Exosomes for the Management of Rotator Cuff Injuries: A Systematic Review

Abstract

Introduction

Rotator cuff injuries (RCIs) are a common musculoskeletal disability affecting daily activities and work performance for millions globally. Current conservative therapies are riddled with limitations. Therefore, this study aims to review the regenerative effects of exosomes derived from mesenchymal stem cells (MSCs) on RCIs and catalogue ongoing clinical trials investigating their effects.

Study Objective

The main aim of this study is to compile findings from in vitro, preclinical, and clinical research articles that evaluate the regenerative potential of exosomes derived from different MSCs on injuries to the rotator cuff. Additionally, the secondary objective is to catalogue ongoing clinical trials registered in various clinical trial databases that explore the impact of exosomes on rotator cuff injuries.

Search Criteria

A systematic search across four extensive online journal databases until March 2024 was conducted following PRISMA guidelines. The search focused on articles in English using specific terms related to exosomes and RCIs. Eligible studies, including basic science, preclinical, and clinical research, and were assessed by two independent reviewers. Additionally, registered trials were identified from three international clinical trial databases.

Results

Four basic science, one basic science and preclinical, and twelve preclinical studies were included in this review. The studies demonstrated that exosomes derived from various stem cells have a strong regenerative capability, whose effects may be further enhanced by adding growth factors, glycoproteins, or other pro-regenerative markers.

Conclusion

In vitro and preclinical studies reveal exosomes’ regenerative potential for RCIs. Further research, including clinical trials, is needed to assess exosomes’ safety and efficacy in RCI management.

Introduction

Rotator cuff injuries are prevalent in various populations, including athletes and older adults, leading to significant impacts on individuals’ quality of life and athletic performance, and are most commonly attributable to repetitive overuse [1]. The high prevalence of rotator cuff injuries underscores the importance of exploring innovative treatment approaches, such as the potential use of extracellular vesicles, specifically exosomes, to enhance the healing process and improve outcomes for individuals suffering from rotator cuff injuries.

Current treatment modalities for rotator cuff injuries face several limitations that impact their effectiveness in managing this common musculoskeletal condition. Traditional approaches such as physical therapy, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroid injections may provide symptomatic relief but often fall short in addressing the underlying pathology, leading to high rates of re-tears and persistent functional deficits [2]. Surgical interventions, while commonly employed for severe cases, are associated with challenges such as the risk of failure, especially in cases of irreparable massive rotator cuff tears [3–5] Additionally, the use of biologics like platelet-rich plasma and stem cells, although showing promise, lacks robust clinical evidence to support their widespread adoption and efficacy in promoting tissue regeneration and functional recovery [6–8]. In light of these limitations, there is a pressing need for further research and innovation to enhance the therapeutic options available for individuals suffering from rotator cuff injuries.

Exosomes are an emerging cell-free method of treatment within the field of orthobiologics. These small extracellular vesicles are secreted by various types of cells and have emerged as promising tools in regenerative medicine. These nanoscale lipid bilayer membrane capsules play a crucial role in intercellular communication by transferring bioactive molecules such as proteins, lipids, and nucleic acids [9]. In the realm of orthopedics, exosomes have shown significant potential in treating a spectrum of degenerative disorders, including osteoarthritis, osteoporosis, intervertebral disc degeneration, and other related ailments [10, 11].

The primary objective of this study is to summarize the outcomes of in vitro, preclinical, and clinical articles assessing the regenerative effects of exosomes derived from various MSCs on RCIs. The secondary objective of this study is to list the ongoing clinical trials listed on different clinical trial registries investigating the effects of exosomes on RCIs.

Purpose

The purpose of the systematic review on exosomes for the treatment of rotator cuff injuries is to evaluate the effectiveness of exosomes derived from different mesenchymal stem cell sources on various rotator cuff injuries. Exosomes have been shown to play a crucial role in promoting the proliferation, migration, and fibrotic activity of multiple other orthopedic pathologies, such as knee osteoarthritis. By comparing the impact of exosomes from different cell origins on the healing of tendons, this review aims to shed light on the underlying mechanisms involved in the regenerative potential of exosomes for rotator cuff injuries. This synthesis of evidence will contribute to advancing our understanding of the therapeutic potential of exosomes in enhancing the healing process of rotator cuff injuries, providing valuable insights for future clinical applications in this field.

Methods

Search Criteria

A systematic search using the online journal databases PubMed (MEDLINE), Embase, Scopus, and Web of Science was conducted, seeking to gather pertinent articles published in English until August 2024. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and guidelines were adhered to throughout the search process for this study, and utilized the following search terms: (‘exosome’ OR ‘microvesicles’ OR ‘extracellular vesicles’ OR ‘secretomes’ OR ‘microsomes’ OR ‘small extracellular vesicles’) AND (‘rotator cuff’ OR ‘rotator-cuff’ OR ‘shoulder’ OR ‘supraspinatus’ OR ‘infraspinatus’ OR ‘teres minor’ OR ‘subscapularis’ OR ‘glenohumeral’ OR ‘acromioclavicular’ OR ‘sternoclavicular’ OR ‘scapulothoracic’ OR ‘glenoid’ OR ‘humerus’ OR ‘acromion’ OR ‘clavicle’ OR ‘sternum’ OR ‘scapula’). Eligibility criteria included studies investigating both acute and chronic rotator cuff injuries and tears. All basic science, preclinical, and clinical studies utilizing exosomes for the treatment of rotator cuff injuries were included in our search strategy. Placebo, injury models, controls, and gold-standard treatment models were compared to the novel exosomal intervention. The study screening process was carried out by two independent reviewers, initiating with title and abstract screening followed by full-text review and extraction of the relevant data, all using reference management software to ensure the inclusion of all possible articles that fit our criteria. Figure 1 illustrates the performed process for filtering through the journal articles.

Fig. 1.

A PRISMA flow diagram outlining the record identification and selection process

To ensure a comprehensive analysis and to capture the full spectrum of relevant studies, our systematic search, as outlined in the preceding section, was intentionally broad. This approach allowed us to include any study that could potentially meet our inclusion criteria. Consequently, we have structured our manuscript into four distinct sections, categorized by study type: basic science, basic science combined with preclinical, preclinical, and clinical. This organization enables a thorough and methodical examination of the available literature, ensuring that each study type is appropriately analyzed and discussed in the context of our research objectives.

To address the secondary objective of this study, clinicaltrials.gov, the Chinese Clinical Trial Register (ChiCTR) and the Clinical Trials Registry—India (CTRI) were searched using the same terms to identify registered trials on the use of exosomes for the management of RCI.

Results

In vitro Studies

Using the aforementioned search strategy, four basic science studies fit the criteria for this systematic review. The results from these studies are summarized below, and will be analyzed in the discussion section of the study.

Li et al. conducted a study assessing the effects of transforming growth factor-beta 1 (TGF-β1)-containing exosomes derived from femoral bone marrow MSCs (BM-MSCs) on RC tenocytes sourced from Sprague–Dawley (SD) rats. After identifying and confirming the typical morphology of the BM-MSCs and BM-MSCs-derived exosomes via transmission electron microscopy (TEM) photographs, flow cytometry was used to characterize the cells. Marker proteins (i.e., CD9, CD63, CD81, and TSG101) were seen to be expressed significantly more in the exosomes compared to the BM-MSCs. BM-MSCs-derived exosomes were found to significantly enhance cell proliferation, migration, and fibrosis of tenocytes, as demonstrated by increased (S + G2/M) phase cells, stronger migration ability, and elevated mRNA and protein levels of Col I, Col III, a-SMA, Scx, and TnC. Focusing on the evaluation of TGF-β1 expression in BM-MSCs-derived exosomes revealed significantly higher mRNA and protein expression levels compared to BM-MSCs alone; the reverse was also found to be true, as suppressing the expression of TGF-β1 via sh-TGF-β1 resulted in decreased cell-building processes. The results of this study suggest that BM-MSCs-derived exosomes, specifically with TGF-β1 expression, have an overall positive impact on tenocyte cell function [12].

Qi et al. study aimed to characterize purified exosome products (PEP) and determined their possible stimulatory outcome on the biology of flexor tendon cells derived from canine models. Four experimental groups were constructed in this experiment: tenocytes cultured in Dulbecco’s modified Eagle’s medium (DMEM), 5% solution of PEP, 10% fetal bovine serum (FBS), and a final group of both 5% PEP and 10% FBS. When determining the proliferative capability of tenocytes, the assay showed that the combined group demonstrated significantly greater values compared to the other single treatment and control cohorts. The migratory effect of the tenocytes showed enhanced cell coverage in the FBS and FBS + PEP groups compared to DMEM and PEP over 30 h, with notable differences between PEP and DMEM groups from 18 to 30 h. At day 1, gene expression was comparable among groups, except for significantly higher DCN in the PEP group; at day 3, the PEP group exhibited significantly elevated levels of SCX, COL1A1, TNMD, DCN, and MKX compared to the blank group, with DCN and MKX also surpassing expression in the FBS group. The FBS + PEP group demonstrated significantly higher DCN and TNMD expression compared to FBS alone. Collagen deposition in the FBS + PEP group was seen to be comparatively thinner and more sparsely distributed than in the single-treatment FBS and PEP groups. Significantly higher absorbance was observed in the PEP group compared to the DMEM group, with the FBS + PEP group exhibiting significantly higher absorbance than the FBS group. The study demonstrates that the use of a PEP matrix product positively influences tenocyte behaviour in vitro, enhancing cell proliferation, phenotype maintenance, collagen deposition, migration, while also mitigating dexamethasone-induced apoptosis. These findings contribute to understanding PEP’s role and potentially pave the way for clinical applications in tendon healing [13].

Thankam et al. investigated the regenerative proteins produced by hypoxia-induced exosomes (HIE) sourced from adipose-derived MSCs (AD-MSCs) and tenocytes from the shoulder joint, using a control, normoxic group and one HIE group. The AD-MSCs-derived exosomes exhibited more than five- and seven-fold upregulation of MMP2 and COL6A transcripts, respectively, compared to the normoxic cells. Furthermore, mean fluorescence intensity (MFI) showed significantly greater protein expression of CTSD and TN-C compared to normoxic cells, while MMP2 and COL6A exhibited increased MFI without achieving statistical significance. Hypoxia-induced exosomes from tenocytes and AD-MSCs displayed distinct protein profiles related to ECM regeneration, suggesting potential translational significance for managing RCIs [14].

Zhang et al.’s controlled laboratory study assessed the homeostatic effect of AD-MSCs-derived exosomes on damaged human RC tendons. After characterizing and successfully identifying the exosomes, they were cultured and assessed for secretion of any pro-inflammatory cytokines, where the researchers found that IL-1β, IL-6, and MMP-9 expression was significantly reduced compared to the control group (i.e., tendons cultured in unsupplemented, complete culture media). When assessing the histological properties of both tendon groups, the AD-MSCs-derived exosomes exhibited increased levels of type I collagen and more superior tendon maturing scores; however, statistical significance of type III collagen was not achieved between the groups. Gene expression was also investigated, with levels of matrix metalloproteinase (MMP) 9 and 13 being decreased in the exosome group compared to the control, while no significant differences in tissue inhibitor of metalloproteinase (TIMP) 1 and 3 levels were seen between the groups. The key findings of this study reveal that AD-MSCs-derived exosomes suppress the synthesis of catabolic cytokines (i.e., IL-1β, IL-6, and MMP-9) and downregulate catabolic gene expression (i.e., MMP-9 and MMP-13), thereby enhancing the characteristics of torn human rotator cuff tendons and supporting the initial inference that these exosomes can promote healing and homeostasis of these damaged tissues [15].

In vitro and Preclinical Combined Studies

Only one study comprising of both an in vitro and a preclinical component fits the inclusion criteria for this systematic review.

Tong et al. investigated the effect of exosomes derived from CD133 + human urine-derived mesenchymal stem cells (HUD-MSCs) with the addition of a hydrogel on healing damaged rotator cuffs via the mediation of BM-MSCs in a rat model. The stem cells and their respective exosomes were isolated, characterized, and subsequently evaluated for their migratory, proliferative, and osteogenic/chondrogenic capability on BM-MSCs via in-vitro assays. The in vivo portion of this study included the local injection of either the hUD-MSCs or the CD133 + hUD-MSCs via an exosome-hydrogel complex, which was further assessed on imaging, histology, and biomechanical capability. CD133 + hUD-MSCs showed positive staining for CD 29, CD33, CD73, CD90, and CD133, and were negative for CD34 and CD45. Histological analysis showed stronger chondrogenic differentiation ability of CD133 + cells compared to the hUC-MSCs, although both interventions showed robust potential for osteogenic, adipogenic, and chondrogenic differentiation. Although both groups included in this study performed well and substantiated favourable outcomes, the researchers observed that the CD133 + cells had a stronger chondrogenic differentiative and bone tendon interface (BTI) healing capacity compared to the hUC-MSC exosomes [16].

Preclinical Studies

Twelve preclinical scientific studies meet the criteria for inclusion in this systematic review, and their results are discussed below.

Cai et al.’s controlled laboratory study investigated the promotion of cartilage formation and collagen maturation for enthesis regeneration in a chronic RC tear in a rat model, utilizing exosomes derived from Kartogenin-preconditioned, BM-MSCs (KGN-BM-MSCs). Identification and characterization of exosomes revealed the presence of untreated exosomes and KGN-BM-MSC exosomes, displaying typical morphology and particle sizes ranging from 100 to 150 nm; positive exosome markers ALIX, TSG101, and HSP70 were detected and negative exosome marker Calnexin was absent. Both groups of exosomes demonstrated a sustained release from the sodium alginate hydrogel, which was loaded into the exosomes, lasting up to 96 h. Notably, the KGN-BM-MSCs exosome group exhibited superior histological scores, increased glycosaminoglycan deposition, and type II collagen expression at 4 and 8 weeks. Additionally, at 8 weeks, the tendon-to-bone interface showed more mature and well-organized collagen fibers with higher ratios of type I/III collagen compared to the control and untreated exosome groups. Biomechanically, the KGN-BM-MSCs exosome group demonstrated the greatest failure load (28.12 ± 2.40 N) and stiffness (28.57 ± 2.49 N/mm) amidst the 3 groups at eight weeks [17].

Davies et al.’s controlled laboratory study explores the secretion of promyogenic exosomes by muscle-derived beige adipose precursors, demonstrating their therapeutic potential in treating RC muscle degeneration in mice. Flow cytometry on UCP1 reporter mouse fibro/adipogenic progenitors (FAPs) displayed a three-mode distribution of UCP1 signal intensity, corresponding to three distinct transcriptomic profiles identified through bulk RNA sequencing. Cells expressing UCP1 were identified by increased mitochondrial gene expression, beige adipose tissue (BAT) markers, and surface markers associated with exosomes. In contrast, UCP1-negative cells exhibited markers associated with fibrogenesis, while UCP1-variable cells demonstrated a distinct enrichment pattern of markers related to white adipose tissue. Exosomes were isolated from UCP1+ and UCP1− FAPs, and when used to treat C2C12 cells and non-myogenic mouse embryonic fibroblasts (MEF) in vitro, they significantly enhanced myotube fusion indices. In an in vivo assessment using a RC tear and nerve denervation injury model in wild-type mice, intramuscular injection of exosomes with intense expression of UCP1 significantly reduced fibrosis development, muscle weight loss, and muscle fiber atrophy compared to exosomes with negative expression [18].

Fu et al. studied the ability of adipose-derived stem cell exosomes (AD-MSCs-Exos) to promote RC repair by stimulating other tendon-derived stem cells (TD-MSCs). The differentiative and proliferative capacity of the exosomes were seen to be directly proportional to the amount of regeneration seen in the rat’s RC. Histopathological analysis was then conducted showing that, at 4 and 8 weeks, both the hydrogel and exosome-hydrogel complex (EHC) groups exhibited reduced inflammation and more orderly aligned collagen fibers compared to the control and phosphate buffered saline (PBS) groups, suggesting the potential of EHC in promoting the healing of RC. Masson staining further highlighted the superior alignment of collagen fibers and muscle bundles in the EHC group, indicating its positive impact on tissue organization. Biomechanical analysis of RC tendons at 4 and 8 weeks post-treatment indicated that, although the maximum tensile forces in the PBS, hydrogel, and EHC groups were lower than the normal group at 4 weeks, the EHC injection promoted increased biomechanical properties compared to other groups, and at 8 weeks, the EHC group demonstrated significantly improved RC tear healing, with upregulated gene expression that codes for proteins involved in the production of tendon-associated molecules contributing to the enhanced healing process. Overall, this study strongly suggests that AD-MSCs-Exos have a beneficial effect on rotator cuff repair by mediating the osteogenic and adipogenic effects of TD-MSCs [19].

Huang et al. investigated the promotive effects of BM-MSCs-derived exosomes on RC BTI healing via the stimulation of neovascularization and regulation of M1 macrophages in 59, 4-week-old, male SD rats. 5 rats were used for BM-MSCs extraction, and 54 rats were involved in constructing a RC reconstruction model. The model comprised two groups: PBS and BM-MSCs-Exos, each consisting of 27 rats. Tissue section analysis involved 18 rats (PBS group = 9; BM-MSCs-Exos group = 9), while the other 18 rats were also equally divided into angiogenesis and biomechanical test groups. BM-MSCs-Exos, at a concentration of 100 μg/mL, demonstrated a significant promotion of human umbilical vein endothelial cell (HUVEC) proliferation (as evidenced by the strong activation of the VEGF growth signalling pathway), migration, and angiogenic tube formation. Furthermore, CCK-8 and EdU assays, Transwell experiments, and tube length analysis, were used to further highlight their positive impact on angiogenesis. The angiogenic effects of these BM-MSCs-Exos were also seen at BTI, evidenced by increased expression of CD31 and endomucin compared to the PBS group. Proinflammatory serum markers (i.e., Interleukin 1-β, 6, 8, and TNF-α) were significantly suppressed at 1, 2, 3, and 4 weeks, indicating the reparative effects of these exosomes; these proinflammatory markers may be stimulated by M1 macrophages, therefore, the researchers concluded that the expression of these macrophages was also attenuated. The biomechanical effects of these exosomes were also assessed, investigating factors such as breaking load and stiffness of the RC, and both of these parameters exhibited significantly superior results in the BM-MSCs-Exo group compared to the PBS [20].

Jenner et al. looked into the potential of umbilical cord mesenchymal stromal cell-derived small extracellular vesicles (UC-MSCs-sEV) on RC healing in 12 ovine models. Immunomodulatory capacity and particle count were confirmed via T-cell proliferation assays and nanoparticle tracking analysis, respectively. The sheep were equally and randomly divided into two groups: one treatment group received the UC-MSCs-sEVs on a base of type I collagen sponge while the control group had the type I collagen sponge only. Macroscopically, fibrotic adhesions were seen to be significantly less in the UC-MSCs-sEVs treated sheep; only one model presented with increased erythema and no surgery sites had evidence of a seroma. Regarding hematologic parameters, on day 1 post-operation, the only parameter significantly increased in the treatment group compared to the control was monocytes, but this was mostly likely due to the surgery and not the intervention, as evidenced by the stabilization of all hematologic parameters for both groups at 6 weeks. MRI demonstrated that inflammation at the surgical site was less prevalent in the treatment group compared to the control. This study demonstrated that a dose of UC-MSCs-sEVs improved the overall regenerative capability of an infraspinatus tear in an ovine model [21].

Tan et al.’s controlled laboratory study explores the effects of BM-MSCs-Exo and AD-MSCs-Exo on RC BTI healing in 63 male mice models. The mice were equally and randomly divided into three groups: an AD-MSCs-Exo group, a BM-MSCs-Exo group, and one control group not receiving any exosome treatment. Various immunofluorescence imaging analyses investigating the amount of fibrovascular granulation tissue and degree of fibrovascular scar organization demonstrated superior histological scores in both of the treatment groups compared to the control, but between the MSC-Exo groups, significance was not achieved. Furthermore, radiographic evaluation of bone surface quality was significantly increased in the treatment groups compared to the control, but no difference was observed between exosome groups at 4 and 8 weeks post-operation. Mechanical testing demonstrated greater stiffness and failure load at 4 and 8 weeks after the intervention in both treatment groups compared to the control. During assessments of migratory, proliferative, and differentiative capacities, exosomes outperformed the control group across all three categories [22].

Wang et al. studied the effects of AD-MSCs-derived exosomes for the prevention of further muscle degeneration in torn RC rat models. Supraspinatus and infraspinatus tenotomies were performed bilaterally to create a rotator cuff tear in 42 female SD rats. They were subsequently equally and randomly allocated to one of three groups: one sham surgery group, one saline injection group, and one AD-MSCs-derived exosome injection group. Wet muscle weight at 8 weeks post-operation showed that the supraspinatus in the exosome and saline group weighed significantly less than the sham surgery group; however, when assessing the infraspinatus weight, the exosomes group weighed less compared to the saline. At 16 weeks, the AD-MSCs-derived exosomes demonstrated less atrophy than the other treatment group. When assessing fatty infiltration and angiogenic progress on histology, the exosome group showed more fatty infiltration compared to the sham surgery, but more than the saline cohort. Regarding inflammatory levels, the exosome intervention led to significantly lower levels of macrophage density compared to the saline group. TUNEL staining was employed to assess the proportion of apoptotic cells within each group; notably, the exosome group demonstrated a significantly lower number of apoptotic cells compared to the other groups at both 8 and 16 weeks. Muscle redevelopment via analysis of the number of centrally nucleated muscle fibres showed that the exosome group had a significantly greater capacity for regeneration than the other groups. The cross-sectional area of the supraspinatus muscle and ultimate failure load was not significantly superior between the saline and exosome group, and the ultimate failure load was greatest in the sham surgery group [23].

A similar study utilizing AD-MSCs-derived exosomes was conducted by this group using a rabbit model. 35 rabbits were randomly assigned to receive either sham surgery (14 rabbits) or bilateral RC tears (21 rabbits). At week 6, only half of the rabbits that were meant to have sham surgery received it, and 7 out of the 21 bilateral RC tear models were euthanized for histological analysis; the remaining 14 RC tear rabbits were randomly and equally assigned to a saline injection group or AD-MSCs-Exo group. Significantly higher fatty infiltration was seen in rabbits with RC tears compared to the sham surgery group. Furthermore, at week 18 the AD-MSCs-Exo group showed significantly decreased fatty infiltration compared to the saline group. Haematoxylin and eosin staining showed significantly fewer inflammatory cells in the AD-MSCs-Exo group compared to the saline group. Fibrocartilage area, type I and II collagen, and tenascin-C were significantly increased in the exosome group relative to the saline. Additionally, the BTI was more evenly spread and contiguous in the exosome group than the saline cohort but displayed less angiogenic evidence and cellularity. Biomechanical testing revealed that the mean ultimate load to failure, stiffness, and stress in the AD-MSCs-Exo group was smaller than the sham surgery group but was greater than the saline group [24].

A further study conducted by this group investigated the effects of AD-MSCs-derived exosomes on BM-derived macrophage polarization in chronic RC tear rat models. A total of 72 mice were included in this study, with 24 being allocated to normal activity and 48 mice underwent a total of 5 weeks of treadmill overuse. Of the treadmill overuse mice, half were injected with AD-MSCs-Exo and the other half with saline. AD-MSCs-Exo inhibited macrophage polarization to M1 but promoted polarization to M2. The intervention of treadmill overuse showed an increase gene expression levels regarding tendon repair, as seen in the AD-MSCs-Exo group, which when compared to saline, demonstrated increased type I collagen levels and decreased type III collagen, potentially improving the tendon’s resilience to stress and rupture by enhancing the type I/III collagen ratio. Additional genetic analyses completed exhibited that the AD-MSCs-Exo group showed decreased expression of MMP 3 and 13 along with increased expression of SOX9 when compared to the saline group. Histological examination of the supraspinatus tendons in mice with treadmill overactivity revealed changes classically associated with chronic overuse tendinopathy, including altered cell morphology, increased cellularity, and erratic deposition of collagen [25].

Xue et al. researched the effects of VEGFA-containing exosomes sourced from TD-MSCs and whether they promote tenocytic differentiation to a fibroblastic phenotype. Two groups were formed in this study: one control group and one group receiving injections of TD-MSCs-Exo. TD-MSCs-Exo significantly enhanced tenocyte growth, cell migration and the upregulated expression of protein and mRNA levels of fibrosis markers (i.e., including type I and III collagen, Scx, α-SMA, and TnC). Similarly, when investigating the means by which these exosomes deliver their growth-promoting impact on tenocytes, increased levels of mRNA and protein VEGFA were seen in the exosome group compared to the control. The study confirmed the influence of exosomal VEGFA on tenocytes, demonstrating that VEGFA promotes tenocyte growth, migration, and transition to a fibroblastic phenotype, and the stimulatory effects of TD-MSCs-Exo on these behaviours may be attributable to the upregulation of VEGFA [26].

Zhang et al. studied the use of BM-MSCs-Exo integrated into an individually customized patch for RCI in a rabbit model. The animals were randomly divided into three groups: one group with direct RC repair designated as the control, repair with the patch only, and repair with the patch loaded with exosomes. The patch loaded with exosomes increased the proliferative and migratory effect of tenocytes significantly more than the patch and the control groups. At 4 weeks, hypervascularity, hypercellularity, BTI healing, and fibroblastic activity were both increased in the augmented patch groups compared to the repair only. Furthermore, at 8 weeks, vascularity and cellularity were decreased in all groups and at 12 weeks was mild, but fibrous tissue arrangement was still superior in the interventional groups in relation to the control at both the time points. Fatty infiltration was seen to be significantly lower at all time points in the exosome group compared to the others. Ultimate load failure was seen to be superior at 4 weeks in the exosome group, but significance was only achieved at 8 and 12 weeks [27].

An additional study conducted by the same group investigated the tendon-protective and anti-inflammatory effects of AD-MSCs-Exo when combined with glucocorticoid therapy in a rat model. Three study groups were included in this study: one control group injected with isotonic saline, one group given only the glucocorticoid (i.e., dexamethasone), and one group receiving combined glucocorticoid-exosome therapy. Dexamethasone significantly reduced the proliferative intensity in rat Rous sarcoma virus-transformed alveolar macrophage-derived (RAW) cell lines over time, and while additional AD-MSCs-Exo treatment did not further decrease proliferation, it counteracted the injurious effect of dexamethasone, resulting in no significant differences compared to the control group at 48 h. Furthermore, dexamethasone reduced rat RAW cell migration at 24 h, and AD-MSCs-Exo treatment countered its effect, demonstrating no significant differences compared to the control group. Regarding the immunomodulatory effects observed in this study, the researchers found that the addition of the exosomes further downregulated pro-inflammatory cytokines (i.e., IL-1α, IL-1β, and TNF-α) and upregulated anti-inflammatory cytokines (i.e., IL4 and IL10) compared to glucocorticoids and the control. Similarly, the addition of exosomes improved the effects of glucocorticoids on the proliferative, migratory, senescent, and apoptotic capability of tenocytes. Moreover, the transcriptive process of reactive oxygen species, degrative enzymes and their respective inhibitors, and tenocyte matrix molecules was also enhanced by the addition of exosomes [28]. This study showed that the complexed use of glucorticoids and exosomes yielded a more robust anti-inflammatory effect that the use of the steroid intervention alone, and also attenuated the chronic, degenerative effects associated with steroid overuse.

Clinical Studies

There are no published clinical studies involving using exosomes for the treatment of RCI.

Ongoing Clinical Trials

As of August 6th, 2024, there are no ongoing clinical trials registered in ClinicalTrials.gov, CTRI, or ChiCTR to study the safety and efficacy of exosomes for the treatment of RCI.

Discussion

The current study assessed the therapeutic ability of exosomes for the management of RCIs. All in vitro, preclinical and clinical studies utilizing exosomes for the treatment of RCIs were included. Based on our pre-defined search and inclusion and exclusion criteria, 17 studies fit the scope of our manuscript.

Qualitative Analysis

Exosomes have demonstrated promising effects on RCI in in vitro studies. These extracellular vesicles derived from MSCs release various cytokines that can prevent inflammation, promote cell differentiation, and reduce fibrosis through intricate signalling pathways [14, 29]. Specifically, exosomes isolated from ADCSs have shown pro-regenerative properties in orthopaedic disorders. Recent studies have indicated that exosomes can slow RC muscle atrophy and degeneration, positively influencing RC repair processes [24]. Moreover, the application of exosomes in RCI has been associated with BTI healing and enhancing the repair of chronic RC tears with degenerative changes [17, 30–32]. Exosomes have been linked to inhibiting inflammasome-related inflammation, inducing the release of IL-1 receptor antagonists, and ultimately promoting tendon-bone healing in RCIs [18]. Factors such as age, overuse, muscle imbalance, and shoulder joint shapes have been implicated in the development of RC tears. Older individuals are particularly susceptible to RC tears due to reduced micro-vessel density in tendons, leading to conditions like fibrovascular hyperplasia, calcification, atrophy, and adiposis which are associated with RCI. Additionally, muscle fibrosis has been identified as a significant contributor to muscle stiffness in RCI [33–37].

Exosomes have also demonstrated significant effects on RCI in preclinical studies. These extracellular vesicles, particularly those derived from BM-MSCs, have been studied for their potential to promote tissue regeneration and reduce degenerative changes associated with RC tears. Research has shown that exosomes from low-intensity pulsed ultrasound (LIPUS)-preconditioned BM-MSCs can enhance fibrocartilage regeneration at the BTI and ameliorate RC fatty infiltration, indicating their possible therapeutic potential in improving RCI healing [38]. Additionally, exosomes isolated from AD-MSCs have been reported to prevent muscle degeneration and fatty infiltration in torn RC, suggesting a role in preserving the function and integrity of muscle damaged in various shoulder joint injuries [24]. Moreover, animal models have played a crucial role in elucidating the pathophysiology of RCI. Studies have indicated that overuse combined with extrinsic compression can lead to more severe injuries compared to either factor alone, highlighting the importance of biomechanical considerations in understanding RCIs, especially tendinosis [39]. Investigations in rodent models have revealed that tear size and nerve injury can contribute to RC muscle fatty degeneration, underscoring the complex interplay of structural and neural factors in the progression of rotator cuff injuries [31]. Additionally, research has explored the role of fibro-adipogenic progenitors in muscle weakness following RCI, emphasizing the significance of understanding cellular mechanisms in addressing functional deficits associated with rotator cuff tears [40].

Limitations

This study has several limitations, those of which will be explained in this section. The research on exosomes is constrained by several factors. While MSC-derived exosomes have been shown to produce regenerative effects that are comparable to or better than standard MSCs, the absence of proper controls, inconsistent dosing, variability in disease outcomes, batch-to-batch differences in exosomal content, and a limited understanding of the off-target effects of exosomal mediators present significant challenges. These issues complicate the ability to make meaningful comparisons across studies. We also recognize that there is considerable difficulty in recommending the use of exosomes for clinical applications, as there is a severely gross lack of literature, represented by the non-existence of clinical studies.

Conclusion

In vitro and preclinical studies have provided valuable insights into the effects of exosomes on RCI, shedding light on their regenerative potential and ability to mitigate degenerative changes in muscle and tendon tissues. These findings underscore the importance of further research in harnessing the therapeutic properties of exosomes for enhancing rotator cuff healing and improving clinical outcomes in patients with RCIs. Future studies, including adequately powered, non-randomized and randomized clinical trials with long-term follow-up periods, are warranted to determine the safety and efficacy of exosomes for the management of RCIs.

Author Contributions

AG conceptualized and supervised the study. AA, SA and AG wrote the initial manuscript draft. MG and AG reviewed and edited the manuscript draft. All authors have read and approved the final manuscript.

Funding

No funding was received to assist with the preparation of this manuscript.

Data availability statement

All data is contained within the manuscript.

Declarations

Conflict of Interest

AA, SA, MG and AG declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human or animal subjects.

Informed Consent

For this type of study informed consent is not required.