As defined by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT^®), mesenchymal stromal cells are required to meet the following minimal criteria: 1. being plastic-adherent under standard culture conditions; 2. expressing CD105, CD73 and CD90 while lacking expression of surface molecules CD45, CD34, CD14 or CD11b, CD79 alpha or CD19 or HLA-DR; 3. possessing multi-differentiation potential into osteoblasts, adipocytes and chondroblasts under the effects of specific culture media (Dominici et al., 2006). Firstly isolated from human and mammalian bone marrow and periosteum, MSCs were officially named “mesenchymal stem cells” (Caplan, 2017). However, due to the limited capacity to differentiate into other cell types and lacking asymmetric division, MSCs are being debated whether they should be classified as “stromal” or “stem” cells. Some researchers state that the term “stem” should be reserved for cells that possess true stem cell properties, such as the ability to self-renew and differentiate into a wide range of cell types (Sipp et al., 2018; Andrukhov, 2021).

Currently, it has been established that MSCs can be derived from almost all postnatal tissues including bone marrow, adipose tissue, peripheral blood, synovial membrane, umbilical cords, dental pulp, Wharton’s jelly, dermis, placenta, amniotic fluid, and other tissues of mesoderm (Shirjang et al., 2017; Najar et al., 2022). Moreover, MSCs may be isolated from perivascular tissue and vascularized tissue since they are found to reside in blood vessel walls (Caplan, 2009). According to the extraordinary secretory, immunomodulatory, self-renewal, and multi-lineage differentiation capacity, MSCs have been widely used in tissue repairing and homeostasis such as bone, cartilage, tendon, muscle, and other mesodermal tissues, as well as immune-related disease therapies, which include acute respiratory distress syndrome (ARDS), OA, graft-versus-host disease (GVHD) and other diseases (van den Akker et al., 2013; Zhou et al., 2021). Furthermore, MSCs exhibit a tolerable safety profile due to the lack of co-stimulatory molecules, including human leukocyte antigen class-II (HLA-II), CD80, and CD86, which helps avoid activation of host T or B lymphocytes and widen the utilization of MSC in vivo (Bassi et al., 2011). Numerous clinical trials of MSC-related therapies have been completed in patients with degenerative, autoimmune diseases and other diseases. However, the selection and preparation of MSCs for studies and applications do not yet have a common standard.

Toll-like receptors (TLRs), a kind of Pattern Recognition Receptors (PRRs), are a family of proteins playing a key role in recognizing altered patterns on microbes, viruses (Pathogen-Associated Molecular Patterns, PAMPs) or endogenous danger signals (Damage/Danger-Associated Molecular Patterns, DAMPs) thus stimulate immunity and host defense that culminate in the expression of inflammatory cytokines, chemokines or antimicrobial products (Fitzgerald and Kagan, 2020). TLRs share a similar domain organization as they are all type Ⅰ single-pass transmembrane proteins with an N-terminal leucine-rich repeat-containing ectodomain, a transmembrane domain, and a cytoplasmic C-terminal Toll-interleukin-1 receptor (IL-1R) homology (TIR) domain. Researchers have identified TLR family members in organisms ranging from corals to humans by genome browsing for proteins that share these features. 10 human TLRs (TLR1-10) and 12 mouse TLRs (TLR1-9, TLR11-13) were identified respectively, while other species vary in number in those evolutionarily conserved proteins (Kawai and Akira, 2011). Most TLRs act as homodimers, while only TLR2 paired with TLR1 or TLR6 function as heterodimers (Oliveira-Nascimento et al., 2012). TLRs that occupy the plasma membrane include TLR1, 2, and 6 which engage bacterial lipoproteins, and TLR4 and 5 which detect lipopolysaccharide (LPS) and flagellin, respectively (Lester and Li, 2014). In contrast, TLRs expressing in endosomes, lysosomes, or endoplasmic reticulum recognize nucleic acids. Specifically, TLR3 detects double-stranded RNA (dsRNA), TLR7, 8 sense single-stranded RNA (ssRNA) and TLR9 recognizes unmethylated CpG containing ssDNA (Chow et al., 2018; Greulich et al., 2019). Considered the only TLR to have the potential role that induces anti-inflammatory activity, TLR10 can homodimerize or heterodimerize with either TL1 or TLR2. The ligands that interact with TLR2 are also considered ligands of TLR10, such as Pam3Cys (Fore et al., 2020). Apart from the PAMPs, TLRs could be activated by endogenous ligands released from damaged tissues or dead cells (Yu et al., 2010).

As for signal transduction pathways, all TLRs except TLR3 use the myeloid differentiation primary response protein 88 (MyD88)-dependent pathway to mediate the production of inflammatory cytokines while TLR3 activated MyD88-independent pathway: TIR-domain containing adaptor inducing interferon (IFN)-β (TRIF or TICAM1) dependent pathway. Interestingly, TLR4 can activate both of them (Sangiorgi and Panepucci, 2016; Abdi et al., 2018; Zhang et al., 2023).

Except for innate immune cells, TLRs also express on MSCs. The exact expression pattern varies from different cell origination: bone marrow (BM), adipose tissue (AT), umbilical cord blood (UC), human placenta (hpl), Wharton’s Jelly (WJ), and other adult or fetal tissues (Guillot et al., 2008; El-Sayed et al., 2017). TLR2-4, however, is irrefutably present on the MSCs due to their responsiveness to the corresponding ligands (Jia et al., 2019). After migration to the site of tissue injury or inflammation, MSCs could be exposed to not only immune cells but also TLR ligands (Mohamad-Fauzi et al., 2023), TLRs greatly impact their functions such as proliferation, migration, angiogenesis, differentiation, and immunomodulatory capacity which lead to the MSC-based treatment in tissue regeneration or inflammation-related diseases more than ever (Figure 1).

The MSCs originated from different tissues possess the capability to undergo differentiation into osteoblasts and terminally the most abundant osteocytes upon exposure to various osteogenic signals, underlying their importance in regenerative medicine (Pipino and Pandolfi, 2015). Moreover, following fracture or bone defects, MSCs transport or migrate to the site of injury via the peripheral blood circulation, thereby augmenting the regenerative capacity of the local MSC population. Besides the bone formation effects, MSCs establish interactions between bone formation and resorption via RANKL (receptor activator of nuclear factor kappa B ligand)/RANK coupling to modulate the activity of osteoclasts (Ikebuchi et al., 2018; Schlundt et al., 2018). Considering the accompanied damage of blood vessels during bone injury, MSCs also promoted angiogenesis by secreting pro-angiogenic factors thus activating endothelial cells (Grote et al., 2013).

To date, several in vitro studies that isolated MSC to detect the expression of TLR profiles are listed in Table 1. Recent research also demonstrated a novel function of TLRs in modulating the osteogenic differentiation potential of MSCs, in which alkaline phosphatase (ALP), osteocalcin (OCN), collagen 1 (Col-I), osteopontin (OPN), osteonectin (ON), osterix (OSX), runt-related transcription factor 2 (RUNX-2) and mineralization assay (alizarin red staining) were used to assess osteogenesis capacity (Loebel and Burdick, 2018). Bioinformatic analyses also identified that various pathways, including the TLR signaling pathway, are associated with the osteogenic differentiation potential of BMSCs (Bone marrow-derived MSCs) (Zhang et al., 2017). The effect of activating TLR3 and TLR4 pathways in MSCs is investigated most extensively, in which most studies conducted the TLR priming protocol by administering Poly (I: C) as TLR3 ligand and LPS as TLR4 ligand. Nevertheless, the osteogenic differentiation varied according to the ligand type and concentration. In addition, age should be taken into consideration as ligation of TLR3 and TLR4 on the BMSCs, that are isolated from young people, showed higher osteogenic differentiation in comparison with the BMSCs isolated from the senior generations (Shirjang et al., 2017). MSCs exhibit different potential according to tissues they are isolated from, BMSCs and adipose tissue-derived MSCs (AMSCs) represented the optimal stem cell source for tissue engineering as they were observed to maintain the strongest differentiative abilities (Heo et al., 2016). Therefore, we categorize and discuss the osteogenic differentiation according to the MSC origin, nevertheless, an identification of the efficient source under a unified standard is needed.

To date, regulation of osteoimmunology has become the central role of recovering functions in defective and diseased bone (Lee et al., 2022). Apart from MSCs which have the potential to become osteoblasts, bone marrow, the semi-solid tissue enclosed in bone, encompasses hematopoietic stem cells (HSCs) and immune cells. Monocytes from hematopoietic origin partly differentiate into osteoclasts, while other groups from HSCs have the crucial function of producing different components of blood (red blood cell et al.) or maintaining the hemostasis of immune microenvironment by differentiating into immune cells such as neutrophils, mast cells, monocytes, T cells, and B cells (Dzierzak and Bigas, 2018). In addition to the immune cells maturation which takes place within the bone marrow, bone formation and immune system also share cytokines and other signal molecules due to their anatomical proximity (Hosseinpour et al., 2022).

Successful bone regeneration requires continuous activation of different types of immune cells and subsequent cytokine patterns. On the other hand, abnormal immune cell function will lead to an imbalance between osteoclasts and osteoblasts, ultimately leading to osteolysis, osteoporosis, osteoarthritis, rheumatoid arthritis, and other diseases. First discovered as early as 1972, in which IL-1 was found to be secreted by leukocytes, supporting activation of osteoclasts (Horton et al., 1972), crosstalk between bone-forming/adsorbing and immune system assists sequential activation of different components in the stages of bone regeneration or bone remodeling. Besides IL-1, one of the primary findings of cytokines, chemokines, and transcription factors involved in osteoimmunology is RANKL/RANK/osteoprotegerin (OPG) signal axis, which mediates osteoblast-controlled osteoclastogenesis and bone degradation (Witkowska-Sedek et al., 2018), has also been found to be expressed by activated T cells (RANKL) under inflammatory conditions thus promoting osteoclastogenesis and subsequent bone loss in various inflammatory and autoimmune conditions (Arron and Choi, 2000). While B cells communicate with osteoclasts and osteoblasts through OPG (Li et al., 2007; Frase et al., 2023). IL-6 was found to be one of the most important mediators of osteoclast formation and function, and over-secretion led to impaired osteogenesis in m-BMSCs (Li et al., 2016c). Nevertheless, IL-6 was reported to participate in TLR-induced osteogenesis of m-AMSCs (Huh and Lee, 2013). SH2-containing inositol phosphatase-1 (SHIP1), an immunomodulatory molecule, activated osteogenesis as a downstream of TLR2 signaling (Muthukuru and Darveau, 2014), while disruption of the modulatory molecules, such as SHP1 and NFATc1, results in mitigation of bone volume by osteoblast deactivation (Lee et al., 2022). A variety of key cytokines released by activated immune cells can activate the function of osteoclasts [OSM (Oncostatin M), IL-6, TNF-α (Fujii et al., 2012)] or exhibit inhibitory effects(IFN- γ, IL-4, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12 and IL-18) (Freire et al., 2016; Mae et al., 2021). Anti-inflammatory factors, such as IL-10, can also promote osteogenesis.

There are two main aspects of immune cells’ impact on bone regeneration: the anabolic function of acute inflammation during bone regeneration and the catabolic function of chronic inflammation during bone resorption (Wu et al., 2014; Kouroupis et al., 2019). Specifically, acute inflammation leads to the production of chemokines or cytokines, which accelerate bone formation in early fracture healing by stimulating the proliferation and osteogenic differentiation of MSCs and progenitor cells. Conversely, chronic inflammation increases cytokines secretion to activate osteoclastogenesis and inhibits the production of factors that stimulate osteoblasts to form new bone, thereby inhibiting bone coupling during inflammatory diseases such as periodontitis and promoting the formation of osteolytic lesions (Karlis et al., 2020; Yang et al., 2021). Together, the immune and skeletal systems crosstalk mutually, suggesting the importance of osteoimmunology not only in bone regeneration but this concept also could be extended to other tissue engineering and diseases.

MSCs release a wide range of paracrine factors into their conditioned media (MSC-CM) in vitro. Intriguingly, the administration of MSC-CM in vivo has been shown to promote tissue healing in various tissues, including bone. This compelling evidence indicates that the secretome of MSCs can trigger the cascade of bone tissue regeneration (Ando et al., 2014). Moreover, MSCs possess immunosuppressive properties and can dampen immune responses. They can inhibit the proliferation and activation of immune cells, suppress the release of pro-inflammatory cytokines, and induce the production of anti-inflammatory factors (Garg et al., 2022). By doing so, MSCs help to create an immunoregulatory microenvironment that is favorable for bone healing and regeneration. MSCs regulate innate immunity in various ways, in which TLR signaling may be of great importance. They are capable of promoting macrophage polarization into the anti-inflammatory and pro-healing phenotype or modulating the maturation of neutrophils, monocytes, macrophages, and nature killer cells (Kota et al., 2014; Jiang et al., 2016). Zhao et al. demonstrated that poly (I: C) enhanced the anti-inflammatory effect of UC-MSCs on macrophages by up-regulating cyclooxygenase-2 (COX-2), indoleamine 2,3-dioxygenase (IDO) and IL-6 (Zhao et al., 2014). Munir et al. reported that LPS-primed h-AMSCs activated neutrophils and promoted NET (neutrophil extracellular trap) formation, which subsequently stimulated macrophage to secrete Transforming growth factor-β (TGF-β) and the continuous activation ultimately led to accelerated wound healing of mice (Munir et al., 2020). Conditioned media (CM) from h-AMSCs activated by TLR5 ligand flagellin (F-CM) were compared to CM from untreated MSCs. F-CM promoted a higher proportion of anti-inflammatory M2 macrophages, inhibited inflammatory cell recruitment, decreased inflammatory factor levels, and finally alleviated acute lung injury (ALI) (Li et al., 2020). Lu et al. stated that TLR4-treated hBMSCs exacerbate the inhibition of natural killer (NK) cell proliferation and function(Lu et al., 2015). On the other hand, MSCs also modulate the adaptive immune system. They proficiently suppress the proliferation of T cells, as observed in animal models of GVHD (Sangiorgi and Panepucci, 2016).

Additionally, MSCs can directly interact with immune cells and promote their transition to anti-inflammatory or regulatory phenotypes. For example, Pre-conditioning of m-BMSCs with poly (I: C) exhibited a notable reduction in the proliferation of CD3⁺ T cells and diminished the differentiation and activation of proinflammatory lymphocytes, specifically Th1 and Th17 (Maria Vega-Letter et al., 2016). The pre-activation of h-AMSCs with Poly:(I: C), enhanced their ability to inhibit the proliferation of T lymphocytes as well (Mancheno-Corvo et al., 2015). Moreover, MSCs can induce the differentiation of T cells into regulatory T cells (Tregs), Rashedi et al. revealed that the generation of Tregs in cocultures of human CD4⁺ lymphocytes and h-BMSCs was enhanced through the activation of either TLR3 or TLR4 in MSCs, and this enhancement was eliminated by gene silencing of TLR3 and TLR4. The amplified induction of Tregs by TLR-activated MSCs was correlated with an upregulation in the gene expression of the Notch ligand, Delta-like 1, while the inhibition of Notch signaling abolished the increased levels of Tregs in the MSC cocultures (Rashedi et al., 2017). In addition, MSCs treated with poly (I: C) exhibited a detrimental impact on the expansion of T-helper type 1/17 (Th1/17) cells while enhancing the suppressive effects of Tregs both in vitro and in vivo. The production of PGE-2 by UC-MSCs in response to poly (I: C) was found to increase the secretion of IL-10 and facilitate the differentiation of Tregs, a process that could be reversed by inhibiting Notch-1 (Qiu et al., 2017). Following a 48-h exposure to LPS, m-BMSCs acquired an anti-inflammatory phenotype, characterized by a substantial enhancement in their immunosuppressive capacity on T cell proliferation in vitro. Moreover, this time-dependent LPS stimulation led to improved therapeutic effects and higher levels of regulatory T cells (Tregs) when compared to unstimulated MSCs (Kurte et al., 2020). MSCs possess the ability to activate B cells, as evidenced by the increased expression of B cell activating factor (BAFF) in the presence of LPS. This observation provided validation for the involvement of signaling pathways such as NF-κB, p38 MAPK, and JNK in the TLR4-mediated induction of BAFF (Yan et al., 2014). IL-17, a pro-inflammatory cytokine, was markedly diminished in the skin-draining lymph nodes of mice that received TLR3-primed WJ-MSCs (Park et al., 2019).

Recent studies have revealed that osteogenesis could be enhanced by activating a proper immune response in the microenvironment. Of the several main immune cells, due to their high plasticity and multi-function, macrophages hold crucial importance as an innate immune cell population involved in the regulation of MSC-based bone regeneration. Macrophages have been extensively characterized into distinct phenotypes based on their receptor expression, cytokine production, effector function, and chemokine repertoires. The two broad categories are M1 and M2 macrophages. M2 macrophages can further be classified into sub-populations, including M2a, M2b, M2c, M2d, and M2f, which are determined by the specific differentiation biomolecules and expressed markers associated with their differentiation process (Murray and Wynn, 2011).

Upon activation, the classically activated M1 macrophage subset demonstrates elevated production of pro-inflammatory mediators such as TNF-α, IL-6, and IL-1β. Conversely, alternatively activated M2 macrophages perform functions related to extracellular matrix (ECM) synthesis and remodeling. They release anti-inflammatory, angiogenic, and growth factors, including IL-10 and TGF-β (Nadine et al., 2022). Early studies demonstrated that sustained activation of M1-type macrophages leads to chronic inflammation and fibrosis, which can have a detrimental impact on the outcome of bone regeneration (Wang et al., 2022). In contrast, the M2 phenotype can reduce inflammation and promote tissue repair. The polarization of macrophages plays a critical role in the regulation of inflammation and serves as a significant target for MSC-based bone regeneration (Hanson et al., 2014). M2 macrophages upregulated IL-4, IL-10 and released higher amounts of BMP-2, vascular endothelial growth factors (VEGF), and TGF-β, thus enhanced osteogenesis and biomineralization of MSCs (Zhu et al., 2021; Wang et al., 2022). Contrary to previous assumptions, recent research has indicated that the presence of M1 macrophages can promote osteogenesis, while an excessive shift towards the M2 phenotype may result in fibrous tissue healing (Mokarram and Bellamkonda, 2014). Cu-incorporated biomaterials exhibited a positive impact on osteointegration, characterized by enhanced expression levels of M1 surface marker CD11c, growth factor BMP-6, as well as osteogenic markers such as OCN and Runx-2 at the interface between the biomaterial and bone tissue in a rat model, suggesting that activating M1 phenotype plays a key role in MSC recruitment and osteogenesis (Huang et al., 2019). Guihard et al. provided evidence that inflammatory M1 macrophages secrete OSM, which promotes osteogenesis of MSCs in an in vitro setting (Guihard et al., 2012). Considering recent studies, it is hypothesized that both M1 and M2 macrophages play crucial roles in the bone healing process. Therefore, precise regulation of the timing of M1/M2 transition rather than the specific phenotype is deemed essential for optimal outcomes in bone regeneration. An ideal paradigm might be activating the inflammatory phase at the early stage of repair, when the macrophages are mainly M1 phenotype, recruiting MSCs and restraining the inflammation to a mild manner. Subsequently, a quick switch to M2 results in an osteogenesis-enhancing cytokine release pattern, helping new bone formation.

Other immune cells also contribute to MSC-intermediated bone formation. Depletion of DCs (dendritic cells) promoted MSC-induced bone formation (Zhao et al., 2021). Infusion of CD4⁺ T cells in mice blocked ectopic bone formation through secretion of TNF-α and IFN-γ, which inhibited MSC differentiation and induced MSC apoptosis, while infusion of CD4⁺ CD25⁺ Treg abolished TNFα and IFN-γ production and improved MSC-mediated bone regeneration (Liu et al., 2011). The secretion of IL-17 by T-lymphocytes represents another crucial aspect during osteogenic differentiation. T-lymphocytes that release IL-17 actively promote the proliferation and osteoblastic differentiation of m-BMSCs (Ono et al., 2016). The dynamic microenvironment of the bone matrix is summarized in Figure 2.

A limited number of TLR-susceptible miRNAs identified in MSCs are involved in the positive regulation of TLR signaling. Fafian et al. reported that stimulation with LPS resulted in an upregulation of miR-21-5p, and inhibition of miR-21-5p led to a decrease in the expression of TLR4 as well as downstream cytokines (IL-6, IL-1β) by m-BMSCs. Moreover, suppression of miR-21-5p resulted in an upregulation of Wnt5a transcription, suggesting that the enhancing effect of miR-21-5p on TLR signaling is mediated by the negative regulation of Wnt (Fafian-Labora et al., 2017). According to Li et al., mAMSC-EVs transfected with miR-301a inhibitor induced M2 macrophage polarization, evidenced by strong elevation of Arg-1, CD163, CD206, and IL-10. Inhibition of miR-301a in mAMSC-EVs also suppressed TLR4 expression in macrophages, suggesting that miR-301a regulates immunomodulatory functions of mAMSC-EVs via the TLR4 pathway (Hsu et al., 2020). Similarly, the overexpression of miR-301a led to an elevation in the expression or secretion of IL-6, IL-8, COX-2, PGE2, IFN-β, and significantly induced the expression of IDO by UC-MSCs (Huang et al., 2013). On the other hand, the majority of miRNAs have a reducing effect on TLR signaling activation, implying their role in downregulating TLR-mediated immune responses in MSCs. In the middle cerebral artery occlusion (MCAO) model, miR-150-5p was found to be reduced while the TLR5 level was augmented. Treatment of hBMSC-EVs ameliorated neurological function, and pathological changes, and reduced inflammatory factors. Enrichment of miR-150-5p in the EVs exerted a protective role against brain ischemia/reperfusion (I/R) injury by inhibiting TLR5. The upregulation of TLR5 counteracts the protective effect of elevated miR-150-5p in brain I/R injury (Li et al., 2022). To mimic the hypoxic microenvironment in vivo, Zhou et al. isolated hBMSC-EVs under hypoxic status (H-sEVs). In intervertebral disc degeneration (IDD) model, by inhibiting the TLR4 pathway and activating the PI3K/AKT signaling pathway, H-sEVs miR-17-5p enhanced the proliferation of human nucleus pulposus cells (HNPCs) and promoted ECM synthesis, contributing to the alleviation of IDD by facilitating the proliferation and synthesis of the HNPCs’ matrix (Zhou et al., 2022). UCMSC-EVs alleviated suprapubic mechanical allodynia and frequent micturition in interstitial cystitis (IC) rats by inhibiting the TLR4/NF-κB pathway (Zhang et al., 2022). M-BMSCs transfected with miR-182-5p overexpressed it in their EVs. The expression of TLR4 in both MSCs and neonatal mouse ventricular myocytes (NMVM) was reduced by miR-182-5p. Treatment of MSC-EVs enhanced cell viability and suppressed the expression of inflammatory cytokines in NMVM posed with H₂O₂. Additionally, they promoted cardiac function in mice with myocardial infarction (MI), inhibited the inflammatory response, and inactivated the TLR4/NF-κB signaling pathway (Sun et al., 2022). Qiu and others used UCMSC-EVs to treat intracerebral hemorrhage following BBB disruption induced by thrombolysis of ischemic stroke with tissue plasminogen activator (tPA). In the MCAO model, the homing of EVs to the ischemic brain was enhanced by tPA. The administration of accumulated EVs demonstrated a reduction in tPA-induced disruption of BBB integrity and alleviation of hemorrhage by suppressing astrocyte activation and inflammation. The underlying mechanism involved the delivery of miR-125b-5p by MSC-EVs, which played a crucial role in maintaining BBB integrity by targeting Toll-like receptor 4 (TLR4) and inhibiting NF-κB signaling in astrocytes (Qiu et al., 2022). MiR-21a-5p, which was reported to activate the TLR pathway, was also found to suppress inflammation by targeting TLR4 and programmed cell death 4 (PDCD4). Furthermore, the therapeutic efficacy of EVs in sepsis was partially diminished when transfected with miR-21a-5p inhibitors (Niu et al., 2023). Liu et al. reported that miR-342-5p secreted by h-AMSC-derived EVs negatively regulated TLR9 to induce autophagy, thereby attenuating inflammation induced by LPS and improving acute kidney injury (AKI) in a mouse model (Liu et al., 2023). hplMSC-EVs could ameliorate LPS-induced inflammation in RAW264.7 cells by regulating TLR4-mediated NF-κB/MAPK and PI3K signaling pathways (Hu et al., 2022). Human tonsil-derived MSC (hTMSC)-EVs were discovered to have regulatory effects in mast cells under TLR7 stimulation. hTMSC-EVs containing miRs reduced inflammatory cytokines, mast cell, and CD-14 cell numbers in IMQ-treated cells and mice (Cho et al., 2022). Gao et al. discovered that UCMSC-derived EVs restrained TLR2/MyD88/NF-κB signaling activation in the spinal microglia, and intrathecal injection reduced chronic constriction injury (CCI)-induced mechanical allodynia, though the exact molecule involved was not studied (Gao et al., 2023). In another severe burn model, the injury significantly amplified the inflammatory response in rats or macrophages exposed to LPS, leading to increased levels of TNF-α and IL-1β, while reducing the levels of IL-10. The administration of UCMSC-EVs successfully reversed this reaction. miR-181c plays a pivotal role in regulating inflammation. The overexpression of miR-181c in UCMSC-EVs more effectively inhibited the TLR4 signaling pathway and alleviated inflammation in burn-injured rats (Li et al., 2016b).

LPS primed-MSCs have shown paracrine effects, including increased trophic support and improved regenerative and repair properties. Therefore, Ti et al. demonstrated that LPS-prestimulated UCMSC-EVs upregulated the expression of anti-inflammatory cytokines and promoted M2 macrophage activation. The let-7b/TLR4 pathway was identified as a potential contributor to macrophage polarization and inflammatory ablation. While in an in vivo setting, LPS pre-EVs demonstrated significant alleviation of inflammation and enhanced healing of diabetic cutaneous wounds (Ti et al., 2015). Monsel and others examined the therapeutic effects of hBMSC-EV in acute lung injury (ALI) in mice. Compared to standard EVs, TLR3-primed EVs elevated the expression of COX2 and IL-10, and decreased expression of TNF-α. The primed EVs also increased the phagocytic index of monocytes and further reduced the BAL fluid bacterial cfu count (Monsel et al., 2015). TLR4-Primed h-MSC (origin not mentioned) EVs exhibited a greater efficacy in counteracting the effects of LPS on microglia compared to EVs derived from MSCs without stimulation. Furthermore, TLR4-primed EVs exhibit greater effectiveness in inhibiting microglia and astrocyte activation, reducing amyloid deposition, preventing demyelination, mitigating memory loss, and improving motor and anxiety-like behavioral dysfunction, which demonstrated the enhanced immunoregulatory potential against neuro-inflammation triggered by LPS (Markoutsa et al., 2022). EVs from MSCs retain antimicrobial capacity as well. Priming hBMSC-EVs with Poly(I: C) enhanced antimicrobial characteristics, which led to the upregulation of EV proteins, out of which 21 miRs have been identified as crucial components in host defense and innate immunity (Pierce and Kurata, 2021).

In conclusion, MSC-derived EVs hold great potential as a cell-free therapeutic approach for bone regeneration through osteo-immunologic modulation although they have not been extensively used. Their diverse cargo and regenerative properties mediated by TLRs make them valuable tools for disease treatment and tissue regeneration, offering new possibilities for precision medicine and personalized therapies.