Mesenchymal stem cell-derived extracellular vesicles, osteoimmunology and orthopedic diseases

Exosomes

Exosomes are cell-derived vesicles that are present in many and perhaps all biological fluids. Their diameter is between 40 and 200 nm, and their density ranges between 1.13 to 1.19 g/ml (Hessvik & Llorente, 2018; Pol et al., 2012). They form through endocytosis of the plasma membrane, then the inner membrane sprouts inward to form multivesicular bodies. These bodies later fuse with the plasma membrane to secrete internal vesicles (Hessvik & Llorente, 2018; Pol et al., 2012). As a result, exosomes are vesicular, membrane-rich cup-shaped structures with a complex composition of protein, nucleic acids, lipids and other metabolites (Hessvik & Llorente, 2018; Mendt, Rezvani & Shpall, 2019; Pol et al., 2012). Exosomes serve as transport vehicles, playing an important role in intercellular communication (Pol et al., 2012; Zhao et al., 2020a). Exosomes contain proteins involved in membrane transport and fusion, such as Rab, annexins, and flotillin, as well as components of the endosomal sorting complex required for transport, such as Alix, tumor susceptibility gene 101, heat shock protein 70, integrins, and tetraspanin molecules CD9, CD63, CD81, CD82 and HSP70 (Kordelas et al., 2014; Pol et al., 2012).

Microvesicles

Microvesicles are present in most biological fluids, their diameter ranges between 200 and 2,000 nm, and their density ranges between 1.16 and 1.19 g/ml (Keshtkar, Azarpira & Ghahremani, 2018; Pol et al., 2012; Vig & Fernandes, 2022; Zhao et al., 2020a). In contrast to exosomes, microvesicles form directly through protrusion and budding of the cell membrane, and they can alter the behavior of target cells by transporting intracellular proteins (Pol et al., 2012). The size ranges of microvesicle and exosomes may overlap, which is important to remember when EVs are isolated from body fluids. Microvesicles contain CD40, matrix metalloproteases (MMP), caspases, selectin, integrins and cytoskeletal protein, and their cell membrane is highly rich in cholesterol, phosphatidylserine and diacylglycerol (Lai, Lichty & Bowdish, 2015; Ratajczak & Ratajczak, 2020).

Apoptotic bodies

When cells undergo apoptosis, they release caspase-3 and rho-related kinase I, then form vesicles called apoptotic bodies or apoptotic vesicles (Ela et al., 2013; Todorova et al., 2017). These bodies produce anti-inflammatory or tolerogenic reactions when absorbed by adjacent cells (Pol et al., 2012). Apoptotic bodies are membrane vesicles that form through lysis or autophagy after apoptosis, they have a diameter of 500–5,000 nm, and their density ranges between 1.16 and 1.28 g/ml (Pol et al., 2012). Specific surface markers of apoptotic bodies include DNA, tumor antigens, phosphatidylserine and histones (Bergsmedh et al., 2001). Inappropriate clearance of apoptotic vesicles is considered to be the primary cause of systemic autoimmune disease (Pol et al., 2012).

Osteoarthritis

Osteoarthritis is an age-related degenerative joint disorder that affects ∼7% of the global population (Hunter, March & Chew, 2020), and it is characterized by articular cartilage destruction, synovial inflammation, sclerosis of subchondral bone, and loss of extracellular matrix (ECM) (Sacitharan, 2019; Taghiyar et al., 2021). The dysfunction of osteoimmunology has been confirmed to be closely related to the occurrence and and progression of osteoarthritis. In the early stage of osteoarthritis, macrophages and neutrophils infiltrate in the synovial and produce inflammatory factors such as IL-1 and TNF-α (Woodell-May & Sommerfeld, 2020). These inflammatory cytokines stimulate chondrocytes to produce matrix degrading enzymes, which can increase matrix degradation and accelerate the progress of osteoarthritis (Griffin & Scanzello, 2019; Woodell-May & Sommerfeld, 2020). In recent years, MSC-derived EVs have emerged as a promising approach to treating osteoarthritis for its immunoregulatory functions (Sokolove & Lepus, 2013). Several studies have shown that EVs can alleviate the development of osteoarthritis by inhibiting inflammation, protecting cartilage and regulating extracellular matrix (ECM) synthesis and catabolism (Kim et al., 2021; Mianehsaz et al., 2019). For instance, exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been found to alleviate cartilage damage, reduce osteophyte formation and synovial macrophage infiltration, inhibit the production of activated pro-inflammatory phenotype (M1) macrophages, and promote the generation of activated M2 macrophages (Zhang et al., 2020a). Another study found that EVs derived from human umbilical cord mesenchymal stem cells (hUCMSCs), by carrying proteins and miRNAs, produced anti-inflammatory and immunomodulatory effects, thus interfering with the occurrence and development of osteoarthritis (Li et al., 2022). EVs can also act through PI3K-Akt signaling to promote polarization of M2 macrophage and reduce levels of pro-inflammatory factors TNF-α, IL-1 and IL-6, giving them strong immunomodulatory potential (Li et al., 2022). In another study, hUCMSC-derived EVs polarized macrophages to the M2 type, based on analysis of the polarization markers CD14, IL-1β, IL-10 and CD206 (Tang et al., 2021). These EVs through PI3K-Akt signaling to stimulate chondrocyte activity and matrix remodeling within the inflammatory environment. MSCs-derived EVs have been shown to contain miRNAs that have been associated with development and progression of osteoarthritis, and some miRNAs from EVs derived from adipose mesenchymal stem cells (ASCs) have been shown to exert anti-inflammatory and protective effects on macrophages, T cells and inflamed chondrocytes in vitro (Ragni et al., 2021). For example, miR-155-5p is crucial for regulatory T cell (Treg) proliferation because it induces the IL-2 receptor; while miR-24-3p overexpression significantly inhibits macrophage activation and M1 polarization (Ragni et al., 2021).

Protecting cartilage is one of the important therapeutic aims in osteoarthritis. BMSCs-derived EVs can promote the proliferation and migration of chondrocytes, while reducing apoptosis by downregulating IL-1β-activated pro-inflammatory signal involving Erk1/2, PI3K-Akt, TAK1 and NF- κB. This alleviates osteoarthritic cartilage injury in vitro (Li et al., 2020). In addition, ASCs-derived EVs can promote the proliferation and migration of chondrocytes and slow the development of osteoarthritis by inhibiting IL-1β and inflammatory responses (Woo et al., 2020).

Taken together, the available evidence indicates that MSC-derived EVs can alleviate the symptoms of osteoarthritis by preventing the apoptosis of chondrocytes while promoting their proliferation and migration, by regulating immune cells and by inhibiting inflammatory responses. Nevertheless, their complex composition and multiple functions need to be further explored.

Osteoporosis

Osteoporosis is a chronic metabolic bone disease that arises through an imbalance between osteogenesis and osteoclastogenesis (Armas & Recker, 2012). With the development of society and longer average life expectancy, this disease has become one of the most widespread and complex skeletal disorders worldwide, especially among postmenopausal women and the elderly (Clynes et al., 2020), yet effective treatments are lacking (Dimitriou et al., 2011; Ensrud & Crandall, 2019). An increasing number of evidence has attributed the pathogenesis of osteoporosis to the dysfunction of immunoregulation (Livshits & Kalinkovich, 2022). Immune cells such as over-activated M1 macrophages, neutrophils, and mast cells release a great quantity of reactive oxygen species (ROS), pro-inflammatory cytokines or chemokines (Dou et al., 2018; Livshits & Kalinkovich, 2022). These inflammatory mediators cause bone loss and subsequent osteoporosis by directly or indirectly inhibiting osteogenic differentiation of BMSCs and inducing apoptosis of osteocytes, osteoblasts and BMSCs.

EVs secreted by MSCs exert important immunoregulatory effects on bone repair in osteoporosis. For example, ASCs-derived EVs can significantly inhibit the osteoclast differentiation of macrophages, promoting the migration of BMSCs (Lee et al., 2021). Those EVs inhibited osteoclast differentiation through osteoprotegerin (OPG), mir-21-5p and let-7b-5p, and they downregulated genes related to bone resorption. In addition, the lncRNA NRON inside BMSCs-derived EVs that have been induced by bioactive glass nanoparticles can activate transcription factors of NFAT family, inhibiting the nuclear translocation of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and thereby the osteoclast differentiation of macrophages (Yang et al., 2022). Another study showed that hUCMSCs-derived exosomes can effectively inhibit the differentiation of macrophages into osteoclasts, enhance bone formation, reduce bone marrow fat accumulation and reduce bone resorption in osteoporotic mice, ultimately reducing bone loss (Hu et al., 2020). ASCs-derived exosomes effectively inhibited osteocyte apoptosis induced by hypoxia and serum deprivation, and they exerted these effects by upregulating Bcl-2/Bax and by suppressing the production of reactive oxygen species, production of cytochrome C, activation of caspases-3/-9 and downregulation of the expression of RANKL (Ren et al., 2019). Tissue engineering has developed rapidly, and MSCs-derived exosomes have shown strong potential in this regard (Shahrezaee et al., 2018; Wang et al., 2020). In one study, MSCs-derived exosomes were modified with polycaprolactone (PCL) and S-nitrosoglutathione (GSNO), and the resulting vesicles significantly reduced the inflammation stimulated by inflammatory macrophages and inflammatory cytokines (IL-6, TNF-α, iNOS, IL-1β). The modification also further accelerated osteogenic differentiation of MSCs (Wang et al., 2020).

The inflammatory response plays a crucial role in bone formation, during which the immune system responds to a variety of cytokines to recruit and activate a variety of cell types, such as MSCs (Kovach et al., 2015). MSCs-derived EVs play an important role in regulating inflammation, so whether MSCs-derived EVs can interfere with the pathogenesis of osteoporosis by regulating inflammation is an important direction for future research.

These results indicate that MSCs-derived EVs may serve as a promising agent for osteoporosis treatment by regulating the differentiation of osteoblasts and osteoclasts and promoting bone regeneration. As the exploitation of EVs expands, studying the various reactions that they mediate will be a direction for future study.

Intervertebral disc degeneration

Intervertebral disc degeneration (IDD) is a pathological condition associated with degeneration of the intervertebral disc, which comprises an inner nucleus pulposus surrounded by an annulus fibrosus (Dowdell et al., 2017). IDD progression is characterized mainly by increased cell death, ECM destruction (Freemont et al., 2002), and accumulation of inflammatory factors (Ding, Shao & Xiong, 2013). Although MSCs themselves were once considered a potential treatment against IDD due to their strong ability to differentiate and modulate immune responses, recent research suggests that MSCs-derived EVs and their miRNAs may exert even stronger therapeutic effects (Lu et al., 2021).

Inflammatory reactions are one of the important pathological drivers of IDD. During development of the disease, IL-1β in the nucleus pulposus significantly increases, followed by an increase in the levels of inflammatory mediators such as COX-2, NO, and NOS (Zhu et al., 2020). MSCs-derived exosomes can interfere with the occurrence and development of IDD by inhibiting apoptosis of nucleus pulposus cells (NPCs); reversing IL-1β-induced secretion of the inflammatory cytokines TNF-α, IL-6, IL-8, IL-12 and IL-18; and activating mitogen-activated protein kinase (MAPK) (Zhu et al., 2020). In a rat model of IDD, EVs secreted by metformin-treated MSCs ameliorated intervertebral disc cell senescence (Cui & Zhang, 2021), and the miR-129-5p within the EVs inhibited apoptosis of nucleus pulposus cells, ECM degradation, and polarization of M1 macrophages (Cui & Zhang, 2021). In addition, BMSC-derived exosomes inhibited activation of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome in NPCs, producing anti-inflammatory effects (Xia et al., 2019). BMSC-derived exosomes can downregulate levels of ROS in NPCs and thereby attenuate their apoptosis, while also downregulating ECM-degrading proteases to protect the ECM; both effects can protect against IDD (Xia et al., 2019). Overproduction of reactive oxygen species is common in degenerative IDD: the oxidative stress enhances matrix degradation and inflammation and reduces the number of viable, functional cells in the IDD microenvironment (Hu et al., 2022). MSC-derived exosomes carrying miR-31-5p act via the ATF6-related ER-stress pathway to inhibit apoptosis and calcification in endplate chondrocytes (EPCs) under oxidative stress (Xie et al., 2020).

These studies have shown that MSC-derived EVs and their miRNAs can significantly mitigate IDD through regulation of inflammatory responses. Additional research should focus on how MSC-derived EVs can modulate inflammatory responses by immune cells as a strategy to delay or even reverse IDD.

Osteonecrosis

Osteonecrosis, also known as ischemic necrosis, is a multifactorial orthopedic disease that is progressive, devastating and refractory (Hernigou et al., 2016). It is characterized by a stereotypical pattern of cell death and a complex repair process involving bone resorption and formation. The earliest pathological feature of osteonecrosis is the necrosis of hematopoietic cells and adipocytes, followed by interstitial bone marrow edema (Shah et al., 2015). Vascular injury, inflammation, mechanical stress and increased intraosseous pressure are considered to be important causes of osteonecrosis (Elgaz, Bonig & Bader, 2020; Loi et al., 2016). With the rapid development of cell-based therapies, MSCs have been extensively studied as a treatment for osteonecrosis.

More recent work suggests that EVs derived from MSCs can treat the disease. For example, MSC-derived EVs prevented zoledronic acid-induced senescence in stem cells, osteoblasts, and fibroblasts, while reducing levels of the inflammatory cytokines IL-6 and IL-8 as well as matrix MMP 1 and 3 (Watanabe et al., 2020). Furthermore, MSC-derived EVs can prevent senescence of cells involved in wound healing and the spread of chronic inflammation around senescent cells, thus promoting angiogenesis and bone regeneration and preventing bisphosphonate-related osteonecrosis of the jaw (Watanabe et al., 2020). Exosomes secreted by BMSCs show potential against osteonecrosis of the femoral head (ONFH) by affecting mainly ONFH osteogenesis (Fang, Li & Chen, 2019), whereas exosomes derived from human-induced pluripotent stem cell-derived MSCs can protect against ONFH by promoting local angiogenesis and preventing bone loss (Liu et al., 2017). BMSC-derived EVs carrying miR-148a-3p were found to improve ONFH by suppressing Smad ubiquitination regulatory factor-1, which in turn increased BMSC osteogenic proliferation and differentiation (Huang et al., 2020). Similarly, exosomal miR-135b alleviated ONFH by reducing programmed cell death protein 4(PDCD4)-induced apoptosis of osteoblasts (Zhang et al., 2020b).

Taken together, the available evidence suggests that MSC-derived EVs loaded with miRNAs can alleviate osteonecrosis progression by promoting the proliferation and differentiation of osteoblasts, while enhancing osteogenesis and angiogenesis and reducing inflammatory responses.

Table 1 and Fig. 1 shows and depicts the immunomodulatory functions of different types of MSCs-derived EVs in aforementioned orthopedic diseases.