Regenerative properties of bone marrow mesenchymal stem cell derived exosomes in rotator cuff tears

Abstrct

Rotator cuff injury (RCI), characterized by shoulder pain and restricted mobility, represents a subset of tendon-bone insertion injuries (TBI). In the majority of cases, surgical reconstruction of the affected tendons or ligaments is required to address the damage. However, numerous clinical failures have underscored the suboptimal outcomes associated with such procedures. Further investigations have revealed that these failures are largely attributable to delayed healing at the tendon-bone interface, excessive formation of vascularized scar tissue, and inadequate integration of tendon grafts within bone tunnels. As a result, the healing process of rotator cuff injuries faces significant challenges.Bone marrow-derived mesenchymal stem cell exosomes (BMSC-exos) have emerged as a prominent focus of research within the field of bioengineering, owing to their remarkable potential to regulate cellular proliferation and differentiation, modulate immune responses, and facilitate tissue repair and regeneration following cellular damage. In this review, we explore the anti-inflammatory, angiogenic, anti-scarring, and bone metabolism-modulating effects of BMSC-exos in the context of rotator cuff injury. Additionally, we address the limitations and ongoing challenges within current research, offering insights that could guide the clinical application of BMSC-exos in the treatment of rotator cuff injuries in the future.

Introduction

In recent years, the growing prevalence of physical exercise and fitness has been accompanied by an escalating risk of musculoskeletal injuries. Consequently, tendon-bone injuries have become increasingly common, particularly in sports and other high-intensity physical activities. Among these, rotator cuff injuries (RCI) represent the most prevalent form of tendon-bone trauma. In the United States alone, approximately 200,000 rotator cuff repairs are performed annually, underscoring the widespread impact of this condition [1,2,3,4,5,6]. Current clinical treatment protocols for RC usually follow a graded treatment strategy with conservative treatment followed by surgical intervention if necessary. Initial management of RC usually includes conservative measures aimed at reducing pain and inflammation while promoting healing and maintaining function. Examples include rest and activity modification, physical therapy, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroid injections, where patients may face complications such as cardiovascular disease, gastrointestinal disorders, or diabetes mellitus when using NSAIDs or corticosteroids for long periods of time [7,8,9]. Conservative treatment of RC has the advantage of being non-invasive, avoids the risk of surgery, and is indicated for mild to moderate injuries, with gradual restoration of function, primarily through therapeutic physics, medications, and injections. However, patients vary from person to person, and severe tears or chronic injuries additionally have limited recovery and higher risk [10, 11]. Surgery may be necessary for patients who do not respond to conservative treatment within 6 to 12 months, or for patients with complete tears. Clinically RC enthesis injuries are usually treated surgically with tendon/ligament reconstruction, which involves inserting a tendon graft into a bone tunnel in order to establish tendon-to-bone healing. In spite of that, re-injuries after surgical reconstruction are extremely high, and 94% of repaired RCs re-tear after surgery [12, 13].

The rotator cuff is an important structure in the shoulder that is vital to the stability and function of the shoulder joint. The rotator cuff consists of the supraspinatus, infraspinatus, teres minor, subscapularis, and their accessory muscles, which work together with the tendons to ensure stability and motion of the shoulder joint [14, 15]. Structurally, the rotator cuff insertions onto the humeral tuberosity are continuous, wide, multilayered, and tightly adherent to the glenohumeral joint capsule, and close to their insertion onto the humeral tuberosity, the teres minor and infraspinatus are tightly attached near their tendon junctions, and the supraspinatus and infraspinatus are fused 15 mm proximal to their insertions. The supraspinatus and subscapularis muscles fuse to form a tunnel for the biceps tendon proximal to the biceps groove. The load on any one of the musculotendinous units is distributed over a wide area, and this structure improves the resistance of the rotator cuff to failure [16, 17]. Fundamentally, Growth of grafts, especially regeneration of fibrocartilage, is critical to adequate tendon-bone healing. At the rotator cuff (RC), the tendon attaches to the bone through a complex structure comprising four distinct zones: Zone I, which is dense connective tissue; Zone II, composed of uncalcified fibrocartilage; Zone III, featuring calcified fibrocartilage; and Zone IV, which is the bone. This gradient of tissue types facilitates a gradual transition that supports effective load transfer and resilience at the tendon-bone interface. Therefore, after rotator cuff injury, type I collagen fibrosis, forming sharpey fibers, which in turn promote tendon healing. However, there is a transition between soft tendon tissue and hard bone tissue that heals slowly due to vascular dysfunction, especially in fibrocartilage [12, 18,19,20,21]. Moreover, the scar tissue formed by excessive fibrosis of type I collagen has a decreased maximum stress threshold since there is no natural gradient structure at the tendon-bone interface and increases the risk of injury again [22, 23]. In order to reduce the re-tearing rate, a variety of different methods have been used clinically, and the results are not satisfactory. In comparison to bone-bone and tendon-tendon healing, tendon-bone healing is particularly difficult since sharpey fiber regeneration is limited.

Bone marrow-derived mesenchymal stem cells (BMSCs), recognized for their pluripotency and capacity for both self-renewal and multilineage differentiation, have garnered significant attention in recent research. Under specific inductive conditions, BMSCs possess the remarkable ability to differentiate into osteoblasts, chondrocytes, and tendon cells, offering considerable potential for the regenerative treatment of rotator cuff injuries (RCI) [24,25,26,27,28]. However, transplantation of BMSCs may also lead to immune rejection and is potentially tumorigenic [29,30,31]. Moreover, direct application of BMSCs to the site of injury will result in a decrease in the number of these cells and a decrease in the associated healing effect, and may lead to ectopic differentiation and vascular blockage [32,33,34,35]. As a result, the development of stem cell-based therapies has faced significant challenges. However, recent studies suggest that the therapeutic benefits of stem cells are primarily attributed to their paracrine signaling mechanisms. Among these Mesenchymal Stem Cell-derived Exosomes (MSC-exos) have emerged as the most pivotal components of the paracrine pathway, playing a crucial role in mediating the therapeutic effects [36]. MSC-exos are a type of extracellular vesicle, typically 30–150 nm in diameter, characterized by a bilayer lipid membrane structure. They contain bioactive lipids, proteins, receptors, mRNA, microRNAs (miRNAs), and long non-coding RNAs, playing essential roles in cell-to-cell communication [37,38,39,40,41]. MSCs-exos, as an effective alternative cell therapy, has stronger in vivo transport and targeting ability with fewer side effects, which makes it particularly suitable for tissues that are difficult to be completely repaired, such as rotator cuff tendon injuries. In addition, MSCs-exos possesses a variety of biological functions such as anti-inflammatory and antioxidant, and a large number of preclinical animal experiments and in vitro studies have demonstrated that MSCs-exos is effective in promoting rotator cuff tissue repair, which has gained a great deal of attention from the medical community [42, 43]. BMSC-exos are one of many MSC-exos, [44]which have the advantages of easy availability and low cost compared to other MSC-exos [45]. Compared with exosomes from other sources, BMSC-exos demonstrate unique advantages in rotator cuff injury treatment due to their low immunogenicity, potent anti-inflammatory and anti-apoptotic abilities, promotion of angiogenesis, and broad tissue adaptability [46,47,48]. It has been reported that ADSC-exos does show good results in wound healing, especially in promoting skin healing and fibroblast proliferation, but in terms of bone regeneration and anti-inflammatory functions, BMSC-exos has more significant advantages [49,50,51]. Extracellular vesicles, or BMSC-exos, are secreted by bone marrow mesenchymal stem cells and contain a range of bioactive substances, including extracellular matrix proteins and growth factors. By stimulating processes like cell proliferation, angiogenesis, and collagen synthesis, these molecules can aid in tissue regeneration and repair at the site of RCI. Among these processes, the RNA or protein in BMSC-exos can play a role in cell homing, regulate cell differentiation, lower inflammatory and immune responses, and ultimately aid in the repair of RCI [52,53,54]. Therefore, BMSC-exos therapy has great promise in the treatment of rotator cuff injury. (Fig. 1).

Fig. 1

Schematic diagram of the extraction, isolation and biological structure of BMSC-exos

At present, ensuring favorable outcomes in the treatment of rotator cuff injuries remains a significant challenge. Consequently, the use of BMSC-exos as a therapeutic strategy has become a prominent focus in the field of biomedicine. In this review, we explore the biology of BMSCs and the physiological structure of the rotator cuff. Furthermore, we delve into the various mechanisms by which BMSC-exos facilitate the healing of rotator cuff injuries. Finally, we address the current limitations of BMSCs-exo-based therapies and propose future research directions to enhance the effectiveness of these strategies in rotator cuff repair.

Pathologic changes in Rci

The rotator cuff is a crucial structure for shoulder stability and function, consisting of the supraspinatus, infraspinatus, teres minor, subscapularis, and their accessory muscles, with tendons that are tightly adherent to the glenohumeral joint capsule and humeral tuberosity, enhancing the shoulder's load distribution and resistance to failure. Factors contributing to rotator cuff disease can be categorized as extrinsic (trauma) and intrinsic (age-related degenerative changes, inflammation, and insufficient blood supply, etc.) [15, 55]. Intrinsically influenced RCI are usually degenerative lesions of the rotator cuff ligaments and tendons. Endogenous tendinopathy is primarily a pathologic change caused by degeneration of the tendon itself, which manifests as structural damage and dysfunction of the tendon. These changes include disorganization of the collagen fiber arrangement of the tendon, increased collagen degradation, increased water in the matrix, and apoptosis. Degenerative changes may also be accompanied by angiogenesis and inflammation, which together result in decreased elasticity, strength, and tolerance of the tendon, ultimately affecting its normal function [56, 57]. Exogenous RCI are usually caused by acute trauma or strenuous exercise, resulting in a direct tear or rupture of the tendon. These injuries cause structural damage to the rotator cuff tendons, which is manifested by rupture of the tendon fibers, hemorrhage, and a local inflammatory response. The site of injury is often associated with tissue swelling and pain, and in severe cases, may lead to bone damage at the point of attachment of the tendon. Post-traumatic pathological changes also include the release of inflammatory mediators, the process of regeneration of collagen fibers, and repair, but incomplete or inappropriate repair may result in poor functional recovery of the rotator cuff [58,59,60].

Isolation, purification and drug loading of bmsc-exos

BMSC-exos are small vesicles, typically ranging from 30 to 150 nm in diameter, secreted by cells and endowed with a wide array of biological functions and promising clinical applications. The isolation of BMSC-exos is commonly achieved through several techniques, including ultracentrifugation, density gradient centrifugation, immunoaffinity capture, nanofiltration, and ultrafiltration [61, 62]. Currently, a variety of methods exist for the isolation of BMSC-exos, yet no single approach has been universally established as the gold standard. Among the most widely employed techniques is ultracentrifugation, which utilizes a stepwise gradient centrifugation process to separate BMSC-exos from cell culture supernatants or biological samples. Initially, the sample is subjected to low-speed centrifugation to eliminate cells and larger debris, followed by medium-speed centrifugation to remove smaller fragments and microscopic particles. The exosomes are then isolated through high-speed centrifugation (typically at speeds exceeding 100,000 × g), during which they sediment at the bottom of the centrifuge tube. Finally, the BMSC-exos are purified by collecting the precipitate and resuspending it, resulting in a purified exosome preparation. Ultracentrifugation is a commonly used method for exosome (e.g., BMSC-Exos) isolation because of its cost-effectiveness, wide applicability, and high yield, but it also has some limitations. The main drawbacks include limited separation purity, susceptibility to co-sedimentation of impurities, and complex and time-consuming operation. In addition, ultrahigh centrifugal force may cause mechanical damage to exosome structures and affect their function [63, 64]. Therefore, although ultracentrifugation is widely used in exosome isolation, studies with high purity and activity requirements usually need to be combined with other purification steps [64, 65]. Methods for loading drugs after exosome isolation include, co-incubation of BMSC-exos with drugs at room temperature, direct embedding, electroporation, gene transfection, and surface modification [66,67,68]. For therapeutic applications targeting shoulder injury, such as promoting anti-inflammatory and angiogenic effects, specific exosome drug loading methods are tailored to each purpose. When exosomes are loaded with different drugs, it is necessary to optimize the selection of methods according to drug properties, therapeutic targets and release time. Small molecules of anti-inflammatory drugs are mostly used in direct incubation and freeze-thawing method, because of its stable structure and easy operation [69]; large molecules of pro-angiogenic factors are suitable for ultrasonication and electroporation to ensure the activity of the drug and promote the penetration; gene drugs or siRNA loading is commonly used in electroporation to maintain the activity of nucleic acids [70], and at the same time can be chemically modified to achieve the targeting of the delivery and the combination of the drug loaded (e.g., anti-inflammatory drugs and pro-angiogenic factors) are suitable for Polymer composite sustained-release systems for long-lasting, stable drug release [71]. However, the membrane structure of exosomes, which consists mainly of a phospholipid bilayer, is hydrophilic, which makes it difficult to load lipid-soluble drugs. To address this challenge, researchers have employed a variety of sophisticated techniques to facilitate the efficient loading of lipid-soluble drugs. One widely utilized approach is the liposome-exosome co-loading method, wherein liposomes encapsulate lipid-soluble drugs and subsequently fuse with exosome membranes, thereby transferring the drug cargo into the exosomes [72, 73]. This strategy capitalizes on the inherent compatibility between liposomes and exosome membranes, significantly enhancing both the loading efficiency and stability of the encapsulated drugs. Another technique that has gained traction involves ultrasound-assisted drug loading, which employs acoustic waves to non-invasively disrupt the exosome membrane, thereby promoting the entry of lipid-soluble drugs [74]. It has been demonstrated that ultrasound can increase the plasticity of the exosome membrane, enabling more efficient encapsulation of these drugs. The lipid-soluble nature of Nordy limits its in vivo efficacy in the water-soluble circulation of the human body. HUA et al. utilized ultrasound to induce microbubbles to load Nordy and successfully achieved the targeted release of Nordy to improve the antitumor efficacy [75]. Small molecule anti-inflammatory drugs, such as NSAIDs or glucocorticoids, are usually more hydrophilic and have low molecular weight. For such drugs, the direct incubation method BMSC-exos is co-incubated with the drug, which is a relatively simple and effective method to load the drug into BMSC-exos, mainly by mixing BMSC-exos with the drug and co-incubating them under certain temperature and time conditions, so as to allow the drug to enter into the interior of the BMSC-exos through a concentration gradient or membrane permeation. It has been shown that exosomes were extracted from hUC-MSCs and then co-incubated with miR-23a-3p, which was demonstrated to promote osteogenesis and angiogenesis and vascularized bone regeneration by delivering miR-23a-3p to activate the PTEN/AKT signaling pathway [76]. In addition to this, curcumin when mixed with exosomes in the presence of 10% ethanol: acetonitrile (1:1) can achieve 18–24% drug loading when incubated at room temperature and formulations stored at − 80 °C are stable for up to 6 months as determined by particle size analysis, drug loading and antiproliferative activity [77]. However, it is worth noting that the efficiency of incubation loading of drugs may be affected by the large amount of proteins and nucleic acids carried by the exosomes themselves. Therefore, the nature of the drug and the characteristics of the exosome need to be considered comprehensively in practical applications in order to optimize the loading conditions and improve the loading efficiency. It is worth mentioning that loading drugs onto MSCs and then isolating exosomes and direct loading onto isolated exosomes each have their own advantages and challenges; the MSC drug loading method can enhance the drug loading capacity and stability of exosomes, however, the survival and drug loading rate of MSCs are affected by cellular metabolism and the microenvironment, and its success rate is low [78, 79]. In contrast, exosome direct drug loading has better biodegradability and biocompatibility, and can achieve precise targeting and controlled drug release through surface functionalization [80,81,82]; therefore, this review demonstrates that exosome direct drug loading is more conducive to targeting therapeutic goals, developing drug properties and technical feasibility.

Mechanisms of bmsc-exos in the treatment of Rci

Immune regulatory mechanism

After surgical reconstruction of rotator cuff injury, rotator cuff structures undergo inflammatory, proliferative, remodeling, and plastic phases [83]. The inflammatory phase is the first stage of rotator cuff injury, and when the graft is implanted into the bone tunnel, it leads to the accumulation of various inflammatory cells. Initially, inflammatory factors recruit macrophages and neutrophils to heal rotator cuffs and remove tissue debris, followed by collagen deposition and fibroblast infiltration [84]. Macrophages are cells that play a major role in the inflammatory mechanism and are divided into M1 type and M2 type according to their function; the former mainly secretes pro-inflammatory factors, and the latter reduces the inflammatory response and exerts tissue repair function [85,86,87]. During the inflammatory phase of rotator cuff injury, massive macrophage accumulation and M1 polarization at the reconstructed site can enhance apoptosis, reduce cell proliferation, and induce fibroblasts to secrete excessive extracellular matrix, leading to peritendinic fibrosis and scar tissue formation [43, 88]. [79] Studies indicate that following acute trauma, there is a disruption in the macrophage M1/M2 balance, unveiling the pivotal immunomodulatory role of macrophages in the acute inflammatory response. This observation underscores the critical function of macrophage polarization in orchestrating immune responses in the early phase of trauma, suggesting their potential as therapeutic targets in the modulation of inflammation [89]. However, Wu et al. investigated whether conditional macrophage depletion increased inflammation, and compared with the control group, mice with macrophage depletion had significantly fewer M1 and M2 macrophages in the surgical joint, but did not reduce the severity of OA in obese mice, on the contrary, induced a systemic inflammatory response [90]. Therefore, the role of macrophages in the inflammatory response requires further investigation. Studies have shown that BMSC-exos can promote rotator cuff healing by polarizing macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, reducing inflammatory responses of transplants after surgical reconstruction. However, the mechanism behind this phenomenon is not yet clear [43, 91]. Huang et al. [92] demonstrated that BMSC-derived exosomes (BMSC-exos) enhance rotator cuff tendon-to-bone healing in rats by modulating M1 macrophages. Their findings revealed that BMSC-exos administration led to improved fracture load and stiffness, alongside enhanced growth at the bone-tendon interface. Further research has demonstrated that the topical application of BMSC-derived exosomes (BMSC-exos) promotes fibrocartilage formation by enhancing M2 macrophage polarization, which in turn improves biomechanical properties [88]. Related studies have also shown that BMSC-derived extracellular vesicles (BMSC-EVs) contribute to tendon healing by reducing inflammation and apoptosis, while simultaneously increasing the presence of tendon-resident stem and progenitor cells [93]. Numerous studies have shown that BMSC-exos can promote tendon-bone healing by polarizing macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype, but the underlying mechanism remains unclear [94,95,96]. The study by Xiang et al. [97] demonstrates that NZ exerts anti-inflammatory effects by suppressing NO production and pro-inflammatory cytokine release via inhibition of the TLR4/MyD88/NF-κB pathway. Similarly, sinigrin has been reported to offer immunomodulatory benefits by inhibiting the NF-κB/MAPK pathway or preventing NLRP3 inflammasome activation [98]. Building on these insights, additional research suggests that GEN-27 exhibits strong anti-inflammatory properties by targeting and suppressing both NLRP3 inflammasome and NF-κB pathway activation [99]. Furthermore, evidence indicates that PM2.5 triggers NLRP3 inflammasome activation through downstream signaling of the TLR4/MyD88 pathway, as well as MAPK and NF-κB pathways, highlighting the complexity of PM2.5-induced inflammatory mechanisms [100]. Expanding this area of study, exosomes have been explored as a therapeutic option for myocardial infarction (MI). Notably, FNDC5 combined with BMSC-derived exosomes has demonstrated significant anti-inflammatory, anti-apoptotic, and M2 macrophage-polarizing effects in vivo [101]. These findings suggest that FNDC5-BMSC-exosomes promote anti-inflammatory responses and facilitate M2 macrophage polarization via the NF-κB signaling pathway and the Nrf2/HO-1 axis. Collectively, these studies underscore the intricate regulation of inflammatory responses by multiple signaling pathways, yet further investigation is essential to pinpoint the dominant mechanisms involved. (Table 1).

Table 1 Study on anti-inflammatory effect of BMSC-exos

Angiogenesis promotion

In contrast to muscle and skin tissue, tendon and ligament vascularization is frequently restricted, especially at tendon-bone insertion. Tendon-bone insertion (TBI) is the transition between soft tendon tissue and hard bone tissue that heals slowly due to vascular dysfunction, especially in the fibrocartilage area [13, 102, 103]. After TBI injury, poor angiogenesis at the tendon-bone junction reduces the utilization of essential nutrients, growth factors, and oxygen levels, resulting in poor biomechanical properties and ultimately tendon-bone healing [104]. More and more studies have shown that neovascularization is essential to promote tendon bone healing. Studies have shown that ACL-derived cells contribute to traumatic brain injury (TBI) recovery by enhancing the brain’s intrinsic angiogenesis capabilities, highlighting the broader therapeutic role of angiogenesis in tissue repair [105]. Extending this concept, ACL-derived CD34( +) cells significantly promote tendon-to-bone healing post-ACL reconstruction via angiogenesis, suggesting a conserved mechanism across injury contexts [106]. This angiogenic function is also critical in rotator cuff repair, where the VEGF-Hippo signaling pathway has been shown to promote tendon-to-bone healing by inducing angiogenesis, underscoring its role in musculoskeletal injuries [107]. Exosomes, known for their rich content of growth factors, have shown promise in regenerative therapy. For instance, BMSC-exos, administered intravenously in rats following rotator cuff reconstruction, not only stimulated angiogenesis at the rotator cuff interface but also enhanced tendon-to-bone healing, highlighting exosomal delivery’s therapeutic potential [92]. Similarly, in a model of MCAO/R-induced brain injury, BMSC-exos promoted tissue repair and angiogenesis by delivering Egr2, which upregulates SIRT6 expression and inhibits Notch signaling, reinforcing the role of exosomes in vascularization and recovery [108]. Further evidence supports that angiogenesis and osteogenesis are closely linked during bone remodeling, with BMSC-derived exosome miR-29a regulating both processes, although the exact pathway remains unknown [109]. Notably, while early neoangiogenesis is essential for tendon healing after rotator cuff reconstruction, excessive vascular scar formation may weaken the tendon interface and raise the risk of retearing [83, 110]. Together, these findings underscore the dual role of angiogenesis in healing and the need for precise regulation to optimize long-term repair outcomes across different tissues.

Research suggests that in vascular-rich tissues such as tendons and ligaments, limiting rather than promoting vascular growth during wound repair may improve long-term healing outcomes and potentially restore full tissue functionality [111]. Similarly, angiogenesis in tissue repair can exacerbate scar formation and reduce tendon stiffness, which may lead to rotator cuff pain and restricted function [112]. Despite extensive studies, the precise signaling pathways regulating angiogenesis remain unclear. For instance, exosome systems targeting nasopharyngeal carcinoma (VM) and angiogenesis, such as iRGD-exo-antagomiR, have shown that exosomes loaded with EBV-miR-BART1-5p-antagomiRs can inhibit VM and angiogenesis in a Spry2-dependent manner, both in vitro and in vivo [113]. In diabetic retinopathy (DR), miR-133b-3p upregulation or FBN1 downregulation has been shown to inhibit angiogenesis, cell proliferation, and migration while promoting apoptosis; BMSC-derived exosomal miR-133b-3p plays an anti-angiogenic role in DR via the FBN1A pathway [114]. Further, studies reveal that overexpression of miR-199a-5p significantly inhibits the proliferation, migration, and neoangiogenesis of HUVECs, with opposing effects observed upon its downregulation. Exosomal transfer of miR-199a-5p from osteosarcoma cells to HUVECs effectively targets VEGFA, thus restraining tumor growth and angiogenesis [115]. While much of the literature focuses on miRNA-carrying exosomes' anti-angiogenic effects in tumors, the mechanisms by which BMSC-exos inhibit angiogenesis in tendon-bone healing are still in early stages and warrant further exploration.(Table 2).

Table 2 Study on the effect of BMSC-exos on promoting angiogenesis

Scarring inhibition

In the early stages of tendon bone tissue repair, fibroblasts proliferate and produce extracellular matrix (ECM), which includes fibronectin, type 3 collagen, and type 1 collagen, which promotes the healing of tendon bone tissue [116,117,118]. There is growing evidence that BMSC-exos may be taken up by fibroblasts, which stimulate collagen production. Zhang et al. [119] studied the mechanism of exosomes of mesenchymal stem cells (MSC-Exos) in repairing damaged tissues, and the results showed that with the increase of hiPSC-MSC-Exos concentration, the secretion of type I and type III collagen and elastin of human dermal fibroblasts and the angiogenesis of human umbilical vein endothelial cells increased. Research has shown that bone marrow mesenchymal stem cell-derived exosome miR-542-3p significantly enhances skin wound healing by promoting collagen1/3 protein expression in treated human skin fibroblasts [120]. Furthermore, topical injections of miR-542-3p exosomes stimulate collagen deposition, fibroblast proliferation, and neovascularization, consistent with extensive findings that mesenchymal stem cell exosomes support fibroblast proliferation and collagen production. However, the role of BMSC-exos in repairing the rotator cuff tendon-bone interface has not yet been conclusively determined [121]. Studies suggest that LIPUS-pretreated BMSC-derived exosomes, as opposed to native BMSC exosomes, can significantly enhance fibrocartilage regeneration at the bone-tendon interface and reduce supraglottic fat infiltration. This improvement is primarily achieved through the targeted delivery of miR-140, which positively modulates prochondrogenic and anti-adipogenic pathways [122]. In tendon-bone repair, excessive fibroblast accumulation and collagen deposition often result in fibrosis and scar tissue formation, compromising tensile strength and increasing the risk of re-tearing [123, 124]. Addressing scar tissue formation is a critical issue in tissue repair. Studies have shown that the antifibrotic effects of MSC-derived exosomes include reduction in type I collagen expression, potentially inhibiting skin fibrosis through mechanisms such as miR-196b-5p [125]. Similarly, BMSC-exos mitigate skin fibrosis by delivering miR-214 to inhibit the IL-33/ST2 axis [126]. Although the antifibrotic capabilities of MSC-derived exosomes are well-documented, the precise signaling pathways involved remain under investigation. Further studies reveal that exosomal microRNAs from umbilical cord-derived mesenchymal stem cells inhibit myofibroblast formation by decreasing α-smooth muscle actin and collagen deposition via the TGF-β/SMAD2 signaling pathway, highlighting a crucial regulatory mechanism in fibrosis control [127]. It has been found that exosomes derived from human adipose mesenchymal stem cells (ASC-Exos) enhance matrix metalloproteinase-3 (MMP3) expression in dermal fibroblasts through activation of the ERK/MAPK pathway. This pathway modulation regulates type III and type I collagen synthesis and directs fibroblast differentiation, thereby reducing scarring and promoting extracellular matrix (ECM) remodeling in skin wound healing [128]. Li et al. [129] demonstrated that extracellular vesicles from adipose-derived mesenchymal stem cells (adMSC-Exos) reduce protein expression of Smad3 and Notch-1, as well as the expression of transforming growth factor β2 (TGF-β2), and upregulate the expression of TGF-β3, thereby reducing collagen protein accumulation. Meanwhile, TGF-β1-induced upregulation of p38 phosphorylation is reversed in fibroblasts. Existing studies have shown that MSC-esos have a variety of pathways to inhibit scarring, but the signaling pathways that play a major role are still uncertain, and the signaling pathways in which BMSC-exos inhibit scarring has not been reported and needs further study. (Table 3).

Table 3 Study on the effect of BMSC-exos on scar inhibition

Regulatory mechanisms of bone metabolism

After surgical reconstruction of rotator cuff injury, the degree to which the tendon graft is inserted into the bone and grows inward determines the strength of the tendon interface after healing [91, 130, 131]. In general, rotator cuff reconstruction has a very slow rate of bone growth, resulting in reduced integration of tendon grafts into the bone tunnel, which in turn reduces the biomechanics of the rotator cuff [130, 132, 133]. Existing studies have pointed out that BMP2-induced transcription factors constitute a signaling network for bone development and osteoblast differentiation. These regulators include Runx2 (Cbfa1/AML3), which has a variety of regulatory activities, including activation or inhibition of gene expression, and integration from biological signals such as BMP/TGF-β, Wnt, and Src signaling pathways that regulate the metabolism of bone tissue [134]. Therefore, RUNT-associated transcription factors-2 (RUNX2) and Osterix (Osx or Sp7) are osteoblast-specific transcription factors that are essential for osteoblast differentiation and are so-called osteogenic differentiation markers [135, 136]. Studies have shown that MSC-exos in rotator cuff injury can affect the proliferation and differentiation of osteoblasts by increasing the expression of transcription factors Osx and RUNX2 [137]. Fu et al. [138] explored the potential ability of adipose-derived stem cell exosomes (ADSC-exos)-mediated tendon stem cells (TDSC) to rotator cuff repair, and the results showed that ADSC-exos-containing hydrogels significantly improved osteogenic and adipogenic differentiation, enhanced the expression of RUNX2, Sox-9, TNMD, TNC and Scx, and the biomechanical properties of rotator cuffs. Similarly, BMSC-exos promote bone action is also a current research hotspot. For instance, studies on osteoporosis reveal that exosomes from BMSCs with upregulated miR-21-5p enhance protective effects more effectively than typical BMSC-exos, while those with downregulated miR-21-5p reduce these cellular benefits. Results indicate that miR-21-5p in BMSC-exos promotes osteoblast differentiation and alkaline phosphatase (ALP) activity by targeting KLF3 [139]. Further research on hyperglycemic bone degeneration demonstrates that exosomes with upregulated miR-140-3p promote the differentiation of diabetic BMSCs (DM-Exos) into osteoblasts by targeting plxnb1, a Sema4D receptor that usually inhibits osteocyte differentiation. This pathway, regulated by miR-140-3p, promotes bone formation by inhibiting Sema4D receptor plexin B1 and the plexin B1/RhoA/ROCK pathway, showing that both normal and miR-140-3p-overexpressed exosomes enhance BMSC osteogenic function [140]. In another study, BMSC-derived exosome miR-206 was found to improve inflammatory response in bone tissues of osteoarthritis (OA) mouse models, increasing expression of osteocalcin (OCN) and BMP2, thereby promoting osteoblast proliferation and differentiation through downregulation of Elf3 [141]. In addition to the above discussion of the effect of bone tunnel healing on the tendon interface, bone resorption can significantly hinder the growth of grafted tendons in the bone tunnel, reduce the extraction strength and stiffness of TBI, and lead to delayed early healing [142,143,144]. Osteoclasts are multinucleated cells with a unique ability to absorb the bone matrix, and their differentiation and activation are strictly regulated by osteoblasts. Therefore, inhibition of osteolysis is another factor affecting osseointegration of tendon grafts to the surface of the bone tunnel [142, 145, 146]. Chen et al. [147] in their study to prevent bone loss in a murine periodontitis model found that exosomes of M2-like macrophages (M2-exos) promote osteogenesis and inhibit osteoclast production through the IL-10/IL-10R pathway, reducing alveolar bone resorption in periodontitis mice. The inhibitory osteolytic effect of BMSC-exos has been shown to promote osseointegration of tendon grafts to the surface of the bone tunnel. Xiao et al. [148] studying exosomes secreted by circulating mechanical stretching (CMS)-treated BMSCs and normally static cultured BMSCs found that CMS_Exos effectively inhibited nuclear factor kappa-B ligand (RANKL)-mediated osteoclastiesis and receptor activators for F-actin loop formation, demonstrating that exosomes from CMS-treated BMSCs inhibit osteoclastiogenesis by weakening NF-κB signaling pathway activity in vitro, thereby mitigating bone loss. Therefore, promoting osteogenesis, inhibiting osteoclast activity or osteoclastic formation can promote the integration of graft tendons and bone marrow tract, thereby enhancing the healing of tendon bone interface, providing new targets for future clinical applications. (Table 4) (Fig. 2).

Table 4 Study on bone metabolism mechanism of BMSC-exos

Fig. 2

Mechanism of BMSC-exos in tendon injury

Prospects and challenges

Exosomes as carriers of miRNAs is generally believed that the anti-inflammatory, angiogenesis, inhibition of scarring, bone metabolism and other effects of exosomes are mainly attributed to small molecules in exosomes, such as proteins and miRNAs. It has been shown above that BMSC-exos promote the repair of rotator cuff injury by inducing macrophages to polarize to M2 type to achieve anti-inflammatory effects in the inflammatory phase after rotator cuff injury, but their specific signaling pathways need to be further explored [149]. In addition, following rotator cuff injury, the inflammation trimming process plays a pivotal role in tissue repair. The initial inflammatory response is triggered by the infiltration of immune cells such as macrophages, neutrophils, and lymphocytes. These cells secrete inflammatory mediators, including IL-6, TNF-α, and IL-1β, which help clear necrotic tissue and pathogens, thereby establishing a conducive environment for healing. Although chronic inflammation may contribute to tissue degeneration, a well-regulated inflammation trimming process facilitates the decomposition and remodeling of the extracellular matrix by modulating the activities of fibroblasts and tenocytes, laying the groundwork for effective tissue repair [150, 151]. Therefore, how to maintain a moderate inflammatory response through BMSC-exos is the key to repairing RCI. Similarly, BMSC-exos equipped with different miRNAs can promote or inhibit angiogenesis, as mentioned above, promoting angiogenesis in the early stage of rotator cuff injury is conducive to the healing of the tendon interface, and inhibiting angiogenesis in the later stage of injury is conducive to improving the biomechanical strength of tendon bone tissue and is more suitable for long-term healing, so determining the time point to promote or inhibit angiogenesis is an urgent problem for us to solve [152,153,154,155]. Equally, in the mechanism of fibrosis, there is a lot of evidence that regulated fibroblast proliferation is conducive to tendon bone healing, but excessive tissue remodeling will lead to fibrosis to form scar tissue, and how to use BMSC-exos to regulate fibroblast proliferation and ECM accumulation is an urgent problem to be solved [156,157,158]. At the same time, the role of BMSC-exos in bone metabolism is also the same mechanism, Huang et al. [159] studied the effect of artesunate (ART) treated BMSC-exos on osteogenesis, and the results showed that ART-BMSC-exos promoted osteogenesis by regulating the transmission of SNHG2 in the TAF7/RUNX15 axis. Xiao et al. [148] found that BMSCs_Exos after mechanical stretching (CMS) treatment effectively inhibits nuclear factor κ-B ligand (RANKL)-mediated osteoclast and receptor activator for the formation of F-actin loops, indicating that exosomes from CMS-treated BMSC inhibit osteoclast production by weakening NF-κB signaling pathway activity in vitro, thereby reducing bone loss. In summary, how BMSC-exos regulate the recruitment and anti-inflammatory mechanism of inflammatory factors, promote angiogenesis and inhibit angiogenesis, promote fibroblast proliferation and inhibit scarring mechanism, and the dynamic balance between osteoblast and osteoclast mechanism to better promote the process of rotator cuff injury is a major challenge we are currently facing [157, 160,161,162]. Overuse of an external force can result in a rotator cuff tendon tear, which can cause trauma and inflammation to the surrounding tissue and cause RCI. Nucleic acid components like microRNAs can regulate gene expression, and proteins and bioactive substances may also affect collagen synthesis, extracellular matrix regeneration, and angiogenesis [163, 164]. Proteins and non-coding RNAs within exosomes have emerged as critical subjects of research for the treatment of rotator cuff injuries, each playing distinct roles and possessing unique potential mechanisms in tissue regeneration and repair. Exosomes play a role in transporting proteins and other materials to recipient cells, and it has been reported that osteobridging protein (OPN), particularly its N-terminal fragment (N-OPN), is encapsulated in β-catenin-controlled renal tubular cell-derived exosomal cargoes, which are subsequently delivered to fibroblasts to promote fibroblast proliferation and activation [165]. Growth factors in exosomes can also reduce symptoms in the injured area, promote cell proliferation, and aid in tissue repair following rotator cuff injury. Exosomes enriched with non-coding RNAs exhibit a wide range of biological activities in the context of rotator cuff injuries, facilitating tendon tissue repair, mitigating inflammatory responses, preventing fibrosis, and promoting angiogenesis. In contrast, proteins within exosomes primarily contribute to tissue repair, immune modulation, and intercellular communication through direct biochemical interactions. Non-coding RNAs, however, exert their effects by modulating gene expression pathways, thereby indirectly influencing cellular functions and repair mechanisms [166,167,168]. Consequently, we propose that the integration of proteins and non-coding RNAs in exosome-based therapies could yield a synergistic effect in treating rotator cuff injuries: proteins drive rapid tissue repair, whereas non-coding RNAs sustain regulatory and anti-inflammatory activities over time. Exosomes have shown great potential in the treatment of RCI, but their collection and purification still face technical problems. However, the amount of miRNA required in BMSC-exos is poor, and it is difficult to meet the criteria required for the complex healing process described above; The content of BMSC-exos also limits its wide application. To overcome the shortage of natural exosomes, loading exogenous miRNAs into natural exosomes can be beneficial for RCI patients. Some scholars have found that cell-derived exosomes can be delivered to hepatocytes or macrophages with exogenous miRNA-155 mimics or inhibitors. At the same time, exosome-mediated delivery of miRNA-155 inhibitors has less cytotoxicity and more effective inhibition than traditional transfection methods. Similar exosome-mounted exogenous miRNA approaches can be used for targeted therapies with targets in vitro and in vivo [169]. Relevant studies have also shown that macrophage-derived exosomes are used as carriers to deliver exogenous miR-21 inhibitors to BGC-823 gastric cancer cells, and hepatocyte-derived exosomes carry miRNA-122 as a communication tool between monocytes and macrophages [170, 171]. The methods of its isolation and synthesis need to be further discussed [172, 173]. The spectrum of drugs that can be encapsulated within exosomes is vast, encompassing a diverse array of therapeutic agents ranging from anti-inflammatory and anti-cancer compounds to gene therapies. The selection of a specific drug is contingent upon the therapeutic objective, the drug’s inherent properties, and the desired efficacy of delivery. Exosomes, in their capacity as drug delivery vehicles, are particularly well-suited for the administration of anti-inflammatory agents, such as glucocorticoids (e.g., dexamethasone, chlortetracycline), owing to their ability to optimize drug efficacy via targeted delivery and controlled, sustained release [174, 175]. This system mitigates the unwanted side effects typically associated with systemic drug distribution. Such agents have demonstrated significant therapeutic potential in the treatment of chronic inflammation, autoimmune disorders, and musculoskeletal injuries, including rotator cuff damage. Moreover, in the realm of oncology, exosomes serve as proficient drug carriers, facilitating the targeted delivery of chemotherapeutic agents like paclitaxel and cisplatin directly to tumor sites [176, 177]. Through the natural biocompatibility and low immunogenicity of exosomes, this approach not only enhances drug accumulation within tumor tissues but also amplifies therapeutic efficacy while minimizing collateral damage to surrounding healthy tissues. The administration routes for exosomes and their associated safety and feasibility vary significantly. The primary methods include intravenous injection, local injection, nasal delivery, oral administration, and transdermal application. Intravenous injection enables systemic distribution but may trigger immune responses or be rapidly cleared from circulation [178]. Local injection increases the concentration at the target site and generally offers a higher safety profile but is applicable only to localized diseases [179]. Nasal administration is non-invasive and suitable for central nervous system disorders, yet its absorption efficiency may be limited [180]. Oral administration is well-accepted by patients but must overcome low bioavailability due to degradation in the gastrointestinal tract [181]. Transdermal delivery is safe and appropriate for skin diseases but is restricted by the skin barrier [182]. In summary, selecting an appropriate delivery modality necessitates balancing the disease type, target tissue, patient compliance, and the physicochemical properties of exosomes to optimize safety and feasibility. In the treatment of rotator cuff injuries, exosomes are predominantly administered through direct local injection into the damaged tendon area. This approach offers several advantages: it ensures efficient delivery and direct therapeutic action at the injury site, avoids the dilution and clearance associated with systemic drug administration, and enhances overall therapeutic efficacy. Moreover, exosomes contain biologically active molecules that promote cell proliferation, migration, and collagen synthesis, thereby accelerating tendon repair. Interestingly, the optimal injection frequency of exosomes has not been determined, and the benefits and harms of multiple injections and single injections have not been known. Li et al. [183] for the treatment of optic neuropathy by intravitreal administration of MSC-exosome found that the treatment of optic neuropathy should minimize repeated injections to ensure safety and efficacy. In addition, the frequency of injections of BMSC-exos in the treatment of different diseases varies and should be determined by extensive research. The application of BMSC-exos encounters numerous challenges, chief among them being the scarcity of clinical trials, the predominance of findings derived from animal models, and the absence of rigorous, standardized criteria for clinical use. A primary obstacle in advancing BMSC-derived exosome therapies is the limited number of clinical trials investigating their effects in human subjects. As of October 2023, research predominantly remains at the preclinical level, focusing on in vitro studies and animal models. For example, while BMSC-exos have demonstrated potential in promoting tissue repair and modulating immune responses in animal models, there are limited clinical studies validating these effects in humans [184]. This paucity of robust clinical trials raises important concerns regarding the safety, efficacy, and potential adverse effects of BMSC exosome therapies in patients. Moreover, most insights into BMSC exosome applications originate from animal studies. For example, BMSC exosomes have demonstrated the ability to promote nerve regeneration in murine models of stroke [185]. However, translating these findings to human settings is challenging, as the mechanisms governing injury response and repair in the human brain may differ markedly from those in rodents. This dependence on animal data contributes to significant uncertainty regarding the clinical efficacy of BMSC exosomes in human diseases.

Conclusion

BMSC-exos offer substantial promise for treating RCI and other musculoskeletal disorders, yet several challenges continue to hinder their clinical translation. Addressing these obstacles through innovative strategies—such as the integration of exogenous miRNA loading, refined isolation techniques, and personalized medicine approaches—could significantly enhance their applicability. Future research must prioritize elucidating the underlying mechanisms, optimizing delivery systems, and conducting rigorous clinical trials to unlock the full therapeutic potential of BMSC-exos.