Extracellular Vesicles in chondrogenesis and Cartilage regeneration

Abstract Extracellular vesicles (EVs), mainly exosomes and microvesicles, are bilayer lipids containing biologically active information, including nucleic acids and proteins. They are involved in cell communication and signalling, mediating many biological functions including cell growth, migration and proliferation. Recently, EVs have received great attention in the field of tissue engineering and regenerative medicine. Many in vivo and in vitro studies have attempted to evaluate the chondrogenesis potential of these microstructures and their roles in cartilage regeneration. EVs derived from mesenchymal stem cells (MSCs) or chondrocytes have been found to induce chondrocyte proliferation and chondrogenic differentiation of stem cells in vitro. Preclinical studies have shown that exosomes derived from MSCs have promising results in cartilage repair and in cell‐free therapy of osteoarthritis. This review will focus on the in vitro and in vivo chondrogenesis and cartilage regeneration of EVs as well as their potential in the treatment of osteoarthritis.


| INTRODUC TI ON
Cartilage is a hydrated tissue with no vascular and neural networks.
They are divided into three major groups: hyaline, fibrous and elastic cartilages. Hyaline cartilage is the most frequent form and is found in synovial joints, ribs, nose, trachea, bronchi, etc 1 The main roles of this articular hyaline cartilage are to tolerate bone load and forming a lubricant environment to enable joint movement. 2 Extracellular matrix (ECM) synthesized by chondrocytes constitutes the main part of each cartilage. Collagen type II is the most frequent ECM molecule in hyaline cartilage and accounts for 90%-95% of total collagen molecules. 3,4 Collagen II forms filamentous structures with collagen IX, responsible for cartilage tensile and shear stress. Proteoglycans, such as aggrecan, and glycosaminoglycans (GAG), such as chondroitin sulphate, are the other components of the articular cartilage ECM. 5,6 The integrity of ECM is vital for the normal function of cartilage. Therefore, changes in ECM elements and composition are the main feature of cartilage diseases. 7 Osteoarthritis (OA) is characterized by progressive cartilage damage and is the most prevalent cartilage-related disease. OA mainly affects elderly people and is more prevalent in women than men. 8 Trauma and pathological conditions such as obesity and congenital abnormalities are the main cause of the disease and usually, knee and hip cartilages are affected. 9 Chondrocytes that produce and secrete ECMs are the main cell source of cartilage; however, they constitute only 2% of cartilage volume. They need a suitable microenvironment for their survival and function. This microenvironment is changed during cartilage diseases results in chondrocyte apoptosis. On the other hand, they have limited replication capacity. 10,11 In addition, inflammation which is one of the main characteristics of OA causes more changes in cartilage ECM and therefore exacerbates tissue damage. 12 Despite the high prevalence of cartilage diseases such as OA, no definitive therapy is available. Surgical and non-surgical therapies are associated with side effects, and their efficacy is not satisfactory. 13 As many cartilage diseases are associated with chondrocyte loss or dysfunction, cell-based therapy has been suggested as an alternative therapy. Stem cells including embryonic stem cells (ESCs), induced pluripotent stem (iPS) cells and mesenchymal stem cells (MSCs) are widely used in tissue engineering and regenerative medicine to restore and repair injured tissues. 14 These cells are mainly used to differentiate into somatic cells of the organ. Furthermore, some types of stem cells, such as MSCs, can induce endogenous progenitors and stem cells to migrate, proliferate and differentiate. 15 Since many functions of MSCs are mediated through paracrine effects and with regard to cell transplantation complications such as immune rejection, cell-free secretome has been proposed for the treatment of cartilage diseases. 16

| THE B I OLOGY AND PATHOPHYS IOLOGY OF E X TR ACELLUL AR VE S I CLE S IN OA
The secretome includes extracellular vesicles (EVs) and is secreted from many cell types. 17 EVs are considered as small bilayer lipids with 30-1000 nm diameters. EVs are usually defined as exosomes, microvesicles and apoptotic bodies. 18 Among them, exosomes and microvesicles share many characteristics, but vary in size as well as protein composition. Exosomes are 30-100 nm cup-shaped vesicles, while microvesicles are heterogeneous with 50-1000 nm in diameter. 19 Exosomes are released from their origin cells by fusion with the plasma membranes, while microvesicles are released through shedding from the plasma membrane. Coding and non-coding RNAs, proteins, antigen-presenting molecules and DNA are the main compositions of exosomes. [20][21][22] Microvesicles consist of bilayer lipids containing mRNAs, miRNAs as well as lipids and cytosol. Because of their size and composition, exosomes are considered to be more important in the field of tissue engineering and regenerative medicine. Almost all of the cells, both in normal and pathological states, including lymphocytes, antigen-presenting cells, platelets, mesenchymal stem cells (MSCs), many mature somatic cells and tumour cells, release exosomes and microvesicles into almost all body fluids. The cargo of each exosome reflects its origin cells. They include cell-specific receptors, heat shock proteins (HSPs), tetraspanins (CD markers), lipid rafts such as flotillin-1 and integrins, which mediate exosomes-cell interactions in a paracrine manner. 19,23,24 Exosomes are present in the synovial fluid (SF); however, the quantity and compositions of SF-exosomes and more importantly their functions are changed in cartilage-related diseases. Studies have shown a higher levels of exosomes in patients with early and late-stage OA than in the normal population. 25 Changes in proteins, miRNAs and lnRNAs have been also observed in the SF of patients with joint diseases. 25,26 In addition, the composition of exosomes differs between various joint disease. 27 In patients with joint diseases, exosomes derived from fibroblast-like synoviocytes (FLS) activate CD4 + lymphocytes and increase the secretion of inflammatory cytokines. 28 Furthermore, these exosomes mediate bone and cartilage degradation through inducing matrix metalloproteinases and promoting the osteoclast function, respectively. 29 Plasma-derived exosomes in patients with RA increase the activity of pro-inflammatory cytokines produced by peripheral blood immune cells. 30 In addition, these exosomes can activate complement systems and increase infiltration of immune cells such as neutrophils and M1 macrophages, which further degrade cartilage. 31 These data suggest the role of exosomes in the joint disease pathology and provide a perspective on the treatment of affected patients.
Recently, many studies have evaluated the potential use of exosomes as diagnostic markers as well as carriers of genes for the therapeutic purpose. [32][33][34] It is also used to suppress immune responses during cell and organ transplantation to avoid immune rejection. 35,36 Exosomes could be used to regenerate and repair tissues including bone and cartilage. 37,38 As exosomes play key roles in the modulation of inflammation and immune responses, they have been widely used by researchers in the treatment of autoimmune and inflammatory diseases such as OA. 39,40 In the following sections, the in vitro and in vivo potential roles of exosomes in chondrogenesis and healing OA are discussed.

| THE IN VITRO CHONDROG ENI C P OTENTIAL OF E VS
In the field of cartilage tissue engineering, iPS and various sources of MSCs have been widely used to differentiate into mature chondrocytes. [41][42][43] Some studies have used growth factors and miRNAs to induce chondrogenic differentiation. 43 46 Cosenza et al showed that MSC-derived exosomes and microparticles can protect osteoarthritis-derived murine chondrocytes in vitro and in vivo. It was found that exosomes from murine bone marrow-derived MSCs (BM-MSCs) increased the expression of chondrocyte markers including aggrecan and type II collagen. On the other hand, exosomes inhibited the expression of immune and inflammatory elements responsible for cartilage degradation such as matrix metalloproteinases (MMPs). The exosome-activated chondrocytes were not able to activate CD4 + and CD8 + T lymphocytes and B lymphocytes in vitro. 47 The same results were also observed in the study of Yubao Liu et al 48 MSC-derived exosomes were found to inhibit apoptosis in chondrocytes and promote their proliferation. The luciferase activity assay showed that exosomal lncRNA-KLF3-AS1 inhibited miR-206 which in turn facilitates G-protein-coupled receptor kinase interacting protein-1 (GIT1) expression in chondrocytes. 48 It is shown that GIT1 mediates chondrocyte proliferation and inhibits apoptosis in chondrocytes. The expression of GIT1 is suppressed by miR-206. 49,50 In fact, exosomes transfer bioactive molecules including miRNAs and growth factors that can affect many cellular processes such as proliferation and differentiation. 51 Exosomes derived from MSCs and chondrocytes contain molecules that direct the chondrogenic differentiation of stem cells/progenitors or promote proliferation and migration of chondrocytes. Therefore, they can be beneficial for in vivo treatment of diseases such as OA that is defined by cartilage degradation. 52 It is well known that exosomal miRNAs mediate many functions of exosomes such as cell proliferation and differentiation as well as inhibition of cell apoptosis. 53 progenitors. This study showed that miR-381-3p directly suppresses TAOK1 (TAO Kinase 1) which in turn suppresses Hippo signalling pathway. 55 Hippo pathway is involved in the promotion of cell apoptosis and inhibition of cell proliferation. 56 Mao et.al reported that exosomes from miR-92a-3p-overexpressing MSCs increase chondrocyte migration and proliferation. It was found that exosomal miR-92a-3p suppresses WNT5A (Wnt family member 5A) that is a key factor in the pathogenesis of OA. 57 Further study revealed that the exosomes isolated from miR-95-5p-overexpressing chondrocytes promote chondrogenic differentiation of MSCs and induce cartilage matrix expression in chondrocytes. miR-95-5p inhibits the expression of histone deacetylase 2/8 (HDAC2/8) that is increased in OA. 58 It is shown that HDAC2/8, HDAC1 and HDAC3 inhibit the expression of COL2A1 (collagen type II alpha 1 chain) and aggrecan. 59

| THE IN VITRO THER APEUTI C S OF E VS
MSCs have been known to promote cartilage repair and chondrocyte differentiation through a paracrine effect via cytokine secretion. These factors including transforming growth factor beta (TGFβ) and hepatocyte growth factor (HGF) constitute a major part of MSCs secretome. 68 Furthermore, MSCs have been found to secrete chemokines and vascular endothelial growth factor (VEGF) into the synovial fluid to promote cartilage repair in OA patients. 69,70 It is shown that the secretome of MSCs has a therapeutic function in the treatment of liver, kidney, skin and other organ injuries. 71 Preclinical studies have shown that exosomes derived from MSCs have promising results in cartilage repair. Yet, the exact mechanisms of tissue repair have not been elucidated. However, it seems that MSCs have a pivotal role in the maintenance of the mesenchymal tissue microenvironment. 11 Here, the therapeutic functions of EVs derived from MSCs and other sources in the treatment of cartilage repair are discussed. Table 2 summarizes the in vivo functions of EVs in the treatment of cartilage repair.   55 Yu et al showed that the controlled release of BMSC-Exos at rat tendons induces the proliferation, migration and differentiation of endogenous tendon stem/progenitor cells (TSPCs) in rat patellar tendon defect model. Following the exosomes-fibrin injection, a neo-tendon with a high expression of mohawk, tenomodulin and type I collagen was formed at the site of the defect. 46 Liu et al also showed that the human MSC-derived exosomes induce the proliferation of chondrocytes and inhibit their apoptosis in an OA rat model.

| Inducing chondrocyte differentiation
They showed that non-coding RNAs including lncRNAs and miRNAs are involved in this phenomenon. 48

| Inducing chondrocyte proliferation
The Mechanistically, exosomes have been found to mediate cell proliferation through Akt and Erk1/2 signalling pathways, 78 and CD73 has a major role in the induction of these pathways. 79 (3) The injected exosomes reduce the inflammation in the damaged cartilage possibly by inhibiting inflammatory molecules such as IL-1, IL-6 and TNFα, which are responsible for cartilage inflammatory diseases such as OA act through kinase receptors involved in the Akt and Erk1/2 signalling pathways, thereby inducing cell proliferation. 81 Zhang et al confirmed the role of the CD73-Akt/Erk pathway in exosome-mediated cell proliferation. They showed that the blocking of this pathway by a CD73 inhibitor (AMPCP) and theophylline (an antagonist of adenosine receptor) decreases the number of chondrocytes; however, the matrix synthesis remains unchanged. 82

| Increasing bioenergetics
The destruction and dysfunction of mitochondria decrease cell bi-

| Reducing inflammation and immune responses
Inflammation is known to be involved in the initiation and development of OA disease. 92 82 Zavatti et al also reported that exosomes derived from amniotic fluid stem cells (AFSC) change the polarization of macrophages into M2 type. They showed that either MSCs or their exosomes reduce the inflammatory cytokines in an animal model. 106 It was also reported that exosomes derived from stem cells from human exfoliated deciduous teeth (SHEDs) inhibited cartilage inflammation by inhibiting mTOR pathway which is mediated by miR-100-5p. 107 Exosomes from other cell sources also could inhibit inflammation and suppress immune reactions in cartilage. 84,87,105,107,108 However, exosomes derived from osteoarthritic chondrocytes have been shown to induce inflammation and increase IL-1 production by macrophages. 108

| E XPRE SS I ON PAT TERN OF E XOSOMAL MIRNA S DURING CHONDROG ENE S IS
Exosomal miRNAs have been found to induce the proliferation and differentiation of stem/progenitor cells into chondrocytes. 55,58 Therefore, it seems that miRNAs are involved in the chondrogen- there are some limitations that should be further considered. The efficient isolation and purification of exosomes are still a challenging task. In some cases, the atrophy of cartilage has been observed following MSC-derived exosome transplantation. Moreover, sufficient cartilage regeneration is challenging when exosomes from various sources were transplanted.

CO N FLI C T S O F I NTE R E S T
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.