Exosomes Immunity Strategy: A Novel Approach for Ameliorating Intervertebral Disc Degeneration

Abstract

Low back pain (LBP), which is one of the most severe medical and social problems globally, has affected nearly 80% of the population worldwide, and intervertebral disc degeneration (IDD) is a common musculoskeletal disorder that happens to be the primary trigger of LBP. The pathology of IDD is based on the impaired homeostasis of catabolism and anabolism in the extracellular matrix (ECM), uncontrolled activation of immunologic cascades, dysfunction, and loss of nucleus pulposus (NP) cells in addition to dynamic cellular and biochemical alterations in the microenvironment of intervertebral disc (IVD). Currently, the main therapeutic approach regarding IDD is surgical intervention, but it could not considerably cure IDD. Exosomes, extracellular vesicles with a diameter of 30–150 nm, are secreted by various kinds of cell types like stem cells, tumor cells, immune cells, and endothelial cells; the lipid bilayer of the exosomes protects them from ribonuclease degradation and helps improve their biological efficiency in recipient cells. Increasing lines of evidence have reported the promising applications of exosomes in immunological diseases, and regarded exosomes as a potential therapeutic source for IDD. This review focuses on clarifying novel therapies based on exosomes derived from different cell sources and the essential roles of exosomes in regulating IDD, especially the immunologic strategy.

Full text

Exosomes Immunity Strategy: A Novel
Approach for Ameliorating
Intervertebral Disc Degeneration
Weihang Li
1
†
, Shilei Zhang
1
†
, Dong Wang
1
,
2
†
, Huan Zhang
1
, Quan Shi
1
, Yuyuan Zhang
3
,
Mo Wang
4
, Ziyi Ding
1
, Songjie Xu
5
*, Bo Gao
1
* and Ming Yan
1
*
1
Department of Orthopedic Surgery, Xijing Hospital, Air Force Medical University, Xi’an, China,
2
Department of Orthopaedics,
Affiliated Hospital of Yanan University, Yanan, China,
3
Department of Critical Care Medicine, Xijing Hospital, Air Force Medical
University, Xi’an, China,
4
The First Brigade of Basic Medical College, Air Force Military Medical University, Xi’an, China,
5
Beijing
Luhe Hospital, Capital Medical University, Beijing, China
Low back pain (LBP), which is one of the most severe medical and social problems
globally, has affected nearly 80% of the population worldwide, and intervertebral disc
degeneration (IDD) is a common musculoskeletal disorder that happens to be the primary
trigger of LBP. The pathology of IDD is based on the impaired homeostasis of catabolism
and anabolism in the extracellular matrix (ECM), uncontrolled activation of immunologic
cascades, dysfunction, and loss of nucleus pulposus (NP) cells in addition to dynamic
cellular and biochemical alterations in the microenvironment of intervertebral disc (IVD).
Currently, the main therapeutic approach regarding IDD is surgical intervention, but it could
not considerably cure IDD. Exosomes, extracellular vesicles with a diameter of 30–150 nm,
are secreted by various kinds of cell types like stem cells, tumor cells, immune cells, and
endothelial cells; the lipid bilayer of the exosomes protects them from ribonuclease
degradation and helps improve their biological efficiency in recipient cells. Increasing
lines of evidence have reported the promising applications of exosomes in immunological
diseases, and regarded exosomes as a potential therapeutic source for IDD. This review
focuses on clarifying novel therapies based on exosomes derived from different cell
sources and the essential roles of exosomes in regulating IDD, especially the
immunologic strategy.
Keywords: exosome, intervertebral disc degeneration, immunologic therapy, vascularization, low back pain
INTRODUCTION
Low back pain (LBP), one of the most severe medical and social problems globally, together with the
causes of complete disability in middle-aged or older adults, has affected nearly 80% of the
population worldwide. It is the most common cause of limited activity in patients younger than
45 (Taylor et al., 1994;Andersson, 1999;Millecamps et al., 2015). Dieleman et al. (2020) reported that
the total cost of back pain, neck pain, and other musculoskeletal disorders comprised a great
proportion of expenditures from 1996 to 2016—approximately $264 billion, which leads to
considerable financial burdens on both society and families of affected individuals. IDD
(intervertebral disc degeneration) is the main cause of LBP, and it occurs frequently in adults. It
is a common musculoskeletal disorder; the progression results in disc herniation, spinal canal
stenosis, and degenerative spondylolisthesis (Jin et al., 2013). While the exact etiology and
degenerative mechanisms remain delineated, existing studies have discovered several factors
Edited by:
Zhongyang Liu,
Chinese PLA General Hospital, China
Reviewed by:
Qizhao Huang,
Southern Medical University, China
Yonggao Mou,
Sun Yat-sen University Cancer Center,
China
Lei Ma,
Third Hospital of Hebei Medical
University, China
Qicong Shen,
Naval Medical Universtity, China
*Correspondence:
Songjie Xu
stu_xusj@163.com
Bo Gao
gaobofmmu@hotmail.com
Ming Yan
yanming_spine@163.com
†
These authors have contributed
equally to this work and share first
authorship
Specialty section:
This article was submitted to
Molecular and Cellular Pathology,
a section of the journal
Frontiers in Cell and Developmental
Biology
Received: 25 November 2021
Accepted: 21 December 2021
Published: 10 February 2022
Citation:
Li W, Zhang S, Wang D, Zhang H,
Shi Q, Zhang Y, Wang M, Ding Z, Xu S,
Gao B and Yan M (2022) Exosomes
Immunity Strategy: A Novel Approach
for Ameliorating Intervertebral
Disc Degeneration.
Front. Cell Dev. Biol. 9:822149.
doi: 10.3389/fcell.2021.822149
Frontiers in Cell and Developmental Biology | www.frontiersin.org February 2022 | Volume 9 | Article 8221491
REVIEW
published: 10 February 2022
doi: 10.3389/fcell.2021.822149

involved in the initiation and progression of IDD, including
aging, loading changes, poor nutrient supply, smoking, and
hereditary aspects (Adams et al., 2000;Mannion et al., 2000;
Freemont et al., 2001;Horner and Urban, 2001;Vogt et al., 2002;
Bibby and Urban, 2004;Wang et al., 2012). Although etiology is
likely multifactorial, mounting lines of evidence have pointed that
genetic factor is considered as a pivotal risk of IDD, which
accounts for more than 70%, with smaller contributions from
environmental factors (Battié et al., 1995;Bijkerk et al., 1999;
Sambrook et al., 1999;Kepler et al., 2013). The pathological basis
of IDD includes the disorders of catabolism and anabolism in the
extracellular matrix (ECM), continuous decrease in nucleus
pulposus (NP) cells, and cellular and biochemical alterations
in the microenvironment of intervertebral disc (IVD) (Wang
et al., 2020a;Binch et al., 2021).
IVD, located between adjacent vertebrae, are
fibrocartilaginous tissues and allow motion between vertebral
bodies; they provide load support, flexibility, energy storage, and
consumption in the spine (Ji et al., 2018). It is a complex avascular
organ consisting of the central NP, peripheral annulus fibrosus
(AF), which envelops the NP, and the upper and lower cartilage
endplates (EP) (Le Maitre et al., 2007). A healthy NP is gelatinous
and is primarily made of proteoglycans (glycosaminoglycan),
type II collagen, and NP cells, while the peripheral AF is a
thick, dense structure, and it is composed of type I collagen
and AF cells; EPs seal the disc, which are cartilaginous structures
that resemble the hyaline cartilage (Kadow et al., 2015). These
distinct anatomical areas formed a special complex structure,
giving it unique biomechanical properties to maintain spinal
flexibility and mechanical stability (Lam et al., 2011). Mature
IVD is made up of avascular tissues, which are inundated with
extensive ECM; the blood supply through peripheral capillaries is
rather limited, and nutrient supply could only be received from
passive diffusion from the EPs (Boubriak et al., 2013). So, it is
easily understood that IVD is prone to degeneration.
Currently, the therapeutic approaches regarding IDD mainly
include conservative treatment and surgical intervention.
Conservative treatment may not alleviate the patients’pain
immediately; patients could only maintain a normal life
mainly through oral painkillers. Surgical interventions attempt
to relieve symptoms rather than restore inherent structure and
function. Discectomy is the most common spinal surgical
treatment of IDD and typically performed in young patients
about 25–40 years old, while the impact of the alteration in
biomechanics and long-term sequelae may be significant
(Hermantin et al., 1999;Yorimitsu et al., 2001). Brinckmann
et al. found that loss of disc tissue resulted in a decrease in disc
height and intradiscal pressure, and an increase in radial disc
bulge (Brinckmann and Grootenboer, 1991). Scoville and Corkill
(1973) reported a 50% incidence of narrowing after disc surgery
at a 3-month follow-up. Tibrewal and Pearcy (1985) also found a
significant narrowing of disc space following disc surgery,
compared to non-operated controls. Patients suffer relapse
after treatment, and the disk degeneration may even accelerate
the degeneration of adjacent segments (Hashimoto et al., 2019),
so recurrent disc herniation might be an inevitable issue for
surgeons and patients; both oral pills and surgical methods could
not considerably cure IDD. Consequently, further explorations
about more effective IDD treatment approaches are of great
significance. Studies have reported that the ideal strategy for
disc regeneration is to restore the functions as well as the integrity
of disc, including biomaterials therapy, native matrices
supplement, mesenchymal stem cell (MSC) therapy, growth
factors therapy, tissue engineering technology,
immunotherapy, and exosome therapy (Longo et al., 2012;Jin
et al., 2013;Richardson et al., 2016;Bowles and Setton, 2017;
Cheng et al., 2018;Du et al., 2019;Sun et al., 2020a); these
methods are currently the most compelling research avenues for
IDD treatment.
Cell–cell communication is an essential way to exchange
information between cells; paracrine signaling is the primary
means of cellular communication, while exosome secretion is a
special mechanism of paracrine regulation, which has been widely
considered for IDD therapy (Lu et al., 2017). In this review, we
focus on the novel approach of exosome therapy, to clarify the
roles in regulating the immunological and inflammatory
pathological process of IDD. This review provides a reference
for elucidating the molecular mechanisms of exosomes in the
treatment of IDD as well as its application prospects.
EXOSOMES
Exosomes were firstly discovered in sheep reticulocytes by Pan
and Johnstone in 1893. During the maturation of sheep
reticulocytes, the release of transferrin receptors into ECM was
correlated with a type of small vesicle (Pan and Johnstone, 1983;
Pan and Johnstone, 1984); such extracellular vesicles (EVs) were
defined as exosomes in 1989 (Johnstone et al., 1989). In the last
decades, a series of EVs have been described, while the definition
of EVs remains confusing among different reports (Hunter et al.,
2008;Skog et al., 2008;Cocucci et al., 2009;Silva et al., 2011). Up
to now, the different kinds of EVs are differentiated based on their
size, content, and formation mechanisms; EVs mainly include
apoptotic bodies, microvesicles, and exosomes (Al-Nedawi et al.,
2009;Raposo and Stoorvogel, 2013), among which, apoptotic
bodies and microvesicles are generated from plasma membrane,
with a diameter of 800–5,000 nm and 200–1,000 nm, respectively
(Camussi et al., 2011;Livshits et al., 2015), and exosomes are
endogenous vesicles with a diameter of 30–150 nm (Mathivanan
et al., 2010;Sampey et al., 2014;Zhang et al., 2019a). Since
exosomes have been firstly discovered, they have been thought of
as a cellular waste product, while in recent years, it has been found
that tiny membrane vesicles contain cell-specific proteins, lipids,
and nucleic acids, which can be delivered to other cells as signal
molecules to change functions or other cells. These findings have
sparked interest in cell secretory vesicles.
The Formation of Exosomes
Exosomes, membrane-bound vesicles, with a diameter of
30–150 nm, are presented in nearly all kinds of biological
fluids; the existence of exosomes has already been found in
saliva, urine, semen, plasma, cerebral spinal fluid, bronchial
fluid, serum, amniotic fluid, breast milk, bile, synovial fluid,
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Li et al. Different Exosomes for IDD Therapy

tears, lymph, and gastric acid (Caby et al., 2005;Akers et al., 2013;
Vojtech et al., 2014;Yoshida et al., 2014;Shi et al., 2015;
Zlotogorski-Hurvitz et al., 2015;Milasan et al., 2016;Yoon
and Chang, 2017;Li et al., 2018a;Dixon et al., 2018;Goto
et al., 2018;Yuan et al., 2018). Exosomes are initially formed
by endocytosis; the cell membrane is internalized to generate
endosomes and then many small vesicles are formed inside the
endosome by invaginating parts of the endosome membrane;
such vesicles are called multivesicular bodies (MVBs). Ultimately,
these MVBs fuse with the cell membrane, releasing the
intraluminal endosomal vesicles into extracellular space by
exocytosis to become exosomes (Heijnen et al., 1999;
Gruenberg and van der Goot, 2006). They are also usually
defined as intercellular communication vectors containing
bioactive substances, including cytokines, proteins, lipids,
mRNAs, miRNAs, non-coding RNAs, and ribosomal RNAs;
the lipid bilayer of the exosomes protect them from
ribonuclease degradation and help improve their biological
efficiency in recipient cells.
The Identification of Exosomes
Generally, the existence of exosomes is authenticated by a series
of identifications to confirm; authentication methods vary from
physical characteristics to surface molecular markers, including
transmission electron microscopy (TEM), nanoparticle tracking
analysis (NTA), and Western blot for molecular marker detection
(Dragovic et al., 2011;Kowal et al., 2017;Jung and Mun, 2018).
Through TEM detection, exosomes are visualized by electron
microscopy after negative staining; exosomes usually appear as
cup-shaped entities by transmission electron microscopy, but as
hemisphere-shaped entities by cryoelectronic microscopy. There
is a high degree of morphological diversity among exosomes
isolated from different kinds of body fluids (Sahoo et al., 2011;
Höög and Lötvall, 2015). Using the NTA method, the Brownian
motion of individual vesicles is tracked, and their size and total
concentrations are calculated using NTA software. NTA could
measure cellular vesicles as small as 50 nm, which is far more
sensitive than conventional flow cytometry (lower limit is
300 nm). Besides, their phenotype could be quickly determined
by combining NTA with fluorescence measurement (Dragovic
et al., 2011). From Western blotting analysis, exosome marker
proteins include a family of four-transmembrane proteins, such
as CD9, CD63, and CD81; cytoplasmic proteins like actin and
annexins; and molecules involved in biological functions,
including apoptotic transfer gene 2 interacting protein X
(Alix), tumor susceptibility gene 101 protein (TSG101), heat
shock protein (HSP70 and HSP90), and cell-secreted specific
proteins, among which CD9, CD63, HSP70, and TSG101 are
commonly used identification proteins for exosomes (Su et al.,
2019).
Communication of Exosomes
Cell–cell communication is an essential way to exchange
information between cells, and exosomes play a pivotal role as
vehicle for carrying information. When exosomes are secreted
from host cells into recipient cells, they could regulate the
biological activities of recipient cells by transferring proteins,
nucleic acids, and lipids. Basically, exosome-mediated
intercellular communications mainly occur in three
mechanisms: First, the exosome membrane proteins could
interact with the receptors, to activate intracellular signaling
pathways of target cells (Munich et al., 2012). Second, in
ECM, exosome membrane proteins could be cleaved by
proteases, and the spliced fragments could act as ligands to
bind to receptors on the cell membrane, thus activating
intracellular signaling pathways (Tian et al., 2013). Third,
exosome membranes directly fuse with recipient cell
membranes, releasing their content such as proteins, mRNA,
and microRNA into the cytosol (Mulcahy et al., 2014).
Compared to traditional gene therapy vectors, exosomes could
protect and transfer bio factors as natural nanocarriers (van
Dommelen et al., 2012). In the clinic, the applications of
MSCs have been predominant due to their stronger functions
than other cells, such as proliferation and differentiation ability
in vitro (Yeo et al., 2013), and MSCs also generate a large amount
of exosomes, which have low immunogenic properties. Several
studies have shown that MSC exosomes may be more appropriate
than MSCs in stem cell-based therapies (Yuan et al., 2020;Krut
et al., 2021;Xing et al., 2021). Currently, numerous studies have
reported the significance of exosomes in the treatment of IDD;
the biological composition and functions of exosomes are based
on different types of cells (Wang et al., 2021).
MicroRNAs (miRNAs) are a class of non-coding single-
stranded RNA with a length of less than 22 nucleotides.
Various studies have reported the associations between
miRNA levels and IDD, including proliferation and apoptosis
of NP cells, ECM regeneration, and inflammation response (Jing
and Jiang, 2015;Li et al., 2015;Wang et al., 2015), and the
dysregulated miRNA expression is widely observed in IDD (Zhao
et al., 2014), which has essential roles in the progression of IDD,
and it attracts much attention in delivering exosome-derived
miRNAs in ameliorating IDD (Saravanan et al., 2019).
MECHANISMS OF EXOSOMES IN THE
TREATMENT OF IDD
In disc degeneration, the main pathological changes are
excessive degradation of ECM and reduction in AF and NP
cells. Since the intervertebral disc is a closed, avascular structure,
intervertebral injection is suggested to be an ideal method for
the treatment of IDD (Hu et al., 2020a). Exosomes have been
demonstrated to affect catabolism and anabolism of ECM by
inhibiting MMPs, and most exosomes play roles in IDD mainly
through releasing miRNAs (Zhang et al., 2019b); the
internalization of PKH67 (most detected)-labeled exosomes
into targeted cells indicates the involvement of exosomes in
modulating these changes. Thus, exosome therapy is a
promising therapeutic approach for IDD, which achieves its
therapeutic effects through continuous release of miRNAs,
proteins, and transcription factors that regulate metabolic
disorders, microenvironment, and cell homeostasis (Wang
et al., 2021). Existing studies have reported the application of
different resources of exosomes in the treatment of IDD; here,
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Li et al. Different Exosomes for IDD Therapy

their main therapeutic mechanisms are illustrated, as shown in
Figure 1.
By Improving Immune Microenvironment
and Inflammation Reactions
As the largest avascular organ in the body, IVDs are located
between vertebras, responsible for the sustainability, durability,
and flexibility of the spine (Ji et al., 2018). NP cells are surrounded
by AF and EPs, and they are trapped in the IVD since the
formation of NP cells; this unique structure isolates NP tissues
from the immune system of the host, and thus, IVDs are
identified as an immune privileged organ (Sun et al., 2020a).
Studies have found that various ingredients of NP induce auto-
immune and inflammation responses after exposure to the host
immune system during IDD (Capossela et al., 2014;Wang and
Samartzis, 2014), and antigen–antibody complexes are
commonly present in herniated NP tissue (Satoh et al., 1999).
The recruitment of immunocytes may lead to deterioration of
IDD, through cell–cell communication and cytokine secretion. As
for immunocyte types, activated T and B cells have been found to
be elevated by autologous NP subcutaneously in a pig model
(Geiss et al., 2007). Murai et al. (2010) have reported that
macrophages and NK cells may recognize autologous NP cells
and display positive cytotoxic effects, according to a comparison
between wild-type mice and immune-deficient mice, and
plasmacytoid dendritic cells, along with few macrophages and
memory T cells, are also found in isolated and extruded discs
(Geiss et al., 2014). Capossela et al. (2014) have also provided
direct evidence of auto-immune response, showing that IgGs are
found specific to collagen type I, II, and V and aggrecan in human
degenerative IVD samples; these findings suggest that
complicated immunocytes participate in auto-immune
response of NP tissues in different stages. Moreover,
inflammatory factors are also increased in the development of
IDD. In an autograft model, Takada et al. (2012) have detected
the high expression of TNF-α, IL-6, IL-8, cyclooxygenase 2, and
macrophage infiltration. Consequently, these reports elucidate
that with the damage of immune privilege in IVD, exposed NP
tissues could promote auto-immune response, which finally
leadstotheactivationofimmunocytesaswellasthe
infiltration of inflammatory factors. Previous research has
also reported the association between autophagy and
miRNAs, showing that dysregulation of the relationship
between autophagy and miRNAs may accelerate the aging
and apoptosis of NP cells (Akkoc and Gozuacik, 2020;Lan
et al., 2021). NP cells firstly activate or repress the expression of
specific miRNAs under stress condition, and then miRNAs
regulate autophagy level by directly targeting ATG and
signaling pathways to meet cellular demands (Zhou et al.,
2016). More studies need to be further conducted, including
the associations between exosomes and immunocytes, and
FIGURE 1 | Mechanisms of exosomes in the treatment of IDD.
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Li et al. Different Exosomes for IDD Therapy

whether these immunocytes could secrete exosomes and play a
role in the treatment of IDD.
By Suppressing Vascularization
Vascularization is widely observed in IDD, which is considered to
play essential roles in the progression of IDD. Blood vessel invasion
would cause activation of immunocytes and inflammatory factors,
increase neuralization, and thereby damage the dynamic balance in
IVD (Sun et al., 2020b). Existing studies have reported the pivotal
roles of exosomes in inhibiting the growth of blood vessels; Cornejo
et al. (2015) and Kwon et al. (2017) have found that soluble factors
from notochordal cellscould suppress endothelial cell invasion and
vessel formation by inhibiting the VEGF signaling pathway;
meanwhile, Sun et al. (2020b) have confirmed the finding that
notochordal cell-derived exosomes could suppress proliferation of
HUVECs; another research has also discovered the regulatory roles
of AF exosomes, showing that degenerative AF exosomes promote
migration of HUVECs and upregulate the expression of
inflammatory factors (Sun et al., 2021). Furthermore, Liang
et al. (2017) have reported that the co-culture system between
PMSCs and endothelial progenitor cells (EPCs) enhances the
angiogenic potential of EPCs through PDGF and the Notch
signaling pathway (Komaki et al., 2017). Collectively, exosomes
play an important role in the regulation of vascularization, different
sources and cells may result in different outcomes, and detailed
information about vascularization is fully discussed below.
By Promoting Synthesis and Inhibiting
Decomposition of ECM
Due to the special structure of AF that consists of 99% ECM and
1% AF cells, the ECM is of great significance to maintain the
avascular structure and homeostasis of IVD. Focal proteoglycan
loss has been noticed to cause alteration of ECM, which facilitates
the growth of nerves and blood vessels (Stefanakis et al., 2012).
Degenerative IVD possesses the common feature of significant
loss of ECM, including COL2 and aggrecan. MMPs are chief
catabolic factors responsible for these pathological changes, and
the dysregulated expression of MMPs is widely observed and
could enhance deterioration in IDD progression, including
MMP-1, MMP-3, MMP-9, MMP-13, ADAMTS-4, and
ADAMTS-5 (Zhang et al., 2021a). Exosomes have been
demonstrated to affect the catabolism of ECM by inhibiting
MMPs (Cabral et al., 2018;Zhang et al., 2019b). Almost all
mechanisms ultimately promote regeneration of ECM and
inhibit ECM degradation for IVD regeneration. Different
nucleic acids transferred from exosomes exhibit their roles
through multiple transductions, aiming at MMPs to modulate
expression of ECM.
POTENTIAL SOURCES OF APPLICATIONS
BY EXOSOMES DURING THE
PROGRESSION AND TREATMENT OF IDD
A number of exosomes from different sources have been
reported, among which exosomes could be divided into two
major parts based on their properties: stem cell-derived
exosomes and non-stem cell-derived exosomes. They could be
further classified according to source, such as BMSC, PMSC,
USC, and ADSC exosomes from stem cell-derived exosomes, and
AF, NP, and NC exosomes from non-stem cell-derived exosomes.
This review aims to focus on the different sources of exosomes to
elucidate the detailed mechanisms in the treatment of IDD; the
whole mechanism is illustrated in Figure 2.
Stem Cell-Derived Exosomes
As a worldwide issue, the treatment of IDD based on stem cells
has been widely studied; the rationales of stem cell therapy are
considered as replenishing disc cells through multipotent
differentiation, promoting proliferation of NP cells, enhancing
immune privilege, and reducing apoptosis and anti-inflammation
(Ma et al., 2015). Studies have already confirmed the potential
therapeutic efficacy of MSC transplantation in the treatment of
IDD, and by inheriting the characteristics of MSCs, exosomes
could also treat IDD through protecting NP cells from apoptosis,
mitigating the inflammatory responses of disc, and promoting the
synthesis of ECM (Lu et al., 2017;Xing et al., 2021).
The usage of exosomes as a cell-free product has
continuously been regarded as substitute therapy against
stem cell transplantation; the applications of exosomes rather
than stem cells have advantages including low risk of
tumorigenesis, malformations, and microinfarctions;
convenience in collection and storage; increase of sustained
biological activity; stability; and minimal immunogenicity when
transplantation (Tao et al., 2018). Exosomes have been
demonstrated to affect the catabolism of ECM by inhibiting
MMPs (Cabral et al., 2018;Zhang et al., 2019b). Consequently,
exosome therapy is a novel promising therapeutic approach for
IDD; the efficacies are achieved from the continuous release of
microRNAs, proteins, and transcriptome factors to regulate
metabolic disorders, microenvironment, and cell homeostasis
(Xing et al., 2021). Different stem cell-derived exosomes are
highly specific in treating IDD; compared to traditional gene
therapy vector, exosomes could be considered as nanocarriers to
transfer specific molecules including miRNA, siRNA, or
AntagomiR to recipient cells through endocytosis and
membrane fusion (Zhou et al., 2016;van den Boorn et al.,
2011). The detailed mechanisms of MSC exosomes in the
treatment of IDD are discussed as follows:
Exosomes Derived From Bone Marrow Mesenchymal
Stem Cells
BMSCs derived from mesoderm are a type of stem cell with
multidirectional differentiation potential. BMSC transplantation,
which serves as a representative cell therapy, is becoming
prevalent in the field of bone regeneration, cartilage repair,
spinal cord injury, and IDD (Sakai and Andersson, 2015;
Hejazi et al., 2021;Jiang et al., 2021;Maldonado-Lasunción
et al., 2021). However, stem cell therapy still has many
limitations such as difficulty in obtaining cells, susceptibility to
aging, potential tumorigenesis, and immune rejection, which
greatly limit the application in the field of regenerative
medicine (Dou et al., 2021).
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BMSC exosomes have advantages of low immunogenicity and
suitability for the IVD microenvironment, and it could provide
cell-free therapy instead of the traditional BMSC therapy. BMSC
exosomes have similar functions to BMSC, such as restoring
tissue damage, inhibiting inflammation, and regulating immune
microenvironment. Existing research has reported that BMSC
FIGURE 2 | Potential sources of applications by exosomes in the treatment of IDD.
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exosomes could modulate the degenerative NP gene toward a
healthy NP gene phenotype (Lu et al., 2017). High levels of
inflammatory factors, like TNF-α, have been demonstrated to
result in excessive apoptosis of NP cells and cause IDD
progression (Risbud and Shapiro, 2014), and growing lines of
evidence have indicated the roles of miRNAs in the progression of
IDD, showing that exosomes transport these miRNAs into IVD
(Lan et al., 2016). Cheng et al. (2018) showed that in an IDD rat
model, BMSC exosomes inhibited TNF-α-induced NP cell
apoptosis, and further analysis suggested that exosomes were
rich in miR-21 and inhibited NP cell apoptosis by specifically
targeting PTEN through transporting miR-21 into NP cells. In a
study of degenerated and normal NP cells, Xia et al. (2019) have
found various proteins related to inflammatory responses in IVD;
the exosomes suppress the progression of IDD by targeting the
activation of inflammatory mediators NLRP3 in NP cells, which
is an effective therapeutic target of IDD. Zhu et al. (2020a) have
suggested highly expressed miR-532-5p in BMSC exosomes,
which could suppress the TNF-α-induced apoptosis, ECM
degradation, and fibrosis deposition in NP cells by delivering
miR-532-5p via targeting RASSF5. RASSF5, a major member of
RASSF5 family, could selectively trigger RAS and function as a
tumor suppressor, which is highly correlated with cell
proliferation, apoptosis, and tumorigenicity among many
neoplasms (Antoniou et al., 1996;Suryaraja et al., 2013).
MMP-13, a regulatory gene of ECM degradation in IDD, has
also been proven to be complementary with miR-532-5p
(Mohanakrishnan et al., 2018). They revealed two therapeutic
targets to attenuate IDD: miR-532-5p in BMSC exosomes could
decrease the apoptosis of NP cells by targeting RASSF5 and
inhibit ECM degradation by targeting MMP-13 in NP cells.
Autophagy is tightly connected to aging as well as apoptosis in
the pathogenesis of disorders, including cancer, osteoarthritis,
and degenerative diseases. Autophagy exists in degenerative disc
and is an effective approach in regulating ECM metabolism in
IVD (Zhang et al., 2021b). Related studies have elucidated that
BMSC exosomes promote proliferation of AF cells by inhibiting
expression of inflammatory cytokines stimulated by IL-1β. The
PI3K/AKT/mTOR signaling pathway is activated by BMSC
exosomes, which is an essential signaling pathway regulating
autophagy (Li et al., 2020), and a previous study has also
confirmed the regulation of exosomes in the PI3K/AKT/
mTOR signaling pathway and that human umbilical cord
mesenchymal stem cell-derived exosomes inhibited apoptosis
of H9C2 cells via autophagy regulation (Liu et al., 2019).
Unfolded protein response (UPR) is an evolutionarily
conservative reaction induced by endoplasmic reticulum (ER)
stress; it is the most typical response among ER stress. When any
abnormal reaction of ER occurs, ER stress could be triggered to
protect the homeostasis of ER (Hetz, 2012). However, severe or
prolonged ER stress could hyperactivate UPR, which results in
excessive degradation of cellular proteins, and eventually leads to
cell demise (Kim et al., 2008). AGEs (advanced glycation end
products), also called the Millard reaction, are non-reducible
substances generated by the polymerization of sugars and
proteins through a series of reactions (Chan et al., 2016).
AGEs commonly accumulate in aging and degenerative
diseases, and are closely related to inflammation, metabolic
dysfunction, and ER stress (Chiang et al., 2016). Both AGEs
and ER stress are highly correlated with IDD; higher levels of
AGEs are detected in degenerative disc, and AGEs are also
connected to Pfirrmann grades of IVD (Fields et al., 2015;
Song et al., 2018). Liao et al. (2019) have demonstrated that
according to an in vitro and rat model, BMSC exosomes reduce
AGE-induced ER stress, inhibit the activation of UPR, and
ameliorate NP cell apoptosis. AGE accumulations are reported
to induce ER stress, leading to cell apoptosis by induction of
prolonged UPR, and the accumulation of CHOP could promote
the cleavage of caspase-3 and caspase-12, resulting in cell
apoptosis (Adamopoulos et al., 2016;Go et al., 2017). Through
detecting the related signaling pathway AKT, ERK, and ER-
related CHOP protein, they elucidated that BMSC exosomes
significantly activate AKT and ERK signaling, which reduced
expression of CHOP, then further inhibited caspase-3 and
caspase-12, and ultimately decreased the apoptosis of NP cells
and catabolism of ECM, thus preventing IDD.
The mitogen-activated protein kinase (MAPK) family consists
of ERK1/2, JNK, and p38 MAPK proteins, and studies show that
ERK is mediated to cell proliferation and inflammation (Sawe
et al., 2008); p38 MAPKs are a type of proinflammatory mediator,
and p38 MAPKs and JNK both modulate cell apoptosis and
inflammation (Yang et al., 2016;Lai et al., 2019). Increasing lines
of evidence have shown that the activation of MAPK transduction
is activated and tightly correlated to ECM degradation, cell aging,
apoptosis, and inflammatory reactions in IDD (Zhang et al.,
2021b). MLK3 belongs to serine/threonine MAPK kinase
(MAP3K) and is aberrantly expressed in mammals, which
mediates cell migration and invasion in various diseases
(Zhang et al., 2014a;Lan et al., 2017;Misek et al., 2017). Zhu
et al. (2020b) have revealed the relationships between miR-142-
3p and IDD, that miR-142-3p is overexpressed in BMSC
exosomes and miR-142-3p excreted from BMSC exosomes
could ameliorate NP cell injury via targeting MLK3, which
further inhibits the activation of the MAPK signaling pathway.
They have proved that exosomal miR-142-3p and the subsequent
MLK3/MAPK cascade transduction could be as an effective
approach in attenuating the progression of IDD.
Exosomes From Urine-Derived Stem Cells
Stem cells are regarded as ideal cells for IVD degeneration, since
they could prevent IVD tissues from aging and apoptosis. The
applications of exosomes from BMSC are widely used in various
diseases including IDD, while the limited sources of BMSC,
expensive costs to obtain BMSC, and the physical trauma in
the acquisition process (Sakai and Andersson, 2015) prompt
researchers to find safer, less expensive, and more effective
exosomes from other sources. Studies have discovered that a
subpopulation of cells isolated from urine have similar
characteristics to BMSCs, such as multidirectional
differentiation capacity, clonogenicity, expansion patterns, self-
renewal capacity, and paracrine properties (Bodin et al., 2010;Wu
et al., 2011a;Bharadwaj et al., 2011;Wu et al., 2011b). These cells
are thereby named urine-derived cells or USCs. Compared to
BMSCs, USCs possess several merits: first, they could be collected
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Li et al. Different Exosomes for IDD Therapy

through a simple, safe, low-cost, and non-invasive manner;
second, the acquisition procedures do not violate ethics; last,
USCs have a low cost of culture and a faster proliferative rate;
besides, they could avoid immunological rejection when using
autologous therapy (Qin et al., 2014;Pavathuparambil Abdul
Manaph et al., 2018). Therefore, USCs, as a novel cell source, may
provide a more effective way in the amelioration of IDD.
IVD tissues are subjected to different levels of mechanical
stress in daily work and life, which have essential roles in spinal
biomechanics. Wang et al. (2013) and Gawri et al. (2014) have
discovered that excessive mechanical stress induces cell apoptosis,
promotes ECM-degrading enzymes, and finally leads to IDD. As
mentioned above, certain levels of UPR in ER stress could prevent
cells from external stimulus and restore cell homeostasis. Proper
biomechanical loads play pivotal roles in the structure and
function of articular cartilage, while abnormal and continuous
biomechanical stimulation could lead to accumulation of
misfolded proteins in ER lumen, resulting in continuous ER
stress and then cell apoptosis (Wang and Kaufman, 2016;
Hunt et al., 2020;Yang et al., 2020). Xiang et al. (2020) have
discovered the therapeutic application of USC exosomes in IDD
under high mechanical loads. They have elucidated that USC
exosomes suppress excessive activation of UPR, cell apoptosis,
and disc degeneration through AKT and ERK signaling pathways,
which is consistent with the above finding that AKT/ERK
transduction is activated in BMSC exosomes and behave in
AGE-induced ER stress (Liao et al., 2019). These findings
imply that AKT/ERK signaling pathways mediated by
exosomes play active roles in AGE-induced or biomechanical
load-induced IDD, leading to caspase reduction by inhibiting
CHOP protein, and thereby reducing ECM degradation and NP
cell apoptosis. Consequently, inhibiting ER stress induced by
AGEs via BMSC or USC exosomes may be a potential therapeutic
target in the treatment of IDD. However, the type of nucleic acids
exosomes carry to mediate the AKT/ERK signaling pathway still
needs to be further analyzed.
COL2 and proteoglycan (chiefly aggrecan, namely, ACAN) are
considered as crucial ECMs for discs to maintain their proper
functions, especially for NP cells (Vo et al., 2013). Guo et al.
(2021) have suggested that the methods of rebalancing disordered
COL2 and ACAN and increasing the synthesis is regarded as a
major point for ameliorating IDD progression. They have found
that USC exosomes could induce cell proliferation of NP cells and
ECM synthesis. TGF-βis a multifunctional cytokine, which
modulates cell fate and plasticity, and MATN3 could be
directly bound to specific integrins, promoting dissociation
and activation of TGF-βand affecting downstream gene
activation (Pullig et al., 2002). Lines of evidence have
demonstrated that multiple cellular reactions induced by TGF-
βare modulated via a canonical SMAD pathway and
noncanonical pathways like PI3K/AKT transduction (Goc
et al., 2011;Hamidi et al., 2017). Guo et al. (2021) have
confirmed that USC exosomes are found to be rich in
MATN3 protein, and exosomal MATN3 could ameliorate IDD
progression by activating the TGF-β/SMAD pathway to promote
the expression of COL2 and ACAN in ECM and NP cells, and by
triggering the TGF-β/PI3K/AKT pathway to function in cell
proliferation and antisenescence (Feng and Qiu, 2018).
Exosomes Derived From Human Placental
Mesenchymal Stem Cells
As mentioned above, although BMSCs are regarded as a gold
standard among other MSCs, the difficulty and efficacies in
obtaining BMSCs have forced scientists to find novel and
multifunctional sources. Among them, the placenta has
attracted much attention as an alternative source of BMSCs,
due to its abundance and ease of availability (Mathew et al., 2020).
It has been reported that a large amount of PMSC could be
collected from a small tissue chunk (In ’t Anker et al., 2004;
Parolini et al., 2008), making the acquisition of PMSC easier,
together with its exosomes. As a transient materno-fetal organ,
the placenta is disposed of after delivery and involves non-
invasive procedures, making it a more convenient source
(Mathew et al., 2020;Gorodetsky and Aicher, 2021). The
applications of PMSC exosomes have been reported to affect
osteogenic and adipogenic differentiation by regulating OCT4
and NANOG in dermal fibroblasts (Tooi et al., 2016). The main
advantage of PMSC exosome-based therapies appears to be the
secretion of a wide range of anti-inflammatory and pro-
regenerative factors, which makes it possible for PMSC
exosomes to treat IDD.
Yuan et al. (2020) have discovered the applications of PMSC
exosomes in IDD; they have found that miR-4450 is highly
expressed in degeneration disc and PMSC exosomes have
therapeutic effects on NP cells, and by inhibiting miR-4450
and thus upregulating downstream gene ZNF121 to alleviate
apoptosis, inflammation, and necrosis of NP cells, it could
even improve gait abnormality in vivo caused by IDD.
ZNF121 has been demonstrated to be the target of miR-4450;
as one of the biggest families of regulatory proteins in human
cells, ZNF121 plays an essential role in the development and
differentiation of various diseases (Ladomery and Dellaire, 2002).
It is modulated by multi-miRNAs and causes diseases including
breast cancer, cell proliferation of childhood neuroblastoma
(miR-1427), and invasion of gastric cancer (miR-204-5p) (Luo
et al., 2016;Wu et al., 2018;Huan et al., 2019). In addition to
finding that PMSC exosomes could directly inhibit miR-4450 and
then upregulate ZNF121, and thus ameliorate the apoptosis of NP
cells and IDD progression, Yuan et al. also successfully delivered
AntagomiR-4450 into exosomes, which made it more effective
than exosomes, further verifying the efficacy of PMSC exosomes
in treating IDD alone and their natural merits as an
oligonucleotide carrier. The method of using the direct role of
siRNA has been considered as an excellent therapeutic approach,
while its low bioavailability limits the full realization of its clinical
potential (El Andaloussi et al., 2013). These findings imply that
the containing of oligonucleotide-based vesicles or drugs opens a
novel avenue for target therapy and that PMSC exosomes could
be regarded as nanocarriers to deliver specific molecules, like
siRNA or AntagomiR, and then transport the contents to target
cells through membrane fusion or endocytosis (Zhou et al., 2016;
Yuan et al., 2020).
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Although PMSC exosomes could be regarded as a vector that
delivers miRNAs, its vascularization characteristic is not suitable
for treating IDD, due to the avascular structure of IVD. The
detailed mechanisms between neovascularization and exosomes
would be discussed later.
Exosomes From Adipose-Derived Mesenchymal Stem
Cells
Like USCs and PMSCs, ADSCs also come from a wide range of
sources and the damage to people is negligible when collected
from the human body (Xing et al., 2021); besides, the applications
of ADSC exosomes have been confirmed to accelerate the
proliferation and inhibit apoptosis of target cells, and they
have anti-inflammatory effects in the treatment of ruptured
tendon (Shi et al., 2018;Zhang et al., 2021c), which suggests
their potential ability in degenerative diseases like IDD. Studies
have obtained exosomes from ADSC immobilized on ECM
hydrogels and then successfully construct an injectable
thermosensitive hydrogel system through mutual crossing of
ADSC exosomes and ECM hydrogels to restore the
microenvironment as well as pyroptosis of IDD (Xing et al.,
2021). They have confirmed that ADSC exosomes could regulate
the expression of MMPs directly, inhibit the catabolism of ECM,
and thus promote the accumulation of aggrecan and COL2.
Besides, ADSC exosomes could also inactivate the
inflammatory factor NLRP3, retard their release, and then
affect the pyroptosis and survival of NP cells. Compared to
traditional exosome release, dECM (decellularization ECM)
exosome combination possesses several merits, such as
preventing immune response, high load rate, slow release of
exosomes, and prolonging their activation time. The
researchers established dECM to load engineered exosomes to
delivery drugs into IVD and NP cells, and eventually enhance the
drug effects of small molecules.
Besides, ADSC exosomes have also been found to activate the
SMAD signaling pathway in tendon healing (Liu et al., 2021a),
which is consistent with the above findings that SMAD
transduction is upregulated by BMSC and USC exosomes.
Thus, further analysis should be expanded about the SMAD
signaling pathway via ADSC exosomes in the treatment of
IDD, and the correlations between SMAD and other different
sources of exosomes should also be further observed.
Non-Stem Cell-Derived Exosomes
In addition to the advantages of stem cell-derived exosomes
mentioned above, they also possess disadvantages: they may
cause hypertrophic differentiation of newly formed tissues,
together with unexpected angiogenesis (Chen et al., 2018),
which are the limitations in such closed and avascular IVD.
However, human cartilage cell-derived exosomes have been
found to facilitate chondrogenesis of cartilage progenitor cells
without such deficiency (Chen et al., 2018), and normal
chondrocytes are located in a similar avascular and closed
environment and share many common features like NP cells
(Rosenthal et al., 2015). Additionally, existing studies have
reported that exosomes from non-stem cells like NP cells
could promote the migration of MSC cells and induce MSC
into IVD differentiation (NP-like phenotype) (Chen et al., 2018);
AF-derived exosomes could also have the same functions. At
present, non-stem cell-derived exosomes that could ameliorate
IDD progression are from NP, AF, and notochordal cells; the
specific mechanisms of these exosomes are discussed as follows:
Exosomes Derived From Nucleus Pulposus
Recent studies of exosomes regarding IDD mainly focus on MSC-
derived exosomes, while the application of NP cells has not been
widely reported yet. Zhang et al. (2021a) have discovered that NP
cells could secrete exosomes and deliver miR-27A to prevent
ECM degradation by targeting MMP-13. Autophagy activation
could promote release of NP exosomes and thereby prevent the
NP cell matrix from degradation, and it also ameliorates
degeneration of IVD, at least partly via exosomal miR-27A,
which targets and inhibits MMP-13 expression. In an in vitro
mutual experiment, Lu et al. (2017) have elucidated the
interaction roles between NP and BMSC cells via exosomes,
that BMSCs could spontaneously migrate across the transwell
membrane to IVD in a dose-dependent manner induced by NP
exosomes, and NP exosomes could induce BMSC differentiation
toward NP-like lineage, suggesting that NP exosomes could
recruit BMSCs and induce BMSC multidirectional
differentiation, and then replenish IVD cells and appropriate
ECM. Besides, they have demonstrated that NP exosomes are
more effective in inducing BMSCs to differentiate toward NP-like
cells than an indirect coculture system of BMSCs and NP cells.
Strassburg et al. (2012) also reported that the formation of gap
junctions and cell fusion are not the predominant mechanisms of
interaction; intercellular transfer of membrane components is the
main interactive mechanism between BMSCs and NP cells. These
findings indicate that direct injection of BMSCs into IVD is not
effective compared to injection of NP exosomes into IVD, which
is consistent with the finding of Sun et al. (2021) that indirect
cell–cell communication and paracrine are predominant in
keeping the avascular condition of IVD. However, the detailed
interactive mechanisms between BMSC and NP cells remain
unclear, and more specific components like proteins, miRNAs,
transcription factors, and lipids are expected for further research.
Furthermore, interactions between NP cells or NP exosomes and
other cells in IVD (such as AF cells and cartilage EP cells) should
be further analyzed (Hampton et al., 1989) to reveal their roles in
the progression of IDD.
Continuing with autophagy mentioned above, autophagy is
considered as a protective procedure for cell survival under stress
situations; in such process, excessive proteins or aging organelles
in cells are degraded to provide extra energies and, in most cases,
restore homeostasis (Mizushima, 2009). Highly conservative
serine/threonine kinase, as a mechanistic target of rapamycin
(mTOR, also known as mammalian target of rapamycin), is a
crucial cell growth regulatory cytokine that connects cellular
metabolism and growth with multiple environment inputs
(Kim and Guan, 2019). mTOR as a core component
autophagic pathway could negatively regulate autophagy.
Several studies have implied that autophagy activation could
maintain the balance of NPC, ECM, and vitality under
inflammation (Jiang et al., 2013;Li et al., 2019). Serving as an
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Li et al. Different Exosomes for IDD Therapy

effective agonist of autophagy and immunosuppressor, the
application of rapamycin has been demonstrated to increase
the release of NP exosomes, upregulate expression of miR-
27A, and target MMP-13 to prevent ECM degradation.
Rapamycin may also overcome the limitation that non-stem
cell exosomes are difficult to collect and reproduce (Zhang
et al., 2021a). Moreover, NP exosomes have been found to be
secreted in an autophagy-dependent manner, and rapamycin has
been confirmed to stimulate NP cell release exosomes via the
RhoC/ROCK2 signaling pathway (Hu et al., 2020b).
Consequently, these findings prove that NP exosomes also
have high therapeutic potentials and provide us with novel
insights into the usage of NP exosomes via rapamycin
activation in the treatment of IDD.
NP cells also secrete different numbers of exosomes in
different degenerative grades; exosomes secreted from high
degenerative levels of NP cells could promote the apoptosis
and inhibit the proliferation of NP cells (Song et al., 2020),
indicating a positive correlation between the functions of
degenerative exosomes and IDD progression, as well as the
duality of exosome application. TGF-βis abundant in NP
exosomes, which is well studied to transform BMSCs into NP-
like cells (Steck et al., 2005), suggesting the direct functions of NP
exosomes to BMSCs and the ability to carry specific genes
through NP exosomes. Circular RNAs (circRNAs) are a class
of covalently closed non-coding RNA molecules generated by
reverse splicing of the exon of precursor mRNA in eukaryotes (Li
et al., 2018b); studies have shown the tight connections between
circRNAs and IDD (Wang et al., 2018). A rat model in Song et al.
(2020) has suggested that circRNA_0000253 is highly expressed
in IDD rat with positive correlations; they could target and absorb
miRNA141-5p, in a negative manner with IDD, and thus
downregulate the expression of Sirt1. Since the continuous
release of exosomes, the degenerative exosomes from NP cells
may secrete and further affect neighboring normal tissues and
thus aggravate the progression of IDD. As a result, there is an
urgent need to discover new approaches targeting degenerative
NP cells, among which circRNA_0000253 and miRNA141-5p
have been demonstrated to be effective, through delivering
siRNA-circRNA_0000253 or mimic-miRNA141-5p into
degenerative NP cells to prevent ECM degradation. Related
signaling pathways are circRNA_0000253/miRNA141-5p/Sirt1.
Given the multiple functions and multiple advantages of
exosomes, exosomes alone or with specific genes or drugs
would be a suitable choice for cell-free strategies in the
treatment of IDD.
Sirt1 (Sirtuin 1) is a nicotinamide adenine dinucleotide
(NAD+)-dependent histone deacetylase, which could reduce
apoptosis in different cells and correlated with various diseases
including cancer, metabolic disorders, COPD, and aging-related
disorders like degenerative and cardiovascular diseases (Dai et al.,
2018). Sirt1 participates in many pivotal cellular biological
processes, such as inflammatory response, oxidative stress, and
mitochondrial functional homeostasis, which are consistent with
the mechanisms in IDD progression in that Sirt1 plays a
protective role in senescence and apoptosis of NP cells (Guo
et al., 2017;Zhang et al., 2019c). Guo et al. (2017) have found that
Pfirrmann grade is negatively correlated with Sirt1 expression in
IDD. In vitro experiments have verified that resveratrol and
quercetin could promote cell proliferation and senescence-
related protein expression (Guo et al., 2017;Wang et al.,
2020b). Shen et al. (2016) and He et al. (2019) have shown
that Sirt1 prevents NP cells from apoptosis via TLR2/Sirt1/NF-kB
transduction, and Sirt1 could ameliorate oxidative stress-induced
senescence of NP cells regulated by the Akt/FoxO1 pathway; a
previous study has also reported that Sirt1 could modulate
autophagy, which may further mediate the status of IVD
(Wang et al., 2020b). These observations imply that Sirt1
could serve as a novel therapeutic target in the treatment of
IDD, and the applications of exosomes mediating Sirt1 have
already been demonstrated in some diseases like brain injury,
photodamage, and neuronal autophagy (Chen et al., 2020;Liu
et al., 2021b;Wu et al., 2021). To the best of our knowledge, the
applications of Sirt1 via exosomes in the treatment of IDD have
not been reported; further relevant studies could focus on
exosomes and Sirt1 to reveal detailed mechanisms.
In terms of osteoarthritic cartilage, osteoarthritic cartilage and
degenerative IVD possess the common feature of significant loss
of ECM, including COL2 and aggrecan; MMPs are chief catabolic
factors responsible for these pathological changes. Recent studies
have discovered that the applications of autophagy agonists like
rapamycin, chloramphenicol, and ozone inhibit MMP-13
expression and thus attenuate OA processes (Zhao et al., 2018;
Ma et al., 2019;Wu et al., 2019). A previous study has reported the
association between autophagy and exosome release in
chondrocytes, that they depend on caspase-3 and Rho/ROCK
transduction (Rosenthal et al., 2015); these findings are consistent
with the autophagy-activated signaling pathway of exosomes
secreted from NP cells mentioned above. From inverted phase
contrast microscopy, NP cells are presented as polygonal shape,
and toluidine blue staining suggests that there are a large number
of notochord cells in IVD. Further IHC analysis also shows the
abundance of COL2; these results all confirm that NP cells
possess cartilage-like characteristics (Zhang et al., 2021a).
These results elucidate the tight connections between
chondrocytes and NP cells, based on the similar features of
chondrocytes and NP cells; the wide research about
chondrocytes and subsequent research regarding NP cells
could focus on the interactions with chondrocytes, to find
novel ideas for treating IDD.
Exosomes Derived From Annulus Fibrosis
AF, the cells enclosing NP, consists of concentric layers composed
of alternatively aligned oblique collagen fibers (mainly type I
collagen) interspersed with AF cells (Kadow et al., 2015), which
serve as a physical barrier to separate the internal NP and external
blood vessels and immune system. Due to the special structure of
AF that consists of 99% ECM and 1% AF cells, the indirect
cell–cell contact or paracrine of AF cells may play pivotal roles in
maintaining avascular conditions in IVD. Vascularization has
been widely observed in IDD and has been considered as a
pathological feature, which mainly occurs in AF tissue, and
blood vessels grow and infiltrate inwards, could cause NP
tissue exposure to immune system, and thereby damage
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Li et al. Different Exosomes for IDD Therapy

immune privilege of IVD (Kim et al., 1981;Di Martino et al.,
2013). Sun et al. (2021) have verified that the exosomes could be
released by both degenerated and non-degenerated AF cells; they
could also be internalized by HUVECs, and degenerative AF
exosomes could enforce HUVEC migration faster than non-
degenerative AF exosomes, elucidating that degenerative AF
cells could induce IVD vascularization via exosome-mediated
effects, while non-degenerative AF tissues could suppress blood
vessels ingrowth as a physical barrier, and its exosomes could also
be regarded as an angiogenesis inhibitor to maintain the healthy
avascular condition of IVD. These results imply that AF
exosomes could serve as one of the mediators in intercellular
communication of IVD and modulate the vascularization
of IVD.
Within the region of IVD, the position of AF and blood vessels
is very close to each other, which may hypothesize the interactive
potential effects between AF and vascular endothelial cells. Pohl
et al. (2016) have discovered that endothelial microparticles
secreted from vascular endothelial cells enhance MMP
expression in AF cells, suggesting that the vascular endothelial
cells could act on AF cells through microparticle delivery, and it
could help blood vessel ingrowth into IVD by promoting ECM
catabolism. Together with the findings that degenerative AF cells
affect vascularization of vascular endothelial cells by delivering
AF exosomes (Sun et al., 2021). Both AF and vascular endothelial
cells secrete microparticles like exosomes, and they endocytose
microparticles secreted from each other, and these reports all
display the essential roles of microparticles/exosomes in AF and
blood vessel communication. Besides, degenerative AF exosomes
could lead to upregulation of IL-6, TNF-α, MMP-3, and MMP-13
as well as VEGF, which are consistent with previous findings that
these factors could cause NP cell apoptosis and IDD progression,
and these indicators could induce the inflammation of IVD.
VEGF is a major proangiogenic factor that could trigger the
growth, expansion, and relocation of endothelial cells, and play
essential roles in vascularization of IVD (Capossela et al., 2018).
These findings suggest the roles of degenerative AF exosomes; if
situations are not promptly treated and intervened, IVD
progression would produce a vicious cycle by degenerative AF
exosomes, leading to further development of IDD.
Up to now, the existence and functions of AF exosomes have
been rarely reported; although the interactions between AF
exosomes and endothelial cell HUVECs have been verified, the
relevant bioactive substances in AF exosomes related to these
effects have not been discovered, and more research should be
conducted to explore the potential molecular mechanisms and
signaling pathways. Besides, only one kind of vascular-related
cells—HUVECs—have been conducted to analyze the
interactions with AF exosomes, while other vascular cells, like
vascular smooth muscle cells, or arterial endothelial cells, have
not been fully understood. Furthermore, due to the special
position of AF and NP cells, whether AF have interactions
with NP via exosomes still need to be further analyzed.
Consequently, the application of AF exosomes is a promising
research orientation whether in IDD or other fields, and these
issues need to be followed up and resolved by researchers.
Exosomes Derived From Notochordal Cells
Notochord is an embryonic structure of chordates; during the
embryogenesis period, the rod-shaped notochord is enclosed
from the vertebral body and develops to the NP tissue soon in
the early stage of fetal life (Cornejo et al., 2015), and blood vessels
recede and vanish slowly from IVD. In human IVD of immature
individuals (embryonic, fetal, and juvenile), NP tissues are mainly
populated by large vacuolated notochordal cells, while IVDs in
adult are populated with small and non-vacuolated chondrocyte-
like cells (Bach et al., 2018), and with the aging of humans, early
IDD begins to happen with the disappearance of NC cells (Risbud
and Shapiro, 2011). Several in vivo experiments have verified the
important roles in maintaining homeostasis of IVD (Bergknut
et al., 2013) and that NC loss coincides with the onset of IDD.
Increasing lines of evidence have also concluded the roles of NC
in the development and functions of IVD: NC could stimulate
proliferation of degenerative NP cells as well as BMSCs (de Vries
et al., 2015;Bai et al., 2017), and it may stimulate chondrogenic
differentiation, reducing natural cell necrosis. It has also been
demonstrated to inhibit angiogenesis and maintain the avascular
state of human IVD (Gruber et al., 2006;Mehrkens et al., 2017).
In conclusion, the functions of NC indicate the essential effects
and the potential roles in maintaining the NP tissues and IVD
healthy, and therefore, NC could be regarded as a promising
source for regenerative and symptom amelioration for IVD
disease.
The existence of NC exosomes has firstly been confirmed by
Sun et al. (2020b); they have found the best appropriate
mechanical stress to stimulate the release of NC exosomes,
and that NC exosomes could be internalized by endothelial
cells. Mechanical stress is of great importance considering the
environment of IVD, which is implicated as the predominant
inductive cause of IDD; they are interconnected and amplify each
other, and cellular physiology is strongly affected by mechanical
loading (Vergroesen et al., 2015). In the early stage of spine
development, NC cells are squeezed into IVD through the thrust
of forming vertebrae, and then they enter the specific
physiological environment and are compressed by mechanical
force. Compressive load cultures have been found to induce the
CK8 phosphorylation and downregulation in NP cells (Sun et al.,
2013a), and NC cells are more resistant to mechanical forces
compared to NP cells (Saggese et al., 2020), while high mechanical
stress may lead to the exhaustion of NC resources (Hong et al.,
2018). These findings hypothesize that the appropriate range of
mechanical force is necessary to maintain the normal survival and
function of NC cells, as well as the secretion of exosomes; 0.5 Mpa
is found to be the suitable mechanical condition for inducing
secretion of NC exosomes and modulating related functions.
NC exosomes have also been unveiled to deliver miR-140-5p
to endothelial cells and then inhibit angiogenesis to maintain the
avascular status of IVD, which are achieved by the Wnt/β-catenin
signaling pathway (Sun et al., 2020b). Among them, miR-140-5p
has been proven to participate in cell migration, proliferation, and
metastasis (Rothman et al., 2016;Fang et al., 2017), and the
exosomal miR-140-5p from NC could suppress the expression of
MMP-2 and MMP-7 and thus ameliorate NP cell apoptosis and
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Li et al. Different Exosomes for IDD Therapy

IDD. Wnt11, one of Wnt family members, has been shown to
stimulate the proliferation, migration, and invasion of various
types of cells via inducing β-catenin (Stefater et al., 2011;Franco
et al., 2016), which is regarded as the pivotal downstream
transduction of Wnt; these are consistent with the interactions
via NC exosomes, that Wnt transduction is involved in
angiogenesis through the modulation of endothelial cell
proliferation and vascular sprouting (Zhang et al., 2017).
Consequently, NC may have anti-angiogenesis ability via the
NC-exosomal miR-140-5p/Wnt/β-catenin axis. According to
their mass spectrometry study, Matta et al. (2017) have
discovered from a non-chondrodystrophic dog notochordal
cell conditioned medium that transforming growth factor
beta1 (TGF-β1) and connective tissue growth (CTGF) are
major hubs in protein interaction networks, which are
essential for the homeostasis regulation of healthy NP tissues.
Interestingly, VEGFA is also predicted to be inhibited among
growth factors in NC cells (Rodrigues-Pinto et al., 2018). The
identification of key biological factors derived from NC cells that
delay the progression of IDD is still at an early stage; based on
these findings, more studies about the functions mediated by NC
exosomes need further exploration.
THE RELATIONSHIPS BETWEEN IVD
VASCULARIZATION, INFLAMMATION AND
IVD CELLS, AND EXOSOMES
As the largest avascular organ, IVDs are composed of three parts,
namely, central NP cells, surrounding AF cells, and the adjacent
cartilage endplates. Generally, blood vessels are confined to the
outer surface of AF cells in normal IVD, while neovascularization
has been widely observed and considered as a common
pathological phenomenon in IDD. The inward growth of
blood vessels causes NP exposure to immune system, leading
to permeation of inflammatory factors and immune cells into NP
tissues, and finally damage to the immune privilege and
homeostasis of IVD (Sun et al., 2021). Additionally, the
ingrowth of blood vessels in IVD enhances neuralization and
pain sensitization (Choi, 2009); it also affects the phenotype and
functions of NP cells by upregulating oxygen concentration in NP
tissue (Wang et al., 2019). Therefore, the maintenance of the
avascular condition of IVD is essential to sustain the homeostasis
and functions of normal IVD (Chu et al., 2018).
In terms of the mechanisms of vascularization in IDD, it is
generally considered to be the breakdown of physical barrier,
such as the fissure of AF tissue, together with the increased
expression of pro-angiogenesis factors, like VEGF and platelet-
derived growth factor (PDGF) (Tolonen et al., 1997;Fujita et al.,
2008), and pro-inflammatory cytokines like IL-1βand TNF-α
(Lee et al., 2011;Risbud and Shapiro, 2014), that finally induce
invasion of blood vessels. Focal proteoglycan loss has been
noticed to cause alteration of ECM, which facilitates the
growth of nerves and blood vessels (Stefanakis et al., 2012);
increased blood vessel ingrowth is correlated with
proteoglycan depletion AF lesion (Moon et al., 2012) and IVD
aggrecan could inhibit migration and invasion of endothelial cells
(Johnson et al., 2005). Furthermore, mechanical loading has also
been proven to influence the capacity of IVD to stimulate the
migration of endothelial cells (Neidlinger-Wilke et al., 2009).
These results demonstrate the pivotal roles of a passive physical
barrier in preventing IVD angiogenesis. As for molecular level,
Wiet et al. (2017) have described the interactions between mast
cells and IVD: that healthy AF culture medium could suppress the
activation of mast cells by downregulating expression of
inflammatory cytokines and then inhibit mast cell-induced
angiogenesis. Cornejo et al. (2015) have provided evidence
that soluble factors derived from notochordal-rich IVD could
suppress angiogenesis via inhibiting VEGF signaling pathways,
and NC-derived ligands are of significance in targeting
neurovascular ingrowth and pain in the degenerative IVD. A
previous study has also reported the importance of Fas–FasL (Fas
Ligand) interaction, showing that FasL could induce apoptosis of
endothelial cells (Sun et al., 2013b); besides, FasL generated by
IVD could mediate the apoptosis of Fas-bearing cancer cells (Park
et al., 2007), and the Fas–FasL network may provide a novel target
for the treatment strategies of IDD. These results all suggest that
these factors or cytokines might be the molecular monitor for
maintaining functions through inducing apoptosis of vascular
endothelial cells in addition to the traditional physical barrier.
In the aspect of cells and related exosomes, a previous study
has displayed that degenerative AF cells could promote
vascularization. Moon et al. (2014) have reported that AF cells
from degenerative IVD stimulate endothelial cells and produce
factors known to induce ECM degradation, angiogenesis, and
innervation (Moon et al., 2014); degenerative NP cells have been
proven to promote blood vessel growth by secreting pro-
inflammatory factors (He et al., 2020). In the early stage of
disc development, notochordal cells existing in NP cells could
inhibit angiogenesis of IVD (Cornejo et al., 2015); additionally, a
previous study has also provided evidence that degenerative AF
exosomes could induce IVD vascularization and inflammation
directly through upregulation of IL-6, TNF-α, and VEGF, and
indirectly through enhancing the invasion of blood vessels via
accumulation of MMPs (Sun et al., 2021). Furthermore, as for
exosomes derived from MSC cells, a report indicated that miR-
125a represses angiogenic inhibitor DLL4 in endothelial cells and
thereby promotes angiogenesis (Liang et al., 2016). In terms of
NP, although NP exosomes have been reported to exist and have
numerous biological effects, they are mainly focused on
stimulating MSC differentiation (Lan et al., 2019;Yuan et al.,
2020); detailed research about NP exosomes and blood vessels
remains limited. As a result, there is an urgent need to clarify the
effects of NP exosomes on vascular endothelial cells.
PMSCs possess several characteristics such as proliferation,
migration, cloning, and immune regulation, and have broad
applications in clinical practice (Mathew et al., 2020). The
numbers of cytokines and chemokines released from PMSC
are a key point to modulate angiogenesis, which facilitates the
possibility of considering PMSC exosomes as a target therapy to
prompt angiogenesis. Liang et al. (2017) have reported that the
co-culture system between PMSCs and EPCs enhances the
angiogenic potential of EPCs through PDGF and the Notch
signaling pathway, and conditioned media that may contain
Frontiers in Cell and Developmental Biology | www.frontiersin.org February 2022 | Volume 9 | Article 82214912
Li et al. Different Exosomes for IDD Therapy

exosomes have significant pro-angiogenesis effects on EPCs and
HUVECs (Komaki et al., 2017). PMSCs generate various kinds of
angiogenic factors like VEGF, bFGF, IL-6, IL-8, and HGF
(Komaki et al., 2017;Liang et al., 2017), and possible
mechanisms of angiogenesis are also reported to include
activation of PAKT and p38MAPK/pSTAT3 that induce
VEGF secretion and recruitment of smooth muscle cells and
pericytes (Chen et al., 2015;Makhoul et al., 2016). Several in vivo
studies have gained promising results, namely, that PMSCs could
enhance vessel density, blood flow, and perfusion in a dose- and
site-dependent manner, especially in ischemia mice models
(Francki et al., 2016;Xie et al., 2016;Zahavi-Goldstein et al.,
2017). Interestingly, a comparative study (König et al., 2015)
revealed that the angiogenic ability of MSCs derived from blood
vessels is stronger than that in avascular sources, indicating the
importance of determining the components of the conditioned
medium from different cell sources. The above comments have
fully discussed the mechanisms in terms of angiogenic potential,
while in the aspect of anti-angiogenic potential of PMSCs,
Alshareeda et al. (2018) have found that the co-culture of
PMSCs along with breast cancer cells could significantly
inhibit the migration, invasion, and tube formation ability of
HUVECs. Zhang et al. (2014b) have shown that PMSCs could
inhibit peritoneal tumorigenesis via downregulating blood vessel
counts, and injection of PMSC into retinopathy mouse models
has been demonstrated to prevent neovascularization through
upregulation of TGF-β1(Kim et al., 2016); these results could
partly be explained by the upregulation of miRNAs like miR-136
under pathology situations that modulate the inhibition of
capillary formation (Ji et al., 2017). Related studies about
PMSC exosomes remain at an early stage; PMSC exosomes are
known to modulate osteogenic and adipogenic differentiation by
upregulating OCT4 and NANOG in dermal fibroblasts (Tooi
et al., 2016), and they could enhance the migration and tube
formation of endothelial cells (Komaki et al., 2017), while Yuan
et al. (2020) have found the applications of PMSC exosomes in
the treatment of IDD and that exosomes could upregulate
ZNF121 and thus ameliorate IDD by delivering miR-4450
inhibitor (AntagomiR-4450). Basically, the applications of
angiogenesis and anti-angiogenesis by PMSC exosomes are a
controversial issue, modulated in a very contextual manner. More
specific miRNAs from PMSC exosomes targeting IDD or
endothelial cell functions as well as their potential roles are to
be further identified.
PROSPECTS OF EXOSOME THERAPY
FOR IDD
Accumulating lines of evidence have already reported the
essential applications of different sources of exosomes in the
treatment of IDD, which have been fully described in this
review. Many studies have already demonstrated the role of
exosomal miRNAs in inhibiting apoptosis of NP cells and
suppressing MMP expression; however, the correlations
among different sources of exosomes and whether the high
expressed miRNAs in one kind of exosome are also detected in
other exosomes remain unknown. These issues all need to be
further addressed.
In addition to the inflammatory environment, the central
portion of degenerative disc also has low cell density, low
glucose, low pH value, low oxygen, high osmotic pressure, and
high mechanical variations (Lu et al., 2017). Although MSC and
MSC-derived exosomes have shown an effective influence on
degenerative IVD, and cell-injection strategy has suggested
promising results, there remain obstacles regarding MSC in
clinical practice, especially how transplanted cells are able to
survive and adapt in avascular IVD conditions and how to inhibit
angiogenesis rather than promote blood vessel ingrowth of IVD
when using exosomes resembling PMSC-derived (Krock et al.,
2015;Sakai and Andersson, 2015); these issues are worth
pondering and need to be resolved by scientific researchers.
Lastly, the most appropriate dose of exosome injection, along
with the most optimal route of administration, remains unclear and
requires further research (Loibl et al., 2019;Forsberg et al., 2020).
The current studies about the administration route mainly focus on
two points: direct intradiscal injection and systemic injection. Due to
the avascular characteristics of IVD, it is hypothesized that direct
injection of exosomes into the disc would be the most effective
approach (Noriega et al., 2017), while an in vivo study of MSC
exosomes administrated through tail vein by Zhang et al. (2020) also
displayed promising results in the treatment of IDD. In the aspect of
systemic injection, current studies only use single dose in vivo,andif
systemic injections are applied, multiple doses may be conducted to
maintain the therapeutic effects, and in this situation, it is essential to
determinesafetyandefficiency, as well as the dose and frequency of
injections. In terms of direct intradiscal injection, the following
questions need to be addressed: Would the puncture needle for
injection cause extra damage to the vertebral body or IVD? What is
the definition of additional hurt to disc? Would the injection
approach be applied in the clinic? To the best of our knowledge,
there is only one clinical trial relating to the treatment of IDD by
exosomes in India, which would be conducted when all participants
are recruited. This clinical trial aims to observe the efficacy of PRP
exosomes by intradiscal injection [Intra-discal Injection of Platelet-
rich Plasma (PRP) Enriched With Exosomes in Chronic Low Back
Pain, https://clinicaltrials.gov/ct2/show/NCT04849429]. Once these
considerations are fully addressed, more clinical trials about IDD
therapy using an exosome approach are needed.
AUTHOR CONTRIBUTIONS
This study was completed with teamwork. Each author had made
corresponding contribution to the study. Conceived the idea: MY, BG,
and WL. Wrote the main manuscript: WL, SZ, and DW. Perpared
figures:WL,HZ,QS,YZ,MW,andZD.Redressedthemanuscript:SX,
MY, BG, and WL. Reviewed the manuscript: SX, MY, BG, and WL.
FUNDING
This study was supported by grants from the National Natural
Science Foundation of China (Nos. 82072475 and 82172475).
Frontiers in Cell and Developmental Biology | www.frontiersin.org February 2022 | Volume 9 | Article 82214913
Li et al. Different Exosomes for IDD Therapy

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Frontiers in Cell and Developmental Biology | www.frontiersin.org February 2022 | Volume 9 | Article 82214920
Li et al. Different Exosomes for IDD Therapy