The results of
Alcian blue staining and the quantitative analysis of
glycosaminoglycan (GAG) showed that GAG synthesis and
matrix deposition decreased in the presence of IL-1β
and was elevated by MLT treatment on day 7 (Fig. 2a,
b), day 14 (Fig. 2c, d), and day 21 (Fig. 2e, f ) (P < 0.05).
These outcomes revealed that IL-1β suppresses the
accumulation of matrix during chondrogenesis of MSCs,
and MLT can rescue the impacts of IL-1β.
To further confirm the effects of IL-1β and MLT on
the process of chondrogenesis, the level of expression of
the typical chondrogenic marker COL2A1 was detected
using real-time RT-PCR and immunohistochemical
(IHC) staining. These findings revealed that IL-1β
dramatically inhibited collagen II expression on day 7
(Fig. 3a, b), day 14 (Fig. 3c, d), and day 21 (Fig. 3e, f )
during chondrogenesis, while the additional MLT
treatment reversed this situation at both mRNA and protein
levels (P < 0.05). These results showed that IL-1β
suppresses gene and protein expression of typical
chondrogenic marker COL2A1 of chondrogenic MSCs. Again,
MLT can counterbalance the effects of IL-1β.
Next, RT-PCR was utilized to explore the impacts of
IL-1β and MLT on the expressions of other
chondrogenic markers, such as ACAN, COL10A1, and SOX9. As
shown in Fig. 4a, b, the level of expression of ACAN and
SOX9 are consistent with the pattern of expression of
COL2A1. IL-1β downregulated ACAN and SOX9
expression, whereas after the addition of MLT to IL-1β,
upregulation of those genes was were observed on days
7 and 14 (P < 0.05). Of note, on day 21 the effects of
IL-1β and MLT on ACAN were consistent with those on
days 7 and 14 (P < 0.05), while the effect of MLT on
SOX9 was gone. COL10A1, a chondrogenic and a
hypertrophic marker, was found to be elevated by
IL-1β treatment and then declined after the addition
of MLT on day 7 (Fig. 4c) (P < 0.05). IL-1β
downregulated COL10A1 expression, whereas MLT reversed the
effect on day 14. The effect of MLT on COL10A1
was also gone on day 21. We also explored the
impacts of IL-1β and MLT on catabolic and
proapoptotic markers like MMP9, MMP13, and ADAMTS4. On
day 7, IL-1β treatment had little effect on MMP9
expression, while MLT treatment decreased the
expression of MMP9; IL-1β then upregulated MMP9
expression on days 14 and 21, while MLT
downregulated the level of expression of MMP9 (Fig. 4d) (P <
0.05). IL-1β upregulated MMP13 and ADAMTS4
expression, whereas the addition of MLT to IL-1β then
downregulated their expression on days 7, 14, and 21
(Fig. 4e, f ) (P < 0.05). The impacts of IL-1β and MLT
on these proapoptotic markers suggested that MLT
could have a part in the chondrogenesis process as an
anticatabolic and antiapoptotic agent.
We performed a TUNEL assay to determine how
IL-1β and MLT influenced the cell fate of chondrogenic
MSCs. IL-1β treatment increased the percentage of
TUNEL-positive cells compared to control, and the
addition of MLT significantly reversed this effect mainly
on day 7 (Fig. 5a, b), day 14 (Fig. 5c, d), and day 21
(Fig. 5e, f ) (P < 0.05). These results confirmed that
IL-1β induces MSC apoptosis during the process of
chondrogenesis and MLT plays the role of an
antiapoptotic agent, reducing MSC apoptosis and rescuing
The NF-κB pathway is one of the most crucial pathways
in apoptosis, and IL-1β is a pivotal ligand of the NF-κB
pathway. We believe that the NF-κB pathway plays a
crucial part in IL-1β’s inhibitory effects in chondrogenesis. To
test this hypothesis, we determined the levels of
expression and activity of key molecules of the NF-κB pathway
of the pellets on day 21 using immunoblotting. As shown
in Fig. 6a, both IL-1β and MLT regulated the
phosphorylation levels of p-P65 and p-IκBα. While the expression of
total P65 was downregulated by IL-1β, MLT then elevated
the P65 level (P < 0.05). Total IκBα remained the same in
different groups (Fig. 6b); however, IL-1β upregulated the
phosphorylation levels of P65 and IκBα, causing increased
NF-κB activation. In contrast, MLT downregulated the
phosphorylation levels of P65 and IκBα, thus attenuating
NF-κB activation (Fig. 6c, d) (P < 0.05). These results
indicated that MLT attenuated IL-1β’s impact on the NF-κB
MSCs have already been intensively examined and
utilized in clinical trials for regenerative therapies in the
skeletal system [
]. Recent studies demonstrate that
MSCs may act as anti-inflammatory agent to allow the
joint to self-repair; MSCs combined with appropriate
scaffolds can form cartilaginous or even osseous
compartments to repair cartilage . However, such
therapies have not been successful thus far and they
did not have the intended impact [
]. A large issue
that researchers encounter is the inflamed
5, 7, 11
]. Our intention is to use the in-vitro
chondrogenesis model to test the possible effect of
MLT on IL-1β-impaired cartilage formation and the
underlying mechanism. We want to prove that MLT
can act as a possible drug that helps to optimize the
cartilage tissue engineering system or even a drug
that helps the cartilage to self-repair under
inflammatory conditions. To the best of our
knowledge, our evaluation is the first to discover that MLT
can function as a proanabolic, anticatabolic, and
antiapoptotic agent for MSCs in the chondrogenic
process under IL-1β challenge.
The majority of previous studies [
] that assessed
either the effect of inflammatory cytokines or MLT have
been based on a single point in time, which disregards
valuable information in the whole process of
chondrogenesis. Liu et al.  investigated the role of MLT on
proinflammatory cytokine-inhibited chondrogenesis in
synovium mesenchymal stem cells and the possible role
of reactive oxygen species in its process. They found that
the chondroprotective effect of MLT was potentially
correlated to decreased ROS, preserved SOD, and
suppressed levels of MMPs. However, in the current study,
we used BMSCs induced for 7, 14, and 21 days to help
monitor the chondrogenic process in a continuous way,
through which we found that on day 7 IL-1β and MLT
barely affected the chondrogenesis in pellet size and
matrix accumulation. Instead, on day 21 the most
obvious effect of IL-1β and MLT was observed among the
different stages. Specifically, on day 7 the induced
cartilage pellets showed no difference in size or perimeter or
Alcian blue staining among different groups, while the
GAG content, COL2A1, ACAN, and SOX9 expression
was altered by IL-1β and MLT treatment. Of note, the
mRNA level of COL10A1, which is a cartilage
hypertrophic marker, was surprisingly upregulated in the
IL-1β group. Surprisingly, MLT downregulated the
expression level of COL10A1. Since hypertrophy is the
terminal stage of cartilage before apoptosis, this result
would lead us to realize that apoptosis of induced MSCs
may be partially responsible for unsatisfactory outcomes
of chondrogenesis in an inflamed environment. On days
14 and 21, pellets treated with IL-1β showed smaller size
and perimeter, decreased matrix accumulation, and
downregulation of typical chondrogenic marker COL2A1
at both mRNA and protein levels, whereas MLT restored
the effects. ACAN shared the same changing patterns
with COL2A1 only on day 14. SOX9, a marker of
cartilage formation, remained at low levels in the IL-1β and
MLT groups, suggesting 21 days is a terminal stage of
chondrogenesis in the presence of IL-1β. However,
MMP9, MMP13, and ADAMTS4, known as catabolic
and proapoptotic markers, were upregulated across all
three stages in the IL-1β group, and addition of MLT
reversed the expression levels of the three genes. Lastly,
the TUNEL assay confirmed the hypothesis that MLT
plays a role in rescuing IL-1β-impaired chondrogenesis
as an antiapoptotic agent.
Our study has some limitations. Firstly, IL-1β is a
highly crucial inflammatory factor, and it has a pivotal
part in the pathogenesis of OA. Apart from IL-1β,
numerous soluble inflammatory mediators (TNF-α, IL-6,
etc.) have been determined to be present in OA joint
tissues and fluids [
]. However, Liu et al. 
demonstrated that IL-1β and TNF-α had an inhibitory impact
on the chondrogenesis of MSCs. IL-1β was found to
have a more potent effect than TNF-α. Thus, we chose
IL-1β as representative to create an inflamed
environment for MSCs. Secondly, our study lacks evidence from
the protein level. MMPs and collagens are important
proteins evolving in the cartilage metabolism. We use
MMP9/MMP13/COL10A1 gene expression as catabolic,
proapoptotic, and hypertrophic markers. We will
continue our work to explore the protein expression and
functions of MMPs and ColX. Thirdly, we did not fully
excavate the underlying mechanisms of MLT’s effect.
Guo et al. [
] demonstrate that MLT inhibits
Sirt1-dependent NAMPT and NFAT5 signaling in
chondrocytes to attenuate OA. However, in the chondrogenic
process with IL-1β presence, MLT’s role is still not clear.
Apoptosis signaling pathways in OA and the potential
protective part of MLT are well established [
NF-κB signaling pathway is crucial in the apoptosis of
chondrocytes. IL-1β is a classical ligand of the NF-κB
pathway. To test this hypothesis, we detected the
expression level and activity of key molecules of the NF-κB
pathway. The results showed that on day 21, IL-1β
upregulated the phosphorylation levels but not the
expression of P65 and IκBα, causing increased NF-κB
activation. In contrast, MLT downregulated the
phosphorylation of P65 and IκBα, thus attenuating NF-κB
signaling activation. These outcomes suggested that
MLT diminished IL-1β-induced stimulation of the
NF-κB signaling pathway. Moreover, further study is
needed to explore the exact mechanism of how MLT
regulates NF-κB and also the possible mechanisms that
involve different pathways in the process. Furthermore,
the growth tendency of chondrogenic pellets in the
control group shrank from day 14 to day 21, which is not
consistent with published results. We speculated that
this was due to different cell sources and cell types and
different methods to induce chondrogenesis. Yang et al.
] used human adipose-derived stem cells and a pellet
culture system to induce chondrogenesis, their results
showing that the pellets in the control groups were
largest on day 7, and shrank from day 7 to days 14 and 21.
We found in our own results that the pellets on day 14
had the most hyaline cartilage characteristics; on day 21
the pellets showed a hypertrophic cartilage phenotype,
which may explain the reason why the pellets shrank.
Lastly, our results were based on in-vitro data only,
which limited the evidence level. We are constructing an
OA rat model to testify the role of MLT, the results (data
not shown) showing that MLT treatment can repair the
cartilage damage caused by OA.
The current evaluation offers evidence that further MLT
treatment can save the IL-1β-impaired chondrogenesis
of MSCs in various ways including pellet size,
glycosaminoglycan accumulation, COL2A1 expression at mRNA
and protein levels, and ACAN, SOX9, and COL10A1
expression levels. Moreover, MLT may achieve this through
rescuing the increased apoptosis of IL-1β-treated MSCs
and the elevated expression of MMP9, MMP13, and
ADAMTS4 in the differentiating process. MLT may have
rescued IL-1β-impaired chondrogenesis of MSCs by
affecting the NF-κB pathway. Additional evaluations have
focused on unraveling the particular mechanisms through
which MLT diminished IL-1β-prompted apoptosis in
MSCs and verified the therapeutic value of MLT in
stem-cell-based therapies for OA.
This research was supported by the National Natural Science Foundation of
China (Nos. 81572134, 81601898), the Natural Science Foundation of
Guangdong Province (No. 2017A030311008), Guangzhou Science and
Technology Program key projects (No. 201704020120), Natural Science Basic
Research Plan in Shaanxi Province (Nos. 2017JQ8056, 2017JM8115), the China
Postdoctoral Science Foundation (No. 2017M613177), and the Postdoctoral
Science Foundation in Shaanxi Province of China (No. 2017BSHQYXMZZ15).
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated
or analyzed during the current study.
DSH and PQS designed the experiments. BG, WJG, ZZW, TFZ, XJQ, XDW, CJL,
YP, AJL, JCQ, YXZ, CXX, and YBL conducted the experiments. BG, WJG, and ZZW
acquired the data. BG, WJG, DSH, and PQS analyzed the data. BG, DSH, and PQS
organized the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the ethics committee of Sun Yat-sen Memorial
Hospital, Sun Yat-sen University, Guangzhou, China.
Consent for publication
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