MSC-EV therapy for bone/cartilage diseases.

Introduction

Mesenchymal stromal cells (MSCs) have been clinically and pre-clinically applied in cell therapy to treat various diseases/injuries ( Akpancar et al., 2016 ; Nitkin and Bonfield, 2017 ; Pittenger et al., 2019 ). Generally, cell therapies using MSCs have demonstrated the potential to provide positive therapeutic effects and to improve symptoms ( da Silva Meirelles et al., 2009 ). Despite such improvements, the mechanisms underlying such therapeutic effects are not fully understood. Initially, due to their ability to differentiate into multiple lineages, MSCs were expected to regenerate tissues by direct differentiation into the tissue cells. However, increasing numbers of reports suggest that MSCs provide their therapeutic activity via secreting paracrine factors such as cytokines, chemokines, and growth factors as well as releasing extracellular vesicles (EVs) ( Caplan, 2017 ; da Silva Meirelles et al., 2009 ; Lukomska et al., 2019 ; Phinney and Pittenger, 2017 ).

EV is a generic term for particles encircled by a bilayered lipid membrane, which are released by cells. These particles can be further classified into subtypes such as exosomes, microvesicles, and apoptotic bodies based on their origin and biogenesis ( Mathieu et al., 2019 ; Théry et al., 2009 ; Théry et al., 2018 ). Exosomes are characterized as smaller EVs ranging from 30 nm to 150 nm in diameter that are formed as intraluminal vesicles by the inward budding of the endosomal membrane inside of multivesicular endosomes (MVEs) ( Mathieu et al., 2019 ; Théry et al., 2009 ; Théry et al., 2018 ). These intraluminal vesicles are extruded into the extracellular space as exosomes by fusing MVEs with the plasma membrane. Microvesicles range in size from 100 nm to 1000 nm in diameter. They are generated by the outward budding and fission of the plasma membrane and subsequently secreted out to the extracellular space ( Mathieu et al., 2019 ; Théry et al., 2009 ; Théry et al., 2018 ). Apoptotic bodies are cellular debris that is produced when cells undergo apoptosis. The size of apoptotic bodies varies from 50 nm to several μm in diameter ( Mathieu et al., 2019 ; Raposo and Stoorvogel, 2013 ; Théry et al., 2009 ; van Niel et al., 2018 ). Despite the recent advance in methods to separate EVs from MSC conditioned media, the physical characteristics of these EV subtypes such as the size or density overlap with each other complicating classification ( Cocucci and Meldolesi, 2015 ; Gould and Raposo, 2013 ; Mathieu et al., 2019 ; Théry et al., 2018 ; van Niel et al., 2018 ; Gardiner et al., 2016 ). Therefore, the Minimal Information for Studies of Extracellular Vesicles (MISEV2018) guidelines proposed by the International Society for Extracellular Vesicles (ISEV) endorses the use of EVs instead of the subtype nomenclature ( Théry et al., 2018 ).

EVs have been demonstrated to contain a complex array of bioactive molecules that includes proteins ( Tkach and Théry, 2016 ), lipids ( Skotland et al., 2017 ), and nucleic acids such as messenger RNA (mRNA) ( Valadi et al., 2007 ), micro RNA (miRNA) ( Nazari-Shafti et al., 2020 ; Scheiber et al., 2022 ), long noncoding RNA (lncRNA) ( Wen et al., 2022 ), transfer RNA (tRNA) ( Baglio et al., 2015 ), genomic DNA( Kahlert et al., 2014 ; Thakur et al., 2014 ), and mitochondrial DNA (mtDNA) ( Guescini et al., 2010 ). EVs mediate intercellular communication by transferring such cargo to each other ( Tkach and Théry, 2016 ; Valadi et al., 2007 ). The cargo has been demonstrated to affect the cellular function of recipient cells ( Skog et al., 2008 ; Uenaka et al., 2022 ). Indeed, it has been demonstrated that proteins, mRNAs, and miRNAs contained in MSC-derived EVs (MSC-EVs) provided therapeutic effects on cartilage by downregulating genes related to inflammatory and matrix degradation ( Duan et al., 2020 ; Liu et al., 2022 ; Lu et al., 2021 ; Mao et al., 2018 ; Wang et al., 2020a ; Xia et al., 2021 ; Ruiz et al., 2020 ; Woo et al., 2020 ; Zhang et al., 2020 ). For instance, mRNA and protein of TGFβ-induced gene product-h3 (TGFBI) in MSC-EVs inhibit catabolic genes such as MMP-13 and ADAMTS5 in chondrocytes ( Ruiz et al., 2020 ). Mir-23a-3p suppresses the level of phosphatase and tensin homolog (PTEN) which results in the upregulation of the Akt signaling pathway in chondrocytes, leading to the promotion of cartilage regeneration ( Hu et al., 2020a ). Other musculoskeletal disorders, such as rheumatoid arthritis, osteoporosis, bone injury, and intervertebral disc degeneration among many others are also under active investigations and numerous effective bioactive molecules have been identified.

As of October 2022, over 1500 clinical trials of cell-based therapies using MSCs are found in ClinicalTrials.gov . Despite this enthusiasm for MSC therapy, MSC-EV therapy has several advantages over conventional MSC therapy. First, EV therapy has no risk of malignant transformation, a major detriment of MSC therapy, since EVs cannot replicate themselves ( Phinney and Pittenger, 2017 ; Théry et al., 2018 ). Second, EVs can be distributed systemically due to their small size, although systemically administered MSCs are mostly trapped in the lung and have potential risks that cause thrombosis and embolization ( Coppin et al., 2019 ; Moll et al., 2019 ; Filippi et al., 2019 ; Kallmeyer et al., 2019 ; von Bahr et al., 2012 ). Third, EVs are relatively stable and can be cryopreserved without losing their functionality, while it has been reported that freshly thawed, cryopreserved MSCs significantly lose their immunomodulatory properties. MSCs must be returned to cultures for 24 h after thawing to fully recover these properties ( François et al., 2012 ; Moll et al., 2014 ). Another advantage is that freshly thawed EVs can be injected directly into patients, making the EV banking system providing off-the-shelf EVs more practical and operational. Thus, compared to MSC therapy, EV therapy could be more accessible to patients who are in hospitals or clinics without GMP facilities.

Despite the promising potential of EV therapy as a cell-free therapy, there are challenges that govern the clinical success of EV therapy ( Nieuwland et al., 2022 ; Phinney and Pittenger, 2017 ; Witwer et al., 2021 ). EVs are heterogeneous with multiple subtypes as mentioned above. Therefore, acquiring EVs of the same quality from different preparations of the same BMSCs is still difficult. It would be much more challenging to obtain EVs with the same quality from BMSCs of different donors. As the reproducibility of therapeutic effects relies on the quality of EVs, it is required to establish methods to isolate EVs with the same quality from MSC conditioned media. In addition to the quality of EVs, the quantity must be considered for clinical usage involving multiple injections throughout the treatment ( Adlerz, 2020 ; Colao et al., 2018 ; Phinney and Pittenger, 2017 ). However, scalable manufacturing of EVs also affects the quality of EVs ( Haraszti et al., 2018 ; Patel et al., 2017 ; Scheiber et al., 2022 ). Therefore, in this review, to overcome these challenges and achieve effective and reliable EV therapy, we highlight the key elements that can affect EV quality followed by a summary of the pre-clinical and clinical EV studies surrounding skeletal diseases.

Key elements that can affect EV quality

Cell sources

While bone marrow and adipose tissues are the sources that MSCs are most commonly isolated from ( Pittenger et al., 2019 ), there are other sources that can harvest MSCs such as the synovium ( De Bari et al., 2001 ), teeth ( Stevens et al., 2008 ), tonsils ( Janjanin et al., 2008 ), the umbilical cord ( El Omar et al., 2014 ), umbilical cord blood ( Lee, 2004 ), and the placenta ( Yen et al., 2005 ). There are many similarities among the immunophenotypes and differentiation abilities of these MSCs from different sources; however, further characterization revealed differences in their gene expression profiles ( Arjmand et al., 2019 ; Elahi et al., 2016 ; Roson-Burgo et al., 2014 ; Secco et al., 2009 ) and proteome/secretome profiles ( Billing et al., 2016 ; Jeon et al., 2016 ; Kozlowska et al., 2019 ; Pires et al., 2016 ), suggesting that their therapeutic potential could be different. Indeed, several studies have reported that differences in the immunomodulatory effects were noticed in MSCs from different sources ( Blanco et al., 2016 ; El Omar et al., 2014 ; Pikuła et al., 2013 ; Strioga et al., 2012 ). Similarly, MSC-EVs from different sources have distinct characteristics that reflect their parent MSCs ( Del Fattore et al., 2015 ; Haraszti et al., 2016 ; Katsuda et al., 2013 ; Li et al., 2020 ; Lopez-Verrilli et al., 2016 ; Maqsood et al., 2020 ; Tang et al., 2021 ; van Balkom et al., 2019 ; Wang et al., 2020b ). In addition to distinct functionality, Haraszti et al. compared three different sources of MSCs (umbilical cord, bone marrow, adipose) for EV production and found that MSCs from different origins produced different amounts and sizes of EVs ( Haraszti et al., 2018 ). It has also been shown that MSCs derived from amniotic fluid produce a significantly greater number of EVs compared to those from bone marrow ( Tracy et al., 2019 ).

In addition to cell sources, cell condition also affects EV content, reflecting the parental cell condition. For example, EVs from MSCs differentiating into osteoblasts contained miRNAs enriched in pathways related to osteogenic differentiation compared to those from undifferentiated MSCs ( Wang et al., 2018b ). On the other hand, EVs from chondrocytes have a reduced chondrogenic ability compared to EVs from MSCs ( Hosseinzadeh et al., 2021 ).

Altogether, these data demonstrate that critical properties such as cell source and cell condition should be always considered when optimizing protocols to collect EVs from MSC culture and when developing reproducible EV therapies.

Donor differences

It has been demonstrated that the proliferative capacity and differentiation ability of MSCs vary from donor to donor ( Phinney et al., 1999 ; Siddappa et al., 2007 ). Considerable variability has been found in the secretome profiles of MSCs isolated from different donors in response to inflammatory stimuli ( Zhukareva et al., 2010 ). When transcriptomes were compared between bone marrow derived MSCs established from five healthy donors using two distinct methods, greater variability was detected among donors rather than between isolation methods ( Otsuru et al., 2013 ; Otsuru et al., 2015 ). These findings suggest that the therapeutic activities of MSCs could differ from donor to donor ( Phinney, 2012 ; Siegel et al., 2013 ). Similarly, donor variability has been identified in MSC-EVs from different donors. Proteomic and transcriptomic analyses of MSC-EVs from different donors demonstrated differences in their contents ( McBride et al., 2021 ; Ragni et al., 2019 ; Scheiber et al., 2022 ). These differences in cargo lead to the functional alteration of MSC-EVs, raising the challenge of reproducibility of EV therapies using EVs from different donors ( Chance et al., 2019 ; Kordelas et al., 2014 ; Kordelas et al., 2019 ).

Moreover, it has been suggested that the age of MSC donors impacts the proliferation ability of MSCs. MSCs from younger donors have greater proliferation potential in culture ( Fickert et al., 2011 ; Mareschi et al., 2006 ; Stenderup, 2003 ; Zhou et al., 2008 ). The age of MSC donors also influences the therapeutic potential of MSC- EVs when they are applied in the treatment of multiple myeloma. MSC-EVs from young donors were shown to have greater potential to inhibit angiogenesis induced by multiple myeloma ( Alexander et al., 2007 ; Alsagaby, 2021 ; Umezu et al., 2017 ).

Therefore, the impact of donor variability including age differences should be considered when selecting EVs for EV therapies.

Culture conditions

Scalable manufacturing of EVs is critical for developing EV therapies for patients. Recent advances in cell culture technology have made great improvements to protocols that rapidly expand MSCs. Fetal bovine serum (FBS) and human platelet lysate (hPL) have been commonly used as supplements in MSC culture media. However, both contain many EVs that are indistinguishable from those released by MSCs. To avoid the contamination of EVs from FBS and hPL, serum-free media are often used to harvest EVs. Serum-free media have been shown to increase EV production per cell and alter proteome in EVs ( Haraszti et al., 2019 ; Li et al., 2015 ). Recently, various chemically defined xeno-free media have become available to grow MSCs. Since there is no raw material derived from animals or humans, such media will be more consistent between lots, which potentially makes EV quality more reproducible. Xeno-free media not only helps the rapid expansion of MSCs but also yields significantly greater amounts of EVs compared to conventional FBS-containing media ( Scheiber et al., 2022 ). However, miRNA profiles in EVs from such media are altered ( Scheiber et al., 2022 ).

The number of passages and the seeding density of MSCs also affect EV production and their therapeutic activities ( Patel et al., 2017 ). Moreover, when MSCs were exposed to hypoxia, EVs from these MSCs significantly promoted angiogenesis compared to those from MSCs in normoxia due to changes in cargo ( Ge et al., 2021 ; Gregorius et al., 2021 ; Zhang et al., 2012 ).

In addition to conventional static 2D culture systems using mono- or multi-layer culture flasks, dynamic 3D culture systems with various types of bioreactors have been developed. Compared to 2D cultures, 3D culture systems grow MSC at higher densities, providing more MSCs from the same volume of culture media ( Mas-Bargues and Borrás, 2021 ). It has been demonstrated that MSCs cultured in a 3D system produce significantly greater amounts of EVs compared to those cultured in conventional 2D systems. Additionally, EVs from 3D culture systems have therapeutic activities equivalent to or superior to those from 2D systems ( Cao et al., 2020 ; de Almeida Fuzeta et al., 2020 ; Haraszti et al., 2018 ; Yan and Wu, 2020 ). Since 3D culture systems can be conducted in a closed system, it is likely to have less risks of bacterial or viral contamination. Although further investigations are needed, these findings suggest that 3D culture systems have the potential to provide scalable production of EVs from MSCs ( Colao et al., 2018 ; Patel et al., 2018 ).

Overall, culture conditions are critical for reproducibility of EV quality and scalable EV production. Technological advances are promising to develop reproducible and scalable protocols for EV production for clinical usage. Given that preclinical studies performed at a laboratory scale often use conventional methods (2D culture with serum-contained media), the changes caused by switching to a scalable 3D culture system need to be considered to reproduce the results of the pre-clinical studies when they are translated into patients.

Separation methods

There is no standardized technique to separate only EVs from biological fluids ( Gardiner et al., 2016 ). Various techniques have been developed for EV separation and purification; however, none of them can achieve high purity and yield at the same time ( Webber and Clayton, 2013 ), suggesting that other EV subtypes and lipids are always co-isolated. Therefore, EV quality and quantity vary depending on separation methods.

Ultracentrifugation is the most commonly used technique for EV separation and concentration ( Busatto et al., 2018 ; Gardiner et al., 2016 ; Gudbergsson et al., 2016 ; Théry et al., 2018 ). Ultracentrifugation can process relatively large volumes for scalable production; however, other techniques such as density gradients or chromatography need to be combined with ultracentrifugation to separate EV subtypes ( Corso et al., 2017 ; Gardiner et al., 2016 ; Théry et al., 2018 ). Alternative methods have been developed to achieve greater purity and recovery for clinical-grade sterile scalable EV production ( Théry et al., 2018 ). Tangential flow filtration is one such method, which allows the processing of a larger volume of biological fluids. It has been demonstrated that tangential flow filtration can more efficiently yield EVs from MSC culture media compared to ultracentrifugation ( Busatto et al., 2018 ; Haraszti et al., 2018 ). Combination of tangential flow filtration with a 3D culture system can achieve a much greater yield as well as more potent EVs ( Haraszti et al., 2018 ).

Therefore, in addition to the proportion of EV subtypes and lipids in MSC conditioned media, the separation methods are critical for the quality and quantity of the final product. The therapeutic effects can differ depending on the types and amounts of EVs after separation.

Storage

To establish an EV banking system that provides off-the-shelf EVs, it is required to store isolated pure EVs rather than crude EVs in the conditioned medium. However, there are no standardized protocols for EV storage ( Lener et al., 2015 ). Similar to cellular cytoplasmic membranes, EV membranes consist of lipids and proteins, which are susceptible to changes in temperature, pH, and osmotic pressure. Corruption of EV membranes could lead to damaging the cargo, resulting in the loss of therapeutic potency. Using EVs from human neutrophilic granulocytes, Lorincz et al. reported that the number of EVs was significantly decreased in a time-dependent manner when EVs were stored at 20 °C or 4 °C after isolation while storage at −20 °C or − 80 °C maintained a stable quantity for 28 days. Light-scattering analysis showed that the size of EVs significantly increased in a time-dependent manner when EVs were stored at −20 °C but not at −80 °C ( Jeyaram and Jay, 2018 ; Lőrincz et al., 2014 ). On the other hand, Sokolova et al. showed that the size of EVs decreased in a time-dependent manner during storage at 4 °C and 37 °C but not at −20 °C ( Sokolova et al., 2011 ). More importantly, the therapeutic function was preserved when stored at −80 °C ( Jeyaram and Jay, 2018 ; Lőrincz et al., 2014 ; Wiklander et al., 2019 ). Additionally, miRNAs in EVs seemed to be stable after storing at 4 °C for 2 weeks. They were well preserved after 5 years of storage at −20 °C and even tolerated repeated freeze and thaw ( Ge et al., 2014 ).

Changes in the pH of the storage buffer cause aggregation and fusion of EVs, resulting in the loss of therapeutic potency ( Lener et al., 2015 ). Therefore, the selection of a storage buffer is critical for stable storage, avoiding pH shifts and the loss of therapeutic activities. Phosphate-buffered saline (PBS) is commonly used for EV storage ( Théry et al., 2006 ); however, supplementation with trehalose into PBS has been demonstrated to prevent aggregation of EVs during storage ( Bosch et al., 2016 ).

Based on the findings from these studies, −80 °C seems to be the current optimal temperature for EV storage. However, the studies examining factors related to the stable storage of EVs are still limited. Further investigations are required to optimize and standardize the protocols of EV storage that can preserve EVs, their contents, and, most importantly, their therapeutic activities. The success of developing a standardized protocol will make EV therapy more accessible for broader patients.

Pre-clinical application of MSC-EV therapy for bone/cartilage diseases

Osteoarthritis (OA)

Worldwide increases in obese and aging populations are increasing the number of OA patients and the overall disease burden ( Murray et al., 2012 ). OA is not a lethal condition but can lower a patient's quality of life by causing intractable pain and restricting daily activities. Thus far, there are no effective treatments that can either halt the progression or regenerate the articular cartilage. Current therapies are restricted to pain relief, rehabilitation, or surgical interventions. Regenerative therapy for OA is highly anticipated and several pre/clinical trials using stem cells have been conducted ( Song et al., 2020 ; Wei et al., 2021b ). Indeed, the regeneration of cartilage tissue and attenuated inflammation by MSC administration have been demonstrated in animal models ( Toh, 2017 ). However, several hurdles of cell therapy such as high costs, risk of tumorigenesis, and inconsistency of therapeutic effects among others still hinder it from becoming a standard treatment of OA. Moreover, the initial expectation that MSCs can engraft and differentiate into cartilage cells has been proven unlikely. Accumulating lines of evidence suggest that MSCs exert therapeutic effects by secreting trophic factors including EVs. Therefore, preclinical studies testing EV therapy for OA as a substitute for MSC therapy have emerged during the last decade ( Table 1 ). Clinical studies have just begun and only one Phase I clinical trial in Universidad de los Andes, Chile is currently listed in ClinicalTrials.gov , which uses a single intra-articular injection of EVs from allogeneic MSCs to treat mild to moderate OA patients. This trial is scheduled to be finished in 2023. Table 1 Summary of the cell origins, culture methods, isolation methods, delivery methods, major in vivo outcomes, and the underlying mechanisms/cargos in representative studies using MSC-EVs for OA. Table 1 References Donor/Cell origin Culture media/method Isolation Animal Models Delivery methods Outcomes and proposed mechanisms Liu et al., 2022 Human urine–derived stem cell (USC) USC medium consisting of 50 % Keratinocyte Serum-Free Medium, 33.75 % DMEM, 11.25 % Ham's F-12 Nutrient Mixture, and 5 % FBS supplemented with 5 ng/ml epidermal growth factor, 50 ng/ml bovine pituitary extract, 0.4 μg/ml hydrocortisone, 5 μg/ml transferrin, 5 ng/ml bovine insulin, 0.18 mM adenine, 2 nM 3,3,5-triiodo-L-thyromine, 100 U/ml penicillin, and 100 μg/ml streptomycin Ultracentrifugation 12w-female SD rats ACL transection and MM resection Multiple intra-articular injections on a weekly interval The first injection was on the 30th day after the surgery Macroscopy: less pathological lesions HE/SO: higher cartilage thickness, chondrocyte number; lower OARSI score IHC: more Col II positive cells, less Col I, VEGFA posivie cells μCT: higher bone volume and BMD, lower trabecular separation, less osteophytes VEGFA upregulated during OA progression, whereas MiR-140-5p directly binded to VEGFA gene and suppress its tranlation and attenuated OA. Lei et al., 2022 Antler stem cell (ASC) αMEM medium +10 % FBS, 0.1 mM NEAA, 1 ng/ml FGF-2, and 1 % P/S exosomes in FBS was removed by ultracentrifugation Ultracentrifugation C57BL/6 J mice ACL transection Weekly intra-articular injections after surgery SO: lower OARSI score IHC: more Ki67 positive cells, less p16 positive cells μCT: higher bone density Higher grip strength ASC-derived exosomes promoted cell division and repressing the senescence-associated inflammation. Huang et al., 2021 Murine BMSC DMEM/F12 + 10%FBS + 1 % P/S Ultracentrifugation + ultrafiltration Mice ACL transection intra-articular injection twice a week HE: lower histological score Tunnel: less apoptotic cells WB: suppressed Elf3, osteocalcin expression μCT: higher bone density Serum ELISA: lower TNF-α, IL-1β and IL-6 MiR-206 delivered by BMSC-EVs targeted Elf3 to promote proliferation and differentiation and inhibits apoptosis of osteoblasts. Jiang et al., 2021 Rat BMSC DMEM +10 % FBS + 1 % P/S Ultracentrifugation SD rats ACL transection induced OA Single intra-articular injection 4 weeks after surgery Treadmill: larger number of electric shocks RT-qPCR and WB: lower TNFα and IL-6; higher Col II, Aggrecan Tunnel: less apoptotic cells IHC: less c-MYC and GLS1 Peripheral plasma ELISA: higher α-KG BMSC-derived exosomes modulated chondrocyte glutamine metabolism. Xu and Xu, 2021 Rat BMSC α-MEM + 10 % FBS, 100 U/ml penicillin, and 10 mg/ml streptomycin ExoQuick-TC™ system SD rats Sodium iodoacetate induced OA Intra-articular injection one week after surgery HE/SO: lower OARSI score IHC: increased GSDMD and cleaved Caspase-1 in cartilage WB: counteracted the repression of STAT1/NF-κB p65 signaling pathway, and the increase levels of pyroptosis-related proteins (NLRP3, ASC, GSDMD, Caspase-1, IL-1β, and IL-18) in cartilage induced by OA BMSC-Exos delivered miR-326 into chondrocytes targeting HDAC3 and STAT1/NF-κB p65. Xia et al., 2021 Human BMSC aspirated from trauma OA patients DMEM/F12 + 10 % FBS Ultrafiltration and ultracentrifugation C57BL/6 mice Mechanical load to the ankle joint Intra-articular injection immediately after surgery RT-qPCR/WB: decreased E2F2, MMP13; increased Col II, Aggrecan MiR-125-5p directly target E2F2. Zhang et al., 2021a Rat BMSC Complete culture media +10 % FBS +1 % P/S Ultrafiltration and ultracentrifugation SD rats OCD AD/CS/RSF hydrogel with exosome was injected into the defect Macroscopy/MRI: regenerated cartilage HE/SO: higher ICRS score IHC: lower Col I, higher Col II; more CD29+, CD11b MSCs migrated in to the hydrogel RNA-sequence analysis of BMSCs treated with extracts of hydrogel: chemokines upregulated RT-PCR: Cxcl6, Plk2, Sphk1 upregulated BMSC-derived EVs stimulated BMSC migration via the chemokine signaling pathways. Fazaeli et al., 2021 AD-MSC vs BMSC DMEM +10 % FBS + 1 % P/S Exocib exosome extraction kit BALB/c mice ciprofloxacin by gastric gavage for 2 weeks Multiple intra-articular injections on 15, 22, 29 day after OA induction HE/SO: lower OARSI score in the BM-MSC group IHC: lower Col I, higher Col II in the BM-MSC group Jin et al., 2021 Human BMSC DMEM +10 % FBS + 1 % P/S Ultracentrifugation Rat complete excision of the MM and resection of the ACL Multiple intra-articular injections weekly until 8th week post-surgery HE/SO/TB/Masson: lower OA score μCT: higher bone preservation IHC: increased osteocalcin, decreased TRAP staining EVs attenuated IL-1β-induced chondrocyte senescence and apoptosis. Liao et al., 2021 Rat BMSC DMEM/F-12 + 10 % exosome-free FBS Ultracentrifugation SD rats ACL transection and MM resection Intra-articular injection and LIPUS application for 4 weeks from week 6 post-surgery HE/SO/TB: lower OARSI score SB/qPCR: higher Sox9, Col II LIPUS strengthened the potential of exosome in promoting chondrogensis and in inflammation-inhibition via the NF-κB pathway Chen et al., 2020 BMSCs from femur bone marrow of traumatized patients DMEM/F12 + 10%FBS Ultrafiltration and ultracentrifugation 5-8w male C56BL/6 mice mechanical load appllied to the ankle joint to create traumatic OA Single intra-articular injection immediately after mechanical induction SO/IHC/WB/RT-qPCR: higher Col II and Aggrecan, lower MMP13 and ELF3 MiR-136-5p promoted chondrocyte migration end ECM secretion via supressing ELF3. He et al., 2020 BMSC isolated from 2-week-old SD rat femurs Low-glucose DMEM +10 % exosome free FBS Ultracentrifugation 10w-male SD rats unilateral intraarticular injection with 8 % sodium iodoacetate Single intra-articular injection one week after OA induction Macroscope: regenerated cartilage HE/SO: Lower OARSI score IHC: higher Col II, lower MMP13 Serum ELISA: lower IL-1β, IL-6, TNF-α, iNOS, CGRP; higher IL-10 Paw withdraw test: less pain sensitivity and hyperalgesia EVs attenuated IL-1β-induced inhibitions on the proliferation and migration of chondrocytes, promoted cell proliferation, reduced apoptosis. Jin et al., 2020 Human BMSCs from ilium of healthy volunteers Human BMSCs complete medium Precipitation (ExoQuick-TC reagent kit) Wistar rats Surgically induced OA One-week intra-articular injection of treatments after surgery HE: lower pathological score IHC: lower MMP3, MMP1 TUNNEL: higher cell viability Serum ELISA: lower IL-1β RT-qPCR: lower PTGS2 expression Human BMSC-derived exosomes transfer miR-26a-5p into synovial fibroblasts to alleviate damage via downregulating PTGS2. Yan and Wu, 2020 Human UC-MSCs 3D culture in FiberCell system (Hollow-fiber bioreactor) Ultracentrifugation New Zealand Rabbits Surgically induced OCD Multiple intra-articular injections at day 0, day 7 and day 14 post-surgery Macroscopy: regenerated cartilage HE/TB/SO/IHC: higher ICRS score, lower Wakitani's score 3D cultured cells secreted more exosomal protein; Exosome treatment promoted chondrocytes viability, migration, reduced apoptosis Zavatti et al., 2020 Pregnant women amniotic fluid stem cell αMEM +20%FBS, 2 mM l -glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin Precipitation (total exosome isolation solution) 8w CD rats nduced OA Intra-articular injection 3 weeks after OA induction (once for Cell and OA groups; twice for Exo group with 10 days interval) PAM–knee joint withdrawal threshold measurement: improved OA symtom HE: lower OARSI score SO: higher composition rate of cartilage Zhang et al., 2020 Rat BMSC from cartilage tissue DMEM/F-12 + 10 % FBS + 1 % P/S Ultrafiltration and ultracentrifugation male SD rats Surgically induced OA Intra-articular injection every 3 days for 4 weeks, starting at week 4 post-operation Exosomes uptake by synovial cells HE/SO: lower OARSI score IHC: more COL II positive cells, less Col X positive cells; reduced macrophage infiltration and promoted M1 to M2 phenotypic change μCT: less osteophytes WB: higher Col II, Sox9; lower Col X, Runx2 Synovial fluid ELISA: reduced IL-1β, IL-6, TNF-α; increased IL-10 Transformation of macrophage polarization maintains chondrogenic characteristics and inhibits hypertrophy of chondrocytes. Zhou et al., 2020 Human BMSCs from polydactyly tissue/human BMSC from bone marrow aspiration LG-DMEM +10 % FBS Precipitation (total exosome isolation kit) 6w-male C57BL/6 J mice Collagenase induced OA Multiple intra-articular injections at day 7, 14 and 21 after collagenase induction HE/SO: lower OARSI score BMP4 expression regulates pBMSC chondrogenic differentiation knockdown of BMP4 in pBMSC exosomes inhibited chondrocyte migration. Yan et al., 2021 Human UC-MSCs cultured in static environment and rotary cell culture system DMEM-F12 + 10 % FBS + 1 % P/S Ultracentrifugation SD rats surgically induced OCD Multiple intra-articular injections on a weekly interval Macoscopy/MRI/T2 mapping: regenerated cartilage, higher ICRS score, higher T2 value HE/TB/SO/IHC(Col II): lower Wakitani's score, higher Col II positive area Leg-withdraw test: less pain sensitivity Mechanical load (rotation) increased exosome production, increased LncRNA H19 expression to enhance chondrocyte proliferation and to inhibit apoptosis. Chen et al., 2019 BMSCs derived from articular cartilage of femoral condyles and tibial plateau of postenatal day 5–6 C57BL/6 mice Serum-free medium Ultrafiltration and ultracentrifugation 6 m-female New Zealand white rabbits surgically induced OCD 3D-printed GelMA/ECM/exosomes scaffold implantation Macroscopic: higher ICRS score MRI: ECM/GelMA scaffold groups with or without exosome exhibited smoother neocartilage and enhanced defect filling HE,SO,Alcian Blue: higher ICRS Visual Histological Scores IHC: lower MMP13, higher COL II in repaired cartilage TEM: reduced mitochondrial vacuoles μCT: higher bone regeneration in the defects Proteomic analysis showed enriched mitochondrial functions in exosomes; Exosome treatment restored mitochondrial mass and mitochondrial DNA content from IL-1β-stimulated degenerative chondrocytes; The scaffold increased chondrocyte migration. Wu et al., 2019 MSC isolated from infrapatellar fat pad and articular cartilage of OA patients DMEM/F12 + 10%FBS and 1 % P/S Precipitation (ExoQuick reagent kit) or ultrafiltration 9w-male C57BL/6 mice Destabilization of MM Multiple intra-articular injections twice a week, starting at week 4 after surgery HE/SO: lower OARSI score IHC: higher Col II, lower ADAMTS5 and MMP13 Catwalk test: improved gait WB: increased authopagy proteins (p65, LC3-I, LC3-II) TEM: increased autophagosome Online-bioinformatic method: predicted microRNAs targeting mTOR RNA Gain-of-function and loss-of-function in vivo experiments demonstrated that of miR-100-5p enhaced autophagy via inhibition of mTOR siganling pathway. Zhang et al., 2019 Immortalized E1-MYC 16.3 human ESC-derived MSCs DMEM +10%FBS Size fractionation and TFF 8w-female SD rats, TMJ—OA MIA injection Multiple intra-articular injections once a week, starting 2 weeks after OA induction HE/TB/IHC(aSMA, MMP13): lower Mankin scores, larger TB positive area, less α-SMA/MMP13 positive cells, less IL-β + and iNOS+ cells, higher PCNA+ cell number, lower CCP3+ cell number μCT: restored bone volume EVs promoted AKT, ERK and AMPK signaling pathways via exosome CD73-mediated adnesine receptor activation. Mao et al., 2018 Human BMSCs from 6 donors (3 males and 3 females, 32–38 years old) α-MEM+ 10 % FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin Ultracentrifugation 6w-female C57B/L10 mice Collagenase-induced OA Intra-articular injections every 3 days for 4 weeks HE/SO/IHC: miR-92a-3p, Col II, Aggrecan upregulate; Wnt5a, MMP13 downregulated MiR-92a-3p directly targets the Wnt5a mRNA to promote chondrocyte proliferation, migration and differentiation. Wang et al., 2018a Commercial rat MSC DMEM +10%FBS, 100 U/ml of penicillin and 100 μg/ml of streptomycin Exosome extraction kit 12w-male SD rats transecting the medial collateral ligament and the medial meniscus Intra-articular injections 100 μl MSC-exosome, 1 × 10 11 MSC-exosome particles Lower OARSI score, higher chondrocytes count TGF-β1 stimulated MSC to secrete exosome derived miR-135b, which down regulated Sp1 expression to promote chondrocyte proliferation. Zhang et al., 2018b Immortalized E1-MYC 16.3 human embryonic stem cell-derived MSCs DMEM +10 % FBS Size fractionation and TFF 8w SD rats surgically induced OCD Weekly intra-articular injections immediately after surgery H&E/SO: Lower Wakitani scores IHC: higher Col II and Col VI stained area, lower Col I stained area more PCNA positive cells (live), less CCP3 positive cells (apoptotic) increased CD163 positive M2 macrophages, decreased CD86 positive M1 macrophage infiltration decreased M1-associated cytokines, IL-1β, and TNF-α MSC exosomes express CD73/ecto-5′-nucleotidase, which converts extracellular AMP to adnosine, and induces AKT/ERK pathway to enhance chondrocyte migration, proliferation and matrix synthesis Cosenza et al., 2017 Mouse BMSC Flat surface culture DMEM, 100 μg/ml penicillin/streptomycin, 2 mmol/ml glutamine and supplemented with 10 % foetal calf serum (FCS) Ultracentrifugation 18,000 g (microparticle) 100,000 g (exosome) 10w C57BL/6 mice Collagenase-induced OA Intra-articular injections of either BM-MSCs (2.5 × 105 cells/5 μl saline), MPs (500 ng/5 μl) or Exos (250 ng/5 μl) at day 7 μCT: less OA change Confocal image 3D reconstructed: higher cartilage volume SO staining: Lower OA score EV treatment increased ACAN, COL1, COL2B expressions, decreased MMP-13, ADAMTS5, iNOS in IL-1β treated chondrocytes from neonatal mice; supressed cell apoptosis, inhibited macrophage differentiation Tao et al., 2017 Human 30 to 35 years old donors SMSCs from knee DMEM/F12 (Hyclone) supplemented with 10 % FBS, penicillin/streptomycin and 250 ng/ml amphotericin B Ultrafiltration and ultracentrifugation 12w-male SD rats transecting the medial collateral ligament and the medial meniscus Intra-articular injection Weekly from 5 to 8 week post-surgery SO & IHC (COL II, Aggrecan, Col I): lower OARSI score, higher number of chondrocytes Wnt5a/b on exosomes activate YAP to enhance chondrocyte proliferation and migration but suppressed the Sox9 expression; miR-140-5p suppressed RalA to enhance Sox9 Wang et al., 2017 Male H1 human ES cell line (ESC) L-DMEM supplemented with 10 % FBS (pre-centrifugated at 110000 g overnight to delete the suspension in FBS) Ultracentrifugation 2 m C57BL/6 mice destabilization of MM surgery Intra-articular injections 4 weeks post-surgery SO & IHC (COL II, ADAMTS5): lower OARSI score EV treatment ncreased Col II positive cells, decreased ADAMTS5 expression in IL-1β treated chondrocytes of neonatal mice Zhu et al., 2017 human iPSC (C1P33)-MSCs vs human SMSC isolated from 22 to 28 years old donors during ACLR surgery iPSC-MSCs: DMEM +10 % FBS, 2 mM l -glutamine, 1 % P/S and 0.1 mM nonessential amino acids SMSC: high-glucose DMEM +10 % FBS and 1 % P/S Ultrafiltration 6w-female C57B/L10 mice Collagenase-induced OA Intra-articular injection of 8 μl iMSC-Exos (1.0 × 1010/ml) or 8 μl SMSC-Exos in PBS (1.0 × 1010/ml) on day 7, 14, 21 Macroscopic: higher ICRS score (iPSC-MSC-Exosome = SMSC-exosome) HE/SO: lower OARSI scores (iPSC-MSC-Exosome < SMSC-exosome) IHC: Col II, Col I EV treatment promoted chondrocyte migration and proliferation (iPSC-MSC-Exosome > SMSC-exosome) Zhang et al., 2016 Human HuES9 human embryonic stem cells (hESCs) N/A tangential flow filtration (TFF) 8w-female SD rats; Surgically induced OCD on both femurs Weekly intra-articular injections of exosomes (100 μg exosomes per 100 μl injection) or PBS control (100 μl) Cartilage regeneration confirmed by HE, SO and IHC (Col II, Col I, Col VI, Col X) Ruiz et al., 2020 Human and mouse BMSCs α-MEM+ 10 % FCS, 100 μg/ml penicillin/streptomycin, 2 mmol/ml glutamine Ultracentrifugation 18,000 g (microparticle) 100,000 g (exosome) 10w C57BL/6 mice Collagenase-induced OA Intra-articular injections of hBMSCs transfected with siCT (control) or siTGFBI at day 7 μCT: less OA change Confocal image 3D reconstructed: higher cartilage volume SO staining: Lower OA score EV treatment increased ACAN, COL1, COL2B expressions, decreased MMP-13, ADAMTS5, iNOS in IL-1β treated chondrocytes from neonatal mice; supressed cell apoptosis, inhibited macrophage differentiation USC: Urine derived Stem Cell; UC-MSC: Umbilical Cord Mesenchymal Stem Cell; BMSC: Bone Marrow-derived Stem/Stromal Cell; AD-MSC: Adipose-derived Mesenchymal Stem Cell; SMSC: Synovial Mesenchymal Stem Cell; iPSC: Induced Pluripotent Stem Cells; ESC: Embryonic Stem Cell; DMEM: Dulbecco's Modified Eagle Medium; αMEM: Minimum Essential Medium α; FBS: Fetal Bovine Serum; P/S: Penicillin and Streptomycin; SD rat: Sprague Dawley Rat; ACL: Anterior Cruciate Ligament; MCL: Medical Collateral Ligament; CFL: Calcaneofibular Ligament Injury; MM: Medial Meniscus; OCD: Osteo-Chondral Defect; MIA: Monosodium Iodoacetate; HE: Hematoxylin and Eosin staining; SO: Safranin-O staining; TB: Toluidine Blue staining; IHC: Immunohistochemistry; ELISA: Enzyme-Linked ImmunoSorbent Assay; OARSI score: Osteoarthritis Research Society International Score; ICRS score: International Cartilage Repair Society Score; Col I: Type I collagen; Col II: Type II collagen; Col X: Type X collagen.

In general, diverse types of donor species (human, rat, mouse, and rabbit) and cell sources [embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), BMSCs, adipose-derived mesenchymal stem cells (AD-MSC), etc.] have been used in preclinical studies. However, there are only a few studies that have made comparisons between species or cell types. For instance, Zhu et al. demonstrated the superior therapeutic efficacy of EVs from human iPSC-derived MSCs to those from human synovial membrane-derived MSCs ( Zhu et al., 2017 ). Fazaeli et al. showed the advantages of BMSC over AD-MSC in treating a ciprofloxacin-induced murine OA model ( Fazaeli et al., 2021 ).

Most of the studies used intra-articular injections for delivery of EVs to specifically target affected joints. Scaffolds are available to either retain EVs in the foci or enhance cell migration ( Liang et al., 2020 ; Zhang et al., 2021a ). All of the identified studies demonstrated the beneficial efficacy of EV treatment in OA models without any side effects, by assessing the histological scores, expression of chondrogenic markers (COLII, Aggrecan, SOX9), matrix degradation markers (MMP3, MMP13, ADAMTS5), hypertrophic markers (COLX), inflammation markers (TNFα, IL1β, IL6, IL10), osteogenic markers (RUNX2, Osteocalcin, COLI), μCT analysis, and serum or synovial fluid ELISA for cytokines. However, the molecular mechanisms underlying these therapeutic actions have not been sufficiently disclosed.

Although it is still controversial whether miRNAs exist in EVs enough to modify the function of target cells ( Albanese et al., 2021 ; Chevillet et al., 2014 ; Qiu et al., 2019 ; Toh et al., 2018 ), miRNAs have been demonstrated to conceivably play an important role in this therapeutic action. To identify key miRNAs and their downstream pathways, miRNAs have been overexpressed or inhibited in EVs using genetic approaches. For example, miR-140 has been suggested to promote chondrogenesis ( Liang et al., 2020 ; Tao et al., 2017 ) by suppressing the expression of the VEGFA gene which is known to contribute to the OA progression ( Liu et al., 2022 ). MiR-136-5p promotes chondrocyte migration and extracellular matrix secretion via suppressing E74-like factor 3 (Elf3) ( Chen et al., 2020 ). MiR-26a-5p alleviates the damage of synovial fibroblasts by downregulating prostaglandin-endoperoxide synthase 2 (PTGS2), leading to a repressive effect on the degradation of cartilage in OA ( Jin et al., 2020 ). EVs containing miR-326 delivered to chondrocytes inhibit pyroptosis of cartilage by targeting HDAC3 and STAT1/NF-κB p65 ( Xu and Xu, 2021 ). MiR-125a-5p ( Xia et al., 2021 ) and miR-206 ( Huang et al., 2021 ) are shown to target Elf3 to promote both chondrocyte and osteocyte proliferation and differentiation. Although promising findings have been obtained in recent studies, many of the above studies utilized ultracentrifuge to isolate EVs ( Table 1 ). Thus, miRNAs bound to carriers other than EVs such as lipoproteins and other protein complexes could be included and the entire picture of beneficial miRNAs within EVs and their molecular regulatory mechanisms is still unclear.

On the other hand, there are a limited number of studies that show the therapeutic effects of proteins within MSC-EVs on OA. Similar to MSCs, MSC-EVs have immunomodulatory properties and inhibit the expression of matrix degradation markers and inflammation markers in chondrocytes and inflammatory cells ( Cosenza et al., 2017 ). It was suggested that TGFBI mRNA and protein in EVs played an important role in this anti-cartilage degradation action; however, the details remain unknown ( Ruiz et al., 2020 ). Interestingly, proteome analysis of EVs from antler stem cells demonstrated that EVs contain “rejuvenation” factors that can revive senescent chondrocytes and alleviate OA ( Lei et al., 2022 ).

These findings suggest that various cargo in MSC-EVs contributes to the therapeutic action in OA; however, further examination is required to fully understand the mechanisms of action of MSC-EVs.

Rheumatoid arthritis (RA)

RA is an autoimmune disease in which chronic synovitis causes bone and cartilage destruction as the disease progresses. This progressive disease causes significant joint destruction, deformity, and pain that affects daily activities if not appropriately managed. Differently from OA, RA is a systematic disease that involves multiple joints, including knee, ankle, shoulder, wrist, and digital joints ( Firestein, 2003 ; Goronzy and Weyand, 2009 ). Therefore, RA therapies need to be systemic to modulate the autoimmune response, which is represented by cytokine-directed therapies such as anti-TNF-α and anti-IL-6 among others. However, these biological drugs are expensive and occasionally cause severe side effects. Thus, novel approaches like stem cell-based therapies are being actively investigated as therapeutic options. While clinical trials of MSC therapies have been conducted for patients with RA, no clinical trials of EV therapy have been listed in Clinicaltrials.gov and only preclinical studies have been performed thus far ( Table 2 ). Table 2 Summary of the cell origins, culture methods, isolation methods, delivery methods, major in vivo outcomes, and the underlying mechanisms/cargos in representative studies using MSC-EVs for RA. Table 2 References Donor/Cell origin Culture media/method Isolation Animal Models Delivery methods Outcomes and proposed mechanism Huang et al., 2022a Human UC-MSCs DMEM/F12 + 12%FBS ExoQuick TC exosome extraction reagent Wistar rats Collagen-induced arthritis model Weekly injection Serum ELISA: lower TNF-α, IL-1β and malondialdehyde; higher superoxide dismutase activity HE/Masson: better histological score, fibrosis score Exosomal miR-140-3p targets SGK1 to inhibit chondrocyte apoptosis, promoted synovial fibroblasts apoptosis Ma et al., 2022 Murine BMSCs (chondrogenic differentiated) DMEM +15 % FBS, 100 U·mL-1 penicillin, 100 μg·mL-1 streptomycin and 2.5 mmol·L-1 l -glutamine for 14 days Chondrogenic medium: high glucose DMEM, 1 × GlutaMax, 100 nM dexamethasone, 200 μM ascorbic acid 2-phosphate, 100 μM sodium pyruvate, 40 μg/ml L-proline, 1 % ITS-X and 100 U/ml PenStrep and 10 ng/ml TGF-β1 Ultracentrifugation C57BL/6 mice Collagen-induced arthritis model Intradermally injected twice per week for 14 days Inflammation score; HE: less leucocyte infiltration in the joints Serum ELISA: lower IL-1β, IL-6 and TNF-α EV treatment inhibited cytokine expression in fibroblast-like synoviocytes through both MAPK and NF-κB signaling pathways; MiR-205-5p from chondrogenic BMSCs exosomes bind to MDM2 mRNA, to attenuate inflammatory response in joints Huang et al., 2022b Rat BMSCs DMEM+10%FBS + 100 u/ml P/S Total Exosome Isolation Kit SD rats Freund's Adjuvant injection N/A HE: less tissue damage and inflammatory cell infiltration Less paw swelling Synovial tissue ELISA: lower IL-1β MiR-223 in BMSCs-secreted exosomes delays the inflammatory responses by targeting NLRP3. Tian et al., 2022 Human gingival MSCs DMEM+10%FBS + 100 μg/ml penicillin and 100 μg/ml streptomycin, 100 μM MEM non-essential amino acids, 550 μM 2-M, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM l -glutamine Ultracentrifugation Mice Collagen-induced arthritis model Single injection via tail vein at the day of boost Better arthritis score Lower paw thickness Less incidence of arthritis HE:less inflammatory cell infiltration μCT: less bone erosion IHC: lower NF-κB p65/p105 expression Flow cytometry of spleen and lymph nodes: decreased the percentages of CD4 + INF-γ + Th1 and CD4 + IL-17A+ Th17 cells, increased the percentages of CD4 + CD25 + FoxP3+ Tregs Serum ELISA: lower IFN-γ, IL-17A, TNF-α, IL-6; higher IL-10 GMSC-derived EVs inhibited IL-17A and promoted IL-10, reducing incidences and bone erosion of arthritis, via inhibiting IL-17RA-Act1-TRAF6-NF-κB signal pathway, increasing regulatory T cell population Duan et al., 2021 Human SMSCs DMEM+10%FBS + 1%P/S, pretreated with LPS Ultracentrifugation C57BL/6 mice Destabilization of MM Intra-articular injection twice a week SO: lower OARSI score IHC: higher Aggrecan, Col II; lower ADAMTS5 Let-7b directly targeted ADAMTS5. Zhang et al., 2021b Human SMSCs High-glucose DMEM+10%FBS Ultracentrifugation DBA/1 J mice Collagen-induced arthritis model Intra-articular injections twice a week lasted from day 26–39 after the first immunization Lower arthritis score μCT: less bone destruction HE: better histological score IHC: lower p-STAT3/STAT3 value, VEGF expression circRNA(hsa_circ_0073244, circEDIL3) targeted miR-485-3p, which suppressed the PIAS3/STAT3/VEGF axis. Chang and Kan, 2021 Human synovial cell line MH7A and human BMSCs MEM + 10%FBS + 1%P/S Ultracentrifugation Lewis rats heat-killed bacteria-induced arthritis model Injection Better sensitivity score, RA score Lower TNF-α, IL-1β, IL-6, and IL-8 by ELISA CircFBXW7 directly sponged miR-216a-3p to upregulate the expression of HDAC4. Li et al., 2021 Murine BMSCs DMEM+10%FBS (EV depleted) Ultracentrifugation BALB/c mice Collagen-induced arthritis model Intra-articular inoculation twice a week Better clinical RA score HE: better histological score μCT: higher BMD Plasma ELISA: lower TRAP activity IHC: lower TRAP staining ELISA of synovial tissue and serum: lower TNF-α, IL-1β, and PGE2 MiR-21 targets TET1 which promotes KLF4 transcription in fibroblast-like synoviovytes. Cosenza et al., 2018 Murine BMSCs DMEM, 100 μg/ml penicillin/streptomycin, 2 mM glutamine and supplemented with 10 % foetal calf serum (FCS) Primed with IFN-γ Ultracentrifugation DBA/1 mice Collagen-induced arthritis model (cuneiform bone) Intravenouse injection at day 18 and 24 post-induction Less paw swelling, lower global clinical score; less CD4 + IL-10+ Tr1 cells in lymph nodes; significant decrease in plasmablast percentage and increase in CD19 + IL-10+ Breg-like populations; downregulated IL-6 and IL-1β in culture supernatants of lymph node cells; IL-10 upregulated when lymph node cells were stimulated with Col II; type II collagen-specific IgG2a/IgG1 antibodies ratios were significantly decreased in serum μCT: higher thickness of cuneiform bone, lower bone degradation area in cuneiform bone Exosomes being more potent than microparticles to suppress arthritis. In vitro findings suggested that MPs and Exos exerted an indirect inhibitory effect on T lymphocyte proliferation through Tr1 and Treg induction; in vivo findings suggested a shift from a Th1-type toward a Th2-type immune response in Exos-treated mice, which was likely due to decreased plasmablast differentiation and generation of Breg cells Casado et al., 2017 Porcine BMSCs DMEM +10%FBS + 5 μl/ml fungizone+1%P/S Ultrafiltration White pig Sequential subcutaenous injection of BSA for immunization followed by intra-articular injection of BSA Intra-articular injection with BSA Synovial fluid assay: less white blood cells and normal fraction, lower TNF-α Kinetic Gait Parameters: normal gait in treatment group UC-MSC: Umbilical Cord Mesenchymal Stem Cell; BMSC: Bone Marrow-derived Stem/Stromal Cell; SMSC: Synovial Mesenchymal Stem Cell; GMSC: Gingival Mesenchymal Stem Cell; DMEM: Dulbecco's Modified Eagle Medium; FBS: Fetal Bovine Serum; P/S: Penicillin and Streptomycin; SD rat: Sprague Dawley Rat; BSA: Bovine Serum Albumin; TRAP: Tartrate-Resistant Acid Phosphatase; Col I: Type I collagen; Col II: Type II collagen; HE: Hematoxylin and Eosin staining; SO: Safranin-O staining; TB: Toluidine Blue staining; IHC: Immunohistochemistry; ELISA: Enzyme-Linked ImmunoSorbent Assay; OARSI score: Osteoarthritis Research Society International Score; BMD: Bone Mineral Density.

Although both OA and RA target articular joints, RA treatments, which need to treat multiple joints simultaneously, are quite different from those for OA. Therefore, most of the studies for RA use intravenous systemic administration instead of intra-articular injections. Human gingival MSCs ( Tian et al., 2022 ) and articular synovial MSCs ( Duan et al., 2021 ; Zhang et al., 2021b ) have been preferentially used with the expectation that they can efficiently target inflammatory synoviocytes. Vanniashinhe et al. used small artificial vehicles (targeted liposomes loaded with anti-arthritic medications) to mimic the EV-cargo system. They successfully treated collagen-induced arthritis by direct binding of these targeted liposomes to synovial fibroblast like cells ( Vanniasinghe et al., 2014 ). Moreover, several studies took advantage of immune cell-derived EVs to efficiently target inflammatory joints, such as EVs derived from M2 macrophages ( Da-wa et al., 2021 ), neutrophils ( Zhang et al., 2022 ; Headland et al., 2015 ), and dendritic cell ( Lee et al., 2021 ).

The evaluation of the therapeutic outcomes in RA is also distinct from OA. Due to its global inflammatory nature, fractionation of immune cells in serum or spleen by flow cytometry, cytokine levels in serum and synovial tissues by ELISA or immunohistochemistry, global clinical score, and the incidence of symptomatic joints are frequently used in studies. In addition, bone destruction is also a feature of RA-affected joints, therefore histological assessment or micro-CT are powerful tools for its detection.

The immunomodulatory properties of MSC-derived EVs have been extensively studied as a potential mechanism of therapeutic effects ( Burrello et al., 2016 ; Gomzikova et al., 2019 ; Robbins and Morelli, 2014 ). In vitro studies showed that MSC-EVs can modulate T cell function by inhibiting CD4+ and CD8+ T cell differentiation ( Blazquez et al., 2014 ; Ji et al., 2019 ), mediating the Th1 to Th2 cell conversion ( Chen et al., 2016 ), and polarizing CD4+ T cells into regulatory T cells ( Zhang et al., 2018a ). The proliferation and differentiation of B cells and natural killer cells can also be modulated by MSC-EVs ( Di Trapani et al., 2016 ; Martínez et al., 2012 ). Furthermore, MSC-EVs can elicit the switch from an M1 to an M2 phenotype in macrophages ( Henao Agudelo et al., 2017 ; Lo Sicco et al., 2017 ). Several cytokines expressed in RA, such as IL-1β, IL-6, and TNFα are suppressed by MSC-EVs, whereas anti-inflammatory IL-10 and TGF-β1 were enhanced in monocytic cells ( Chen et al., 2016 ).

Recent comprehensive in vitro and in vivo studies identified several key miRNAs underlying the versatile functions of EVs. MiRNA let-7b can directly target ADAMTS5 to reduce its expression and secretion ( Duan et al., 2021 ). MiR-140-3p is shown to target serum- and glucocorticoid-inducible kinase 1 (SGK1) to inhibit chondrocyte apoptosis and promote synovial fibroblast apoptosis ( Huang et al., 2022a ). MiR-205-5p from chondrogenic BMSC-EVs binds to mouse double minute 2 (MDM2) mRNA and attenuates inflammatory response in joints ( Ma et al., 2022 ). MiR-223 in BMSCs-secreted exosomes delays the inflammatory responses by targeting NLR family pyrin domain containing 3 (NLRP3) ( Huang et al., 2022b ).

Osteoporosis

Bone is an organ exhibiting dynamic metabolic activity. Bone formation and bone resorption are strictly regulated by osteoblasts, osteoclasts, and osteocytes to maintain overall bone quality and quantity. However, situations like aging and menopause cause an imbalance in bone metabolism, resulting in osteoporosis. The decline in quality of life associated with osteoporosis has become a major social and medical problem in the global aging society. The interaction between bone cells (osteoblast, osteoclast, and osteocyte) is complex and not fully elucidated. Numerous therapeutic agents have been developed, and many of them exhibit prominent efficacy. These therapeutic agents target one of those bone cells; however, it seems that targeting a single cell type is not sufficient to correct the imbalance of bone metabolism ( Ukon et al., 2019 ). Given that MSC-EV therapy as well as MSC therapy can target multiple cell types simultaneously, the efficacy of EVs is being actively investigated in well-established preclinical models ( Table 3 ). Table 3 Summary of the cell origins, culture methods, isolation methods, delivery methods, major in vivo outcomes, and the underlying mechanisms/cargos in representative studies using MSC-EVs for osteoporosis. Table 3 References Donor/Cell origin Culture media/method Isolation Animal Models Delivery methods Outcomes and proposed mechanisms Hu et al., 2022 Human AD-MSC DMEM/F12 + 15%Exo-free human serum Ultracentrifugation BALB/c proteoglycan-induced ankylosing spondylitis (PGIA) model Weekly intraspinal injection μCT: higher vertebral BMD TRAP staining: less osteoclasts MiR-21 inhibited osteoclast activation and decreased IL-6 expression in mice spine. Cui et al., 2022 Human iPSC derived MSCs MSC culture medium Ultracentrifugation C57BL/6 Ovariectomy Intravenously injected once a week for 6 weeks through the tail vein μCT: higher trabecular bone mass, higher mineral apposition rate IHC: more type H vessel formation TRAP staining: less osteoclasts Serum ELISA: higher SLIT3 and PINP; lower RANKL, TRAP5b, β-CTX Osteoblasts could take up bone-targeting-exosomes in a periostin-dependent manner. EV Loaded siShn3 was directly delivered to osteoblasts, and promoted osteogenesis and angiogenesis, but suppressed osteoclast activation. Zhang et al., 2021d Human BMSCs DMEM/F12 + 10%FBS + 0.2%P/S Ultracentrifugation SD rats Ovariectomy Twice a week via intracavitary injections μCT: higher trabecular bone mass MiR-935 inhibits STAT1 expression and promotes osteoblast proliferation and differentiation. Zhang et al., 2021c Rat AD-MSCs N/A Ultracentrifugation SD rats high glucose exposure and streptozotocin injection Intravenouse injection via tail vein every two days μCT: higher trabecular bone mass Serum ELISA: lower Cathepsin, TRACP-5b level AD-MSCs-derived exosomes suppressed NLRP3 inflammasome activation in osteoclasts. Yahao and Xinjia, 2021 Human UC-MSC Normal Evs (EV1): DMEM/F12 + exosome-free 10%FBS Osteogenic Evs (EV2): DMEM/F12 + 10 % exosome-free FBS, with 50 M vitamin C, 10 mM beta-phosphoglycerol, and 0.1 M dexamethasone Ultracentrifugation C57BL/7 J mice Ovariectomy Intraperitoneally injected every 3 days for 6 weeks HE: denser cancellous bone in tibia μCT: increased bone mass in trabecular area Hsa-mir-2110 and hsa-mir-328-3p gradually increased with prolonged osteogenic differentiation. RNAseq data showed that exosomal miRNAs derived from osteogenic induced-MSCs are involved in bone development and differentiation, such as osteoclast differentiation and the MAPK signaling pathway. Sadat-Ali et al., 2021 Autologous BMSCs Autologous BMSCs derived osteoblasts Autologous BMSCs derived osteoblasts-EVs N/A N/A Rabbits Ovariectomy Injected in the saphenous vein HR-pQCT: Osteoblast treatment showed the highest bone mass Yang et al., 2020 Human UC-MSC a-MEM + 10 % exosome-free-FBS + 100 mg/ml P/S Ultracentrifugation SD rats Tail suspension Intramuscular injections HE/μCT: reversed osteoporotic changes Exosomal miR-1263 could bind to the Mob1 to activate YAP expression (miR-1263/Mob1/Hippo signaling pathway). Hu et al., 2020b Human UC-MSC DMEM/F12 + 10%FBS + 1 % GlutaMAX+100 U/ml penicillin and 100 μg/ml streptomycin Ultrafiltration C57BL/6 mice Ovariectomy Intravenously administrated via the tail vein twice a week for 21 days μCT: higher bone mass Three-point-bending test: higher bone strength HE/IHC: less adipocytes, higher mineral apposition rate, more osteoblasts, less osteoclasts Serum ELISA: lower CTX-I Proteomic analysis: highly expressed CLEC11A CLEC11A (C-type lectin domain family 11, member A) delivered by EVs enhanced the shift from adipogenic to osteogenic differentiation of BMSC and inhibited osteoclastogenesis Yang et al., 2019 Human BMSCs DMEM/F12 + 10%FBS Ultracentrifugation+ultrafiltration BALB/c mice Ovariectomy Periosteal injection into the marrow cavity of the femur twice per week μCT: higher bone mass MSCs-derived exosomal MALAT1 enhanced osteoblast activity in osteoporotic mice by mediating the miR-34c/SATB2 axis. UC-MSC: Umbilical Cord Mesenchymal Stem Cell; BMSC: Bone Marrow-derived Stem/Stromal Cell; AD-MSC: Adipose-derived Mesenchymal Stem Cell; iPSC: Induced Pluripotent Stem Cells; DMEM: Dulbecco's Modified Eagle Medium; αMEM: Minimum Essential Medium α; FBS: Fetal Bovine Serum; P/S: Penicillin and Streptomycin; SD rat: Sprague Dawley Rat; BMD: Bone Mineral Density; TRAP: Tartrate-Resistant Acid Phosphatase; HE: Hematoxylin and Eosin staining; IHC: Immunohistochemistry; ELISA: Enzyme-Linked ImmunoSorbent Assay; HR-pQCT: High-Resolution peripheral Quantitative Computed Tomography.

Ovariectomy is a widely used, reproducible post-menopausal osteoporotic model that can be applied to various species including mice ( Cui et al., 2022 ; Hu et al., 2020b ; Li et al., 2016 ; Yahao and Xinjia, 2021 ; Yang et al., 2019 ; Yu et al., 2021 ), rats ( Zhang et al., 2021d ), and rabbits ( Sadat-Ali et al., 2021 ). Other interesting models such as a fatigue loading model ( Xun et al., 2021 ), a disuse model by tail-suspension ( Yang et al., 2020 ), a proteoglycan-induced ankylosing spondylitis model ( Hu et al., 2022 ), and an LPS-induced osteolysis model ( Ren et al., 2022 ) have also been identified. In general, dysfunction of osteoblasts and/or over-activation of osteoclasts are observed in these models, causing decreased bone mass and bone strength. Therefore, most of the studies targeted these cell types using the cargo in EVs.

MSC-EVs from various parental sources have been examined. Interestingly, cells from younger animals (humans) were frequently used in studies and exhibited promising results in this aging-associated disease. Human umbilical cord MSCs are one of the most investigated cell types among treatments for osteoporosis ( Hu et al., 2020b ; Yahao and Xinjia, 2021 ; Yang et al., 2020 ). Yang et al. have demonstrated that miR-1263 contained in EVs from human umbilical cord MSCs prevents apoptosis by inhibiting the Hippo pathway, leading to the elimination of bone loss ( Yang et al., 2020 ). EVs from human umbilical cord MSCs have also been shown to contain miR-328-3p and miR-2110 which are known to enhance osteogenesis ( Yahao and Xinjia, 2021 ).

Human BMSC-derived EVs contain miR-935 which can directly inhibit STAT1 expression to promote osteoblast proliferation and differentiation ( Zhang et al., 2021d ). Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in human BMSC-derived EVs enhanced osteoblast activity in osteoporotic mice by mediating the miR-34c/Special AT-rich sequence-binding protein 2 (SATB2) axis ( Yang et al., 2019 ). In osteoclasts, miR-21 from human AD-MSCs inhibited the activation of osteoclasts and decreased the expression of IL-6 ( Hu et al., 2022 ). Rat BMSC-derived EVs suppress NLRP3 inflammasome activation in osteoclasts ( Zhang et al., 2021c ). Recent in vitro studies have suggested some cargo exists with negative roles in osteogenesis. MiR-424-5p from BMSC-derived EVs hindered osteoblast differentiation via binding to Wnt inhibitor-1 (WIF1) to suppress Wnt/β-catenin axis ( Wei et al., 2021a ); Ad-MSC derived EVs deliver lncRNA-KCNQ1OT1, which promoted osteoblast apoptosis ( Wang et al., 2021 ). Downregulating these molecules rescued the phenotypes, suggesting that they are potential therapeutic targets for osteoporosis. Numerous cargos targeting osteoporosis have been investigated in recent literature and are well summarized in other reviews ( Li et al., 2022 ; Yun et al., 2021 ).

Summary and future direction

MSC-EV therapy has the potential to become a beneficial treatment not only for bone/cartilage diseases but also for diseases involving other tissues. However, there are challenges, such as reproducibility and scalability of the production of EVs, that need to be overcome to make it a standard treatment. Recent developments in cell culture technologies enable us to rapidly expand EV production, although reproducing EVs remains an obstacle. As described here, various elements including but not limited to “Cell sources”, “Donor differences”, “Culture conditions”, “Separation methods”, and “Storage” affect the quality of EVs. To make MSC-EV therapy more consistent and reproducible, these elements should be considered when studies are performed. Additionally, it is required to develop quick and easy assays to test the identity and therapeutic potency of EVs for quality control. Although the detailed mechanisms of action by MSC-EVs remain unknown, assays based on the biological activity related to the therapeutic effects of MSC-EVs will enable us to select potent EVs from the EV banking system for treatment ( Gimona et al., 2021 ; Witwer et al., 2019 ). Once developed, these assays will accelerate the clinical translation of MSC-EV therapy.

Currently, numerous efforts are being made to elucidate the mechanisms underlying the therapeutic actions of EVs in various diseases. Once the key molecules necessary for therapeutic activities are identified, EVs can be engineered to contain a sufficient amount of the molecules. Artificially synthesized targeted nanoparticles that contain only the necessary molecules may be developed as the next generation cell-free therapy in the future although further elucidation of the mechanisms of EV therapy will be required ( Esmaeili et al., 2022 ; Johnson et al., 2021 ; Watson et al., 2016 ).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH, R21AR077654).

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Data availability

No data was used for the research described in the article.