Therapeutic potential of mesenchymal stem cell-derived exosomes in skeletal diseases

Skeletal diseases impose a considerable burden on society. The clinical and tissue-engineering therapies applied to alleviate such diseases frequently result in complications and are inadequately effective. Research has shifted from conventional therapies based on mesenchymal stem cells (MSCs) to exosomes derived from MSCs. Exosomes are natural nanocarriers of endogenous DNA, RNA, proteins, and lipids and have a low immune clearance rate and good barrier penetration and allow targeted delivery of therapeutics. MSC-derived exosomes (MSC-exosomes) have the characteristics of both MSCs and exosomes, and so they can have both immunosuppressive and tissue-regenerative effects. Despite advances in our knowledge of MSC-exosomes, their regulatory mechanisms and functionalities are unclear. Here we review the therapeutic potential of MSC-exosomes for skeletal diseases.


Introduction
The prevalences of musculoskeletal ailments are increasing with the average life expectancy (Pourakbari et al., 2019;Malekpour et al., 2022).Around 1.71 billion people globally have musculoskeletal conditions in 2019, necessitating increasingly complex treatment modalities (Cieza et al., 2021).Osteoarthritis (OA), osteoporosis (OP), intervertebral disc degeneration (IDD), fracture, bone defects, and rheumatoid arthritis (RA) are among the most prevalent skeletal diseases; these not only impose a considerable financial burden on patients but also diminish their overall wellbeing (Li et al., 2018;He et al., 2021;Ding et al., 2023;Torrecillas-Baena et al., 2023).A systematic review pointed out that the global prevalence of OP was reported to be over 23.1% in women (Salari et al., 2021).In China, about 20.6% of females above 40 years old were suffering from OP (Wang et al., 2021).For OA, the mean cost of working-age OA patients is reported to be $14,521 per year.Moreover, the estimated prevalence in adult population is up to 26% by 2040 (Lo et al., 2021).Research in tissue bioengineering has led to the development of liposomes, dendrimers, micelles, and inorganic nanoparticles, which augment the effectiveness of drugs while mitigating their systemic toxicity.However, the clinical application of these agents is hampered by their cytotoxicity and poor biodegradability.Therefore, it is important to develop novel therapeutics that can overcome the above limitations and ameliorate skeletal diseases.
Mesenchymal stem cells (MSCs) have therapeutic potential for skeletal diseases (Wang et al., 2022;Yu et al., 2022;Torrecillas-Baena et al., 2023).The multipotent characteristics of MSCs enable them to undergo self-renewal and differentiate into multiple lineages.Furthermore, they migrate toward injured areas and secrete growth factors, thereby facilitating wound healing (Gholami Farashah et al., 2022;Malekpour et al., 2022).MSC transplantation therapy (MSCT) shows considerable promise for the treatment of diverse ailments.However, the hypopermeability and low blood circulation of bone impedes the application of MSCT for skeletal diseases (Shang et al., 2021).In addition, the survival of engrafted MSCs is poor, and the effects of MSCT are characterized by the paracrine release of cytokines, and exosomes rather than the direct actions of the cells themselves (Vitha et al., 2019;Wang et al., 2022).
Exosomes, characterized as scale extracellular vesicles with diameters ranging from approximately 30-150 nm (Kubiatowicz et al., 2022), are released by various cellular entities.They transport biologically active endogenous and exogenous factors such as nucleic acids, proteins, lipids, oligonucleotides, therapeutic RNAs, and small molecules, thereby modulating physiological and pathological processes (Shang et al., 2021;Gholami Farashah et al., 2022).As natural nanocarriers, they transport endogenous factors and have a low immune-clearance rate, good barrier penetration, and allow targeted delivery of therapeutics.MSC-derived exosomes (MSC-exosomes) modulate the functionality of recipient cells by conveying information in the form of constituents of MSCs (Tan et al., 2020;Torrecillas-Baena et al., 2023).Bone marrow-derived MSCs are typically used to produce MSC-exosomes.Compared to MSCT, therapies based on MSC-exosomes have enhanced safety and more convenient storage, transportation, and administration.Consequently, the clinical potential of MSC-exosomes warrants further investigation.The isolation, engineering, cargo loading, and boosting of exosomes have been investigated (Zhang et al., 2019;Wang et al., 2023).This review primarily concentrates on the recent advancements in the utilization of MSC-exosomes for the therapeutic intervention of skeletal disorders including OP, OA, RA, fracture, and IDD.In addition, we discuss barriers to their clinical application (Figure 1).

The biogenesis and composition of exosomes
multivesicular bodies (MVBs) that harbor intraluminal vesicles (ILVs) (Wang and Thomsen, 2021;Wang et al., 2022).The initial plasma membrane invagination gives rise to a structure that contains cell-surface and extracellular proteins, leading to the de novo creation of early sorting endosomes (ESEs), which subsequently become late-sorting endosomes (LSEs) and ultimately generate MVBs.The endoplasmic reticulum also plays a role in the synthesis and content of ESEs.MVBs can undergo fusion with lysosomes, leading to their degradation, resulting in the release of enclosed ILVs as exosomes (Vig and Fernandes, 2022).There are different up-take mechanisms, including fusion, internalization by endocytosis, phagocytosis, etc. (Figures 1, 2).
Exosomes are present in diverse bodily fluids, including blood, saliva, amniotic fluid, hydrocephalus, and urine et al., and serve as vehicles for intercellular information exchange (Behera and Tyagi, 2018;Huang et al., 2022).The markers of exosomes include CD81, CD9, CD63, tumor susceptibility gene 101 (TSG101), heat shock protein 70/90 (HSP70/90), major histocompatibility complex (MHC), and lysosomal-associated membrane protein 1 (LAMP1) et al., which varies among different MSC types.In addition, the cargo of exosomes also shows significant differences, in terms of quantity and diversity, according to the originate cells from which they are derived (Malekpour et al., 2022;Kushioka et al., 2023).Upon binding to recipient cells, exosomes unload their cargo into these cells, thereby mediating intercellular signaling and material exchange, and ultimately modulating the functionality of the recipient cells (Zeng and Xie, 2022).Exosomes, including MSCexosomes, markedly influence the immune response and inflammation (Huang et al., 2022).The membranes of MSCexosomes protect their contents, and naturally occurring or artificially altered biomacromolecules on the exosomal surface facilitate the identification of target cells or tissues.Overall, the diagnostic and therapeutic potential are achieved profoundly via substantial transportation of bioactive agents within MSC-exosomes (Bellavia et al., 2018;Li et al., 2018) (Figure 2).

Therapeutic application of MSC-exosomes for skeletal diseases MSC-exosomes in OA
OA, the most prevalent chronic disease of the joints, affects a substantial proportion of individuals ≥50 years of age.Given the aging population and the escalating rate of obesity, it is anticipated that its incidence will double over the next three decades (Bao and He, 2021;Cheng et al., 2022).OA is distinguished by the degeneration of cartilage, thickening of subchondral bone, and the development of osteophytes (Chen et al., 2023;Zou et al., 2023).Most interventions aim to manage pain, stiffness, and swelling, and arthroplasty is the only option for late-stage OA.Regrettably, despite the temporary relief provided by physical or drug therapy, restoration of joint function is challenging (Duan et al., 2020;Foo et al., 2021;Ghafouri-Fard et al., 2021).Most research on the clinical potential of MSC-exosomes has been on OA (Yuan et al., 2022;Zeng and Xie, 2022;Zhang et al., 2022;Chen et al., 2023) (Table 1; Figure 3).Exosomes derived from bone marrow MSCs and those from adipose stem cells (ADSCs), infrapatellar fat pad (Wu et al., 2019), human umbilical cord (Li et al., 2022;Cao et al., 2023), synovial fluid (Xu et al., 2021), embryonic stem cells (Wang et al., 2017), and synovial membrane (Tao et al., 2017) have been used to generate exosomes for the treatment of OA.Among these sources, ADSCs and bone marrow-derived MSCs are relatively easy to obtain, which may be conducive to future therapeutical applications.Cosenza et al. showed that MSC-exosomes and MSC-derived microparticles have similar in vitro and in vivo chondroprotective effects in OA, thus reproducing the main therapeutic effects of bone marrow MSCs (Cosenza et al., 2017).MSC-exosomes protect cartilage and bone by inhibiting catabolic and inflammatory cytokines, suppressing macrophage activation, and preventing chondrocyte apoptosis (Huang et al., 2022;Kwon et al., 2022).Zhu et al. compared induced MSC (iMSC) and synovial MSC (SMSC) exosomes in mice OA model and found that the former had the greatest therapeutic effect in OA.The OARSI score of OA samples was significantly decreased after iMSC-exosomes treatment (Zhu et al., 2017).
Several OA studies have combined bioengineering approaches, typically biomaterials, with MSC-exosomes.Zeng Overview of the therapeutic application of MSC-exosomes in OA, including the cell sources of MSC-exosomes, the active components/cargos, and the possible mechanism/pathways during the alleviation of OA using MSC-exosomes.Up-arrow (red), upregulation; down-arrow (green), downregulation.2023).Others have reported similar findings (Zhang et al., 2022;Pang et al., 2023), suggesting that combinations of novel biomaterials and MSC-exosomes have potential as cell-free therapeutics for OA.

MSC-exosomes in OP
Under normal physiological conditions, the equilibrium between bone resorption and bone formation preserves the integrity and quality of bone tissue.This delicate balance is Overview of the therapeutic application of MSC-exosomes in OP, including the cell sources of MSC-exosomes, the active components/cargos, and the possible mechanism/pathways during the alleviation of OP using MSC-exosomes.Up-arrow (red), upregulation; down-arrow (green), downregulation.Overview of the therapeutic application of MSC-exosomes in RA, including the cell sources of MSC-exosomes, the active components/cargos, and the possible mechanism/pathways during the alleviation of RA using MSC-exosomes.Up-arrow (red), upregulation; down-arrow (green), downregulation.disturbed in a number of bone disorders.OP, one of the most prevalent skeletal disorders worldwide, disproportionately affects the elderly, particularly women (Tan et al., 2020;Xie et al., 2020).Hormonal, nutritional, behavioral, and genetic factors may contribute to its development and progression, although aging and deficiencies in estrogen are the primary causes.Perturbations in bone metabolism, such as an imbalance in the activities of osteoclasts (OCs) and osteoblasts (OBs), are also implicated in its pathogenesis.During recovery from OP, OBs secrete osteoids to facilitate bone regeneration (Li et al., 2018;Yang et al., 2022).Currently available treatments (including anti-resorptive and anabolic drugs) control OP by promoting bone formation, impeding adipocyte development, or inhibiting OC differentiation.However, the potential adverse effects, which include fever, nausea, bone pain, and cancer, are nonnegligible (Duan and Guan, 2021;Li et al., 2022;Huo et al., 2023).

MSC-exosomes in RA
RA is a chronic autoimmune disease that affects up to 2.5% of the population in each country, causing cartilage destruction and bone erosion (You et al., 2021).Current treatments focus on suppressing inflammation, but side effects like bone loss and long-term toxicities remain a challenge (Xu et al., 2022).Defective immune regulation leads to autoreactive T and B lymphocytes activation and differentiation, leading to the produce of autoreactive antibodies, activation of inflammatory responses and cartilage degeneration (Cosenza et al., 2017;Chang et al., 2022;Heydari et al., 2023).MSC-exosomes have not only antiinflammatory but also immunomodulatory effects, which is why they help mitigate joint destruction (Heydari et al., 2023;Zhao et al., 2023).

MSC-exosomes in fracture and bonedefect healing
Fractures and bone defects are common musculoskeletal issues, and approximately 5%-10% of patients experience delayed union or nonunion as a result of inadequate bone regeneration (He et al., 2021;Smolinska et al., 2023).Bone regeneration involves various cell types, including OBs, OCs, endothelial cells, chondrocytes, and MSCs.Bone regeneration can be mediated by intramembranous ossification or endochondral ossification.MSC-derived OBs directly contribute to the calcification of bone via intramembranous osteogenesis, whereas endochondral ossification is an intricate process regulated by different cells, including chondrocytes.MSCs promote bone regeneration, an effect mediated by several key factors, including exosomes (Liu et al., 2020;Huang et al., 2022;Smolinska et al., 2023).Indeed, MSC-exosomes promote the repair of fractures (Table 4) and bone defects (Table 5).
Furuta et al. investigated MSC-exosomes in a CD9 −/− mouse femur fracture model and found that bone union was significantly accelerated by MSC paracrine signaling (Furuta et al., 2016).Liu et al. showed that exosomes from hypoxia-preconditioned hucMSCs have a greater effect on fracture healing than those from normoxiapreconditioned hucMSCs, emphasizing the importance of mimicking normal physiological conditions (Liu et al., 2020).Unlike OP, OA, and RA, MSC-exosomes for the healing of fractures and bone defects are typically administered in a hydrogel-based local implant or by local injection.Ma et al. combined small intestinal submucosa hydrogels with bone marrow-derived MSC-exosomes and fusion peptides to enhance the osteogenesis-promoting role of exosomes in a calvarial defect model (Ma et al., 2022).Chen et al. incorporated miR-375-carrying ADSC exosomes in a hydrogel to achieve the slow and controlled release of miR-375, which had a marked bone-healing effect in a rat model of a calvarial defect (Chen et al., 2019).

MSC-exosomes in IDD
The intervertebral disc (IVD), including the nucleus pulposus (NP) and annulus fibrosus (AF), is an important load-bearing component of the spinal column.The accumulation of advanced glycation end products (AGEs) causes endoplasmic reticulum (ER) stress in the IVD.Subsequently, IDD is initiated by the apoptosis of NP cells (NPCs) and increased pro-inflammatory cytokine production and disruption of the ECM (Widjaja et al., 2022;Xia et al., 2022).IDD is the main reason for low back pain in over 90% of people over 50 (Krut et al., 2021).Surgical therapy and pain relief medication are the main treatments, but their effectiveness is uncertain (Xiao et al., 2022).

Obstacles to the use of MSC-exosomesbased therapeutics in skeletal diseases
Research has focused on the therapeutic potential of MSCs and exosomes for skeletal diseases.Besides exhibiting similar therapeutic effects, MSC-exosomes could overcome the safety and ethical concerns associated with MSCs injection.In addition, exosomes have less-stringent storage requirements than MSCs.Their efficacy and ability to be targeted to bone marrow, cartilage, and macrophages can be enhanced by bioengineering and preconditioning.These characteristics make exosomes good candidates for the treatment of skeletal diseases.
Currently, the existing clinical trials of MSC-exosomes-based therapies are mainly focused on cardiovascular disease, liver cirrhosis, psoriasis, macular holes, dry eye disease, diabetes mellitus, pneumonia, sepsis, wound healing and cancer (data from http://clinicaltrials.gov).For example, Dehghani et al. reported that no post-interventional adverse effects were observed following intraparenchymal implantation of MSCexosomes in five ischemic stroke patients (Dehghani et al., 2022).There are also several ongoing clinical trials investigating the role of MSC-exosomes in skeletal diseases.Matas et al. are conducting an interventional clinical trial to compare the safety and efficacy of MSC-exosomes injection in patients with mild to moderate symptomatic OA (Phase I, No. NCT05060107, 2021-10-05~2023-10-05).In another clinical trial, researchers are comparing injection of SF-MSC-exosomes with SF-MSCs in degenerative meniscal injury (Phase II, No. NCT05261360, 2022-03~2025-03).Also, autogenous MSC culture medium that containing exosomes are being studied in a clinical trial to enhance the osteogenesis of bone grafting (Phase I/ II, No. NCT04998058, 2023-12-15~2024-12-30).
However, whether MSC-exosomes can replace MSCs, and whether stem cells and exosomes in combination have synergistic therapeutic effects, is unclear.In addition, the long-term hazards of MSC-exosome therapy are unknown, necessitating evaluation of their effect on the immune system and the bone microenvironment.Moreover, compared with the existing MSCs-related therapies, the standardized methods of isolation, characterization and purification of MSC-exosomes need to be improved in terms of their reliability, cost, yield, and reproducibility.The ability of exosomes to deliver therapeutics for skeletal diseases also merits further investigation.

FIGURE 2
FIGURE 2 Overview of the biogenesis and composition of exosomes.(A) The molecular structure of exosomes.(B) The common sources of exosomes.(C) The production of exosomes.(D) Example of the uptake of exosomes.

FIGURE 5
FIGURE 5 2019) showed that bone marrow MSC-exosomes administered intravenously did not ameliorate OVX-induced OP in a mouse model.They conjugated a bone marrow MSC-specific aptamer to the surface of bone marrow-derived MSC-exosomes to target bone marrow, which enhanced bone regeneration in OVX mice.Similarly, Cui et al. functionalized iPSC exosomes by modifying a bonetargeting peptide.The exosomes were loaded with the Shn3 siRNA and showed therapeutic potential for OP by enhancing bone and vessel formation and inhibiting OCs(Cui et al., 2022).Ge et al. compared the functions of hucMSCexosomes from normal culture and those produced via osteogenic differentiation preconditioning.

TABLE 1
Representative in vivo studies of MSC-exosomes in OA.

TABLE 2
Representative in vivo studies of MSC-exosomes in OP.

TABLE 3
Representative in vivo studies of MSC-exosomes in RA.

TABLE 5
Representative in vivo studies of MSC-exosomes in bone defect.

TABLE 6
Representative in vivo studies of MSC-exosomes in IDD.