The interplay between mesenchymal stem cells and the immune microenvironment in rotator cuff tendon-to-bone healing: current progress and future directions

The tendon-to-bone interface of the rotator cuff, or enthesis, is a specialized structure that facilitates load transfer between tendon and bone. As Figure 1 depicts, it comprises four distinct zones: (1) tendon, (2) unmineralized fibrocartilage, (3) mineralized fibrocartilage, and (4) bone (13 –15).

Each zone features unique cells, matrix components, and mechanical roles:

This four-zone architecture develops after birth, with collagen and cell phenotypes gradually establishing a spatial gradient along the interface (14, 15). This organization ensures efficient load transmission and mechanical durability.

Following injury and repair, this native structure is rarely restored. The interface is often replaced by disorganized fibrous scar tissue lacking zonal features, with disrupted collagen alignment, reduced chondrocyte presence, and impaired mechanical strength (13, 17, 18). Animal studies show that the regenerated interface typically resembles a single fibrous layer with limited function (15, 17, 18).

Recent tissue engineering strategies have attempted to replicate the zonal structure using growth factors, scaffolds, and stem cells. Some animal models have achieved partial regeneration of multilayered tissue with improved outcomes, but clinical success remains limited (14, 16, 19).

The absence of native enthesis architecture remains a major barrier to durable repair.

Following rotator cuff injury and surgical repair, the restoration of the native tendon-to-bone interface (enthesis) is rarely achieved. Instead, healing is typically characterized by the formation of disorganized fibrovascular scar tissue, which lacks the highly specialized zonal architecture and mineralized fibrocartilage of the native enthesis (20, 21). This aberrant repair tissue exhibits inferior mechanical properties, including reduced load-to-failure and decreased stiffness, predisposing the repair site to retear under physiological loading (1, 15).

Histological and ultrastructural analyses reveal that the repaired interface fails to recapitulate the gradation of collagen types, mineral content, and cellular morphology seen in the normal enthesis (16, 17). The absence of these gradients results in a biomechanical mismatch between tendon and bone, further compromising the integrity of the repair (17, 21). Animal studies consistently demonstrate that the newly formed interface is mechanically weaker than the native insertion, with lower ultimate tensile strength and increased susceptibility to failure (22, 23).

Current repair techniques, including suture and anchor-based methods, do not adequately address the biological requirements for enthesis regeneration, often resulting in persistent biomechanical deficits (1, 22). While scaffold augmentation and biologic strategies such as growth factor delivery and stem cell incorporation have shown promise in preclinical models for enhancing fibrocartilage formation and improving mechanical strength, translation to consistent clinical benefit remains limited (3, 24). Thus, inadequate reformation of the enthesis after injury remains a principal cause of biomechanical weakness and high retear rates following rotator cuff repair (15, 17).

Immediately after tendon reattachment, the healing site undergoes an acute inflammatory response. Neutrophils, macrophages, and lymphocytes infiltrate the repair interface, releasing pro-inflammatory cytokines and chemokines that initiate debris clearance and recruit reparative cells (27). Upregulation of HMGB1 and activation of the NLRP3 inflammasome have been implicated in amplifying inflammation and extracellular matrix (ECM) disorganization during this period. Capillary proliferation and increased cellularity are observed within the first week, peaking around 7–10 days post-repair. This early inflammatory milieu is essential for subsequent healing, but excessive or prolonged inflammation can impair matrix organization and enthesis regeneration (28, 29).

The proliferative phase is marked by robust fibroblast proliferation, neovascularization, and synthesis of new ECM, predominantly type III collagen. Growth factors such as TGF-β1 and TGF-β3 are upregulated, promoting cell proliferation and matrix deposition. This phase typically spans from the end of the first week to several weeks post-injury (27, 30). The repair tissue remains highly cellular and disorganized, with ongoing angiogenesis and granulation tissue formation. The transition from type III to type I collagen synthesis begins during this phase, setting the stage for subsequent tissue maturation (27, 31).

During the remodeling phase, which can extend from several weeks to months, the initially disorganized collagen matrix is gradually replaced by more organized, aligned type I collagen fibers. Cellular density decreases, and the vascular network regresses (20, 27). The enthesis undergoes slow maturation, with partial restoration of zonal architecture and mineralization, although the regenerated interface rarely achieves the structural and biomechanical properties of the native enthesis. The ratio of type I to type III collagen increases, and the tissue becomes more resistant to mechanical loading, but persistent differences in organization and strength compared to uninjured tendon-bone insertions remain (30, 31).

Overall, the incomplete recapitulation of the native enthesis structure and the persistence of scar-mediated healing contribute to the biomechanical weakness and high retear rates observed after rotator cuff repair (27, 31).

Following rotator cuff injury and repair, neutrophils are among the first immune cells to infiltrate the tendon-to-bone interface, rapidly clearing debris and releasing cytokines that recruit additional immune cells (32). Macrophages follow, performing phagocytosis, secreting growth factors, and orchestrating the early inflammatory response (33). Both tendon-resident and circulating macrophages are involved, with chemokine receptor CCR2 playing a critical role in their recruitment and function (34). These early innate immune responses are essential for debris clearance and the initiation of tissue repair, but their magnitude and duration must be tightly regulated (35).

While acute inflammation is necessary for healing, persistent or dysregulated inflammation impairs tendon-to-bone interface regeneration (36, 37). Prolonged macrophage activation and sustained pro-inflammatory signaling can lead to excessive scar formation, extracellular matrix disorganization, and biomechanical weakness at the repair site (38). The balance between pro-inflammatory (M1) and anti-inflammatory (M2) macrophage phenotypes is particularly important; a shift toward M2 polarization is associated with improved healing outcomes, while persistent M1 activity is linked to fibrosis and poor integration (39). Biologic adjuvants and stem cell-derived exosomes have been shown to promote M2 polarization and attenuate chronic inflammation, thereby enhancing tendon-to-bone healing (40).

Adaptive immune cells, including T cells and dendritic cells, accumulate at the repair site and in draining lymph nodes during the subacute and remodeling phases (33, 34). CD4+ T cells and dendritic cells coordinate the immune response, influence macrophage polarization, and regulate the transition from inflammation to tissue remodeling (36, 37). B cells and CD8+ T cells also increase over time, suggesting a sustained adaptive response that may impact long-term healing quality. The coordinated actions of these immune cell populations are essential for optimal tendon-to-bone healing, and dysregulation at any stage can compromise repair integrity (40).

BM−MSCs are most used; they show tri−lineage differentiation and trophic, proangiogenic, and immunoregulatory (anti−inflammatory, macrophage−polarizing) effects that support tendon–bone healing (42, 43, 48).

AD−MSCs are high−yield and minimally invasive (43), retain BM−MSC−like multipotency but greater proliferation and anti−inflammatory paracrine activity (2), aiding inflammation resolution, matrix remodeling, and interface regeneration (44).

Synovial MSCs are highly proliferative/chondrogenic, and tendon−derived MSCs are tenogenic; both modulate macrophage polarization and curb pro−inflammatory signaling, favoring targeted tendon/enthesis repair (46).

Across sources, MSCs are multipotent, yielding tendon− and bone−relevant lineages. Through trophic factor/EV secretion they recruit cells, promote angiogenesis and ECM remodeling, and via immunomodulation (↓ pro−inflammatory, ↑ anti−inflammatory, immune−phenotype control) they establish a regenerative milieu that improves tendon–bone healing (44).

MSCs show source−dependent phenotypes and functions that shape regeneration. AD−MSCs are strongly immunomodulatory and tolerate inflammatory niches (49 –51), whereas BM-MSCs have superior osteogenic capacity, supporting bone and enthesis repair (52, 53). Synovial MSCs are chondrogenic, while tendon−derived MSCs are tenogenic, aligning with cartilage and tendon repair, respectively (54 –57). These traits likely drive outcome differences in preclinical models and support source–tissue–context matching.

MSCs possess multilineage differentiation potential, enabling them to give rise to tenocytes, chondrocytes, and osteoblasts under appropriate microenvironmental cues (45, 58). This plasticity is critical for reconstructing the transitional zones of the tendon-bone interface, which require the regeneration of fibrocartilaginous and osseous tissues (59). In vivo and in vitro studies have demonstrated that MSCs can integrate into injured sites and differentiate along these lineages, contributing to the restoration of native tissue architecture and biomechanical properties (59, 60).

Beyond differentiation, MSCs exert significant paracrine effects by secreting a repertoire of bioactive molecules, including transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and interleukin-6 (IL-6) (58). These factors modulate the local microenvironment by promoting cell proliferation, suppressing inflammation, and enhancing extracellular matrix (ECM) synthesis. The MSC secretome, including exosomes, has been shown to influence tenocyte migration, angiogenesis, and matrix remodeling, further supporting tissue repair (8, 61).

MSCs facilitate neovascularization at the repair site, primarily through the secretion of angiogenic factors such as VEGF. Enhanced vascularity is essential for nutrient delivery and waste removal, supporting the metabolic demands of regenerating tissues (46, 62). Additionally, MSCs promote ECM remodeling by upregulating the synthesis of type I collagen and other matrix proteins, and by modulating the balance between matrix metalloproteinases and their inhibitors, thereby improving the quality and organization of the repair tissue (8, 61).

MSCs can recruit and activate endogenous progenitor cells through the release of chemotactic and immunomodulatory factors. This recruitment amplifies the regenerative response by increasing the pool of reparative cells at the injury site (46, 58). Furthermore, MSCs modulate the inflammatory milieu, creating a pro-regenerative environment that supports the survival and function of both transplanted and resident progenitor cells (59, 61).

Collectively, these mechanisms underscore the therapeutic potential of MSCs in tendon-to-bone healing. However, challenges remain regarding the optimization of cell source, delivery methods, and standardization of protocols to maximize clinical efficacy (47, 60, 63).

Recent preclinical studies have demonstrated that MSCs and their derivatives can significantly enhance tendon-to-bone healing in rotator cuff injury models. In rat and rabbit models, local delivery of MSCs—whether as cell suspensions, exosomes, or cell sheets—has been shown to improve fibrocartilage formation, collagen maturation, and biomechanical strength at the enthesis (64, 65). For example, exosomes derived from kartogenin-preconditioned MSCs or loaded with kartogenin have been shown to promote chondrogenesis, collagen organization, and superior biomechanical properties compared to controls (66). Menstrual blood-derived MSCs encapsulated in platelet-rich gel facilitated new bone and fibrocartilage formation, with improved mechanical properties in a rabbit chronic tear model (65). Similarly, umbilical cord MSC-derived extracellular vesicles and cryopreserved adipose-derived stem cell sheets have demonstrated efficacy in large animal and rabbit models, respectively, supporting the translational potential of off-the-shelf or cell-free approaches (7, 67).

Genetic modification of MSCs, such as Msx1 overexpression, has also been explored to enhance proliferation and migration, resulting in improved tendon-bone integration and mechanical strength in rat models (11). The use of biomaterial scaffolds, such as 3D-printed polycaprolactone loaded with basic fibroblast growth factor and MSCs, further augments osteogenesis and immunomodulation, leading to improved enthesis healing (68). Conditioned medium from human bone marrow-derived MSCs has been shown to promote tendon-bone healing via immunomodulation, particularly through macrophage polarization (8). Collectively, these studies provide strong mechanistic and functional evidence for the role of MSCs in enhancing tendon-to-bone healing in preclinical models.

Clinical translation of MSC-based therapies for rotator cuff repair remains in early stages, with limited but growing evidence from human studies. Early-phase clinical trials and case series suggest that MSC augmentation may reduce retear rates and improve structural outcomes after rotator cuff repair, but results are heterogeneous and often limited by small sample sizes, lack of standardized protocols, and short-term follow-up (2). Safety profiles are generally favorable, with no major adverse events reported in the available literature (2).

Key limitations include variability in MSC source (bone marrow, adipose, umbilical cord, menstrual blood), cell processing methods, delivery vehicles, and dosing regimens. Clinical heterogeneity in MSC-based therapies arises from several factors beyond cell source and delivery. Variations in surgical technique—such as single- vs. double-row repair and open vs. arthroscopic approaches—can affect MSC retention and local biomechanics, influencing treatment outcomes (69, 70). Patient-related variables including age, diabetes, smoking, and baseline inflammation also impact MSC responsiveness, with evidence that aging and metabolic disorders reduce MSC efficacy (71, 72). Differences in processing—such as cryopreservation, passage number, and preconditioning—as well as culture conditions contribute to product variability and functional inconsistency (72 –74). These factors are rarely standardized across studies, complicating data comparison and contributing to inconsistent results. Harmonizing protocols and improving reporting practices are essential for enhancing reproducibility in MSC research.

Furthermore, the optimal timing, route of administration, and patient selection criteria remain to be defined. The lack of large, multicenter randomized controlled trials with standardized outcome measures and long-term follow-up precludes definitive conclusions regarding efficacy (64). Regulatory, ethical, and logistical challenges also hinder widespread clinical adoption. Ongoing research is focused on optimizing cell-free approaches (e.g., exosomes, conditioned medium), biomaterial scaffolds, and strategies to recruit endogenous MSCs, which may address some of these barriers and facilitate broader clinical translation (7, 8).

In summary, robust preclinical evidence supports the potential of MSC-based therapies to enhance tendon-to-bone healing in rotator cuff repair, but clinical evidence remains preliminary. Further high-quality, standardized clinical trials are needed to establish efficacy, safety, and best practices for MSC application in this context.

Acute inflammation is characterized by a rapid influx of neutrophils and pro-inflammatory macrophages, which are essential for debris clearance and the initiation of tissue repair. However, a timely resolution of this response is necessary to prevent chronic inflammation, which is associated with persistent infiltration of inflammatory cells, elevated levels of pro-inflammatory cytokines, and impaired tendon-to-bone healing (37, 39). Chronic inflammation, often observed in aged or degenerative rotator cuff tears, leads to a fibrotic microenvironment that impairs MSC function and promotes scar tissue formation rather than regeneration (1, 38, 39).

Macrophages are central regulators of the immune microenvironment, exhibiting plasticity between the pro-inflammatory M1 phenotype and the anti-inflammatory, pro-regenerative M2 phenotype. M1 macrophages secrete high levels of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β), promoting inflammation and tissue degradation. In contrast, M2 macrophages produce interleukin-10 (IL-10) and transforming growth factor beta (TGF-β), supporting resolution of inflammation, matrix remodeling, and tissue regeneration (8, 38, 42). The polarization state of macrophages at the tendon-bone interface is a key determinant of MSC fate and function. Recent studies demonstrate that MSC-derived secretomes and exosomes can shift macrophage polarization from M1 to M2, thereby creating a more favorable environment for tendon-to-bone healing (38, 39, 75).

The cytokine milieu at the injury site is a major modulator of both immune cell and MSC behavior. Elevated levels of TNF-α and IL-1β are hallmarks of the pro-inflammatory phase and are associated with impaired healing and increased fibrosis (38, 75). Conversely, anti-inflammatory cytokines such as IL-10 and TGF-β promote the resolution of inflammation, enhance MSC-mediated tissue repair, and support the transition to a regenerative microenvironment (8, 38, 39). The balance between these cytokine profiles is dynamic and can be therapeutically modulated. For example, administration of MSC-conditioned medium or exosomes has been shown to suppress pro-inflammatory cytokines and upregulate anti-inflammatory mediators, thereby improving tendon-to-bone healing in preclinical models (1, 8, 38, 75).

In summary, the immune microenvironment after rotator cuff injury is a critical determinant of MSC function and tendon-to-bone healing. Acute inflammation is necessary for initiating repair, but chronic inflammation impairs regeneration. The polarization of macrophages and the balance of cytokine profiles are central to this process, and therapeutic strategies that modulate these factors—such as MSC-derived secretomes—hold promise for improving clinical outcomes (1, 8, 38).

MSC-induced M2 macrophage polarization is a key mechanism by which MSCs promote tissue repair. MSCs secrete soluble factors such as prostaglandin E2 (PGE2), TGF-β, and IL-10, which drive macrophage polarization toward the anti-inflammatory M2 phenotype (8, 76, 78). M2 macrophages, in turn, secrete cytokines and growth factors that support matrix remodeling and regeneration, while reducing pro-inflammatory mediators. This polarization is further enhanced by MSC-derived extracellular vesicles, which can transfer regulatory microRNAs and proteins to macrophages (39, 78).

MSC-mediated suppression of T cell activation and proliferation occurs via the release of immunomodulatory molecules including indoleamine 2,3-dioxygenase (IDO), nitric oxide, and TGF-β, as well as through direct cell-cell interactions (76, 77). These mechanisms reduce effector T cell responses and promote the expansion of regulatory T cells, thereby dampening local inflammation and preventing immune-mediated tissue damage (1, 42).

Bidirectional effects of the inflammatory milieu are evident, as acute inflammation can "license" MSCs, enhancing their immunosuppressive and regenerative functions, while chronic or excessive inflammation impairs MSC viability and differentiation potential (76). Pro-inflammatory cytokines such as TNF-α and IFN-γ can inhibit MSC osteogenic and tenogenic differentiation, whereas anti-inflammatory conditions or M2 macrophage presence promote MSC-mediated tissue integration (1, 39).

Secreted factors including PGE2, IDO, TSG-6, and HLA-G are central to MSC-immune crosstalk. PGE2 and IDO are critical for macrophage polarization and T cell suppression, while TSG-6 and HLA-G contribute to the anti-inflammatory and immunosuppressive milieu that supports tissue regeneration (8, 76, 77). The coordinated action of these factors underpins the therapeutic potential of MSCs in modulating the immune microenvironment for optimal tendon-to-bone healing.

Brief priming with IFN−γ—alone or combined with TNF−α—elevates IL−10, IDO, PGE₂ and CXCR4/CCR2, thereby strengthening T−cell suppression, Treg/M2 skewing and homing to inflamed sites (79, 80). Inflammation−responsive IL−10 transgenes give a similar context−specific anti−inflammatory boost without constitutive expression (80). These immunoengineering tactics overcome immune barriers in rotator−cuff repair while preserving MSC osteo− and tenogenic potential (81).

MSC-EVs are lipid bilayer-enclosed particles that encapsulate a diverse cargo of proteins, lipids, and nucleic acids, mirroring the therapeutic properties of their parent cells while minimizing risks associated with cell transplantation, such as immune rejection and tumorigenicity (84, 85). Preclinical studies have demonstrated that MSC-EVs can modulate both innate and adaptive immune responses, suppressing pro-inflammatory cytokine production, promoting regulatory T cell expansion, and attenuating macrophage activation (83, 86, 87). These properties are particularly advantageous in musculoskeletal repair, where excessive inflammation impedes tendon-to-bone healing. Additionally, MSC-EVs exhibit favorable pharmacokinetics, low immunogenicity, and the capacity to cross biological barriers, further supporting their translational potential (85, 86, 88). Despite these advantages, challenges remain regarding large-scale production, standardization, and precise targeting of EVs in clinical applications.

The immunomodulatory effects of MSC-EVs are largely attributed to their cargo of microRNAs (miRNAs) and proteins, which orchestrate complex regulatory networks within recipient immune cells (83, 85, 89). Key miRNAs, such as miR-21, miR-146a, and miR-155, have been shown to downregulate inflammatory signaling pathways (e.g., NF-κB, STAT3), inhibit pro-inflammatory cytokine release, and promote the polarization of macrophages toward an anti-inflammatory (M2) phenotype (86, 87). In addition, MSC-EVs are enriched in immunoregulatory proteins, including TGF-β, HGF, and galectins, which further suppress immune activation and foster a regenerative microenvironment (85, 86). These molecular mediators collectively contribute to the attenuation of local inflammation, enhancement of tissue repair, and prevention of fibrosis in tendon-to-bone healing models (83, 89).

Precisely engineered MSC−derived extracellular vesicles (MSC−EVs) are emerging acellular immunomodulators. Hypoxia, IFN−γ, TNF−α or kartogenin preconditioning enrich cargo (miR−21/146a/155, TGF−β, HGF, galectins), amplifying M2 macrophage polarization, T−reg expansion and NF−κB/STAT3 suppression (90 –92). Surface RGD/mannose ligands or SPIO labelling further improve homing to the tendon–bone interface (82, 93). These modifications boost efficacy, cut batch variability and favor scalable "off−the−shelf" translation for rotator−cuff repair (93).

In summary, MSC-EVs represent a versatile and potent cell-free therapeutic platform with robust immunomodulatory capabilities, mediated by their unique repertoire of miRNAs and proteins. Ongoing research is focused on optimizing their production, characterization, and delivery to maximize their clinical utility in musculoskeletal and other regenerative applications.

The co-delivery of cytokines, particularly interleukin-4 (IL-4), has been shown to shift macrophage polarization toward the M2 phenotype, which is associated with anti-inflammatory and tissue-reparative functions. In preclinical models, MSC-derived secretomes and conditioned media have demonstrated the capacity to inhibit pro-inflammatory M1 macrophages and promote M2 polarization, thereby enhancing tendon-bone healing and reducing fibrotic scar formation. The beneficial effects of MSCs in this context are mediated, at least in part, by the regulation of macrophage phenotype via paracrine signaling pathways, including Smad2/3 activation (8, 39, 78).

Pharmacologic agents such as corticosteroids and statins have been investigated for their ability to modulate the immune microenvironment and synergize with MSC therapy (1). These agents can suppress excessive inflammation and promote a milieu conducive to MSC-mediated repair (39). While corticosteroids are well-established anti-inflammatory agents, their use must be balanced against potential inhibitory effects on tissue regeneration (95). Statins, in addition to their lipid-lowering properties, exhibit pleiotropic immunomodulatory effects that may enhance MSC survival and function in the context of tendon healing (47). However, the optimal dosing and timing of these agents in combination with MSCs require further investigation in translational models (96).

Genetic engineering of MSCs represents a promising strategy to augment their immunomodulatory and regenerative properties. Approaches include overexpression of anti-inflammatory cytokines, enhancement of paracrine factor secretion, and modification of surface molecules to improve immune evasion and homing (94). Preclinical studies have demonstrated that genetically modified MSCs can more effectively suppress local inflammation, promote M2 macrophage polarization, and facilitate tendon-bone integration (47, 96). The use of exosome-based delivery systems derived from engineered MSCs further amplifies these effects by providing a cell-free platform for targeted immune modulation (96).

Engineering MSCs to express IL−10, TSG−6, HLA−G, HGF or to up−regulate CXCR4/CCR2 augments M2 skewing, T−cell inhibition, angiogenesis and targeted enthesis homing (97, 98)[2−3]. Short IFN−γ priming further elevates IDO activity without safety loss (99, 100)[5−6]. These approaches consistently depress TNF−α/IL−1β, boost IL−10/TGF−β and enhance fibrocartilage strength and load−to−failure outcomes (97, 98)[2−3].

Collectively, these immunoengineering strategies highlight the critical role of modulating the immune microenvironment to fully harness the therapeutic potential of MSCs in rotator cuff tendon-to-bone healing. Continued research is essential to optimize these approaches and facilitate their clinical translation.

Hydrogels, particularly those based on natural or synthetic macromolecules, provide a three-dimensional, extracellular matrix-mimetic environment that supports MSC viability, proliferation, and differentiation while enabling localized, sustained release of bioactive factors (102, 105, 106). Decellularized extracellular matrix (dECM) hydrogels further enhance MSC retention and integration by closely recapitulating native tissue architecture and minimizing immunogenicity (106). Hybrid hydrogels combining dECM with proteins or polysaccharides can further optimize mechanical and biological properties for tendon-to-bone interface repair (106).

Microsphere-containing hydrogels and composite scaffolds allow for precise spatial and temporal control of MSC and cytokine delivery. For example, polylactic-glycolic acid (PLGA) microspheres embedded in hydrogels can sequentially release chemotactic and immunomodulatory peptides, first recruiting endogenous MSCs and then modulating macrophage polarization to favor tissue regeneration (107, 108). Such systems have demonstrated enhanced osteogenesis and improved immune response regulation in preclinical models (107, 108). Electrospun scaffolds, especially when integrated with biological polymers, further improve MSC adhesion, paracrine signaling, and engraftment, supporting robust tissue remodeling (109).

Injectable micro-fragmented nanofiber-hydrogel composites (mfNHCs) have shown promise as minimally invasive carriers for MSC delivery, promoting host macrophage infiltration, pro-regenerative polarization, and angiogenesis in both small and large animal models (110). These advances collectively underscore the importance of biomaterial design in controlling the local microenvironment and optimizing MSC-based therapies for tendon-to-bone healing (101, 102, 108, 109).

The immune response to implanted biomaterials is a critical determinant of healing outcomes. Recent strategies focus on engineering immune-responsive biomaterials that actively modulate the local inflammatory milieu to promote constructive tissue remodeling (103 –105). Hydrogels and scaffolds can be functionalized with immunomodulatory agents or designed to present specific physical and chemical cues that direct macrophage polarization toward a pro-regenerative (M2) phenotype, thereby reducing chronic inflammation and fibrosis (107, 108, 111).

Sequential release systems, such as those delivering LL37 and W9 peptides, exemplify how biomaterials can orchestrate the transition from an initial pro-inflammatory response (necessary for debris clearance and cell recruitment) to a subsequent anti-inflammatory, regenerative phase (107). The physicochemical properties of hydrogels—including crosslinking density, degradation rate, and surface chemistry—can be tuned to modulate immune cell infiltration and phenotype (101, 103, 105, 111). Standardized in vitro pathways for evaluating immune responses to biomaterials are being developed to ensure safety and reproducibility in regenerative applications (103, 104).

Engineered MSCs and cargo−enriched EVs—achieved through IL−10/TSG−6/HLA−G or CXCR4/CCR2 overexpression, brief IFN−γ priming, and miRNA−boosting preconditioning—further reinforce M2/T−reg immunity and fibrocartilage formation, but evidence is still largely preclinical and awaits protocol standardization (83, 93).

Overall, incorporating immunomodulatory design principles into biomaterial platforms is crucial for optimizing MSC–immune interactions and improving tendon-to-bone healing outcomes.

The co-administration of MSCs with growth factors, or genetic modification of MSCs to overexpress trophic factors (e.g., hepatocyte growth factor [HGF]), has been shown to improve cell survival, paracrine signaling, and regenerative capacity in preclinical models (112, 114, 115). HGF gene-modified MSCs, for example, demonstrate enhanced anti-inflammatory and pro-angiogenic effects, which are critical for tendon-bone interface healing (114). Preconditioning MSCs with bioactive substances or optimizing culture conditions further augments their therapeutic potential (97, 115). These approaches are under active investigation for musculoskeletal repair, including tendon injuries (42, 112).

Combining MSCs with regulatory T cells (Tregs) has shown synergistic immunomodulatory effects, leading to superior attenuation of inflammation compared to either cell type alone (116, 117). In models of traumatic injury, MSC+Treg therapy more effectively suppresses neuroinflammation and modulates systemic immune responses, suggesting potential for improved tendon-to-bone healing by promoting immune tolerance and reducing chronic inflammation (116, 118). This strategy is particularly relevant in settings where immune-mediated tissue damage impedes regeneration.

Integrating MSC therapy with structured physical rehabilitation protocols—termed "regenerative rehabilitation"—can enhance functional recovery by promoting graft integration, neural plasticity, and tissue remodeling (112, 119). Rehabilitation modalities may optimize the local microenvironment, facilitating MSC engraftment and paracrine activity (112). Preclinical studies in musculoskeletal and spinal cord injury models support the use of combinatorial regimens to maximize anatomical and functional outcomes (120, 121).

Engineered MSCs combined with regulatory T cells enhance immune tolerance and curb chronic inflammation, but evidence remains limited to animal and early−phase studies (122). Designer EVs enriched with IL−10 or miR−146a suppress inflammation, drive M2/Treg responses, and, when paired with rehabilitation or bFGF−loaded scaffolds, boost angiogenesis and graded mineralization (87, 123). Translation is hampered by dose, delivery, standardization, and safety challenges (93). Advancing MSC-based therapies for rotator cuff repair increasingly relies on integrated strategies, which hold significant promise but require further investigation to establish effective clinical protocols and pathways for translation.

Advances in single−cell transcriptomics and multiplex cytokine profiling enable patient immune stratification and MSC sub−typing (74, 141). Matching low−inflammatory MSC subsets to hyper−inflammatory patients—or licensing cells exvivo with IFN−γ to normalize IL−10/IDO output—could minimize heterogeneity and maximize benefit (79 –81).

Humanized organoids and bioprinted constructs now recreate tendon–bone zonal ECM, permitting high−throughput screening of MSC–immune–biomaterial interactions (142 –144). However, current 3D−bioprinted enthesis models still fail to reproduce gradient mineralization, zonal load transfer and anisotropic tendon−bone integration (145 –147). Fabricating multiphasic scaffolds with precise spatial control over ECM, stiffness and mineral composition remains technically difficult, impeding functional replication of native enthesis and slowing clinical translation (142, 148).

Consequently, these unresolved biomechanical gaps underscore that present 3D-bioprinted enthesis constructs continue to face substantial mechanical constraints. These include inadequate reproduction of gradient mineralization, impaired zonal load transfer, and insufficient anisotropic integration between tendon and bone tissues (145 –147). Moreover, fabricating multiphasic scaffolds with precise spatial organization of ECM, stiffness, and mineral content remains technically difficult (142, 148). These challenges continue to hinder the functional replication of native enthesis and represent major barriers to the clinical translation of 3Dbioprinting in tendon−to−bone repair.

Integrating single−cell transcriptomes, cytokine arrays and proteomics with machine−learning enables prediction of MSC potency, identification of immune subtypes linked to poor healing and optimization of donor selection and licensing parameters (149, 150). These AI−derived signatures promise to reduce therapeutic variability and guide personalized immuno−engineering strategies, advancing data −driven precision therapy (151, 152).

Artificial−intelligence tools offer promising means to integrate high−dimensional immune data—such as single−cell transcriptomics, cytokine profiles, and proteomics—for predictive modeling and personalized MSC therapy optimization (152, 153). By uncovering complex patterns across omics datasets, AI algorithms can stratify patients, predict MSC potency, and identify immune subtypes linked to poor healing (154, 155). These approaches also guide donor selection, cell processing, and immunomodulatory strategies, helping to reduce therapeutic variability and enhance clinical outcomes in tendon−to−bone repair.

Together, these emerging strategies tackle cellular−immune heterogeneity and translational barriers, complementing the immuno−engineering and biomaterial approaches detailed earlier. Their convergence is expected to deliver reproducible, patient−tailored MSC/EV therapies for rotator−cuff tendon−to−bone repair.