International Journal of

Molecular Sciences
Review

Potential of Exosomes for Diagnosis and Treatment of Joint
Disease: Towards a Point-of-Care Therapy for Osteoarthritis of
the Knee
Miki Maehara 1,2 , Eriko Toyoda 1,2 , Takumi Takahashi 1,2 , Masahiko Watanabe 1,212*

Citation: Maehara, M.; Toyoda, E.;
Takahashi, T.; Watanabe, M.; Sato, M.
Potential of Exosomes for Diagnosis
and Treatment of Joint Disease:
Towards a Point-of-Care Therapy for
Osteoarthritis of the Knee. Int. J. Mol.

and Masato Sato 1,2, *Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya,
Isehara 259-1193, Kanagawa, Japan; m-maehara@tsc.u-tokai.ac.jp (M.M.); etoyoda@tokai-u.jp (E.T.);ttakahashi@tokai-u.jp (T.T.); masahiko@is.icc.u-tokai.ac.jp (M.W.)
Center for Musculoskeletal innovative Research and Advancement (C-MiRA), Tokai University Graduate
School of Medicine, 143 Shimokasuya, Isehara 259-1193, Kanagawa, Japan
Correspondence: sato-m@is.icc.u-tokai.ac.jp; Tel.: +81-463-93-1121 or +81-463-96-4404

Abstract: In the knee joint, articular cartilage injury can often lead to osteoarthritis of the knee (OAK).
Currently, no point-of-care treatment can completely address OAK symptoms and regenerate articularcartilage to restore original functions. While various cell-based therapies are being developed toaddress OAK, exosomes containing various components derived from their cells of origin haveattracted attention as a cell-free alternative. The potential for exosomes as a novel point-of-caretreatment for OAK has been studied extensively, especially in the context of intra-articular treatments.
Specific exosomal microRNAs have been identified as possibly effective in treating cartilage defects.
Additionally, exosomes have been studied as biomarkers through their differences in body fluid
composition between joint disease patients and healthy subjects. Exosomes themselves can be utilized
as a drug delivery system through their manipulation and encapsulation of specific contents to bedelivered to specific cells. Through the combination of exosomes with tissue engineering, novelsustained release drug delivery systems are being developed. On the other hand, many of thefunctions and activities of exosomes are unknown and challenges remain for clinical applications. In
this review, the possibilities of intra-articular treatments utilizing exosomes and the challenges inusing exosomes in therapy are discussed.

Sci. 2021, 22, 2666. https://doi.org/
10.3390/ijms22052666

Keywords: exosome; point-of-care therapy; osteoarthritis of the knee; cartilage regeneration

Academic Editor: Kurt A. Jellinger
Received: 2 February 2021
Accepted: 3 March 2021
Published: 6 March 2021

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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Cartilage has poor self-repair and regeneration ability once injured, as first reported
in 1743 by William Hunter [1]. Thus, the regeneration of articular cartilage has been along-standing challenge for physicians and researchers in the field of orthopedics. Articularcartilage consists of highly elastic hyaline cartilage, which is mainly composed of waterand extracellular matrix (ECM), such as proteoglycans and collagen fibers. The cellularcomponent of articular cartilage makes up only about 5% of its wet weight and <10% of its
tissue volume. Articular cartilage has poor self-renewal ability due to the extremely low
infiltration of oxygen, nutrients, and progenitor cells, as it is avascular [2,3].
Osteoarthritis of the knee (OAK) is one of the most common joint diseases. It is achronic, progressive disease in which the articular cartilage gradually degenerates due
to complex interactions between various factors including trauma due to accidents andsports, obesity, skeletal structure, heredity, and age-related overload on the joints. The
global prevalence of OAK is estimated to be approximately 3.8%, and it is observed
more in women (mean, 4.8%) than in men (mean, 2.8%) [4]. Initially, partial-thickness
articular cartilage defects with wear and tear on the cartilage surface are observed. As
the pathological condition progresses, the subchondral bone becomes exposed, leading tofull-thickness articular cartilage defects. One of the most serious symptoms is pain during

Int. J. Mol. Sci. 2021, 22, 2666. https://doi.org/10.3390/ijms22052666

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daily activities such as walking and climbing the stairs, leading to decreased physicalactivity and quality of life.
Non-surgical treatments for articular cartilage injury include non-pharmacologicaltreatments such as exercise therapy, weight loss therapy, and orthotic therapy. Pharmacological treatments include utilization of analgesics or non-steroidal anti-inflammatory
drugs (NSAID) and intra-articular injection of corticosteroids or hyaluronic acid. Currently,
these are the limited options that are widely available for point-of-care treatment for OAK,
and they fail to reverse the progress of cartilage degeneration. For patients who show
no improvements with non-pharmacological and pharmacological treatments, surgicaltreatment options are considered.
Surgical options for injuries with small defects include subchondral drilling [5], microfracture [6,7], and mosaicplasty [8,9]. However, the cartilage regenerated through thesemethods is often fibrocartilage, which is mechanically inferior to the hyaline cartilage that
constitutes the original articular cartilage. For larger cartilage defects, autologous chondrocyte implantation (ACI) [10] has been utilized. Currently, products such as JACC® [11,12],
CaRes® [13,14], Hyalograft® C [15], and BioSeed® C [16] are on the market and used worldwide. However, the regenerated cartilage is reported to be either fibrocartilage or a composite of fibrocartilage and hyaline cartilage, and these methods are not yet applicable to
OAK [17,18]. In Japan, cell sheet transplantation with high tibial osteotomy [19–21] is a celltherapy that has been approved for the treatment of OAK as an advanced medical care.
However, the treatment is costly and time consuming. After other options are exhausted,total knee arthroplasty (TKA) is applied. Although the treatment outcomes of TKA are
remarkable [22], artificial joints may need revision after 15 to 20 years. Thus TKA is often
applied to people over the age of 65.
To bridge the gap between traditional pharmacological and surgical treatments,
cell therapies such as mesenchymal stem cells (MSCs) [23,24] and platelet rich plasmas(PRPs) [25–27] have been in use as a point-of-care treatment for OAK. Patients can receive
these treatments through intra-articular injections at relatively low costs in select places.
However, therapeutic effects attributed to various modes of action including cytokines andgrowth factors can be variable [28–31]. In addition, risks of inappropriate transformationof the transplanted cells and inflammatory reactions exist [32–36]. Thus, a cell-free therapy
that maximizes the therapeutic effect through the selected administration of identified
factors is ideal.
To address such issues, extracellular vesicles (EVs), especially exosomes, are attracting
attention as a novel tool that can be applied to treat cartilage and bone disease [37,38].
The therapeutic effects of MSCs have also been partially attributed to exosomes carrying
specific cargos [39–43]. With recent advancements, exosomes are used as efficient drugdelivery systems that can be manipulated to carry specific cargo such as micro ribonucleic
acids (miRNAs). As such, exosomes may be used as a point-of-care treatment for joint
disease, especially through intra-articular injection. In addition, exosomes may be useful asbiomarkers to detect joint disease such as OAK at early onset. More recently, a combinationof exosomes with tissue engineering methods has been investigated to prolong and improvethe efficacy of exosomes. In this review, we present the recent concepts of exosomes thatcan be applied to the development of point-of-care treatment for joint disease such as OAK.
2. Extracellular Vesicles
Historically, membrane vesicles released by cells have been thought of as non-functional,
inactive “debris” resulting from cell damage or turnover of the cellular membrane. However, in 1983, the existence of a 100-nm functional endoplasmic reticulum comprised of lipid
bilayer membranes secreted from cells was confirmed [44]. Subsequent research identified
membrane vesicles secreted by various cells, which were named according to their features,such as ectosomes, microvesicles, oncosomes, exosomes, and apoptotic bodies [45–52]. To
resolve confusion, the International Society for Extracellular Vesicles (ISEV) suggested
using the term “extracellular vesicles” (EVs) as “the generic term for particles naturally

Int. J. Mol. Sci. 2021, 22, 2666

identified membrane vesicles secreted by various cells, which were named according totheir features, such as ectosomes, microvesicles, oncosomes, exosomes, and apoptotic
3 of 16
bodies [45–52]. To resolve confusion, the International Society for Extracellular Vesicles(ISEV) suggested using the term “extracellular vesicles” (EVs) as “the generic term forparticles naturally released from the cell that are delimited by a lipid bilayer membraneandcannotreplicate,containby
a functional
nucleus”[53]. Various
subtypesofreleasedfromthe cell i.e.,
thatdoarenotdelimiteda lipid bilayer
membraneand cannot
replicate,
EVs
share
similarcharacteristics,
andamethodtoaccurately
classify
EVs
and
their
funi.e., do not contain a functional nucleus” [53]. Various subtypes of EVs share similar chardamentalroles is[54].classify
Therefore,suggested biologicala classification
acteristics,biologicaland a method
tounclearaccurately
EVs theandISEV
theirhas
fundamental
roles
ofsubtypesEVs according
to thecharacterisis theunclear[54]. of
Therefore,
the ISEV
hasfollowing
suggestedcharacteristics:
a classification(a)ofphysicalthe subtypesof EVstics,
such as(“small EVs”
(sEVs, <100–200nm) and“medium/large(m/lEVs,
accordingto sizethe following
characteristics:(a) physicalcharacteristics,
suchEVs”
as size(“small
>200
nm))
or
density(low,
middle,high);(b)biochemicalcomposition(clusterofdifferenEVs” (sEVs, <100–200 nm) and “medium/large EVs” (m/lEVs, >200 nm)) or density (low,tiationEVs, Annexin
A5-stainedetc.); or (c) descriptionsof condimiddle,(CD)63+/CD81+high); (b) biochemicalcomposition(clusterEVs,of differentiation(CD)63+/CD81+
EVs,
tions
or
cell
of
origin(podocyteEVs,hypoxic
EVs,
large
oncosomes,
apoptotic
bodies)Annexin A5-stained EVs, etc.); or (c) descriptions of conditions or cell of origin (podocyte[53].
EVs, hypoxic EVs, large oncosomes, apoptotic bodies) [53].
Biogenesis,Composition,
Composition,and
andIsolation
Isolationof
ofExosomes
Exosomes
3.3.Biogenesis,Thenomenclatureof
exosomeswassuggesteda subtypeof
The nomenclature exosomes was suggestedinin1987
1987[46].
[46].Exosomes
Exosomesarearea subtype
EVs
that
have
a small
diameterofof
50–150nm.
They
aresecreted
from
most
of of
thethe
cells
in
of
EVs
that
have
a small
diameter
50–150nm.
They
aresecreted
from
most
cells
the
body
and
areareobserved
in in
body
fluidsandcellculturemedia[47,52].
in
the
body
and
observed
body
fluidsandcellculturemedia[47,52].
Exosomesare
areknown
knowntoto
formedendocytosis(Figure1). Early
endosomes
Exosomes
bebe
formedby byendocytosis(Figure1). Early
endosomes
trantransition
tolateendosomes.
Then
a
large
numberofintraluminal
membranevesiclessition to late endosomes. Then a large number of intraluminal membrane vesicles (ILVs)(ILVs)
are formed
by internalof the endosome
membrane.
Late endosomesareformedby internalbuddingbuddingof the endosome
membrane.
Late endosomes
are calledarecalledmultivesicularbodies(MVBs).
When
the
MVBs
fuse
with
the
cellthemembrane,
multivesicular bodies (MVBs). When the MVBs fuse with the cell membrane,internal
the internal
ILVsinto
are thereleased
into theextracellularspace
exosomes[55] (Figure1).
ILVs
are released
extracellularspace
as exosomes[55]as(Figure1). Endosomal
sortEndosomal
sortingcomplexes
required
for transport(ESCRT) andareinvolved
thought
ing
complexes
required
for transport(ESCRT)and tetraspaninsaretetraspaninsthought to betobeinvolved
intheformationofendosomes[47,56,57].
The
mechanisms
foruptakein the formation of endosomes [47,56,57]. The mechanisms for uptake of the exosomesoftheexosomes
intotarget
cells
and
the
deliveryoftheexosomecontents
areunclear.
into target cells and the delivery of the exosome contents are unclear. Many studies have
Many studies
have suggestedthat
endosomes
areoftheputative location
EV-content
suggestedthat endosomes
are theputativelocation
EV-content
delivery,ofandcontents
delivery,andcontents
arereleasedintothecytoplasmofthetargetcells
byfusingwithare released into the cytoplasm of the target cells by fusing with endosome membranes
endosomemembranes[47,55].
Othermechanismsofcontent
deliverytotargetcells,[47,55]. Other mechanisms of content delivery to target cells, such as interaction withsuchtheasinteraction
withthereceptorspresentonthecellmembraneanddirectfusionwith
thereceptors present on the cell membrane and direct fusion with the cell membrane, havecell membrane, have also been proposed [47,55].
also been proposed [47,55].

Figure 1.1.Formation
Formationofofexosomes
exosomes
anduptakeintotargetcells.
Early
endosomes
are formedby endocytosis
andtranthey
Figureanduptakeintotargetcells.
Early
endosomes
are formedby endocytosis
and theytransition
tolateendosomes.
These
are
calledmultivesicularbodies(MVBs),
withintraluminal
membranevesicles(ILVs).
sition to late endosomes. These are called multivesicular bodies (MVBs), with intraluminal membrane vesicles (ILVs). ILVs
ILVs that are released from MVBs into the extracellular space are called exosomes. Released exosomes are taken up by thetarget cells through endocytosis, interactions with receptors on the cell membrane, or direct fusion with the cell membrane.

Exosomes are comprised of a lipid bilayer membrane containing lipid raft constituents,
such as cholesterol, sphingomyelin, and ceramide, and contain messenger RNAs (mRNAs),
miRNAs, deoxyribonucleic acid (DNA), and various proteins (Figure 2) [58–61]. As ofthat are released from MVBs into the extracellular space are called exosomes. Released exosomes are taken up by the target
cells through endocytosis, interactions with receptors on the cell membrane, or direct fusion with the cell membrane.
Int. J. Mol. Sci. 2021, 22, 2666

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Exosomes are comprised of a lipid bilayer membrane containing lipid raft constituents, such as cholesterol, sphingomyelin, and ceramide, and contain messenger RNAs(mRNAs), miRNAs, deoxyribonucleic acid (DNA), and various proteins (Figure 2) [58–61].
As of2021,
January2021,
9769 proteins,
11163408
lipids,
3408 mRNAs,
andmiRNAs
2838 miRNAs
have
January9769
proteins,
1116 lipids,
mRNAs,
and 2838
have been
been
registeredinthe
ExoCarta
database(http://www.exocarta.org/,
accessedon
2
Januregistered in the ExoCarta database (http://www.exocarta.org/, accessed on 2 Januaryary2021),
which
catalogues
exosomecontents.
Althoughvariousproteins
are associated2021),
which
catalogues
exosomecontents.
Althoughvariousproteins
are associated
with
with
the
lipid
membraneofexosomes,noclear
biomarker
hasbeen
identified.
Proteins,
the lipid membrane of exosomes, no clear biomarker has been identified. Proteins,
such
such
as majorhistocompatibility
complex(MHC)
and classII, shockheat shockproteins
as majorhistocompatibility
complex(MHC)
class class
I andIclass
II, heatproteins(e.g.,(e.g.,
heatshock
protein(Hsp)
70,
Hsp90),
flottilin-1,
andactin,
have
been
used
as
“exoheat shock protein (Hsp) 70, Hsp90), flottilin-1, and actin, have been used as “exosome
somemarkers”
in thepastandbeenhave
been toshown
to be inpresent
in alltypes
of EVs[62]. In
markers”
in the past
and
have
shown
be present
all typesof EVs[62].
In addition,
addition,theco-expressionoftetraspanins(e.g.,
CD9,
CD63,
CD81)
and
proteins
associthe co-expression of tetraspanins (e.g., CD9, CD63, CD81) and proteins associated
with
ated
with
MVB
formation(e.g.,
ALG-2-interacting
protein
X(ALIX),
tumor
susceptibility
MVB formation (e.g., ALG-2-interacting protein X (ALIX), tumor susceptibility gene 101gene
101 (TSG101))been suggested
2)not
[62].
is notwhether
yet clearwhether haveexo(TSG101))
have beenhave
suggested(Figure 2) (Figure[62]. It is
yetIt clear
exosomes
somes
specific
foundin whether
other EVs
or whether
they canbe reliably disspecifichave
functionsnotfunctions
found in not
other
EVs orthey
can be reliably
distinguishedandtinguishedand separated
separated fromother EVs.from other EVs.

FigureFigure 2.
2. Structure
Structure ofof exosomes.
exosomes. Exosomes
Exosomes have
have aa lipid
lipid bilayer
bilayer membrane
membrane composedcomposed ofof lipid
lipid raft
raft constituents,
constituents, tetraspaninstetraspanins(such as CD9, 63 and 81), and proteins (such as integrin, cell specific receptors, MHC class I and class II, and flottilin-1).
(such as CD9, 63 and 81), and proteins (such as integrin, cell specific receptors, MHC class I and class II, and flottilin-1).
Exosomes contain cellular components such as proteins, DNA, mRNAs, miRNAs, proteins associated with MVB formation
Exosomes contain cellular components such as proteins, DNA, mRNAs, miRNAs, proteins associated with MVB formation(ALIX, TSG101), and heat shock proteins (Hsp70, Hsp90).
(ALIX, TSG101), and heat shock proteins (Hsp70, Hsp90).

EVs are a heterogeneous population with a mixture of cell vesicles of various fracEVs are a heterogeneous population with a mixture of cell vesicles of various fractions.
tions. In
In orderorder toto useuse exosomes
exosomes forfor researchresearch andand treatment,
treatment, aa technique
technique forfor isolating
isolating only
only
“exosome
fractions”
inlarge
quantities
and
with
high
purityisrequired.
Various“exosome fractions” in large quantities and with high purity is required. Various exosome
exosomeisolation[63–65]. The
method isisisolation methods
methods have
have been
been developed
developed toto thisthis dayday [63–65].
The most
most classic
classic method
ultracentrifugation. This method is still the most widely used [66] and has become the gold
standard for exosome isolation. In addition, there are various methods such as ultrafiltration, polymer precipitation, size exclusion chromatography, immunoaffinity purification,and microfluidics-based techniques. Furthermore, various exosome isolation kits suchas exoEasy Maxi kit (QIAGEN, Venlo, Nederland), ExoQuick (System Biosciences, Palo

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Alto, CA, USA), qEV size exclusion columns (Izon Science, Oxford, United Kingdom), andMagCapture™ Exosome Isolation Kit PS (Wako Pure Chemical corporation, Osaka, Japan)
are on the market. Each isolation method has advantages and disadvantages, and thequality and amount of exosomes obtained vary greatly depending on the isolation method
selected [63–65]. In order to isolate the exosome fraction with the desired activity, it isimportant to select the optimal exosome isolation method for each objective.
4. Potential of Exosomes for Diagnosis and Treatment of Joint Diseases
The potential for exosomes and their derivatives to be delivered through intra-articular
injection opens up new possibilities for the treatment of joint diseases such as OAK.
Exosomes contain specific information about the cells from which they are released, andmay have the ability to deliver molecules to specific organs or tissues at a distance [61,67,68].
Studies have shown that exosomal miRNAs play an important role in joint homeostasis.
Furthermore, the imbalances in exosomes created during joint disease can be utilized as
diagnostic biomarkers. Studies have also shown the potential for exosomes to be utilized
as a drug delivery system (DDS) to deliver specific cargo to localized areas. In combination
with tissue engineering, studies have utilized exosomes in a sustained release drug delivery
system (SRDDS) to further improve the efficacy. Here, we discuss such topics in regards totheir applications to the diagnosis and treatment of joint diseases.
4.1. Exosomal miRNAs
MiRNAs are small non-coding RNAs that have approximately 22 nucleotides. These
induce gene silencing by sequence-complementary binding to target sites located within
the three prime untranslated regions (3’UTR) of the target mRNA and regulate multiple
biochemical pathways [69]. Many miRNAs associated with chondrocyte function andhomeostasis have been reported to contribute to cartilage repair [70,71]. For example, miR140, a promising miRNA for OAK treatment, is expressed specifically in articular cartilage
and plays an important role in cartilage development and metabolic balance of the cartilage
matrix by inhibiting a disintegrin and metalloproteinase with thrombospondin motifs
5 (ADAMTS5) and matrix metalloproteinase 13 (MMP13) [72,73]. Ukai et al. examined
cartilage tissues from pediatric patients with polydactylism, young patients with anterior
cruciate ligament injury, and elderly patients with OAK and identified miRNA-199a-3p,
193b, and 320c as markers that suppress age-related cartilage metabolism [74]. The discovery that mRNAs and miRNAs are packaged in exosomes that are transported between
cells and function in target cells was a breakthrough [59,75–77]. Exosomal miRNAs areprotected from endogenous RNase activity by the exosome membrane and can exist stably
in body fluids and cell culture media. Therefore, exosomal miRNAs can be collected andadministered through intra-articular injections. This section introduces the current researchon intra-articular treatments with miRNAs (i.e., exosomal miRNAs) secreted extracellularly
via exosomes (Table 1).
Studies have shown that exosomal miRNAs may influence cartilage differentiationand homeostasis. Sun et al. performed a microarray analysis of exosomes secreted fromboth human bone marrow-derived MSCs (hBMSCs) undifferentiated and hBMSCs differentiated into cartilage [78]. Through the comparative analysis, they identified 35 miRNAs,
including miR-1246, miR-1290, miR-193a-5p, miR-320c, and miR-92a, that were upregulated in exosomes derived from hBMSCs differentiated into cartilage. In addition, they
demonstrated that exosomes derived from miR-320c-overexpressing hBMSCs upregulate
SRY-box transcription factor 9 (SOX9) and downregulate MMP13 in OA chondrocytes, andenhance chondrogenesis of hBMSCs [78]. Mao et al. demonstrated that exosomes secreted
by OA cartilage express significantly less miR-95-5p compared to normal cartilage [79].
In addition, they showed that exosomes derived from miR-95-5p-overexpressing primarychondrocytes promote cartilage development and cartilage matrix expression by hBMSCs
and chondrocytes, and that miR-95-5p may be effective in the treatment of OA by directly
targeting histone deacetylase (HDAC) 2/8. Li et al. showed that chondrocyte-derived

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exosomal miR-8485 promotes the chondrocyte differentiation of BMSCs [80]. The study
suggested that miR-8485 may suppress the production of glycogen synthase kinase 3 beta
(GSK-3β) by regulating the expression and activity of GSK-3β, activate the Wnt/β-catenin
pathway through phosphorylation of GSK-3β by targeting disheveled binding antagonist
of beta catenin 1 (DACT1), and promote chondrocyte differentiation.
Table 1. Studies on exosomal miRNAs regulating chondrocyte function and homeostasis for the purpose of intraarticular treatments.
ExosomeOrigin Cell
SourceStudy Design

AnimalModelsmiRNA

Result(s)Target(s)Reference

Human BMSCs

in vitro-

miR-320c

Upregulates SOX9 and downregulates
MMP13 expression in OA chondrocyte.

Not mentioned[78]

HDAC2/8[79]

Human BMSCs

in vitro-

miR-955p

Enhances histone H3 acetylation andmaintains the function of articularchondrocytes. Promotes SOX9, COL2A1
and Aggrecan expression and enhances
cartilage development.

Humanchondrocytes

in vitro-

miR-8485

Activates Wnt/β-catenin pathways.
Promotes chondrogenic differentiationof hBMSCs.

GSK3B,DACT1[80]

Human BMSCs

in vitro
and in vivo

MicemiR-92a3p

Promotes cartilage proliferation. In both
MSCs and PHCs, promotes matrix genes
expression and inhibits WNT5A
expression.

WNT5A

[81]

Human BMSCs

in vitro
and in vivo

Rats

miR-26a5p

It was shown that the damage ofsynovial fibroblasts is suppressedin vitro, and the OA damage isalleviated in vivo.

PTGS2

[82]

Human IPFP
MSCs

in vitro
and in vivoMicemiR-1005p

Protects cartilage from damage andameliorate gait patterns of
DMM-induced OA mice.

mTOR

[83]

Human SMSCs

in vitro
and in vivo

Rats

miR-1405p

Enhances the proliferation andmigration of ACs, and the progressionof early OA and prevents severe damage
to knee articular cartilage in the OA rats.

RalA

[84]

hBMSC: human bone marrow-derived MSC, IPFP: infrapatellar fat pad, SMSC: synovium-derived MSC, MSC: mesenchymal stem cell,
PHC: primary human chondrocytes, WNT5A: Wnt family member 5A, OA: osteoarthritis, SOX9: SRY-box transcription factor 9, MMP13:
matrix metalloproteinase 13, COL2A1: Collagen type II alpha 1, DMM: destabilization of the medial meniscus, AC: articular cartilage,
PTGS2: Prostaglandin-endoperoxide synthase 2, HDAC: histone deacetylase, mTOR: mammalian target of rapamycin, RalA: Ras-related
protein, GSK-3β: glycogen synthase kinase 3 beta, DACT1: dishevelled binding antagonist of beta catenin 1.

Other studies have shown the potential of exosomal miRNAs in various animal models. Mao et al. showed that miR-92a-3p directly targets Wnt family member 5A (WNT5A)
and promotes proliferation and migration of chondrocytes and the differentiation of MSCs
into chondrocytes [81]. In addition, they showed that exosomes derived from miR-92a-3poverexpressing hBMSCs inhibit the progression of early osteoarthritis (OA) and prevent thesevere damage of cartilage in an OA model mouse induced with collagenase VII. Jin et al.
found that the expression of miR-26a-5p decreases and the expression of prostaglandinendoperoxide synthase 2 (PTGS2) increases in OA patients and in synovial fibroblasts
treated with interleukin-1 beta (IL-1β) [82]. In addition, they showed that hBMSC-derived
exosomes provide miR-26a-5p to synovial fibroblasts and alleviate the damage of OA
synovial fibroblasts by controlling PTGS2 expression. Moreover, articular injection of
exosomes derived from miR-26a-5p-overexpressing hBMSC into an OA rat model prevents
OA damage by alleviating the inflammation of synovial tissues and decreasing apoptosis. Wu et al. demonstrated that infrapatellar fat pad (IPFP) MSC-derived exosomes

Int. J. Mol. Sci. 2021, 22, 2666

7 of 16(MSCIPFP -Exos) can deliver miR-100-5p to chondrocytes [83]. The miR-100-5p specificallytargets the 30 UTR region of mammalian target of rapamycin (mTOR) and significantly
enhances autophagy levels in chondrocytes, inhibits cell apoptosis, enhances anabolism,
and represses catabolism in IL-1β-treated OA chondrocytes. In addition, MSCIPFP -Exos
improved the pathological degree of severity and foot gait patterns in a destabilization of
the medial meniscus (DMM)-induced OA mouse model. Tao et al. showed that exosomes
derived from synovium-derived MSCs (SMSCs) activate Yes-associated protein (YAP) andpromote the proliferation and migration of chondrocytes, but decrease ECM secretion [84].
Therefore, exosomes derived from miR-140-5p-overexpressing SMSCs (SMSC-140-Exos)
affected proliferation and migration of chondrocytes without ECM secretion being influenced. Moreover, the administration of SMSC-140-Exos to an anterior cruciate ligamenttransection (ACLT) OA rat model demonstrated the inhibition of severe damage of thearticular cartilage and the progression of OA.
In addition, exosomes are thought to be deeply involved in the development ofvarious physiological phenomena and pathological conditions, and further research on
exosomal miRNAs may lead to the elucidation of the mechanism of onset and progressionof joint diseases such as OAK. Ni et al. showed that exosome-like vesicles from OA
chondrocytes inhibit the expression of autophagy related 4B cysteine peptidase (ATG4B)
via miR-449a-5p [85]. This result suggests that inflammation of the synovial membrane
may be promoted by the inhibition of autophagy of macrophages and the increase inproduction of mature IL-1β, leading to progression of OA diseases.
4.2. Exosomes as Biomarkers
As OAK patients experience few symptoms during early onset and because of the
difficulty in providing treatment as the disease progresses, early diagnostic methods using
tools such as biomarkers are desired. Exosomes are anticipated to be useful as novel
biomarkers as they contain specific information from the released cells and can stably carryencapsulated substances. In the diagnosis of lung cancer, Exo Dx™ Lung (ALK) (Exosome Diagnostics, Inc., Waltham, MA, USA) is the first Clinical Laboratory Improvement
Amendments-validated exosome-based clinical liquid biopsy test that isolates exosomal
mRNAs in blood to diagnose cancer [86,87]. For the diagnosis of OAK, exosomes isolated
from blood or joint fluid have been identified as useful to date.
For instance, several studies have shown that the expression of specific exosomal
miRNAs is altered in patients with joint disease. Kolhe et al. showed that synovial fluid
exosomal miRNAs may be altered in OAK patients [88]. In particular, exosomal miRNAs
expressed specifically in female OAK patients are responsive to estrogen and target the
Toll-like receptor (TLR) signal pathway. Meng et al. confirmed that the expression of
exosomal miRNA-193b-3p was decreased in the plasma of OAK patients compared to thatof healthy subjects [89].
In addition, Zhao et al. mentioned the possibility that long noncoding RNAs (lncRNAs) can be used as biomarkers for OAK. They conducted an analysis of plasma-derived
and synovial fluid-derived exosomal lncRNAs to assess the progression of OAK [90]. As
a result, the expression of plasma-derived exosomal lncRNAs showed no significant difference; however, the expression of synovial fluid-derived exosomal lncRNAs was higher
in early OA and late-stage OA than in healthy subjects. Specifically, exosomal lncRNA
prostate-specific transcript 1 (PCGEM1) was much higher in late OA than in early OA andmuch higher in early OA than in healthy subjects.
Other studies have examined the use of exosomes as biomarkers to differentiate
joint diseases. Chen et al. reported that specific plasma-derived exosomal miRNAs wereconnected to the common pathogenesis of psoriatic arthritis, psoriasis vulgaris, rheumatoid
arthritis (RA), and gouty arthritis. An analysis of the potential target genes revealed
that these miRNAs are related to immune disorders and bone injury [91]. In addition,
Tsuno et al. demonstrated that serum exosomes of patients with active RA possess different

Int. J. Mol. Sci. 2021, 22, 2666

Other studies have examined the use of exosomes as biomarkers to differentiate joint
diseases. Chen et al. reported that specific plasma-derived exosomal miRNAs were connected to the common pathogenesis of psoriatic arthritis, psoriasis vulgaris, rheumatoidof 16
arthritis (RA), and gouty arthritis. An analysis of the potential target genes revealed8 thatthese miRNAs are related to immune disorders and bone injury [91]. In addition, Tsuno
et al. demonstrated that serum exosomes of patients with active RA possess different protein profiles compared to patients with inactive RA, patients with OA, and healthy donors
protein profiles compared to patients with inactive RA, patients with OA, and healthy[92].
donors [92].
Together, these results indicate that differences in exosome profiles in patients with
Together, these results indicate that differences in exosome profiles in patients withjoint
joint diseases
diseases andand healthy
healthy subjects
subjects cancan bebe investigatedinvestigated using
using body
body fluids
fluids such
such asas synovial
synovial
fluidandplasma,
which
may
lead
to
the
developmentofvarioustests
andfluid and plasma, which may lead to the development of various tests and diagnostic
diagnostic
methods
methods forfor OAKOAK andand otherother joint
joint diseases
diseases inin thethe future.
future.
4.3.
4.3. Exosomes
Exosomes as
as aa Drug
Drug Delivery
Delivery System
System (DDS)(DDS)
If
If specificspecific miRNAs
miRNAs andand otherother molecules
molecules areare identified
identified asas having
having a therapeutic effectforfor thethe treatment
treatment ofof OAK,
OAK, they
they cancan bebe packaged
packaged inin exosomes
exosomes thatthat can
can act
act asas a DDS totransporttargettissuesor organs[68,93].
ThereThere
are twomethtransportspecificspecificcontents
contentstototargettissuesor organs[68,93].
aretypestwo oftypes
ofods
to enclose
the target
in exosomes:
pre-loadingand post-loading
methods
methods
to enclose
the substancestarget substancesin exosomes:
pre-loadingand post-loading[94](Figure3).(Figure 3).
methods[94]

Figure 3.
3. Intra-articular
Intra-articulartreatments
treatmentsutilizing
utilizing
modifiedexosomesa drugdeliverysystem.
The loadingof specificFiguremodifiedexosomes
as aasdrugdeliverysystem.
The loadingof specificcomcomponents
into exosomes
be conducted
either before
exosomeisolation(pre-loadingor after exosome
ponents
into exosomes
can becan
conducted
either before
exosomeisolation(pre-loading
method)method)or after exosome
isolation(post-loading
method). method).
isolation (post-loading

Pre-loading methods collect exosomes released by the modified cells after cell modifiPre-loading methods collect exosomes released by the modified cells after cell modication is performed by miRNA or small interfering RNA (siRNA) transfection or by the
ficationis performedby miRNA
small
interfering
RNA (siRNA)transfection
or by
the
uptake oftarget substances
fromortheculturemedia during
cell culture(Figure 3).
Manyuptakeoftargetsubstancesfromtheculturemedia
duringcell
culture(Figure3).
Manystudies have confirmed the ability of cartilage repair by exosomes collected from cells overstudies
have
confirmed
the [71,74,76].
ability of In
cartilage
repairbyetexosomesfrom cells
expressing
specific
miRNAsaddition,Wang
al. showedcollectedthat the expression
overexpressing
specific
miRNAs[71,74,76].
In
addition,
Wang
et
al.
showedthat
exof miR-135b within the released exosomes can be upregulated by stimulating MSCsthe
usingpressionofmiR-135b
withinthereleased
exosomes
canbeupregulatedbystimulatingtransforming growth factor beta 1 (TGF-β1) [95]. The pre-loading method is a relatively
simple procedure that relies on cells to package exosomes. However, it can be difficult tocontrol the loading of specific target molecules in exosomes. A further understanding of
the mechanisms by which intracellular miRNAs, mRNAs, and proteins load exosomes
efficiently with specific target molecules is necessary.
In contrast, post-loading methods extract exosomes from cells and body fluids, andthe target molecules are encapsulated in the exosomes through disruption of the exosome

Int. J. Mol. Sci. 2021, 22, 2666

9 of 16

membrane (Figure 3). Post-loading methods include physical induction by electroporation, freeze/thaw method, sonication, etc. and chemical induction by saponin treatment,
transfection reagents, etc. In the post-loading method, the target molecules can be directly
loaded into the exosomes, so the loading efficiency is expected to be higher than that ofpre-loading methods. However, physical disruption may affect the size and shape of exosomes. Chemical induction has been shown to be more efficient in loading target molecules
compared to physical induction methods [96]. However, chemicals used for the loading ofmolecules may be toxic, so removing them in the process is essential.
In terms of applications to joint therapy, post-loading methods may allow the targetingof specific tissues, such as cartilage, subchondral bone, or synovium, to treat the various
symptoms. Liang et al. reported on the loading of specific miRNAs through electroporation into engineered exosomes [97]. They engineered chondrocyte-affinity peptide (CAP)
exosomes by fusing CAP with lysosome-associated membrane glycoprotein 2b (Lamp2b)
on the surface of exosomes. Furthermore, they loaded miR-140 into the CAP exosomes
using electroporation (CAP exosome/miR-140). The CAP exosomes/miR-140 allowed
for the specific delivery of chondrocytes. Moreover, in a DMM-induced OA rat model,
they demonstrated that the progression of OA can be reduced by CAP-exosome/miR-140.
Through modification of exosomes and post-loading methods, they were able to simultaneously achieve tissue-specific delivery and inclusion of target molecules into exosomes.
These improvements in exosome modification technology could enhance the effectivenessof exosome-based intra-articular treatments.
4.4. Potential of Exosomes in a Sustained Release Drug Delivery System (SRDDS)
Exosomes are relatively stable in vivo, but multiple administrations may be required
to overcome the natural elimination of exosomes in the joint. As such, to prolong andenhance the effect of joint treatment methods, studies have combined exosomes withvarious biomaterials to be used in a SRDDS [98–101]. Chen et al. produced a 3D-printed
cartilage ECM/gelatin methacrylate (GelMA)/exosome scaffold with radially oriented
channels through a desktop-stereolithography technology [102]. This 3D printed scaffold
was shown to continuously release exosomes for 14 days in vitro. When the 3D printed
scaffold was transplanted through an invasive procedure in a rabbit osteochondral model,
the scaffold retained exosomes for at least 7 days in vivo and significantly promoted
cartilage repair.
Other studies have combined exosomes with injectable materials that can be administered through intra-articular injection. Liu et al. utilized stem cell-derived exosomes incombination with photoinduced imine crosslinking hydrogel glue to produce a cell-free
exosome tissue patch [103]. The tissue patch continuously released exosomes for 14 days.
In vivo studies showed that the new cartilage tissue regenerated by the tissue patch was
hyaline cartilage-like in a rabbit articular cartilage defect model. Alternatively, Hu et al.
produced a Gelma/nanoclay hydrogel (Gel-nano-sEVs) with embedded human umbilicalcord MSC-derived small extracellular vesicles (hUC-MSCs-sEVs) [104]. In this study, they
showed that miR-23a-3p, which is highly expressed in hUC-MSCs-sEVs, activates the phosphatase and tensin homolog deleted from chromosome 10 (PTEN)/AKT serine/threonine
kinase (AKT) signaling pathway and promotes cartilage regeneration. The Gel-nanosEVs were demonstrated to release exosomes continuously for approximately 30 days andcontribute to cartilage regeneration. Further studies are expected in this field.
5. Advantages and Challenges of Developing Treatments Utilizing Exosomes
Treatments utilizing exosomes have remarkable potential to target and affect specifictissues. With proper control, specific cargos such as miRNAs can be packaged to be delivered to various tissues affected by OAK, such as cartilage, synovium, and subchondralbone. Unlike cell-based therapies, exosomes have a lower immunogenicity and an inability to directly form tumors [105,106]. Cell-based therapies rely on a more complicatedmode of action such as the production of humoral factors that may be influenced by lo-

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cal conditions [34,107]. While exosomes may also be influenced by local conditions as
well, with proper manufacturing they can carry and deliver a consistent set of cargos atdesired concentrations.
On the other hand, many challenges remain, such as the large number of cells required
for the manufacturing of exosomes, the multiple steps and time required for collection, andthe unclear risks in ensuring safety and efficacy [39,41,43,55]. In addition, when exosomes
are used clinically, the possible transmission of infectious diseases, due to contamination
with viruses, bacteria, and fungi, harmful effects of components other than exosomes thatare simultaneously administered, and potential risks of inconsistent efficacy and quality
must be considered.
Exosomes possess complicated characteristics such as their size, the cargos they hold,and their destinations, which must be properly characterized in addition to the originsof the exosomes. As such, setting quality standards in the manufacturing process will bea challenge. When manufacturing exosomes for treatments, the quality of cells used as
raw materials and their constituents and the management of the manufacturing process
will be important additions to the quality control of the exosomes themselves. The ISEV
proposed Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines
in 2014, which was updated in 2018, suggesting a comprehensive yet evolving guideline
for studies using extracellular vehicles, including exosomes [53,108]. To secure the quality,
efficacy, and safety of exosomes, strict quality control and manufacturing control must
be implemented.
6. Conclusions
EVs, including exosomes, present new possibilities for treatment, diagnosis, and drug
development for patients with joint diseases including OAK. Exosomes from body fluids
such as joint fluid and plasma can provide important prognoses of disease to administer
appropriate treatments and other preventive measures. The use of exosomes for treating
OAK through intra-articular injection provides a novel treatment method to bridge the gap
between pharmacological and surgical procedures. However, many unclear aspects of thefunctions and mechanisms of EVs, including exosomes, and their roles in treatments ofjoint diseases such as OAK, remain to be elucidated. For the realization of exosome therapy,
it is essential to clarify the risks of the treatment methods, implement strict manufacturingcontrols, and prepare appropriate guidelines and laws.
Further research in this field and scientific evidence of safety and efficacy may greatly
contribute to the point-of-care treatment and cell-free therapy of joint disease.
Author Contributions: Conceptualization, M.M. and M.S.; writing—original draft preparation,
M.M., E.T., T.T. and M.S.; writing—review and editing, M.M. and T.T.; supervision, M.W. and M.S.;
funding acquisition, M.M. and M.S. All authors have read and agreed to the published version of
the manuscript.
Funding: Authors received financial support for the authorship and publication of this article by
JSPS KAKENHI Grant Number JP19K18509 and TERUMO LIFE SCIENCE FOUNDATION.
Conflicts of Interest: The authors declare no conflict of interest.

Int. J. Mol. Sci. 2021, 22, 2666

11 of 16Abbreviations
30 UTRACIACLTADAMTS5
AKT
ALIX
ATG4B
CAP
CD
DACT1
DDS
DMM
DMOAD
DNA
ECM
ESCRT
EV
GelMA
GSK-3β
hBMSC
HDAC
Hsp
hUC
IL-1β
ILV
IPFP
ISEV
Lamp2blncRNA
MCH
MCS
miRNA
MISEV
MMP13
mTOR
MVB
NSAID
OAK
PCGEM1
PHC
PTEN
PTGS2
RA
RNA
siRNA
SMSC
SOX9
SRDDS
TGF-β1
TKA
TLR
TSG101WNT5A
YAP

three prime untranslated region
autologous chondrocyte implantation
anterior cruciate ligament transection
a disintegrin and metalloproteinase with thrombospondin motifs 5
AKT serine/threonine kinase 1
ALG-2-interacting protein X
autophagy related 4B cysteine peptidasechondrocyte-affinity peptide
cluster of differentiation
dishevelled binding antagonist of beta catenin 1
drug delivery system
destabilization of the medial meniscus
disease-modifying OA drugs
deoxyribonucleic acidextracellular matrix
endosomal sorting complexes required for transport
extracellular vesicle
gelatin methacrylate
glycogen synthase kinase 3 beta
human bone marrow-derived MSC
histone deacetylase
heat shock protein
human umbilical cordinterleukin-1 beta
intraluminal membrane vesicle
infrapatellar fat pad
International Society for Extracellular Vesicles
lysosome-associated membrane glycoprotein 2blong noncoding RNA
major histocompatibility complex
mesenchymal stem cell
microRNA
Minimal Information for Studies of Extracellular Vesicles
matrix metalloproteinase 13
mammalian target of rapamycin
multivesicular bodynon-steroidal anti-inflammatory drugs
osteoarthritis of knee
prostate-specific transcript 1
primary human chondrocytes
phosphatase and tensin homolog deleted from chromosome 10
prostaglandin-endoperoxide synthase 2
rheumatoid arthritis
ribonucleic acidsmall interfering RNA
synovium-derived MSC
SRY-box transcription factor 9
sustained release drug delivery systemtransforming growth factor beta 1
total knee arthroplasty
Toll-like receptor
tumor susceptibility gene 101
Wnt family member 5A
Yes-associated protein

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