Regenerative biologics modulating inflammation and promoting tenogenesis in equine superficial digital flexor tendonitis: from molecular pathways to clinical translation

The SDFT is highly susceptible to tendonitis due to its role as an energy-storing tendon and exposure to mechanical overload that exceeds the structural tolerance of the tissue [27]. This overload may result from sudden excessive stretching or, more commonly, from the cumulative effects of repetitive strain [28]. Microdamage accumulates gradually within the collagen matrix, and most lesions develop subclinically before clinical signs appear [29]. Clinically, acute SDFT injuries present with variable lameness, and in severe cases, fetlock hyperextension may be observed due to loss of tendon integrity [18]. Although clinical signs may resolve, inflammation persists at the molecular level, indicating ongoing cellular pathology [30].

Both ageing and repeated mechanical loading disrupt tendon structure by inducing collagen disorganization and matrix protein imbalance [31, 32]. Studies have revealed age-related accumulation of degradation fragments and altered glycosaminoglycan profiles, contributing to reduced mechanical resilience and higher reinjury risk [33, 34].

Tendon repair begins with type III collagen deposition, forming a mechanically weaker matrix than native type I collagen. During remodeling, partial replacement by type I collagen occurs, aided by fibroblast-mediated contraction and alignment [35]. However, the repaired tissue seldom restores full strength or organization, making controlled, programmed exercise essential to guide fiber alignment and enhance functional recovery [10].

In addition to the canonical cytokine-mediated inflammation, several intracellular signaling pathways play pivotal roles in orchestrating the inflammatory response during tendinopathy. Among these, the NF-κB pathway is the most extensively studied and serves as a powerful pro-inflammatory signaling pathway. Upon activation by cytokines such as IL-1β and TNF-α, NF-κB translocates to the nucleus and promotes transcription of pro-inflammatory genes, including IL-6 and cyclooxygenase-2 (COX-2), thereby sustaining the inflammatory milieu and contributing to matrix degradation and fibrosis [43, 46]. Under hypoxic conditions, NF-κB activation stabilizes hypoxia inducible factor-1α (HIF-1α) and promotes reactive oxygen species (ROS) production, forming a self-amplifying inflammatory loop that disrupts matrix homeostasis and drives chronic tendon degeneration [41]. Recent in vivo evidence demonstrated that canonical NF-κB activation persists beyond the inflammatory phase and promotes myofibroblast survival during the remodeling stage of tendon healing, thereby contributing to fibrotic matrix deposition rather than true regeneration [30].

The nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome represents another critical inflammatory mechanism. Upon stimulation by danger signals such as high mobility group box 1 (HMGB1), mitochondrial dysfunction, or ionic flux (K⁺ efflux, Ca²⁺ influx), NLRP3 assembles with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 to promote the maturation of IL-1β and IL-18 [47, 48]. This pathway contributes to excessive ECM remodeling and inflammatory cell recruitment, and its overactivation has been associated with poor tendon healing and fatty infiltration of tenocytes [49].

The p38 mitogen-activated protein kinase (MAPK) pathway is activated by mechanical stress and oxidative signals, and modulates cellular responses by promoting transcription of TNF-α, IL-6, and IL-8 [50]. Persistent activation contributes to heterotopic ossification and chronic inflammation, while pharmacologic inhibition has shown promise in reducing these adverse outcomes in preclinical models [51, 52].

The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway also plays a dual role in tendon inflammation. While it may contribute to fibrosis and senescence when persistently activated [53], it is also essential for mediating the anti-inflammatory effects of IL-10 [54, 55]. Activation of the interleukin-10 receptor/Janus kinase/signal transducer and activator of transcription 3 (IL-10R/JAK/STAT3) axis downregulates NF-κB activity and promotes M2 macrophage polarization, thereby limiting excessive ECM deposition and supporting regenerative remodeling [56].

A key player in the tendon healing process is the macrophage, due to its ability to dynamically switch phenotypes in response to environmental cues [57]. Initially, pro-inflammatory M1 macrophages dominate the injury site to clear debris and promote inflammation, but a timely transition to the anti-inflammatory M2 phenotype is critical for resolving inflammation and promoting tissue repair. However, disruption of this transition can lead to chronic inflammation [57 –59]. Importantly, M2 macrophages secrete anti-inflammatory mediators such as IL-10 and interleukin-1 receptor antagonist (IL-1Ra), which are key to suppressing the early inflammatory response, preventing further tissue degradation, and initiating a reparative immune response [24]. This reparative response attracts growth-promoting factors and may continue throughout the proliferative and remodeling stages, providing a supportive microenvironment for tissue repair [22].

The tendon microenvironment is characterized by cross-talk between immune cells and tendon-resident cells, including tenocytes and progenitor/stem cells, through exchange of exosomes and soluble mediators [60]. This intricate communication plays a central role in controlling the overall healing process [61, 62]. In an autologous indirect tenocytes co-culture with peripheral blood mononuclear cells (PBMCs), the presence of PBMCs led to a marked upregulation of proinflammatory cytokine gene expression, including IL-1β, TNF-α, and IL-6. This suggests that the crosstalk between tenocytes and immune cells occurs via secreted factors [63]. Experimental data have shown that tenogenic markers such as scleraxis, along with cell proliferation capacity, were significantly reduced when MSCs were cultured in the presence of immune cells [64].

Additional evidence has identified diverse immune components within the tendon microenvironment, including tenophages, mast cells, T and B lymphocytes, and natural killer cells, all of which contribute to the regulation of inflammation during tendon healing [66, 71, 72].

Tendon cells, including tenocytes and tendon stem/progenitor cells, are highly mechanosensitive; they transduce external loading into intracellular signals that regulate cell behavior, matrix synthesis, and immune responses [73]. When exposed to physiological loading, such as that induced by controlled exercise, they enhance the synthesis of collagen and activate cross-linking enzymes, thereby improving tendon tensile strength [74]. In contrast, mechanical unloading, which may result from immobilization or inactivity, suppresses the expression of key extracellular matrix components, ultimately compromising tendon structural integrity [75]. These load-dependent cellular behaviors are orchestrated through mechanotransduction pathways involving the actin cytoskeleton and transcriptional regulators, which translate matrix stiffness into gene expression programs that promote either tissue stiffening or elasticity [76]. As such, maintaining a balanced mechanical environment is essential, not only for directing tenogenic responses but also for preventing pathological outcomes like fibrosis [73].

The tendon microenvironment operates as a highly integrated regulatory network, where immune, stromal, and mechanical signals interact through overlapping pathways [26, 77]. This complexity underlies the limited efficacy of single-target therapies and supports the development of multimodal strategies that address the diverse regulatory axes involved in tendon healing [78].

Conventional anti-inflammatory treatments like Non-steroidal anti-inflammatory drugs (NSAIDs), cold therapy, compression, and topical Dimethyl sulfoxide (DMSO) are widely used to treat intratendinous inflammation and relieve pain, but these primarily suppress inflammation rather than promote its natural resolution [81]. The use of corticosteroids remains controversial due to their potential to impair tenocyte function, and if used, they should be limited to the early inflammatory phase and applied peritendinously rather than intratendinously [10].

Emerging evidence suggests that transitioning from merely suppressing inflammation to actively modulating and resolving it represents a more physiological and regenerative approach to tendonitis treatment [20]. Recent findings highlight that specific elements of the inflammatory cascade are essential not only for initiating repair but also crucial for proper resolution of tissue injury [82]. In this context, ACS has shown promise due to its enrichment with IL-1Ra, which competitively inhibits IL-1 binding to its receptors [83, 84]. This targeted blockade disrupts IL-1–mediated recruitment of M1 macrophages and reduces the expression of matrix-degrading enzymes such as MMPs [85].

Although ACS does not directly induce macrophage polarization toward the reparative M2 phenotype, its capacity to attenuate the pro-inflammatory environment facilitates a shift toward tissue regeneration [61]. This permissive environment allows immune and progenitor cells to regain regulatory function, leading to the secretion of exosomes and anti-inflammatory mediators such as IL-10 and transforming growth factor beta (TGF-β), which further promote M2 polarization and resolution of inflammation [62].

ACS has shown promise in enhancing tendon healing, particularly in experimental models and some clinical studies in equine patients [84]. In animal models, ACS treatment led to improved histological healing, increased type I collagen expression, and accelerated recovery of tendon structure, although improvements in ultimate tendon strength were not consistently observed within the study periods [86 –88]. Notably, a single intralesional injection of ACS in horses diagnosed with SDF tendonitis was shown to reduce lameness and swelling, improve ultrasonographic and histological outcomes, and enhance collagen type I expression [89]. Similarly, APS, which shares a comparable biological profile with ACS, demonstrated protective effects against IL-1-mediated matrix degradation [85]. Moreover, APS treatment significantly reduced collagen type III expression in an equine SDF tendonitis model, further supporting its potential role in enhancing tendon matrix composition and mechanical integrity [90]. On the other hand, an in vitro study on equine tenocytes demonstrated that ACS did not fully reverse the detrimental effects induced by combined IL-1β and TNF-α stimulation [40].

While experimental and clinical data suggest that ACS can enhance histological repair, increase type I collagen expression, and alleviate clinical signs in equine tendon injuries [84], its clinical utility remains a topic of debate among equine practitioners [91]. The biological effect of ACS relies primarily on IL-1Ra–mediated blockade of the IL-1 pathway, which limits its capacity to modulate other key inflammatory cascades such as NF-κB [40]. This narrow target profile may contribute to persistent inflammation or the inconsistent improvements in ultimate tensile strength reported in some studies [86 –88]. Furthermore, histological improvements often precede measurable recovery of mechanical properties, and the absence of cellular scaffolds may reduce long-term regenerative potential compared with treatments like I-PRF [90]. Finally, the short follow-up periods in most equine studies restrict the ability to draw strong conclusions on recurrence rates or long-term performance outcomes.

Other biological therapies, such as PRF, have been shown to enhance antioxidant defenses in tenocytes and to promote macrophage polarization toward the anti-inflammatory M2 phenotype, as demonstrated in controlled macrophage cultures [92 –94]. However, direct evidence confirming these immunomodulatory effects within the tendon microenvironment, particularly on resident tendon macrophages, remains limited.

Similarly, exosomes derived from MSCs have been reported to promote macrophage polarization toward a reparative M2 phenotype and downregulate pro-inflammatory and apoptotic markers [95 –99]. In human PBMCs, MSCs-derived exosomes suppress key pro-inflammatory cytokines (IL-1β, TNF-α) while enhancing anti-inflammatory mediators such as IL-10 and TGF-β [100]. Priming MSCs with IL-1β further augments these effects by enriching exosomes with microRNA (miR) such as miR-147b, which suppresses IL-1β/TNF-α expression and inhibits NF-κB signaling [101, 102]. Complementary explant and co-culture studies support translational relevance, showing that MSCs-derived exosomes reduce IL-6 and MMP-3, and exert anti-inflammatory effects in tenocyte–macrophage co-cultures [103, 104]. However, an in vitro study reported limited anti-inflammatory activity when equine tenocytes were directly stimulated with IL-1β and TNF-α [40].

While MSCs-derived exosomes have demonstrated promising results in preclinical tendonitis models, the absence of clinical studies in naturally occurring equine tendon injuries limits the strength of current recommendations [105]. Their occasional limited efficacy in highly inflammatory environments may be related to the inability to modulate all key inflammatory pathways, particularly under strong IL-1β and TNF-α stimulation [40]. Considering these constraints, exosomes derived from tenogenic-primed MSCs or primed with IL-1β may represent a promising alternative or adjunct, offering greater functional specificity and a more targeted pro-regenerative effect in tendon repair [106].

Biological therapies currently represent the most commonly employed strategy for promoting tenogenesis in equine SDF tendonitis. These approaches rely on products derived from blood or tissues to elicit a regenerative healing response [15]. Although the definitive goal of tissue regeneration remains unachieved with current strategies [10], numerous experimental and clinical studies have demonstrated improved healing quality and favorable clinical outcomes following the application of these biological therapies [96, 107 –116].

Most biologics used fall into two major categories, growth factor-based products and cellular therapies. While individual growth factors such as IGF-1 have been explored, platelet-derived products (PDPs) and various preparations of ACS are more commonly applied due to their content of a broader mixture of bioactive molecules [13, 84, 85, 90].

Among PDPs, PRP has received significant attention and shown beneficial effects in experimental models [13, 14]. However, its efficacy in equine tendon healing remains controversial both experimentally and in clinical trials [10]. Although several individual studies have reported favorable histological or clinical improvements following PRP administration in equine tenodesmic lesions [15, 16], the recent meta-analysis pooling fifteen trials found no definitive evidence of benefit over controls [17].

Autologous conditioned plasma (ACP), a leukocyte-reduced platelet concentrate prepared stall-side by a simplified single-spin centrifugation, offers a moderate platelet enrichment (~ 1.2–2.5× baseline) with markedly lower white blood cell content compared to many PRP protocols [117, 118]. In equine practice, a small case series reported clinical improvement following intralesional ACP injections for severe tendinitis; however, the absence of controls and standardized outcome measures limited the strength of these findings [119]. In a surgically induced SDF Tendonitis model, two ACP injections did not yield significant improvements in ultrasonographic, histological, or biomechanical outcomes over saline controls at 24 weeks, despite modest biochemical changes [120].

Variability in PRP preparation protocols, including platelet concentration, leukocyte content, and activation methods, significantly influences the biological activity and subsequent healing outcomes [121]. For instance, leukocyte-rich PRP may provoke a heightened inflammatory response, potentially delaying regeneration, whereas leukocyte-poor formulations might favor anti-inflammatory effects but lack sufficient growth factor release [122]. Methodological limitations in existing studies, including small sample sizes, lack of proper controls, and inconsistent outcome measures, limit the reliability of positive findings and underscore the need for well-designed, standardized clinical trials [12]. Compared to other biologics like MSCs may fall short in providing consistent regenerative benefits [123].

PRF, an advanced platelet-derived product, has gained attention as a favorable substitute to PRP. It can be simply prepared stall-side from autologous blood without the need for anticoagulants or sophisticated laboratory tools. Compared to PRP, PRF provides a greater amount of cytokines and growth factors and ensures a more prolonged release, lasting up to fourteen days versus nearly four days in PRP [124, 125]. Moreover, its fibrin-rich network facilitates cellular migration and serves as a supportive scaffold, while being free of chemical additives that may hinder its therapeutic efficacy [126]. The liquid form of platelet-rich fibrin, also known as injectable PRF (I-PRF), permits intratendinous administration, unlike the conventional clotted PRF. Our previous study utilized I-PRF in the treatment of naturally occurring superficial digital flexor tendonitis in donkeys and demonstrated significant improvements in clinical outcomes compared to saline-treated controls [127].

A key element for effective tendon repair and regeneration is the availability of a suitable cellular source. Such cells are essential to leverage their proliferative capacity, contribution to intercellular signaling, secretion of bioactive molecules, and support of ECM synthesis [128]. Stem cells, in particular, have attracted considerable interest owing to their intrinsic multipotency [129].

Among the different stem cell types, MSCs are an attractive cell source due to their high proliferative ability and capacity to differentiate into multiple cell types. MSCs are commonly derived autologously, especially from bone marrow or adipose tissue [114, 123]. Allogeneic MSC products have also been safely employed in clinical settings [112, 130], though concerns persist regarding potential immune responses to non-autologous sources [131, 132]. In addition, stem cells have also been isolated from tendon tissue itself across several species, including horses [133]. These tendon-derived stem cells have shown promising regenerative potential and may offer advantages in terms of tenogenic differentiation when applied to tendon repair [134].

Despite their regenerative potential, the clinical application of MSCs in tendon therapy faces several limitations. Maintaining MSCs' viability after administration is difficult, particularly in the harsh inflammatory tendon microenvironment, with studies showing less than 5% survival of MSCs within 10 days post-injection [105, 135]. There are also concerns about immune rejection and potential tumorigenicity, particularly with prolonged culture or repeated administration [131, 132]. Multiple clinical studies show that intralesional MSCs can improve return-to-racing rates and reduce reinjury [113, 114, 116]. However, a recent meta-analysis found their effectiveness to be inconclusive, largely due to heterogeneity in cell sources, preparation and priming methods, timing of administration, and outcome measures, as well as generally small sample sizes, short follow-up periods, and high risk of bias in most studies [12].

Various experimental model studies have demonstrated that MSCs-derived exosomes enhance tendon repair [99]. These exosomes regulate the balance between ECM synthesis and degradation and promote tendon regeneration by enhancing the proliferation and migration of endogenous tendon stem/progenitor cells, increasing tendon marker expression, collagen I deposition, and improving biomechanical strength [139, 140]. Their cargo activates pro-survival signaling pathways, thereby reducing apoptosis and fostering a regenerative microenvironment [96]. In a rat model of Achilles tendinopathy, exosomes derived from MSCs enhanced tendon healing by increasing type I collagen expression and improving biomechanical properties. These effects were partly linked to the enrichment of miR-29a, miR-21-5p, and miR-148a-3p, which are known to regulate collagen remodeling and promote tissue regeneration [97]. Engineering MSCs-derived exosomes to overexpress miR-29a further amplified these effects [141].

Adipose-derived SVF comprises a heterogeneous mix of cells, stem/progenitor cells, pericytes, and endothelial cells, obtained directly from adipose tissue without cell culture expansion, offering a rapid, cost-effective, and minimally manipulative alternative to culture-expanded MSCs [142]. In vitro studies in equine models have shown that SVF exhibits elevated expression of key growth factors such as insulin-like growth factor 1 (IGF-1) and TGF-β, and its conditioned medium promotes tenocyte chemotaxis and ECM-related gene expression, positioning SVF as a potent trophic mediator in tendon healing [143]. Experimental in vivo studies, including collagenase- and surgically-induced SDFT lesions in horses, suggest that SVF administration can enhance collagen fiber organization and promote neovascularization [144]. A small clinical case report involving three Thoroughbreds with naturally occurring SDF tendinitis found that intralesional autologous SVF injections correlated with marked clinical improvement and a shortened rehabilitation period [145]. However, SVF exists in distinct forms (enzymatically vs. mechanically isolated), each with differing cell compositions and bioactivity [144]. This heterogeneity, compounded by batch-to-batch variability influenced by donor tissue characteristics and processing protocols, poses a major challenge for standardization.

BMAC, represent a culture-free cellular product containing a mixture of mesenchymal stem/progenitor cells, hematopoietic cells, endothelial progenitors, leukocytes, and platelets, along with an array of bioactive cytokines and growth factors [146]. BMAC can be prepared stall-side through point-of-care centrifugation of autologous bone marrow aspirates, offering a rapid and minimally manipulative alternative to culture-expanded MSCs [147]. In vitro, BMMNCs exhibit anti-inflammatory, trophic effects, and enhancement of ECM synthesis [148]. Clinically, multiple equine case series and retrospective analyses have reported improved ultrasonographic lesion resolution, reduced reinjury rates, and higher return-to-performance in horses treated with intralesional BMAC compared with conventional therapies or PRP [149, 150].

Combining MSCs with PRP has demonstrated synergistic potential in tendon healing. They improve histological architecture, upregulate tenogenic markers, and enhance biomechanical properties in both preclinical rat models and clinical studies in humans [151 –156]. In horses with SDF tendonitis, co-administration of MSCs and PRP led to superior healing outcomes compared to either therapy alone [109, 157 –159], with meta-analyses suggesting reduced reinjury rates, although evidence regarding return to performance remains inconsistent [12].

In donkeys, combining ACS with I-PRF improved clinical and ultrasonographic parameters over I-PRF alone in cases of naturally occurring SDFT lesions [127], further supporting the promise of multi-modal biologic strategies in equine tendon repair.

The clinical translation of regenerative therapies for equine tendonitis is hindered by intersecting biological, methodological, and technical challenges. Equine tendons, particularly the SDFT with its energy-storing function, operate under extreme mechanical strain that modulates both cellular behavior and immune signaling, and they possess a sparsely vascularized ECM with a distinct immune milieu, resulting in reparative responses that differ fundamentally from those of experimental models or simplified in-vitro systems [160, 163, 164]. Even experimental lesions within equine patients themselves (e.g., collagenase or surgically induced injuries) fail to fully replicate the degenerative, multifactorial nature of naturally occurring lesions [90].

Furthermore, the strength of clinical studies is weakened by practical barriers inherent to equine research, including high inter-individual variability in healing response, small sample sizes, owner-driven treatment preferences, and the limited feasibility of conducting randomized controlled trials [10]. Addressing these barriers through equine-specific protocols and long-term mechanistic studies is crucial to enhancing the consistency, safety, and clinical efficacy of regenerative therapies.

Effective tendon regeneration depends on targeted modulation of the inflammatory microenvironment, rather than indiscriminate suppression, together with regenerative stimuli [165]. Despite the increasing use of biologic therapies, achieving complete and sustained resolution of tendon inflammation remains challenging [10]. Current biologics often focus on single cytokines, yet tendon inflammation is driven by complex and redundant signaling pathways [166].

Given the multifactorial nature of tendon inflammation and repair, combination biologic therapies may offer a more comprehensive strategy by providing multi-target, optimized modulation of inflammation while simultaneously delivering regenerative cues. Early studies suggest that such synergistic approaches could enhance therapeutic outcomes beyond what is achievable with single-modality treatments [12]. Future research should therefore explore rational combinations of biologics, optimized in both timing and dosing, to maximize their efficacy and clinical relevance.

Although stem cell-based therapies are now well established in equine tendinopathy, key challenges remain, particularly related to poor cell retention and limited understanding of their mechanisms of action [6]. In this context, increasing attention has been directed toward extracellular vesicles, such as exosomes, which mediate many of the paracrine effects of stem cells. However, their composition is highly variable and requires better product characterization, as well as optimization of dose and timing [167, 168]. Therefore, specific regulatory standards are needed to ensure safety, efficacy, and responsible clinical use.

Advancing the clinical application of regenerative biologics for equine tendonitis requires stronger evidence supported by well-designed studies. Although randomized controlled clinical trials remain the gold standard for demonstrating therapeutic efficacy, their feasibility in equine medicine is often limited by ethical concerns, owner preferences, and logistical challenges associated with enrolling sufficiently large and unbiased populations [10]. As a practical alternative, large-scale prospective cohort studies, with clearly defined inclusion criteria, standardized treatment protocols, and long-term follow-up, can offer a more achievable and ethically sound pathway for generating meaningful clinical evidence.