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RESEARCH ARTICLE
Exosomes from M2c macrophages alleviate intervertebral disc
degeneration by promoting synthesis of the extracellular
matrix via MiR-124/CILP/TGF-β
Yi Liu
1,2
| Mintao Xue
1
| Yaguang Han
1
| Yucai Li
2
| Bing Xiao
1
|
Weiheng Wang
1
| Jiangming Yu
2
| Xiaojian Ye
2
1
Department of Orthopaedics, Second
Affiliated Hospital of Naval Medical University,
Shanghai, People's Republic of China
2
Department of Orthopedics, Tongren
Hospital, Shanghai Jiao Tong University School
of Medicine, Shanghai, People's Republic of
China
Correspondence
WeiHeng Wang, Department of Orthopedics,
Second Affiliated Hospital of Naval Medical
University, No. 415, Fengyang Road, Shanghai
20003, People's Republic of China.
Email: chinawangweiheng01@163.com
Jiangming Yu and Xiaojian Ye, Department of
Orthopedics, Tongren Hospital, Shanghai Jiao
Tong University School of Medicine, No. 1111,
Xianxia Road, Shanghai 200336, People's
Republic of China.
Email: yjm_st@163.com and yexj2002@
163.com
Funding information
Interdisciplinary Program of Shanghai Jiao
Tong University, Grant/Award Number:
YG2021ZD34; National Key R&D Program of
China, Grant/Award Number:
2020YFC2008404; National Natural Science
Foundation of China, Grant/Award Number:
82102605; Natural Science Foundation of
Shanghai, Grant/Award Number:
80ZR1469800
Abstract
Immuno-inflammation is highly associated with anabolic and catabolic dysregulation
of the extracellular matrix (ECM) in the nucleus pulposus (NP), which dramatically pro-
pels intervertebral disc degeneration (IVDD). With the characteristics of tissue remo-
deling and regeneration, M2c macrophages have attracted great attention in research
on immune modulation that rebuilds degenerated tissues. Therefore, we first demon-
strated the facilitating effects of M2c macrophages on ECM anabolism of the NP
in vitro. We subsequently found that exosomes from M2c macrophages (M2c-Exoss)
mediated their metabolic rebalancing effects on the ECM. To determine whether
M2c-Exoss served as positive agents protecting the ECM in IVDD, we constructed an
M2c-Exos-loaded hyaluronic acid hydrogel (M2c-Exos@HA hydrogel) and implanted it
into the degenerated caudal disc of rats. The results of MRI and histological staining
indicated that the M2c-Exos@HA hydrogel alleviated IVDD in vivo in the long term.
To elucidate the underlying molecular mechanism, we performed 4D label-free prote-
omics to screen dysregulated proteins in NPs treated with M2c-Exoss. Cartilage inter-
mediate layer protein (CILP) was the key protein responsible for the rebalancing
effects of M2c-Exoss on ECM metabolism in the NP. With prediction and verification
using luciferase assays and rescue experiments, miR-124-3p was identified as the
upstream regulator in M2c-Exoss that regulated CILP and consequently enhanced the
activity of the TGF-β/smad3 pathway. In conclusion, we demonstrated ameliorating
effects of M2c-Exoss on the imbalance of ECM metabolism in IVDD via the miR-124/
CILP/TGF-βregulatory axis, which provides a promising theoretical basis for the appli-
cation of M2c macrophages and their exosomes in the treatment of IVDD.
KEYWORDS
cartilage intermediate layer protein, exosomes, intervertebral disc degeneration, M2c
polarization, macrophages, miRNAs
Yi Liu, Mintao Xue, and Yaguang Han contributed equally to this work.
Received: 15 July 2022 Revised: 16 January 2023 Accepted: 27 January 2023
DOI: 10.1002/btm2.10500
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2023 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.
Bioeng Transl Med. 2023;8:e10500. wileyonlinelibrary.com/journal/btm2 1of22
https://doi.org/10.1002/btm2.10500
1|INTRODUCTION
Low back pain, numbness or paralysis of the lower limb, and spinal
instability are severe clinical manifestations of spinal degenerative dis-
eases, which is significantly prevalent with the aging of the population
and places a heavy burden on social public health.
1–4
As the primary
pathological condition of spinal degenerative diseases, intervertebral
disc degeneration (IVDD) causes compression of the neural pathway,
which leads to peripheral neural disorders.
5,6
The general treatment for
IVDD-caused spinal diseases is surgical resection of herniated discs to
relieve neural compression, with solid interbody fusion and fixation.
Although this surgical therapy generally achieves efficacious pain relief,
it alters the distribution of stress on the spinal sequence and impairs
intervertebral motion function, which increases the risk of adjacent
segment disease.
7,8
Therefore, surgical discectomy and interbody
fusion are not a perfect treatment for spinal degenerative disease.
The pathology of IVDD is highly related to the particular anatomi-
cal structure of the intervertebral disc. As the core of the interverteb-
ral disc, the nucleus pulposus (NP) is a gelatinous tissue in which most
water is anchored by a network skeleton of proteoglycan and type II
collagen (Col II). Therefore, the NP effectively withstands and buffers
the pressure load from the vertebral body.
9
Under normal conditions,
an isolation barrier composed of the annulus fibrosus and endplate
keeps the NP isolated from the immune system.
10
Minor damage to
the cartilage endplate and annulus fibrosus caused by daily vertebral
motion induces micro-vascularization and the infiltration of inflamma-
tory cells into the NP.
11,12
TNF-α, IL-1, and other pro-inflammatory
cytokines produced by inflammatory cells abnormally inhibit SOX-9
expression, which further aggravates the apoptotic loss of NPCs, stim-
ulates the oversecretion of metalloproteinases and results in the
degeneration of the NP extracellular matrix (ECM).
13–16
Although
NPCs can proliferate and remodel the ECM, cell vitality is impaired by
disturbances in the metabolic and inflammatory microenvironments.
17
To break through the current bottlenecks in IVDD therapy, it is critical
to perform novel theoretical exploration on restoration of the self-
inflammation-induced imbalance of matrix metabolism and activation
of NP cells to the phenotype of ECM remodeling.
As vital immune-regulating cells, macrophages may be polarized
into distinct subtypes at different stages of inflammation.
18,19
Macro-
phages are polarized with LPS stimulation to the pro-inflammatory
M1 type in the early stage of inflammation, which serves as the initia-
tor of inflammation to remove adverse substances that are hindering
regeneration.
20–22
Macrophages are stimulated by IL-4 and polarized
to the M2a type in the advanced stage of inflammation, which limits
the extension of inflammation via the secretion of anti-inflammatory
cytokines to regulate inflammation. Macrophages stimulated by IL-10
may be polarized into the M2c type, which contribute to tissue remo-
deling and regeneration.
23,24
The different types of macrophage polar-
ization conversion are mutually transformed under distinct
microenvironments.
23
Dynamic maintenance of the temporal–spatial
balance between different macrophage subtypes is beneficial to tissue
regeneration.
18
The infiltration of M1 macrophages positively corre-
lated with the degree of IVDD, but the infiltration of M2-type
macrophages did not show a significant association with IVDD.
25,26
Therefore, the variant distribution of M1 and M2 macrophages in the
intervertebral disc may be a special indicator of the progression of
IVDD. M1 macrophages aggravate degeneration of intervertebral disc
via activating HMGB1/Myd88/NF-κB pathway and forming NLRP3
inflammasome,
27
while M2a macrophages secrete CHI3L1 to promote
ECM metabolic imbalance via ERK/JNK pathways, and transformed
NPCs to a degenerative phenotype.
28
Notably, no study investigated
the effects of M2c macrophages on the metabolism and phenotype
of NPCs.
Exosomes are nanoscale vesicles that are secreted autonomously
by host cells into the intercellular space and absorbed by neighboring
cells or distant tissue.
29,30
Exosomes transport the contained bioactive
molecules to establish an informatic exchange between the original
cells and recipient cells, which serve as a bridge for intercellular regu-
lation.
31
After fusing with recipient cells, exosomes release various
cytokines, long noncoding RNAs and miRNAs derived from original
cells, which affect the signaling pathway activation and transcription
of recipient cells.
32
Several studies found that exosomes played vari-
able regulatory roles in promoting regeneration and delaying the aging
of intervertebral discs. For example, exosomes derived from bone
marrow mesenchymal stem cells effectively regulate endoplasmic
reticulum stress in NP cells and inhibit their apoptosis.
33–35
Although
exosomes derived from M2 macrophages exert diverse effects in tis-
sue degeneration, regeneration, and tumorigenesis,
36
whether M2
macrophages affect NP metabolism and alter the process of IVDD via
the exosomal transfer of key cytokines or miRNAs is not clear.
Based on the above review, we presumed that there was a poten-
tial relationship between the ECM metabolism of the NP and
exosome-mediated remodeling of M2c macrophages. To support this
hypothesis, we first observed the influence of M2c macrophages and
secreted exosomes on the vitality and metabolism of NPCs. We evalu-
ated how the isolated exosomes from M2c macrophages improved
matrix metabolism and the activity of NP cells to promote the regen-
eration of IVDD combined with hyaluronic acid (HA) hydrogel. We
used proteomics to screen and verify the key miRNA/protein/path-
way axis mediating the effects of M2c macrophage exosomes on the
ECM-related phenotype of the NP. The miR-124-enriched exosomes
derived from M2c macrophages improved the metabolism of the NP
matrix by inhibiting CILP in the present study, which activated the
TGF-β/smad3 signaling cascade and alleviated IVDD. This finding pro-
vides a theoretical and practical basis for IVDD therapeutic strategies
based on the metabolic regulation of exosomes from immunomodula-
tory cells.
2|EXPERIMENTAL MATERIALS AND
METHODS
The Ethics Committee of Naval Medical University approved all ani-
mal experiments, which were performed in accordance with the
guidelines of the Institutional Animal Care and Use Ethics Committee
of Naval Medical University.
2of22 LIU ET AL.
2.1 |Generation of M2c macrophages
Bone marrow-derived macrophages (BMDMs) were prepared as
described previously.
37,38
Briefly, BMDMs were extracted from the
tibia and femur of 8-week-old Sprague–Dawley rats and cultured in
RPMI-1640 (Gibco) medium supplemented with 10% fetal bovine
serum (Gibco), 1% penicillin–streptomycin (Gibco), and 10 ng/mL rat
M-CSF (Peprotech) in vitro. After incubation for 7 days, BMDMs
without polarization (M0 macrophages) were obtained. M2c polariza-
tion of BMDMs was induced via the addition of 10 ng/mL recombi-
nant Rat IL-10 (Peprotech) o the medium and continuous incubation
for 2 d.
2.2 |Flow cytometry
To identify the M2c polarization of BMDMs, flow cytometry was per-
formed to detect CD11b (a general marker of macrophages) and
CD163 (a specific marker of M2c macrophages). After dissociation
and resuspension, M2c macrophages were labeled with anti-CD11b-
FITC and anti-CD163-PE (eBioscience) for 1 h. The positive rates of
CD11b and CD163 on the surface of M2c macrophages were ana-
lyzed using flow cytometry (BD, USA).
2.3 |Isolation and culture of rat NPCs
NP tissue was dissected from the coccygeal intervertebral disc of
rats and cut into pieces. The tissues were digested in 0.2% type II
collagenase (Gibco) for 3 h, filtered and washed in PBS. The isolated
NPCs were centrifuged and resuspended in DMEM/F12 medium
with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin
(Gibco). The culture medium was replaced every 3 days. NPCs at the
third passage were used in the following procedures. In addition, for
simulating pathological conditions of IVDD or stimulating
TGF/smad3 pathway, recombinant Rat IL-1β(Peprotech) and TNF-α
(Peprotech), or recombinant Human TGF-β(Peprotech) were used to
treated NPCs.
2.4 |Isolation, identification, and internalization of
exosomes from M2c macrophages
The M2c macrophages were incubated in medium with 5% exosome-
free FBS (SBI) for 48 h prior to exosome isolation. The medium of
M2c macrophages was harvested and centrifuged at 2000 gfor
30 min at 4C. After removing the residual debris, the supernatant
was filtered through 0.22-μm membrane filters and ultracentrifuged
at 120,000 g for 2 h using an Optima XPN-90 ultracentrifuge
(Beckman). The sediment was resuspended in 200 μL of precooled
PBS and stored at 80C.
The nanoparticle tracking analysis (NTA) for detecting the size of
exosomes was performed using a ZetaView PMX 110 System (Particle
Metrix). The morphology of exosomes was observed using a transmis-
sion electron microscope (JEM-1400Flash). Specific markers of exo-
somes, including TSG101, CD9, and CD63, were tested using
Western blotting.
To observe the internalization of exosomes by nucleus pulposus
cells (NPCs), we added 2 μM PKH26 to the M2c-Exos suspension and
incubated it for 5 min at room temperature. The labeled exosomes
were filtered through a 0.22-μm filter and added to the medium of
NPCs (3 10
4
cells/well in a 24-well plate). After incubating for
12 and 24 h, the NPCs were fixed with 4% polyformaldehyde, and
their nuclei were stained with 4',6-diamidino-2-phenylindole. The
fluorescent exosomes in the cytoplasm of NPCs were photographed
to show absorption into NPCs.
2.5 |EdU staining
To assess the influence of M2c macrophages and their exosomes on
the proliferation of NPCs, we seeded NPCs in 24-well plates at a den-
sity of 1 10.
4
After co-culture with M2c macrophages in Transwell
chambers (pore size: 0.4 μm) or treatment with M2c-Exoss for 24 h,
NPCs were incubated in medium with a 20 μM EdU working solution
(5-ethynyl-20-deoxyuridine, Servicebio) for 6 h. After fixation with
4% polyformaldehyde, NPCs were incubated with an EdU fluorescent
solution for 30 min in the dark. The percentage of EdU+NPCs was
calculated based on images obtained using fluorescence microscopy.
2.6 |Cellular immunofluorescent staining
Prior to co-culture with M2c macrophages or treatment with M2c-
Exoss, NPCs were cultured on slides at a proper density. NPCs were
fixed, blocked with 4% polyformaldehyde and 5% BSA and incu-
bated with primary anti-Col II (Abcam, catalog: ab34712) and anti-
MMP13 (Abcam, catalog: ab39012) at 4Covernight.NPCswere
washed with PBS and incubated with the secondary fluorescent
antibody in the dark for 2 h. After the nuclei of NPCs were stained
with 4',6-diamidino-2-phenylindole, we obtained fluorescent
images using fluorescence microscopes (IX71; Olympus).
2.7 |Migration of NPCs detected by crystal violet
staining
To evaluate the influence of M2c macrophages and their exosomes on
the migration of NPCs, we seeded NPCs on the polycarbonate mem-
brane of Transwell chambers (pore size: 0.4 μm) at a density of 1 10.
4
NPCs were co-cultured with M2c macrophages or treated with M2c-
Exoss. Cell migration was detected at 12 and 24 h. After treatment, NPCs
in the upper chamber were fixed with 4% polyformaldehyde and stained
with 0.1% crystal violet (Servicebio) for 20 min. We used a cotton swab
to wipe off the nonmigrated NPCs in the upper chamber. We used an
optical microscope (Olympus, Japan) to obtain images for cell counting.
LIU ET AL.3of22
2.8 |Western blotting
Proteins from NPCs and M2c-Exoss were obtained using RIPA lysis
buffer (Servicebio, China) and quantified using a BCA assay. Pro-
teins were separated using SDS-PAGE and transferred to a polyvi-
nylidene fluoride membrane (Servicebio, Wuhan, Hubei, China).
Membranes were blocked with TBST buffer containing 5% skim milk
for 1 h and incubated with the following primary antibodies over-
night at 4C: anti-TSG101 (Abcam, Cambridge, UK, catalog:
ab125011), anti-CD9 (Abcam, catalog: ab236630), anti-CD63
(Abcam, catalog: ab134045), anti-Col II (Abcam, catalog: ab34712),
anti-aggrecan (Abcam, catalog: ab36861), anti-MMP13 (Abcam, cat-
alog: ab39012), anti-ADAMTS5 (Abcam, catalog: ab41037), anti-
β-actin (Cell Signaling Technology, catalog: #4970), anti-CILP
(Thermo Fisher, catalog: PA5-18553), anti-Smad3 (Cell Signaling
Technology, catalog: #9513), and anti-phosphorylated Smad3 (Cell
Signaling Technology, catalog: #9520). The membranes were incu-
bated with HRP-conjugated secondary antibodies for 1.5 h. Protein
bands were visualized using an ECL kit (Servicebio), and gray values
were quantified using ImageJ software.
2.9 |Fabrication and release detection of the M2c-
Exos@HA hydrogel
First, 1 g of HA powder (Sigma, catalog: 924474) was dissolved with
then 100 mL of deionized water and evenly stirred on a magnetic stir-
rer. Then 1 mM hydrochloric acid solution was slowly added to the
prepared HA solution until its pH dropped to about 4.7. Afterward,
0.4 g of adipate diphthalide was added to the solution and mixed
evenly, then 0.4 g EDCI was added and stirred for 30 minutes. After
its pH was adjusted to about 7 by slowly adding 1 mM sodium bicar-
bonate solution, HA solution became hydrogel. After being on dialysis
for 2 days, HA hydrogel was preserved by rapid freezing and drying.
To fabricate HA hydrogels containing M2c-Exoss, 0.01 g of freeze-
dried HA hydrogel and 1 mL suspension of M2c-Exoss (0.5 mg/mL)
mixed and kept stirring in an ice bath for 30 min.
The internal morphology and structure of HA hydrogel were
observed using scanning electron microscopy (S-3400N, Hitachi). The
storage modulus (G0), loss modulus (G00) and viscosity of M2c-Exo-
s@HA hydrogel was measured by rheological test (Physica MCR302,
Anton Paar).
Before the construction of the M2c-Exos@HA hydrogel, PKH26
was used to label M2c-Exoss as mentioned above. The fluorescence
intensity on the surface of the M2c-Exos@HA hydrogel was observed
under a fluorescence microscope at different time points (0, 1, 3, 6,
12, and 24 h) to reveal the release of M2c-Exoss from the HA hydro-
gel in the short term.
To test the long-term cumulative release of M2c-Exoss from
the HA hydrogel, 0.5 mL per well of the M2c-Exos@HA hydrogel
was placed in a 24-well plate and covered with 0.5 mL PBS buffer.
The PBS buffer above the M2c-Exos@HA hydrogel was collected
and restored at different time points (2-day intervals for 60 days).
The cumulative release curve of M2c-Exoss was tested using the
BCA method.
2.10 |Animal experiment
Eighty Sprague–Dawley rats (12 weeks old, male) were randomly
divided into four groups: sham group (n=20), IVDD group (n=20),
HA hydrogel group (n=20), and M2c-Exos@HA hydrogel group
(n=20). Rats were anesthetized via an intraperitoneal injection of 2%
pentobarbital (0.3 mL/100 g weight), fixed on the operating platform,
and their tails were sterilized with ethanol. The segment of the caudal
intervertebral disc (Co9/10) was percutaneously punctured at a depth
of 5 mm with a 20-gauge needle and rotated for 30 s. For the sham
group, the puncture depth was approximately 0.5 mm, which main-
tained annulus fibrosus integration. At 2 weeks post-initial surgery
when IVDD was established, rats in each group were anesthetized
again and received corresponding injections in the degenerated seg-
ment of caudal intervertebral disc: 5 μL PBS for IVDD group; 5 μLHA
hydrogel for HA hydrogel group; and 5 μL M2c-Exos@HA hydrogel
for M2c-Exos@HA hydrogel group. This puncture injection was per-
formed using a 10-μL micro-syringe.
2.11 |In vivo tracing of M2c-Exos
To trace M2c-Exoss in the caudal intervertebral discs of rats, M2c-
Exoss were labeled with PKH26 as described earlier. The labeled
M2c-Exoss were suspended in PBS or mixed into HA hydrogel and
observed using an in vivo imaging system (IVIS Lumina LT Series III) at
different time points (0.5, 1, 3, 7, 14, and 28 days).
2.12 |MRI examination
The present study used a 7.0-T magnetic resonance imaging (MRI)
system (Bruker BioSpec 7T/20 USR; Bruker AXS GmbH, Karlsruhe,
Germany) to scan the caudal intervertebral disc of rats 4 and 8 weeks
after exosomal intervention. After gas anesthesia, the rats were
placed on the MRI platform, and the Co8/9, Co9/10, Co10/11 seg-
ments of the intervertebral discs were located. T2-weighted phase
sections in sagittal and cross-sectional plane were acquired with the
following settings: repetition time 2500 ms, echo time 16 ms; field of
view 3.0 cm; and layer thickness 0.7 mm. The obtained MRI images
were analyzed using Radiant Dicomviwer software. The T2 signal
intensity in the NP was measured, and the MRI index of the NP was
calculated to assess the degree of IVDD.
2.13 |Histological staining
Four and 8 weeks after intervention, the rats were sacrificed, and the
Co9/10 intervertebral discs were harvested. Discs were fixed,
4of22 LIU ET AL.
decalcified, and embedded into paraffin blocks. Sagittal sections of
IVDD samples at a thickness of 5 μm were obtained using radial
microtomes and fixed on slides. Hematoxylin & eosin and safranin-O/
fast green staining was performed to stain all slides according to the
manufacturer's instructions. An optical microscope (Olympus, Japan)
was used to obtain histological images. According to a previous
study,
39
a scoring system for grading the histological degeneration of
intervertebral discs was applied in the present study. Briefly, the cellu-
larity and morphology of intervertebral disc were evaluated by calcu-
lating scores from such aspects: count and morphology of cells in NP,
count of fibroblast and morphology of fibers in annulus fibrosus, and
the morphology of border between NP and annulus fibrosus. Higher
histological scores significantly predicted worse IVDD.
2.14 |Immunohistochemical staining
To evaluate the expression of Col II and MMP13 in the discs of each
group of tissues, the sections were incubated with 0.01 M citrate
buffer for 15 min at 95C and blocked with 5% skimmed milk for
30 min at 37C. Tissue sections were incubated at 4C overnight with
primary antibody (anti-Col II, 1:200, Abcam; MMP13, 1:500, Abcam).
The sections were incubated with HRP-conjugated secondary anti-
bodies (Abcam) for 1 h at 37C. The nucleus was stained with hema-
toxylin. IHC images were obtained using an optical microscope
(Olympus). The IHC Profiler of ImageJ software was used to deter-
mine the score of the positive staining area.
40
2.15 |4D label-free proteomics
2.15.1 | Sample Preparation
NPCs were treated with M2c-Exoss or M0-Exos (150 μg/mL) for 48 h
and lysed using SDT buffer. The lysate was sonicated and boiled for
15 min. After centrifugation at 14,000 gfor 40 min, the superna-
tant was quantified using the BCA method. Proteins from NPCs were
prepared as peptides for subsequent mass analysis in FASP
digestion.
41
2.15.2 | Mass spectrometry analysis
Peptides were analyzed on a nanoElute (Bruker, Bremen, Germany)
coupled to a timsTOF Pro (Bruker). Through a 25 cm 75 μm analyti-
cal column and 1.6 μm C18 beads with a packed emitter tip
(IonOpticks, Australia), peptides were separated at 300 nL/min using
a linear gradient as follows: 3% buffer B for 3 min, 3%–28% buffer B
for 70 min, 28%–38% buffer B for 7 min, 38%–100% buffer B for
5 min, and hold in 100% buffer B for 5 min. The peptides were ana-
lyzed using a timsTOF Pro system (Bruker) in parallel accumulation
serial fragmentation (PASEF) mode: mass range 100–1700 m/z, 1/K0
start 0.6 Vs/cm
2
end 1.6 Vs/cm
2
, ramp time 100 ms, lock duty cycle
to 100%, capillary voltage 1500 V, dry gas 3 L/min, and dry tempera-
ture 180C. The following PASEF settings were used: 10 MS/MS
scans (total cycle time 1.16 s), charge range 0–5, active exclusion for
0.4 min, scheduling target intensity 20,000, intensity threshold 2500,
and CID collision energy 42 eV.
2.15.3 | Data analysis
The MS data were analyzed using MaxQuant software (Version
1.6.14.0). All peptide sequences were aligned to the NCBInr database
downloaded from NCBI (ncbi-blast-2.2.28 +win32.exe), and only
the sequences in the top 10 with an E-value ≤1e3 were retained.
The cutoff of the global false discovery rate (FDR) for peptide and
protein identification was set to 0.01. Protein abundance was calcu-
lated on the basis of the normalized spectral protein intensity (LFQ
intensity). Proteins with jFold changej>2 and adj-pvalue <0.05 were
considered differentially expressed proteins. The GO term of proteins
with top Bit-Score in Blast2GO was selected. The annotation from
GO terms to proteins was completed using Blast2GO Command Line.
After elementary annotation, InterProScan was used to add the func-
tional motif information to proteins to improve annotation. Pathway
analysis was performed using the KEGG database. Fisher's exact test
was used to enrich GO terms and pathways.
2.16 |Real-time quantitative PCR
Total miRNAs from NPCs and M2c-Exoss were collected using an
Exosome DNA/RNA Extraction Kit (Guidechem). We used cDNA syn-
thesis kits (Toyobo, Osaka, Japan) to reverse transcribe cDNA. We
performed real-time PCR in a StepOnePlus real-time PCR system
(Applied Biosystems) using SYBR Green PCR Master mix (Applied Bio-
systems). The 2ΔΔCT method was used to evaluate the relative
expression of miRNAs. The primers used in the present study are
listed in Table S1.
2.17 |Transfection of plasmid
Plasmids containing the pcDNA 3.1-CILP sequence, miR-124 inhibitor
or negative control vector were constructed by GenePharma (Shanghai).
NPCs were seeded on 24-well plates at a density of 4 10
4
cells/well
and incubated until they reached 50% confluence. We transfected NPCs
with the plasmid using Lipofectamine
®
2000 (Invitrogen) for 48 h
according to the manufacturer's instructions. The effect of CILP overex-
pression was validated using Western blot analysis.
2.18 |Luciferase reporter assay
To construct the CILP 30-UTR reporter, the 30-UTR sequence (WT),
and miR-124 binding mutant sequence (MUT) of CILP mRNA were
LIU ET AL.5of22
synthesized and cloned into the pmirGLO luciferase vector (Promega,
Madison, WI, USA) by GenePharma (Shanghai) (pmirGLO-WT-CILP
and pmirGLO-MUT-CILP, respectively). HEK293 cells (ATCC) were
transfected with miR-NC OE, and miR OE and seeded in 12-well
plates at a density of 3 10
5
cells/well. The cells were transfected
with pmirGLO-WT-TLR4 or pmirGLO-MUT-TLR4 using Lipofecta-
mine
®
2000 (Invitrogen) for 48 h. The cells were treated with miR-
124 mimics for 48 h via transfection with plasmid. Luciferase activity
assessment was performed using a dual luciferase reporter detection
kit (Promega).
2.19 |Statistical analysis
The measurement data are presented as the mean ± standard devia-
tion. SPSS (Version 25.0, IBM, USA) was used to perform statistical
analyses. For data with a normal distribution, Student's ttest, one-
way, or two-way ANOVA was used for comparisons. For data without
a normal distribution, the Mann–Whitney U test was used for com-
parisons. A pvalue <0.05 was selected as the cut-off for statistical
significance.
3|RESULTS
3.1 |M2c macrophages enhanced the
proliferation, migration, and ECM synthesis of NPCs
First, M0 macrophages were polarized into M2c macrophages by IL-
10 and TGF-β. Immunofluorescence staining revealed that the expres-
sion of CD11b (pan-marker for macrophage) was similar between
M2c and M0 macrophage, while the expression of CD163 (specific
marker for M2c macrophage) was significantly elevated in M2c mac-
rophages, compared with that in M0 macrophages (Figure S1A). Flow
cytometry showed consistent difference in expression of CD163
between M2c and M0 macrophages (Figure S1B). After successful
polarization had been identified, we established a co-culturing system
of M2c macrophages in the top chamber and NP cells in the bottom
chamber for 24 h. EdU staining was used to investigate the influence
of M2c macrophages on the vitality of NPCs in vitro. As shown in
Figure 1a, the EdU fluorescence intensity of NPCs was significantly
increased under M2c macrophage co-culturing compared to the con-
trol group. We re-cultured NPCs with M2c macrophages in the Trans-
well system (NPCs in the upper chamber and M2c macrophages in the
bottom chamber) to observe the effect of M2c macrophages on the
migration of NPCs. Figure 1b shows that the number of NPCs posi-
tively stained with crystal violet in the M2c macrophage co-culturing
group was significantly higher than the M0 macrophage co-culturing
group after 12 and 24 h. These results indicated that M2c macro-
phages significantly enhanced the proliferation and migration
of NPCs.
To examine whether M2c macrophages affected the expression
of ECM proteins (Col II and aggrecan) and metalloproteinases
(MMP13 and ADAMTS5) in NPCs, we extracted proteins from NPCs
co-cultured with M2c macrophages for 48 h and detected the pro-
teins using immunofluorescence staining and Western blotting.
Figure 1c–eshows that the expression of Col II and aggrecan in NPCs
co-cultured with M2c macrophages was significantly up-regulated
compared to the control group, and the expression of MMP13 and
ADAMTS5 was significantly down-regulated. This finding indicated
that M2c macrophages regulated the balance of ECM metabolism and
promoted the secretion of matrix proteins in NPCs.
3.2 |Inhibition of exosome secretion impeded the
effects of M2c macrophages on ECM synthesis
in NPCs
Various cytokines and exosomes relay intracellular effects via a para-
crine mechanism. Therefore, we indirectly examined whether M2c
macrophages stimulated the self-renewal, migration, and ECM meta-
bolic regulation of NPCs by delivering exosomes. The present study
pretreated M2c macrophages with the exosome-secreting inhibitor
GW4869 for 24 h then co-cultured the cells with NPCs to observe
changes in proliferation and migration. Figure 2a,b shows that regard-
less of GW4869 pretreatment, M2c macrophages still significantly
increased the EdU fluorescence intensity and crystal violet positive
rates of NPCs. There was no significant difference in the proliferation
or migration of NPCs between the pretreated and nonpretreated
group groups. Therefore, these results indicated that the mechanism
by which M2c macrophages promote the proliferation and migration
of NPCs was independent of their derived exosomes, which may be
mediated by other paracrine cytokines.
We further examined the effect of GW4869-pretreated M2c
macrophages on the ECM metabolism of NPCs. As shown in
Figure 2c–e, although the expression of Col II and aggrecan in NPCs
was higher in the co-culturing group in which M2c macrophages were
pretreated with GW4869 than the control group (no macrophage co-
culture), it was significantly lower than in the co-culturing group in
which M2c macrophages were not pretreated. The expression of
MMP13 and ADAMTS5 in NPCs co-cultured with pretreated M2c
macrophages exhibited a similar dysregulated pattern, which was sig-
nificantly higher than the control group and lower than the nonpre-
treated group. Therefore, after inhibition of exosomal secretion, M2c
macrophages had weaker stimulating effects on the expression of
matrix proteins and inhibiting effects on the expression of metallopro-
teinases in NPCs. These results indirectly indicate that M2c macro-
phages may promote the secretion of ECM proteins and inhibit
metalloproteinases in NPCs by secreting exosomes.
3.3 |Extraction and identification of exosomes
from M2c macrophages
The present study isolated exosomes from the median supernatant of
M2c macrophages (M2c-Exoss) using ultracentrifugation and
6of22 LIU ET AL.
FIGURE 1 Paracrine effects of M2c
macrophages on nucleus pulposus cells
(NPCs) in vitro. (a) The proliferation of NPCs
co-cultured with M2c macrophages was
detected using EdU staining. (b) Migration of
NPCs co-cultured with M2c macrophages
was detected using crystal violet staining at
12 and 24 h. (c, d) The expression of Col II,
aggrecan, MMP13, and ADAMTS5 in NPCs
co-cultured with M2c macrophages was
assessed using immunofluorescent staining
and Western blotting. Scale bar =100 μm.
*p< 0.05, **p< 0.01.
LIU ET AL.7of22
FIGURE 2 Pretreatment with
GW4869 impaired the promoting
effects of M2c macrophages on
extracellular matrix (ECM) synthesis of
nucleus pulposus cells (NPCs).
(a) Proliferation of NPCs co-cultured
with M2c macrophages pretreated with
GW4869 was detected using EdU
staining. (b) Migration of NPCs co-
cultured with M2c macrophages
pretreated with GW4869 was detected
using crystal violet staining at 12 and
24 h. (c, d) The expression of Col II,
aggrecan, MMP13, and ADAMTS5 in
NPCs co-cultured with M2c
macrophages pretreated with GW4869
was assessed using immunofluorescent
staining and Western blotting. Scale
bar =100 μm. *p< 0.05, **p< 0.01.
8of22 LIU ET AL.
characterized the exosomes using transmission electron microscopy
(TEM), NTA, and Western blotting. First, the expression of the exo-
some markers CD9, CD63, and TSG101 was verified using Western
blotting (Figure S2A). TEM revealed that the exosomes were morpho-
logically intact and uniform in size and showed typical round shapes
(Figure S2B). Finally, NTA (Figure S2C) showed that the diameter of
M2c-Exoss appeared to be normally distributed and reached a single
peak at 125 nm, which was consistent with the feature size of exo-
somes. These results suggested that the M2c-Exoss isolated by ultra-
centrifugation conformed to the normal morphological characteristics
of general exosomes.
We labeled M2c-Exoss with PKH26 to test internalization by
NPCs. After incubation with M2c-Exoss for 24 and 48 h, the red fluo-
rescence of PKH26 was significantly enriched in the cytoplasm of
NPCs, which indicated that M2c-Exoss were absorbed into NPCs
(Figure S2D).
3.4 |M2c-Exoss mimicked the function of M2c
macrophages on ECM synthesis of NPCs in a
concentration-dependent manner
To directly demonstrate the phenotypic effects of M2c-Exoss on the
viability of NPCs, the cells were treated with M2c-Exoss at different
concentrations (0, 50, 100, and 150 μg/mL). After incubation for 24 h,
EdU staining showed that the number of proliferating NPCs was not
significantly different between the M2c-Exos-treated group and the
control group (Figure 3a). Figure 3b shows that after 12 and 24 h of
continuous intervention, different concentrations of M2c-Exoss did
not significantly influence the positive rate of crystal violet staining in
NPCs. These results indicated that M2c-Exoss did not stimulate the
proliferation or migration of NPCs, which was consistent with the
results of GW4869 pretreatment mentioned above.
We treated NPCs with M2c-Exoss at the same concentration to
investigate how M2c-Exoss regulated the expression of ECM pro-
teins and metalloproteinases. As shown in Figure 3c–e, the expres-
sion of Col II and aggrecan in NP cells increased significantly with
increasing concentrations of M2c-Exoss. The expression of MMP13
and ADAMTS5 in NPCs showed a significant and opposite trend to
the ECM proteins and decreased with increasing M2c-Exos concen-
trations. The effects of M2c-Exoss on ECM synthesis in NPCs
reached a maximum at 150 μg/mL. These results indicated that
M2-Exos promoted the expression of ECM proteins and inhibited
the synthesis of metalloproteinases in NPCs in a concentration-
dependent manner.
Moreover, we intended to investigate the potential effects of
M2c-Exoss on ECM metabolism of NPCs in pathological conditions of
IVDD. For simulating the pathological microenvironment in vitro, pro-
inflammatory cytokines, IL-1β(10 ng/mL), and TNF-α(25 ng/mL),
were used to treat NPCs for 48 h, combined with or without 150 μg/
mL of M2c-Exos. As shown in Figure S3, M2c-Exoss can significantly
mitigate the downregulating effects of TNF-αand IL-1βon Col II and
Aggrecan expression and upregulation on MMP13 and Adamts5 in
NPCs. This means the M2c-Exoss also exert protective effect on ECM
metabolism of NPCs in pathological conditions of IVDD.
3.5 |Characterization of HA hydrogel loaded with
M2c-Exoss
Hyaluronic acid (HA) hydrogels have excellent biocompatibility and
capacity of loading and transferring biomolecules in vivo application.
Given the rapid clearance of exosomes in vivo, we chose HA hydro-
gels as carriers for encapsulating exosomes. The general view of
M2c-Exos@HA hydrogel was shown in Figure 4a. The internal mor-
phology of M2c-Exos@HA hydrogel was characterized with three-
dimensional porous network structure, in which M2c-Exoss adhered
to the inner surface of the HA hydrogel (Figure 4b). The elastic mod-
ulus of M2c-Exos@HA hydrogels was studied by oscillatory rheolog-
ical experiment. As shown in Figure 4c, the storage modulus (G0)and
loss modulus (G00) of M2c-Exos@HA hydrogels increased uniformly
with the increase of angular frequency, which indicated it was stable
viscoelastic gel. In addition, the viscosity of M2c-Exos@HA hydro-
gels decreased with the increase of shear rate (Figure 4c), which was
consistent with the storage modulus (G0) and loss modulus (G00)of
hydrogels, confirming that hydrogel had eligible rheological
properties.
To evaluate whether the HA hydrogel had the advantage of a
slow M2c-Exos release, we constructed HA hydrogels loaded with
PKH26-labeled M2c-Exoss, and the short-term release of M2c-
Exoss from HA hydrogels was observed in vitro. The fluorescence
on the surface of the HA hydrogel gradually increased within 24 h of
loading with M2c-Exoss (Figure 4d). We continuously detected for
60 days and plotted the cumulative curve of M2c-Exos protein to
reflect their cumulative release from HA hydrogel. As shown in
Figure 4e, the efficiency of M2c-Exos release was maximal 7 days
after gelation of the hydrogel (the slope of the tangent line in the
cumulative curve at 7 days was the highest) and gradually slowed at
16 days. The ratio of aggregate M2c-Exos release reached 90% at
35 days after gelation. This result suggested that the HA hydrogel
can continuously release M2c-Exoss for approximately 6 weeks
in vitro.
To probe the retention of M2c-Exoss loaded in HA hydrogel
in vivo, we punctured the coccygeal discs of rats to establish an ani-
mal model of IVDD and injected PKH26-labeled M2c-Exos@HA
hydrogels into the punctured disc in a volume of 5 μL. The same vol-
ume of PKH26-labeled M2c-Exoss was injected into the control
group. In vivo imaging showed (Figure 4f) that the fluorescence signal
of M2c-Exoss without HA hydrogel decreased significantly 3 days
after injection and completely disappeared at 14 days, but the fluores-
cence signal of M2c-Exoss loaded in HA hydrogel was maintained
until 28 days after injection. This finding indicated that the HA hydro-
gel delayed the metabolic clearance rate of M2c-Exoss in vivo and sig-
nificantly prolonged the effective time of M2c-Exoss in intervertebral
discs, which is helpful to provide essential stimulation for the regener-
ation of IVDDs.
LIU ET AL.9of22
FIGURE 3 M2c-Exoss enhanced
extracellular matrix (ECM) synthesis of
nucleus pulposus cells (NPCs). (a) The
proliferation of NPCs treated with M2c-
Exoss (0, 50, 100, and 150 μg/mL) was
detected using EdU staining. (b) Migration
of NPCs treated with M2c-Exoss (0, 50,
100, and 150 μg/mL) was detected using
crystal violet staining at 12 and 24 h. (c, d)
The expression of Col II, aggrecan, MMP13,
and ADAMTS5 in NPCs treated with M2c-
Exoss (0, 50, 100, and 150 μg/mL) was
assessed using immunofluorescent staining
and Western blotting. Scale bar =100 μm.
*p< 0.05, **p< 0.01.
10 of 22 LIU ET AL.
3.6 |M2c-Exoss@HA hydrogel alleviated IVDD by
promoting ECM synthesis and inhibiting its
degradation in vivo
To assess the therapeutic effects of M2c-Exoss combined with HA
hydrogels on IVDD, we injected PBS, HA, and M2c-Exos@HA into the
degenerated coccygeal disc 4 weeks after establishment of the IVDD
model. We performed MRI examination 4 and 8 weeks after injection
and measured the MRI index, which reflected the change in T2 signals
and structure during the development of IVDD. As shown in
Figure 5a,b, the MRI index of the degeneration group was 55.45
± 13.21, which was significantly lower than the HA group (73.61
± 7.85) and the M2c-Exos@HA group (85.49 ± 9.93) at 4 weeks.
There was no significant difference between the HA group and the
M2c-Exos@HA group in the MRI index at 4 weeks. However, the MRI
index of the M2c-Exos@HA group (81.37 ± 6.91) was significantly
higher than the degeneration group (43.97 ± 4.74) and HA group
(58.29 ± 6.62) at 8 weeks.
According to H&E staining and safranin O-fast green staining, we
measured the histological degeneration score of the IVDD to evaluate
the degree of IVDD between the different groups 4 and 8 weeks after
injection (Figure 5c,d). Four weeks after injection, the degeneration
FIGURE 4 Characteristics of
HA hydrogels loaded with M2c-
Exoss. (a) A photograph of the
M2c-Exos@HA hydrogel. (b) SEM
image of M2c-Exos@HA
hydrogel. (c) Rheological analysis
of M2c-Exos@HA hydrogel.
(d) The release of PKH-26-labeled
M2c-Exoss from HA hydrogels in
24 h. (e) Cumulative curve of
M2c-proteins released from HA
hydrogels assessed using the BCA
assay. (f) Retention of M2c-Exoss
loaded with HA hydrogels in
caudal discs of rats at different
time points after implantation.
LIU ET AL.11 of 22
group exhibited a disordered ECM structure with a large number of
infiltrating inflammatory cells around the NPCs. The HA and M2c-
Exos@HA groups showed moderate infiltration of inflammatory cells
and less ECM loss than the degeneration group. However, these two
groups had similar degenerated morphology, ECM preservation, and
histological degeneration scores (HA group: 7.24 ± 0.92, M2c-Exo-
s@HA group: 7.18 ± 1.62) at 4 weeks, although they were better than
the degeneration group. Eight weeks after injection, the structural
disorder and loss of ECM in the degeneration group deteriorated and
were replaced by a large amount of fibrous tissue. Although the HA
group and the M2c-Exos@HA group also showed some fibrosis, the
structure and ECM of the NP were well preserved. The M2c-Exo-
s@HA group exhibited a more orderly and clearer structure of the NP,
less fibrosis, and more retention of the ECM than the HA group.
Although the histological scores of the HA group and the M2c-Exo-
s@HA group were significantly lower than the degeneration group
FIGURE 5 Administration of the M2c-Exos@HA hydrogel alleviated IVDD in vivo. (a) Representative MRI scans of caudal vertebral discs
4 and 8 weeks after implantation in different groups. (b) Quantification of MRI scans in various groups using the MRI index. (c) Longitudinal H&E
and safranin O staining of caudal vertebral discs in different groups 4 and 8 weeks after implantation. (d) Comparison of IVDD histological scores
between different groups. *p< 0.05, **p< 0.01. Scale bar: 200 μm.
12 of 22 LIU ET AL.
(12.77 ± 1.02), the M2c-Exos@HA group (7.26 ± 0.86) was signifi-
cantly lower than the HA group (8.61 ± 1.37). These results indicated
that M2c-Exos@HA did not have a significant therapeutic effect in
the early stage of degeneration, but it significantly delayed the pro-
gression of IVDD in long-term degeneration.
We performed immunohistochemical staining to evaluate the
expression of Col IIand MMP13 in degenerated intervertebral discs
after intervention. As shown in Figure 6a–d, the IHC score of collagen
type II in the HA and M2c-Exos@HA groups was significantly higher
than the degeneration group 4 weeks after intervention, and the IHC
score of MMP13 was significantly lower than the degeneration group.
However, there was no significant difference in the IHC scores of Col
II and MMP13 between the M2c-Exos@HA and HA groups. Notably,
the IHC scores of Col II in the degeneration and HA groups were sig-
nificantly lower than the M2c-Exos@HA group 8 weeks after inter-
vention, and the scores of MMP-13 in these two groups were
significantly higher than the M2c-Exos@HA group. These results sug-
gest that the M2c-Exos@HA hydrogel improved the secretion of ECM
proteins, inhibited metalloproteinases, and promoted the regeneration
of degenerated NP in the long-term process.
3.7 |4D-LFP proteomic analysis of
M2c-Exos-treated NPCs
To explore the mechanism by which M2c-Exoss promote regenera-
tion of IVDD via regulation of the ECM anabolism and catabolism of
NPCs, we used 4D-LFP label-free proteomics analysis to detect the
high-throughput changes of protein in NPCs after intervention with
M2c-Exoss. As shown by heatmaps (Figure 7a), the expression pat-
terns of proteins in groups treated with different exosomes
(M0-Exos and M2c-Exoss) were distinctly clustered, which sug-
gested significant M2c-Exos-induced dysregulation of protein
expression in NPCs. We set jfold-changej>2 and a BH-adjusted p
value <0.05 as the criteria to screen for differentially expressed pro-
teins in M2c-Exos-treated NPCs. As shown by the volcano plot
(Figure 7b), 98 proteins in NPCs were significantly changed.
Twenty-four proteins were down-regulated, and 74 proteins were
up-regulated. GO and KEGG analyses were performed on differen-
tially expressed proteins to further classify their functions and iden-
tify the signaling pathways involved, which may reflect the
influence of M2c-Exoss on the metabolic phenotype and signal
FIGURE 6 Administration of
the M2c-Exos@HA hydrogel
improved the synthesis of Col II
and inhibited the secretion of
MMP13 in IVDD in vivo. (a, b)
IHC staining of Col II and MMP13
in different groups 4 and 8 weeks
after implantation. (c, d)
Quantification of Col II and
MMP13 IHC staining in different
groups. *P< 0.05, **P< 0.01.
Scale bar: 200 μm.
LIU ET AL.13 of 22
transduction of NPCs to a certain extent. The differentially
expressed proteins were primarily enriched in integrin binding,
extracellular space, oxidoreductase activity, the cytokine–cytokine
interacting pathway, and the NF-κB pathway (Figure S4), which sug-
gests that M2c-Exoss affect the above-mentioned biological func-
tions and pathways of NPCs.
3.8 |M2c-Exoss promoted the synthesis of the
NPC matrix by regulating CILP
The present study further screened the down-regulated proteins in
NPCs treated with M2c-Exoss and found that cartilage intermediate
layer protein (CILP) was the most significantly inhibited (jfold-
FIGURE 7 Proteomic analysis
revealed that M2c-Exoss
promoted extracellular matrix
(ECM) synthesis in nucleus
pulposus cells (NPCs) by
regulating cartilage intermediate
layer protein (CILP). (a) Heatmap
of the 4D-LFP proteome in NPCs
treated with M2c-Exoss.
(b) Volcano plot showing NPCs
treated with M2c-Exoss. (c–e)
Western blot analysis detecting
the expression of CILP, Col II,
aggrecan, MMP13, and
ADAMTS5 in NPCs treated with
CILP-overexpressing plasmid or
M2c-Exoss. *p< 0.05, **p< 0.01.
14 of 22 LIU ET AL.
changej=2.74, adj-pvalue =0.003). Several studies found that CILP
overexpression was associated with IVDD.
42–44
Therefore, we used a
plasmid-based approach to overexpress CILP in NPCs that were
simultaneously treated with M2c-Exoss to verify whether CILP played
a critical role in M2c-Exos-mediated metabolic regulation of the ECM
of NP. Western blot results showed that treatment with M2c-Exoss
dramatically inhibited the expression of CILP in NPCs (Figure 7c–h).
Overexpression of CILP dominantly down-regulated Col II and aggre-
can and up-regulated MMP13 and ADAMTS5. Notably, the combined
intervention of M2c-Exoss partially offset the above-mentioned
effects of CILP overexpression on ECM proteins and metalloprotei-
nases. Based on these findings, we selected CILP as the key molecule
mediating M2c-Exos-induced anabolic and catabolic ECM rebalance
in the NP and focused on CILP to further examine the upstream regu-
latory molecules in M2c-Exoss and the downstream ECM metabolic
pathways in NPCs.
3.9 |CILP was targeted and inhibited by
M2c-Exos-transferred miR-124
Exosomes generally regulate the phenotype of receptor cells by deliv-
ering active substances, such as miRNA. Due to the heterogeneity of
exosomal RNAs, the different miRNAs may have opposite effects on
the phenotypic regulation of recipient cells.
26,45
Therefore, accurately
predicting and verifying the function of exosomal RNA is critical in
research on exosome-induced regeneration. Based on the findings
mentioned above, we used CILP as the core of the M2c-Exos-
dependent mechanism and further used the TargetScan database to
predict the potential miRNAs targeting CILP mRNA. Eight miRNAs,
miR-18a, miR-7a, miR-452, miR-124, miR-186, miR-17, miR-1, and
miR-191a were screened depending on the context score. Validation
using qPCR showed that mir-124 was significantly enriched in M2c-
Exoss compared to M0-Exos (Figure 8a). The expression of mir-124 in
M2c-Exos-treated NPCs was significantly higher than M0-Exo-treated
NPCs (Figure 8b). These results indicated that M2c-Exoss evidently
delivered miR-124 into NPCs.
MiRNAs bind to specific sequences at the 30end of targeted
mRNAs and promote the degradation of target mRNAs, which inhibits
the expression of target proteins.
46
To further investigate whether
miR-124 targeted the 30end of CILP mRNA and inhibited its expres-
sion, we constructed a dual-luciferase reporter system (including nor-
mal and variant sequences of CILP) and transfected it into HEK293
cells. As shown in Figure 8c, the luciferase activity was significantly
reduced after intervention with miR-124. When the CILP sequence
was mutated, the effect of miR-124 on luciferase activity was partially
inhibited. This finding indicates that miR-124 targets and degrades
CILP mRNA.
To further demonstrate that miR-124 mediated the regulation of
M2c-Exos on CILP in NPCs, we detected the expression of CILP, ECM
protein, and metalloproteinase in NPCs under the combined interven-
tion of an miR-124 inhibitor and M2c-Exos intervention. Western
blotting showed (Figure 9a) that the miR-124 inhibitor did not
significantly affect the expression of CILP in NP cells. However, when
combined with M2c-Exoss, the miR-124 inhibitor partially offset the
inhibitory effect of M2c-Exoss on CILP expression and reversed the
pro-expressing effect of M2c-Exoss on Col II and aggrecan and its
FIGURE 8 M2c-Exoss inhibited cartilage intermediate layer
protein (CILP) expression in nucleus pulposus cells (NPCs) by
transferring miR-124. (a) Comparison of miRNAs potentially targeting
CILP in M2c-Exoss and M0-Exos using qPCR. (b) Comparison of
miRNAs potentially targeting CILP in NPCs treated with M2c-Exoss
and M0-Exos using qPCR. (c) Luciferase reporter assay demonstrating
that miR-124 specifically bound to the 30UTR of CILP mRNA.
*p< 0.05, **p< 0.01.
LIU ET AL.15 of 22
down-regulating effect on MMP-13 and ADAMTS5 (Figure 9b–f).
These results suggested that M2c-Exoss inhibited CILP in NPCs by
delivering miR-124 to improve the anabolism and catabolism of ECM
in the NP.
3.10 |M2c-Exoss activated the TGF-β/Smad
3 pathway indirectly by suppressing CILP
It has been reported that the pro-degenerating effects of CILP on the
NP we related to the hindrance of the TGF-βsignaling pathway.
43
Therefore, we used the miR-124 inhibitor, M2c-Exos (150 μg/mL) and
TGF-β(10 ng/mL) to jointly intervene in NPCs and detected the activ-
ity of the TGF-βsignaling pathway using Western blotting. As shown
in Figure 10a–c, M2c-Exoss significantly up-regulated the phosphory-
lation of Smad3, but the miR-124 inhibitor had no significant influence
on the phosphorylation of Smad3. Intervention with the miR-124
inhibitor significantly impaired the phosphorylation effect of M2c-
Exoss on Smad3. These results indicated that M2c-Exoss indirectly
promoted the phosphorylation of Smad3 and enhanced the transduc-
tion of the TGF-βpathway by inhibiting the expression of CILP
protein.
4|DISCUSSION
The imbalance of ECM metabolism and inflammatory regulation in
intervertebral discs is the fundamental mechanism of IVDD. Treat-
ments to regulate this disorder and restore the normal metabolic
microenvironment of the NP are crucial to the repair of IVDD. There-
fore, an immune-modulating-based intervention strategy for IVDD
was introduced in the present study (Figure 10d). The exosome-
FIGURE 9 MiR-124
transferred by M2c-Exoss
promoted extracellular matrix
(ECM) synthesis in nucleus
pulposus cells (NPCs) by
inhibiting cartilage intermediate
layer protein (CILP). (a) Western
blot analysis detecting the
expression of CILP, Col II,
aggrecan, MMP13, and
ADAMTS5 in NPCs treated with
miR-124 inhibitors or M2c-Exoss.
(b–f) Quantification of the
Western blot analysis mentioned
above. *p< 0.05, **p< 0.01.
16 of 22 LIU ET AL.
mediated intercellular transfer of miR-124 between M2c macro-
phages and NPCs regulated the CILP/TGF-βaxis and subsequently
alleviated IVDD by enhancing synthesis and inhibiting degeneration
of the ECM.
Autoimmune responses to the NP play a critical role in promoting
IVDD, in which a variety of pro-inflammatory cytokines and cells
inhibit the anabolism of NP matrix proteins (e.g., proteoglycan and
type II collagen) and induce secretion of matrix metalloproteinases
(e.g., MMP and ADAMTS families), which worsen the inflammatory
response and matrix degeneration.
47–49
M2-polarizing macrophages
promote tissue regeneration by antagonizing the pro-inflammatory
factors mentioned above. Therefore, different subtypes of M2 macro-
phages (M2a, M2b, and M2c) probably have reversal effects on NP
degeneration. M2a macrophages, a subtype of M2 polarization
induced by IL-4, aggravated the degeneration of the NP matrix by
secreting the anti-inflammatory cytokine IL-13.
28
This contradictory
effect of M2a macrophages in IVDD may be attributed to their inac-
tion on tissue remodeling.
21
Moreover, M2b macrophages are charac-
terized as simultaneously having inflammatory regulating function and
effects of promoting tumor growth and invasion.
50
After being
induced by combined immune complexes as well as TLR or IL-1R ago-
nists, M2b macrophages produce IL1, IL6, IL10, and TNFα, which are
harmful factors for intervertebral discs.
51
Therefore, we selected M2c
macrophages, which are induced by IL-10 and TGF-βand function as
a remodeling phenotype, as a potential pivot to investigate their inter-
cellular communicating effects on NPCs. M2c macrophages promote
the vitality of NP cells and ameliorate ECM dysmetabolism. Therefore,
the relevant mechanisms by which M2c macrophages stabilize the
metabolism of the NP ECM should be further explored in future
research.
FIGURE 10 Inhibition of cartilage intermediate layer protein (CILP) by M2c-Exoss activated extracellular matrix (ECM) synthesis in nucleus
pulposus cells (NPCs) by enhancing the TGF-β/Smad3 pathway. (a) Western blot analysis detecting the expression of CILP, Smad3, and P-Smad3
in NPCs treated with TGF-β, miR-124 inhibitors or M2c-Exoss. (b, c) Quantification of the Western blot analysis mentioned above. (d) Schematic
illustration of the mechanism by which M2c-Exoss promote the synthesis of ECM in NPCs via the miR-124/CILP/TGF-βaxis.
*p< 0.05, **p< 0.01.
LIU ET AL.17 of 22
Exosomes are an important carrier for the intercellular exchange
of information and effective substitutes for simulating the bio-
regulating functions of original cells.
52
The modulating potential of
exosomes from various cells for the regenerative microenvironment
has been widely explored in the field of intervertebral disc rebuilding.
Exosomes from bone marrow mesenchymal stem cells inhibited the
assembly of the NLRP3-mediated inflammasome and alleviated apo-
ptosis of NP cells induced by oxidative stress and inflammation.
53,54
Urine-derived stem cells promote the proliferation of NP cells and the
secretion of matrix protein via the exosomal transport of protein
MATN3 to NP cells.
55
Exosomes from notochords cells prestimulated
by pressure inhibited vascularization and inflammation via the miR-
140/Wnt/β-catenin regulatory axis.
56
M2c macrophages possess
functions similar to stem cells in immune modulation and homeostasis
of the microenvironment.
57
It is rationally presumed that exosomes
mediate the intercellular regulation of M2c macrophages on NPCs to
enhance vitality and matrix protein synthesis. Therefore, the present
study further demonstrated this presumption in vitro and in vivo. Exo-
somes are intraluminal vesicles that are precursors of exosomes,
which are generally derived from intracellular in-folding of the mem-
brane mediated by ceramide.
58
The small molecule compound
GW4869 selectively antagonizes ceramides and blocks the formation
of intraluminal vesicles, which terminates the secretion of exo-
somes.
59
Therefore, GW4869 was selected to block the exosomal
secretion of M2c macrophages in the present study and indirectly
demonstrate the presumption mentioned above. As shown in the
results, M2c macrophages pretreated with GW4869 promoted the
proliferation and migration of NPCs. However, pretreatment with
GW4869 significantly antagonized the upregulation of matrix proteins
(e.g., Col II and aggrecan) and the inhibition of matrix proteases
(e.g., MMP-13 and ADAMTS5) induced by M2c macrophages. These
results suggest that M2c macrophages stimulate the anabolism of the
ECM and inhibit its degeneration by transferring exosomes to NPCs.
According to this clue, we treated NP cells with different concentra-
tions of M2c macrophage exosomes and found that M2c macrophage
exosomes significantly up-regulated the expression of Col II and
aggrecan in the NP cells and down-regulated the expression of MMP-
13 and ADAMTS5, with an optimal effective concentration of
150 μg/mL. These results indicate that M2c macrophage-derived exo-
somes effectively ameliorate the degeneration of the NP matrix by
regulating its metabolism instead of enhancing the vitality of NPCs
in vitro.
Compared to the uncomplicated environment in vitro, the in vivo
microenvironment combined with multiple factors restricts external
exosomes from exerting pro-regenerative efficacy.
60
Because of the
rapid clearance and nonspecific internalization of exosomes by the
microcirculation and other cells, the effective duration of exosomes is
reduced significantly in vivo and barely covers the entire process of
regeneration.
61
The main strategy to deliver exosomes accurately and
adequately in vivo is the use of biomaterials as effective carriers of
exosomes, which ensures the stability and long-term release of exo-
somes in specific damaged tissue.
62
Hydrogels are the most com-
monly used bioactive material for the loading and delivery of
exosomes to promote tissue regeneration.
63,64
The present study
selected HA hydrogel as the carrier of M2c macrophage exosomes
because the internal structure of HA after gelatinization was similar to
the NP tissue, which is propitious for M2c macrophage exosomes to
exert modulating effects. The decomposition of HA hydrogel lasts
more than 1 month, which makes the release of M2c macrophage
exosomes sufficiently cover the inflammatory process of the interver-
tebral disc.
65,66
In the construction of the HA hydrogel, we used the
gelation strategy of directly mixing HA powder and a suspension of
M2c macrophage exosomes without a crosslinker to avoid unknown
effects caused by the crosslinker. As shown in the present study, HA
hydrogel effectively loaded M2c macrophage exosomes and pro-
longed the continuous release of M2c macrophage exosomes in coc-
cygeal intervertebral discs of rats for up to 28 days. Therefore, HA
hydrogels are capable of providing favorable conditions for M2c mac-
rophage exosomes in vivo. According to the results of MRI, histologi-
cal staining, and IHC in the present study, HA hydrogel loaded with
M2c macrophage exosomes showed an excellent capability of pre-
serving the height, hydration, and histological structure of the coccy-
geal intervertebral disc after puncture-induced degeneration in
advanced stages after intervention (8 weeks). As shown by the IHC
results, the M2c-Exos@HA hydrogel also significantly promoted the
expression of collagen II and inhibited the expression of MMP13,
which improved the anabolism and remodeling of the NP ECM. How-
ever, the M2c-Exos@HA hydrogel was not superior to the HA hydro-
gels alone in terms of reversing the IVDD mentioned above at the
early stage after implantation (4 weeks). Given the effects of attenuat-
ing inflammation at early stage of IVDD,
67,68
HA hydrogels probably
covered the antidegeneration effects of M2c-Exos. While HA hydro-
gels degraded completely at 4 weeks after intervention, its blanketing
effects on M2c-Exos vanished, which further resulting in the reveal of
M2c-Exos therapeutic effects. Therefore, M2c macrophage exosomes
combined with HA hydrogels have long-term therapeutic effects of
improving the metabolism of the NP matrix and promoting IVDD
regeneration in vivo.
4D-LFQ proteomics was used to analyze the differential proteins
in NP cells treated with M2c-Exoss, and we found that CILP, which
was significantly down-regulated by M2c macrophage exosome inter-
vention, was a potential key molecule mediating the metabolic regula-
tory effects of M2c-Exoss on the ECM of NPCs. CILP is a
chondrocyte-secreted protein that is primarily deposited in the inter-
territorial matrix of articular cartilage.
69,70
The abnormal expression of
CILP is highly associated with the progression of IVDD.
42,71
Trans-
genic mice overexpressing CILP exhibit a tendency toward IVDD.
43
Long-term cyclic tension induces the expression of CILP protein in
human NPCs, which resulted in the downregulation of Col II and
aggrecan.
72
The present study found that M2c macrophage exosomes
were potentially dependent on CILP downregulation to produce long-
term improvement of matrix anabolism, synthesis of Col II and aggre-
can, and inhibition of matrix metalloproteinases in NPCs.
A variety of noncoding RNAs contained in exosomes may mediate
the regulation of M2c macrophages in the metabolism of NPCs via tar-
geted regulation of CILP. To accurately determine the up-stream
18 of 22 LIU ET AL.
regulatory molecules of CILP in M2c-Exoss, we used TargetScan data
to perform backward prediction of miRNAs and subsequently identi-
fied miR-124, which potentially acts on CILP. MiR-124 was also identi-
fied. According to qPCR validation and luciferase reporter assays, miR-
124 was delivered by M2c-Exoss into NPCs and bound to a sequence
of the 30end of CILP mRNA, which inhibited its translation. This find-
ing indicates that miR-124 transferred by M2c-Exoss specifically
inhibits the expression of CILP depending on the classical miRNA-
induced mechanism of mRNA silencing.
46
In subsequent rescue experi-
ment of present study, the reversal effects of miR-124 inhibitor on
ECM metabolic regulation of M2c-Exoss further demonstrated that
miR-124 promoted the synthesis of ECM proteins and inhibited metal-
loproteinases by regulating the expression of CILP in NP cells. How-
ever, it was intriguing that the single miR-124 inhibitor had no
influence on CILP expression. We speculate that there might be a rea-
son accounts for this paradoxical phenomenon. Accordingly, CILP is
regulated by various miRNAs and endogenous signals, such as miR-
330-5p and miR-542-3p, which are endogenously expressed in NPCs
and synergistically maintaining the low expression level of CILP in
NPCs.
73,74
Only inhibition of miR-124 can hardly increase CLIP level.
The molecular mechanism by which CILP promotes disc degenera-
tion is significantly related to its inhibitory function of the TGF-
β/Smad3 pathway, which is critical in maintaining the natural metabo-
lism of the NP matrix.
43,44
The CILP protein has similar domains as the
TGF-βreceptor, and CILP hinders the activation of the TGF-βreceptor
by competitively binding to TGF-βand inhibiting the phosphorylation
of Smad3.
75
Activation of the TGF-β/Smad3 pathway also promotes
the expression of CILP protein.
76
This phenomenon is the negative
feedback regulatory mechanism of the TGF-β/Smad3 signaling path-
way mediated by CILP. The present study confirmed that Smad3 phos-
phorylation was enhanced in NPCs under intervention with M2c-
Exoss, which suggests that M2c macrophage exosomes regulate the
activation of the TGF-β/Smad3 pathway. After blocking the mRNA
silencing effects of mir-124, the up-regulated CILP significantly inhib-
ited TGF-β-induced phosphorylation of Smad3, which led to inhibition
of ECM protein expression and enhancement of metalloproteinase
secretion in NPCs. Additional intervention with M2c-Exoss in NPCs
reversed the blockade of Smad3 phosphorylation and the imbalance in
ECM metabolism caused by the miR-124 inhibitor. Therefore, the
above-mentioned phenomenon accounts for the molecular mechanism
of M2c-Exoss in metabolic improvement of the NP matrix, in which
miR-124 was delivered to indirectly activate the TGF-β/SMad3 path-
way via the targeted regulation of CILP. Notably, the proliferation of
NPCs is activated by the TGF-β/Smad3 pathway.
77
However, M2c
macrophage exosomes had no significant influence on the proliferation
of NP cells with activation of the TGF-β/Smad3 pathway in the present
study. This phenomenon was likely attributed to the undiscovered pro-
liferative regulation of NP cells by other underlying bioactive molecules
from M2c-Exoss, which should be explored in future research.
There are several limitations in the present study. First, an acu-
puncture model of a rat coccyx disc cannot thoroughly mimic the
pathological process of IVDD. IVDD in humans is associated with
adaptive changes in the NP and cartilage endplate induced by long-
term multiaxial pressure. The acupuncture model of rat coccyx discs
cannot reflect the degeneration induced by mechanical loading.
Breakage of the annulus fibrosus using acupuncture leads to man-
made changes in the mechanical microenvironment of the interverteb-
ral disc rather than natural degenerating processes.
78
Therefore, to
strictly simulate the mechanical mechanism of IVDD, it is urgent to
explore new animal models of IVDD in future research. Second, prote-
omic analysis identified CILP as the core of the mechanism by which
M2c-Exoss regulated the metabolism of the NP matrix. MiR-124 from
M2c-Exoss was backward speculated and verified as the upstream
regulator of CILP. This research strategy was conducive to quickly
identifying the CILP-centered regulating axis comprised exosomal
miR-124 and the TGF-β/Smad3 pathway, by which M2c-Exoss
affected the phenotype of NP cells. However, the forward strategy
based on M2c-Exos transcriptomic analysis was used to screen the
regulatory noncoding RNAs in M2c-Exoss more extensively than the
above-mentioned backward research. Therefore, other potential non-
coding RNAs regulating NP metabolism should be mined in future
research.
In conclusion, exosomes from M2c macrophages may serve as
therapeutic agents for IVDD via the miR-124/CILP/TGF-β/Smad3
regulatory axis. HA hydrogel may be used as an effective carrier for
the long-term conservation and sustained release of M2c macrophage
exosomes in degenerated discs in vivo. The present study demon-
strated that exosomes derived from M2c macrophages combined with
biomaterials were a prospective strategy for regulating intervertebral
disc matrix metabolism and promoting degenerative regeneration.
AUTHOR CONTRIBUTIONS
Yi Liu: Conceptualization (lead); data curation (lead); formal analysis
(lead); software (lead); validation (equal); visualization (equal); writing –
original draft (lead); writing –review and editing (lead). Mintao Xue:
Conceptualization (equal); data curation (equal); formal analysis (equal);
investigation (equal); methodology (equal); software (equal); writing –
original draft (equal); writing –review and editing (equal). Yaguang
Han: Conceptualization (equal); data curation (equal); formal analysis
(equal); investigation (equal); methodology (equal); software (equal);
writing –original draft (equal); writing –review and editing (equal).
Yucai Li: Data curation (supporting); investigation (supporting); meth-
odology (supporting); software (supporting); writing –original draft
(supporting). Bing Xiao: Data curation (supporting); investigation (sup-
porting); methodology (supporting); validation (supporting); visualiza-
tion (supporting). Weiheng Wang: Data curation (supporting);
investigation (supporting); methodology (supporting); software (sup-
porting); visualization (supporting). Jiangming Yu: Conceptualization
(equal); funding acquisition (equal); project administration (equal);
resources (equal); supervision (equal). Xiaojian Ye: Funding acquisition
(lead); project administration (lead); resources (lead); supervision (lead).
FUNDING INFORMATION
This study was supported by National Key R&D Program of China
(Grant No. 2020YFC2008404), National Natural Science Foundation
of China (Grant No. 82102605), Natural Science Foundation of
LIU ET AL.19 of 22
Shanghai, China (Grant No. 80ZR1469800), Interdisciplinary Program
of Shanghai Jiao Tong University (Grant No. YG2021ZD34).
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
DATA AVAILABILITY STATEMENT
The datasets used and/or analyzed during the current study are avail-
able from the corresponding authors on reasonable request.
ORCID
Yaguang Han https://orcid.org/0000-0003-3861-3358
Xiaojian Ye https://orcid.org/0000-0001-5978-2439
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SUPPORTING INFORMATION
Additional supporting information can be found online in the Support-
ing Information section at the end of this article.
How to cite this article: Liu Y, Xue M, Han Y, et al. Exosomes
from M2c macrophages alleviate intervertebral disc
degeneration by promoting synthesis of the extracellular
matrix via MiR-124/CILP/TGF-β.Bioeng Transl Med. 2023;8(6):
e10500. doi:10.1002/btm2.10500
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