microRNAs delivered by small extracellular vesicles in MSCs as an emerging tool for bone regeneration

Bone regeneration is a dynamic process that involves angiogenesis and the balance of osteogenesis and osteoclastogenesis. In bone tissue engineering, the transplantation of mesenchymal stem cells (MSCs) is a promising approach to restore bone homeostasis. MSCs, particularly their small extracellular vesicles (sEVs), exert therapeutic effects due to their paracrine capability. Increasing evidence indicates that microRNAs (miRNAs) delivered by sEVs from MSCs (MSCs-sEVs) can alter gene expression in recipient cells and enhance bone regeneration. As an ideal delivery vehicle of miRNAs, MSCs-sEVs combine the high bioavailability and stability of sEVs with osteogenic ability of miRNAs, which can effectively overcome the challenge of low delivery efficiency in miRNA therapy. In this review, we focus on the recent advancements in the use of miRNAs delivered by MSCs-sEVs for bone regeneration and disorders. Additionally, we summarize the changes in miRNA expression in osteogenic-related MSCs-sEVs under different microenvironments.


Introduction
Bone tissue is a highly vascularized tissue, with abundant vessel networks that transport nutrients and oxygen (Anada et al., 2019).Bone repair after injury occurs in three stages: acute inflammation, bone repair, and remodeling (Claes et al., 2012).However, excessive inflammation negatively affects the osteogenic potential of cells.Therefore, the promotion of bone regeneration relies on angiogenesis, osteogenesis, and anti-inflammatory effect (Bucher et al., 2019).
Interestingly, the small extracellular vesicles (sEVs) released by MSCs are considered as the executors of this paracrine effect and have potential to replace MSC-based treatments in bone tissue engineering (Hade et al., 2021).Compared to organ transplantation and stem cell therapy, sEVs induce less immunological rejection and provide greater stability for application, transportation, and storage (Liu F. et al., 2019;Kim et al., 2021;Zheng et al., 2021;Tsai et al., 2022).The regenerative functions of MSCs-derived sEVs (MSCs-sEVs) depend on the proteins, lipids, DNA, RNA, and miRNAs they carry.Importantly, miRNAs are associated with bone homeostasis and angiogenesis.However, miRNA-based treatments have encountered challenges due to limited in vivo delivery efficiency.Recently, MSCs-sEVs have emerged as a viable tool for delivering therapeutic miRNAs (Zhu et al., 2020).As a natural delivery system, sEVs not only contain abundant miRNAs internally but also serve as carriers, enhancing the stability of encapsulated cargo, prolonging circulation periods, and facilitating transmembrane delivery.However, several challenges persist in the realm of MSCs-sEVs, including the presence of endogenous miRNAs, their potential impact on bone regeneration, and the successful encapsulation of osteogenic miRNAs within sEVs.
This review highlights the delivery of miRNAs by MSCs-sEVs to enhance angiogenesis, reduce inflammation, and promote osteogenesis (Figure 1).To begin with, we provide a concise overview of the biogenesis and functions of sEVs, which serve as nanocarriers for miRNAs.Next, we summarize the alterations in the expression of osteogenic-related miRNAs within MSCs-sEVs, observing diverse microenvironments.Lastly, we discuss the latest research findings concerning the delivery of miRNAs by MSCs-sEVs in the context of bone regeneration and bone-related diseases.
2 sEVs as nanocarriers for miRNAs 2.1 The biological properties and functions of sEVs Extracellular vesicles (EVs) are intricate structures with doublelayer lipid membrane, ranging in diameter from 40 to 160 nm.They are abundantly found in diverse body fluids being secreted by various cell types including bone mesenchymal stem cells (BMSCs) (Li X. et al., 2020), adipose mesenchymal stem cells (ASCs) (Fang and Liang, 2021), human umbilical cord mesenchymal stem cells (huc-MSCs) (Yang J. et al., 2020), and tumor cells (Kalluri and LeBleu, 2020).EVs can be classified into three distinct groups based on their size: large, medium, and small EVs.Moreover, they can be categorized according to their origin as microvesicles, apoptotic bodies, and exosomes.Microvesicles emerge through cell membrane budding, while apoptotic bodies generate during cell apoptosis (Tkach and Théry, 2016).Exosomes are produced via the formation of intracellular multivesicular bodies (MVBs) that encompass intraluminal vesicles (ILVs).These exosomes are then released through the fusion of MVBs with the plasma membrane and subsequent exocytosis (Gandham et al., 2020).In line with the latest guidelines recommending the use of "sEVs" instead of "exosomes," this review will adopt the term "sEVs".The isolation of sEVs can be achieved through various techniques, including differential ultracentrifugation, density gradient centrifugation, microfluidics (Cheng et al., 2021), size exclusion chromatography (Koh et al., 2018), immunoaffinity capture (Stam et al., 2021), or sEVs isolation kits (Macías et al., 2019).To characterize sEVs, their structures, size, and surface markers are examined using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting (Zhao et al., 2020).
sEVs have emerged as valuable tools for both diagnosis and therapy in the treatment of various diseases.Their composition, including proteins, lipids, DNA, RNA, and miRNAs, contributes to their unique properties.sEVs are promising non-invasive diagnostic biomarkers for cancer, diabetes mellitus (Huang et al., 2022), and bone diseases (Li R. et al., 2022).For example, urinary sEVs have been utilized for the early detection of gastric cancer (Chen et al., 2022), hepatocellular carcinoma (Li et al., 2022c), and head and neck cancer (Hofmann et al., 2022).Additionally, the identification of differentially expressed miRNAs within sEVs offers illuminating insights into various processes related to bone diseases such as osteoarthritis, femoral head necrosis, and bone fracture healing (Li et al., 2022b).Consequently, the detection of sEVs has become a simple, non-invasive, highly sensitive, and cost-effective method for monitoring the emergence and progression of diseases.
In the treatment of various diseases, sEVs have demonstrated significant advantages due to their biocompatibility, high bioavailability, and ability to deliver therapeutic cargo to target cells.Stem cell-derived sEVs possess remarkable regenerative potential, making them highly applicable in therapeutic applications.Notably, MSCs-sEVs have been explored as nanotherapeutics for autoimmune and neurodegenerative disorders.MSCs-derived sEVs, for instance, have shown their ability to reduce demyelination and neuroinflammation (Riazifar et al., 2019).Meanwhile, ASCs-derived sEVs have accelerated wound healing by promoting re-epithelialization and reducing inflammation (Zhou et al., 2022b), while BMSCs-derived sEVs have exhibited potential in improving osteoarthritis by promoting cartilage repair and alleviating knee pain (He et al., 2020).Huc-MSCs-derived sEVs have exhibited potential in repairing Parkinson's disease by crossing the blood-brain barrier (BBB), reducing apoptosis, and preventing the loss of substantia nigra dopaminergic neuron (Chen et al., 2020).sEVs have also been detected in various body fluids.For example, bovine milk-derived sEVs have demonstrated the ability to alleviate colitis symptoms by modulating intestinal inflammatory responses (Han et al., 2022).Plasma-derived sEVs have shown the potential to promote the proliferation and migration of BMSCs while inhibiting inflammation-induced chondrocyte degeneration (Zhang Y. et al., 2022).Furthermore, saliva-derived sEVs have exhibited potential in promoting cutaneous wound healing by stimulating the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) (Mi et al., 2020).In addition, there is growing interest in using sEVs as delivery tools for therapeutic miRNAs, proteins, and drugs.For instance, miR-31-5p mimics loaded in milk-derived sEVs have demonstrated efficacy in promoting diabetic wound healing (Yan et al., 2022).Zha et al. (2020) reported that encapsulated VEGF plasmid gene within sEVs elevated vascularized osteogenesis in vivo.Qian et al. (2022) found that sEVs derived from neural stem cells inhibited glioma by transferring miR-124-3p.Moreover, doxorubicin-loaded neutrophil-derived sEVs have shown potential in the treatment of glioma, brain diseases, and solid tumors (Wang J. et al., 2021).Overall, sEVs possess biocompatibility and exhibit extended circulation time by evading macrophage capture and clearance (Kamerkar et al., 2017).MSCs-derived sEVs offer significant potential in promoting angiogenic and osteogenic differentiation, making them a promising cell-free therapy for bone repair and regeneration (Heris et al., 2022).

sEVs transfer miRNAs into cells
miRNAs are initially transcribed into pre-miRNAs by RNA polymerase II and further processed by Drosha/DGCR8 to generate pri-miRNAs.These pri-miRNAs are exported to the cytoplasm through exportin-5 and mature into functional miRNAs.These mature miRNAs bind to the 3′ untranslated region (3′UTR) of target mRNAs, regulating various physiological and pathological processes through post-transcriptional silencing (Kim et al., 2009).However, the therapeutic potential of miRNAs is hindered by the lack of safe, effective, and stable delivery systems that protect them from degradation and facilitate cellular uptake.
Studies indicated that sEVs contain multiple miRNAs that can be transferred to target cells, influencing their functions.sEVs present potential advantages over other miRNA delivery strategies, including enhanced delivery efficiency and reduced degradation rates (Liang et al., 2021).Factors such as cell source, culture conditions, and sEV isolation techniques can influence the number of miRNAs in sEVs.Loading miRNAs into sEVs can be achieved either by modulating donor cells (endogenously) or by loading cargoes into sEVs in vitro (exogenously).Endogenous transfection methods involve modifying source cells to alter miRNA levels (Shojaati et al., 2019;Lou et al., 2020), while exogenous techniques include electroporation, co-incubation, sonication, and lipofectamine for sEVs derived from body fluids like blood, urine, saliva, and breast milk (Asadirad et al., 2019).Notably, the clinical implementation of electroporation (Zhang et al., 2017) for the direct transfer of miRNA mimics or inhibitors into sEVs encounters challenges such as exosome destruction, aggregation, and low loading efficiency (Wei Z. et al., 2021).Exo-Fect transfection has demonstrated high effectiveness, with over 50% transfection efficiency and lower co-localization with lysosomal and early endosomal compartments compared to other methods like heat shock or cholesterol modification of miRNAs (de Abreu et al., 2021).Nevertheless, since sEVs already naturally contain miRNAs and proteins, the efficient encapsulation of additional miRNAs remains unclear.To overcome these challenges and enhance the clinical translation of sEVs for miRNA delivery, further research is needed to optimize their utilization.

Variations in osteogenic-associated miRNAs expression within MSCs-sEVs under different microenvironments
Altered miRNA expression has been observed in stem cells derived from different sources and cultured under various conditions, as identified through miRNA microarray or highthroughput sequencing techniques.This article provides a summary of effects of osteogenic induction, hypoxic preconditioning, cellular senescence, and chemical or biomaterial microenvironments on the expression of osteogenic-associated miRNAs in MSCs-sEVs.

Osteogenic induction
Despite the potential of MSCs for multi-lineage differentiation, their application in tissue-engineering is limited due to low survival rates and differentiation efficiency.Osteogenic induction medium (OIM) can improve the stability, calcified nodules, and levels of ALP, OCN, OPN, and Runx2 in MSCs.Moreover, it affects the expression of miRNAs in MSCs-derived sEVs.For example, osteogenic induction of huc-MSCs resulted in 67 upregulated and 64 downregulated miRNAs in MSCs-derived sEVs during extended culture.These miRNAs target genes associated with bone growth and function, which are silenced.Notably, the gradually increasing expression of miR-2110 and miR-328-3p promoted osteogenesis by inhibiting the MAPK and PI3K-AKT-mTOR signaling pathways (Yahao and Xinjia, 2021).In BMSCs-sEVs, 8 miRNAs were downregulated, and 16 miRNAs were upregulated under osteoinductive culture, closely linked to bone formation by regulating the balance between Bmpr2/Acvr2b and smad1/5/9 phosphorylation (Liu A. et al., 2021).Moreover, OIM significantly altered miRNA expression over time, with miR-455-3p (Ma et al., 2022) and miR-27a-3p (Ren et al., 2021) continuing to increase on the 7th and 14th days in OIM, resulting in the downregulation of downstream targets HDAC2 and CRY2/ERK1/ 2. In conclusion, osteogenic induction can alter miRNA expression in sEVs, thereby impacting the osteogenic differentiation ability of sEVs.

Hypoxia preconditioning
Hypoxic pretreatment promotes the viability, proliferation, plasticity, and differentiation of BMSCs, while decreasing their apoptosis via the upregulation of HIF-1α (Luo et al., 2019) and downregulation of stress response-related genes p16 and extracellular signal-regulated kinase (Tsiapalis and Zeugolis, 2019).Recent studies have unveiled the capability of hypoxia preconditioning to elevate miRNA expression in sEVs derived from BMSCs (Shen et al., 2022).For instance, hypoxia preconditioning upregulated miR-126 in MSCs-sEVs via HIF-1α activation.This activation improved bone fracture healing through the miR-126/SPRED1/Ras/Erk signaling pathway (Liu et al., 2020a).Moreover, hypoxic MSCs-derived sEVs stimulate osteogenesis and promote new blood vessel growth in mice with bone deficiencies.During hypoxia, miRNA sequencing analysis demonstrated that elevated levels of miR-210-3p in sEVs.The upregulation of miR-210-3p facilitated vascularized bone regeneration by inhibiting the expression of EFNA3 and activating the PI3K/AKT pathway (Zhuang et al., 2022).Furthermore, MSCs-sEVs derived from the hypoxic preconditioning microenvironment induced the polarization of M1 to M2 phenotype by enriching miR-216a-5p and activating the TLR4/NF-κB/PI3K/AKT axis (Liu et al., 2020b).It is important to note that prolonged periods of hypoxia or excessively low oxygen concentrations may impair the function of MSCs, despite the effectiveness of hypoxic preconditioning in optimizing the regenerative and therapeutic potential of MSCs.

Senescence
Senescence induces irreversible cell-cycle arrest, which in turn contributes to age-related bone fragility and loss.The composition of sEVs and their miRNAs in the bone marrow microenvironment may vary with age.Previous studies have revealed that BMSCs-sEVs from young and aged mice are rich in miRNAs, but the miRNA profile differs significantly, contributing to the dysfunction of stem cells associated with aging.Particularly, aged sEVs exhibited a significant increase in miR-183-5p expression.Transfection of miR-183-5p mimics into BMSCs induced osteoblast dysfunction by downregulating heme oxygenase-1 (Hmox1) activity (Davis et al., 2017).Xu et al. (2018) discovered elevated levels of miR-31a-5p in BMSCs-derived sEVs from aged rats compared to those from their younger counterparts.The enrichment of miR-31a-5p in sEVs regulated osteoblastic and osteoclastic activities, promoting bone resorption and inhibiting bone formation.This observation presents miR-31a-5p as a potential therapeutic modulator for age-related bone loss.Furthermore, sEVs derived from aged bone matrix stimulated adipogenesis and vascular calcification during bone resorption by upregulating the expression of miR-128-3p (Xu et al., 2020), miR-483-5p, and miR-2861 (Wang ZX. et al., 2022).In conclusion, as time progresses, sEVs enriched with specific miRNAs hinder bone formation, enhance bone resorption, and stimulate adipogenesis in bone marrow microenvironment.These molecules hold promise as valuable biomarkers for age-related bone diseases.

Chemical or biomaterials microenvironment
Both chemical elements and biomaterial structures can influence cell-to-cell communication, altering miRNA profiles and target gene expression in sEVs.For example, the incorporation of lithium (Li) into bioactive materials enhanced the proliferation, migration, and tube formation of HUVECs, thereby promoting angiogenesis during bone remodeling.This effect was achieved through the upregulation of miR-130a and activation of the PTEN/AKT signaling pathway in BMSCs-derived sEVs (Liu L. et al., 2019).Similarly, biocompatible titanium alloys supported the attachment of mineralized bone matrix and promoted cell-free bone regeneration by upregulating the expression of miR-146a-5p, miR-503-5p, miR-483-3p and miR-129-5p, while downregulating the expression of miR-32-5p, miR-133a-3p, and miR-204-5p.Coating cell-free titanium alloy scaffolds (Ti-scaffolds) with MSC-sEVs facilitated bone-forming outcomes comparable to those achieved with MSC-seeded Ti-scaffolds (Zhai et al., 2020).In another study, Fe 3 O 4 nanoparticles were used to manufacture BMSCs-sEV, which were found to promote osteogenesis and angiogenesis by modulating miR-1260a/ HDAC7/COL4A2 (Wu et al., 2021a).Similarly, the utilization of strontium (Sr) -containing biomaterials prompted the production of pro-angiogenic miR-146a cargoes within BMSCs-sEVs.These cargoes, in turn, inhibited the expression of Smad4 and NF2, leading to the development of engineered Sr-sEVs with dualfunctional regulation for promoting both osteogenesis and angiogenesis in the context of vascularized bone regeneration (Liu L. et al., 2021).Additionally, 3D printing biomaterials loaded with human gingival MSCs-sEVs increased the expression of osteogenic and angiogenic markers such as RUNX2, VEGFA, OPN, and COL1A1, alongside enhanced expression of miR-2861 and miR-210 (Pizzicannella et al., 2019).Taken together, these studies suggest that even tiny bioactive elements or structures can significantly impact the expression of miRNAs in MSCs-sEVs, thereby playing a pivotal role in bone repair and regeneration.

miRNAs delivered by MSCs-sEVs in bone regeneration
Osteogenesis, angiogenesis, and inflammation are all essential processes involved in the healing of bone defects.miRNAs delivered by MSCs-sEVs and the associated signaling pathways play crucial roles in regulating the above processes (as depicted in Figure 2).These small molecules facilitate intercellular communication, enabling cells to exchange information and participate in the regulation of osteogenesis.Therefore, targeting sEVs-miRNAs and their related signaling pathways holds potential therapeutic opportunities for the treatment of bone injuries and diseases.The formation of blood vessel networks is crucial to supply the necessary nutrients and oxygen required for regulating bone remodeling.The expression of sEVs-miRNAs affects angiogenesis, thereby influencing osteogenesis (Yang et al., 2021).Furthermore, during the inflammatory phase of bone healing, researchers discovered that sEVs-miRNAs enhanced their immunomodulatory properties, leading to the suppression of pro-inflammatory markers and elevation of anti-inflammatory markers (Kang M. et al., 2022).These findings emphasized the importance of the pro-angiogenic and anti-inflammatory abilities of sEVs-miRNAs in promoting bone regeneration.

Facilitating osteoblastic differentiation
Researchers have been investigating the potential application of miRNA mimics or inhibitors to stimulate the proliferation, migration, and differentiation of osteoblasts in stem cells.They have discovered that incorporating these miRNAs into MSCs-sEVs is an effective method for promoting bone formation.For example, BMSCs-sEVs containing miR-21-5p enhanced the differentiation of osteoblasts and increased ALP activity in hFOB1.19 cells (You et al., 2022).Similarly, miR-126-5p and miR-150-5p enriched apical papilla-derived sEVs (SCAP-sEVs) facilitated migration and tube formation of HUVECs, promoted differentiation of MC3T3-E1 cells, and improved bone regeneration (Jing et al., 2022).BMSCs-sEVs were also found to support hBMSCs migration and osteogenesis by transporting miR-20a (Liu et al., 2021d).

Promoting angiogenesis
The growth of blood vessels and the high expression of angiogenic factors are crucial for bone repair (Zhang L. et al., 2020).sEVs play a significant role in promoting vascularized bone regeneration by modulating the expression of miRNAs and multiple signaling pathways.BMSCs-sEVs can promote the expression of proangiogenic cytokines, including VEGF, platelet endothelial cell adhesion molecule-1 (CD31), and HIF-1α, leading to improved vascular development and regeneration in bone defects through the upregulation of the miR-21/NOTCH1/DLL4 signaling axis (Zhang et al., 2021c;Hu H. et al., 2022).The overexpression of miR-130a in BMSCs-sEVs facilitates bone formation and enhances the pro-angiogenic potential of HUVECs by stimulating the PTEN/ AKT signaling pathway (Liu L. et al., 2019).The high expression of BMSCs-sEVs-miR-126 enhances angiogenesis by targeting PIK3R2, which activated the PI3K/Akt signaling pathway (Zhang L. et al., 2021).Encapsulation of BMSCs-derived sEVs in scaffolds extends their delivery and released time.sEVs-loaded hydrogels release miR-21, which targets SPRY2 and accelerates both osteogenesis and angiogenesis (Wu et al., 2021b).However, sEVs-miR-214-3p results in impaired angiogenic potential and decreased bone mineral density (Wang X. et al., 2021), highlighting the importance of downregulating this miRNA in sEVs to promote vascularized bone regeneration.Overall, these findings shed light on the regulatory roles of sEVs containing miRNA in angiogenesis during the process of bone remodeling, further emphasizing their potential in bone tissue engineering.

Reducing inflammation
sEVs carrying miRNAs hold significant potential in modulating the bone healing process by regulating the polarization state of host macrophages.The polarization of macrophages plays an essential role in bone healing.M1-type macrophages induce persistent inflammation and tissue degradation, while M2-type  et al., 2022).The capacity of sEVs to transport and transfer miRNAs renders them a promising avenue for biomedical research.Studies have demonstrated the potential of sEVs in attenuating inflammation and promoting bone tissue repair.For instance, sEVs derived from BMSCs transfected with miR-23a-3p mimics promoted M2 macrophage polarization, reduced inflammation by inhibiting the IRF1 and NF-κB pathways, and improved tendon-bone healing.This was further evidenced by an increased ratio of bone volume to total volume (BV/TV), upregulated collagen type II alpha 1 levels, and improved bone healing outcomes (Li Z. et al., 2022).Similarly, ASCs-sEVs enriched with miR-451a significantly promoted bone healing and facilitated the shift from M1 macrophages to M2 macrophages, thereby inhibiting inflammation via miR-451a/MIF signaling pathway (Li R. et al., 2022).Furthermore, BMSCs-sEVs overexpressing miR-181b facilitated M2 macrophage polarization and osteointegration by suppressing PRKCD while activating p-AKT (Liu et al., 2021e).Additionally, gingival MSCs-derived sEVs containing miR-1260b inhibited osteoclastogenesis and periodontal bone resorption by inducing anti-inflammatory M2 macrophage polarization and repressing Wnt5a/RANKL pathway (Nakao et al., 2021).Dental pulp stem cells derived sEVs (DPSC-sEVs) enhanced T-cell modulatory function, restored the balance between Th17 cells and Treg cells, suppressed inflammation, and accelerated alveolar bone healing by transferring miR-1246 and inhibiting Nfat5 expression (Zhang et al., 2021b).Lastly, huc-MSCs-derived sEVs containing miR-378a-5p (Cai et al., 2021) and miR-326 (Wang et al., 2020) inhibited the expression of interleukin (IL)-18, IL-1β, Caspase-1, and NLRP3 inflammasomes.These findings suggest that miRNAs enriched in MSCs-sEVs possess the capability to regulate macrophage polarization and suppress inflammation, thereby promoting bone regeneration.

miRNAs delivered by MSCs-sEVs in bone-related diseases
MSCs and their sEVs have shown promising potential as therapeutic agents for bone-related diseases.They can transfer various types of information, regulate immune responses, inhibit cell apoptosis, induce differentiation, and promote tissue regeneration (Malekpour et al., 2022).The miRNAs found in MSCs-sEVs play a crucial role in maintaining the balance between osteoblasts and osteoclasts, promoting angiogenesis, and aiding in bone restoration.Table 2 outlines the important roles of miRNAs from MSCs-sEVs in various bone-related conditions such as bone fractures, osteoporosis, osteoarthritis, and osteonecrosis of the femoral head.

Bone fracture
Bone fracture healing is a complex process, involving the coordinated actions of osteoclasts for bone resorption and osteoblasts for bone formation.Recent studies have highlighted the role of miRNAs encapsulated within BMSCs-sEVs in enhancing neovascularization and bone formation at the fracture site through various complicated signaling pathways.For example, BMSCs-sEVs carrying miR-136-5p promoted osteoblast differentiation and facilitated fracture healing by targeting LRP4 and activating the Wnt/β-catenin signaling pathway (Yu et al., 2021).Another miRNA, miR-19b, abundant in BMSC-sEVs, facilitated bone cell mineralization, and enhanced neovascularization at the fracture site through the WWP1/ Smurf2/KLF5/β-catenin signaling pathway (Huang et al., 2021a).Additionally, BMSC-sEVs secrete miR-25 (Jiang et al., 2020), miR-29b-3p (Yang et al., 2022), and miR-335 (Hu H. et al., 2021), which have demonstrated the ability to enhance the proliferation, migration, and differentiation of osteoblasts in vitro.In vivo, these miRNAs accelerate bone fracture healing through three distinct pathways: SMURF1/Runx2, VapB/Wnt/β-catenin, and PTEN/PI3K/AKT axes, respectively.Collectively, these findings suggest that MSCs-sEVs-encapsulated miRNAs may offer valuable insights into the disappearance of fracture lines, callus formation, and overall fracture healing process.

Osteoporosis
Osteoporosis (OP) is a degenerative bone disease caused by an imbalance in bone remodeling cycle, resulting in an increased risk and susceptibility to bone fractures (Brown, 2017).Recent in vitro experiments have demonstrated that certain miRNAs carried by MSCs-sEVs could impact the prognosis of OP.In postmenopausal osteoporotic rats, the expression of BMSCs-sEVs-miR-27a-3p and miR-196b-5p was relatively reduced (Lai et al., 2022), but these miRNAs actually accelerated osteogenesis and reduced bone resorption in OP.Upregulating BMSCs-sEVs containing miR-150-3p (Qiu et al., 2021), miR29a (Lu et al., 2020), andmiR-21-5p (You et al., 2022) also stimulate angiogenesis and osteogenesis, presenting a novel therapeutic strategy for treating OP.Conversely, downregulating miR-146a in BMSCs-sEVs enhance the viability of MC3T3-E1 cells and prevent their apoptosis (Cao et al., 2020).BMSCs-derived sEVs containing MALAT1 boost osteoblast activity in osteoporotic mice by inhibiting the expression of miR-34c and promoting the expression of SATB2 (Yang et al., 2019).Mechanistically, miR-186 loaded BMSCs-sEVs facilitate osteogenesis in osteoporotic rats through the Hippo signaling pathway (Li L. et al., 2021).Further research revealed that sEVs derived from huc-MSCs prevent OP by inhibiting BMSCs apoptosis and regulating the miR-1263/Mob1/Hippo signaling pathway (Yang BC. et al., 2020).Overall, MSCs-sEVs carrying specific miRNAs have emerged as a promising therapy for OP, as they promote bone damage recovery and reduce bone resorption through various signaling pathways (Wang Y. et al., 2022).

Osteonecrosis of the femoral head
Osteonecrosis of the femoral head (ONFH) is a bone disease caused by impaired blood supply and necrosis of the marrow in the femoral head.Recently, studies have shown that the levels of miRNAs in sEVs might be altered during the progression of ONFH (Li et al., 2018).For example, miRNA sequencing revealed decreased expression of miR-185-3p and miR-1b-5p, while miR-129b-5p and miR-223-5p were upregulated in sEVs from femoral tissue in ONFH patients.These changes closely related to classical osteogenesis-related signaling pathways, including PI3K/Akt and Wnt signaling pathways (Zhu et al., 2022).Furthermore, elevated expression of sEVs-miR-100-5p in ONFH inhibits osteogenesis and angiogenesis through the BMPR2/ SMAD1/5/9 signaling pathway (Yang et al., 2021).In contrast, in ONFH, the expression of miR-224-3p is downregulated in BMSCs-sEVs, which leads to increased angiogenesis by upregulating FIP200 (Xu et al., 2019).Overall, miRNAs promoting disease progression are upregulated, while those inhibiting disease progression are downregulated in ONFH.

Conclusion and future perspectives
In summary, bone repair and regeneration rely on the development of bone and blood vessels.MSCs show significant potential for bone regeneration due to their self-renewal and multilineage differentiation capacity (Huang CC. et al., 2020).However, MSCs therapies face obstacles such as high costs, limited sources, and strict storage requirements.Therefore, it is crucial to develop cell-free or acellular approaches to promote bone regeneration.
miRNAs play a crucial role in regulating osteoblast-osteoclast interactions and offer substantial clinical potential (Hu et al., 2022c).However, the lack of effective delivery systems has limited the use of miRNA-based therapeutics.sEVs have emerged as ideal delivery systems due to their ability to maintain miRNA stability during storage.However, sEVs are quickly cleared in vivo, which hampers their ability to reach the target site.To overcome this challenge, scaffolds can provide long-term preservation and sustained release of sEVs.For instance, MSCs-sEVs were lyophilized on a microporous bio-glass scaffold, resulting in controlled release, heightened expression of osteogenic-related markers, and enhanced bone repair efficiency (Liu A. et al., 2021).To ensure prolonged retention and controlled release of sEVs, researchers have developed cell-free metal-organic frameworks functionalized with hASCs-sEVs (Kang Y. et al., 2022).These sEVsloaded composite scaffolds have shown the ability to accelerate blood supply, osteogenic differentiation, and bone reconstruction over an extended period.
However, a major challenge in sEVs research is the impurity and low abundance of conventionally produced sEVs.Threedimensional (3D) cultures have proven more effective than twodimensional (2D) cultures in producing sEVs by preventing cell adhesion to culture flask surfaces.sEVs produced from 3D-cultured MSCs exhibit potential in suppressing inflammation and enhancing therapeutic effects in bone regeneration by the upregulation of miRNAs.The upregulation of miRNAs is thought to be caused by the hypoxic conditions in the center of the 3D spheroidal structure (Zhang et al., 2021b).Other findings have shown that combining tangential flow filtration (TFF) with 3D cell cultures can increase the concentration of sEVs in cell culture supernatants, resulting in higher yields of biologically active sEVs and improved transferability of therapeutic siRNAs (Haraszti et al., 2018).Furthermore, the creation of specific sEVs-mimetics (EMs) through sequential mechanical extrusion of cells offers a rapid method for producing large quantities of sEVs, thereby enhancing manufacturing efficiency compared to traditional methods (Zha et al., 2020).
Ultimately, challenges in using sEVs for bone regeneration are their uncertain distribution and lack of targeting ability in the bone microenvironment.Recent research suggested that click chemistry, physical surface modification, and genetic engineering can help sEVs accumulate at the target site and enhance their therapeutic efficacy (Jiang et al., 2022).Several bone-targeting delivery strategies have been developed, including attaching the bone-targeting peptide SDSSD to the membrane of sEVs for specifically delivery to osteoblasts and the promotion of bone formation (Cui et al., 2022).Additionally, C-X-C motif chemokine receptor 4 (CXCR4) positive bone-targeted sEVs could be recruited by BMSCs and released miR-188 to promote osteogenesis and decrease cortical bone porosity for age-related bone loss (Hu Y. et al., 2021).Although the isolation, delivery, and targeted modification of sEVs are relatively well-documented, further research is necessary to understand the mechanisms by which sEVs deliver functional miRNAs to recipient cells.Therefore, it is important to consider good manufacturing practices, stability, loading efficiency, and targeted delivery of sEV-encapsulated miRNAs for bone repair and regeneration.Frontiers in Bioengineering and Biotechnology frontiersin.org16 Liu et al. 10.3389/fbioe.2023.1249860

FIGURE 2
FIGURE 2MSCs-sEVs deliver miRNAs by transfection and their applications.The miRNA mimics or inhibitors were transfected into stem cells to overexpress or knock down the expression of miRNAs in MSCs-sEVs.Then, miRNAs loaded in MSCs-sEVs enhance angiogenesis and bone regeneration.

TABLE 1
MSC-sEVs delivered miRNAs can regulate many signaling pathways to facilitate osteogenic differentiation.TABLE 2 miRNAs delivered by MSCs-sEVs in bone-related diseases.