Biological tendon regeneration, are we almost there or is it still a stretch?

1.

Tendon injuries are a major clinical challenge, affecting millions worldwide and often resulting in impaired mobility, chronic pain, and high rates of re-injury. The adult tendon’s poor vascularity and low cellularity limit its regenerative capacity, frequently leading to disorganized repair and fibrotic scar formation rather than functional tissue regeneration. Current therapeutic approaches, including orthobiologics and mesenchymal stromal/stem cells (MSCs), offer only modest benefits due to issues such as donor variability, short-lived effects, and poor reproducibility. Moreover, MSCs are often driven toward fibrotic or non-tenogenic lineages within the injury microenvironment, further hindering effective repair.

Recent advances in stem cell biology have enabled the generation of induced pluripotent stem cell (iPSC)-derived tenocytes (iTenocytes), which show promise as a scalable, lineage-committed, and potentially hypoimmunogenic cell source. These cells can be integrated with engineered biomaterials, such as bioresorbable 3D-printed scaffolds, to enhance structural support, guide tissue organization, and promote host-mediated repair. Mimicking developmental signaling pathways has improved tenogenic differentiation, but challenges remain in optimizing maturation stages and minimizing off-target lineage formation. Moving forward, combining iTenocytes with mechanically robust, cell-laden scaffolds and precise biomechanical cues may enable scarless tendon regeneration and full functional recovery, addressing a longstanding unmet clinical need in musculoskeletal medicine.

2. Why does tendon healing remain a clinical challenge?

Tendon trauma constitutes over half of the musculoskeletal injuries in the US and approximately affecting 10% of the population by the age of 45 and 1 million athletes around the globe, while it results in over $40 bn loss in medical treatment [1,2]. In addition, the estimated global incidence of tendon ruptures each year is 6 to 7 million cases. The adult tendon is characterized by low vascularity and cellularity, which significantly constrain their intrinsic regenerative potential. Consequently, the formation of a functional provisional matrix is impaired, thus leading to disorganized tissue repair and fibrotic scar formation.

It is well-established that there are three phases in the tendon healing process: the inflammatory phase, the proliferative phase, and the remodeling phase [3]. When injury occurs, the extracellular matrix (ECM) of the tendon is disrupted, immune cells are recruited to the site and acute inflammation ensues [4]. These immune cells secrete matrix remodeling cytokines, like transforming growth factor-β (TGF-β), which mainly causes resident fibroblasts to differentiate into myofibroblasts, in the proliferative phase [5]. In addition, mechanical cues concurrent with paracrine signaling between pericytes, tendon cells such as tendon sheath and progenitor cells, and myofibroblasts contribute to the repair process through proliferation, migration, and angiogenesis, playing complementary roles in tissue regeneration. During this phase, the collagen type I and type III, decorin, and periostin-rich provisional matrix begins to be deposited, serving as a structural scaffold that supports neo-tendon formation and facilitates vascular ingrowth. This matrix also recruits mesenchymal cells to the injured site to promote healing [6]. In optimal regenerative tendon healing, vascularization gradually regresses, and the initially enlarged tendon – adapted to reduce local mechanical stress – remodels into a thinner, collagen type I-rich structure with high tensile strength. This process involves a shift from a stem cell-rich niche to a stable population of mature tenocytes. However, in adult tendons, this regenerative trajectory is often disrupted. Instead of restoring cellular homeostasis characteristic of healthy tendon tissue, healing is hindered by persistent vascularization, excessive deposition of highly crosslinked collagen type III, and an accumulation of fibrotic cells. Elevated signaling from factors such as TGF-β3, Colony stimulating factor 1 (CSF1), and Interleukin 33 (IL-33) sustains a chronic inflammatory environment, impairing matrix remodeling and preventing clearance of excessive scar tissue – ultimately leading to tendon fibrosis [7]. Persistent fibrosis can also create a permissive environment for aberrant differentiation of infiltrating mesenchymal cells. Prolonged TGF-β signaling and mechanical dysregulation have been implicated in promoting the trans-differentiation of fibrotic or tenogenic progenitor populations into chondrocyte-like or osteoblast-like phenotypes [8]. This can lead to ectopic formation of fibrocartilaginous tissue or, in more severe cases, heterotopic ossification within the tendon matrix. These lineages extensively contribute to loss tendon elasticity and tendon gliding, thus leading to restricted mobility, and a high risk of re-injury.

3. Orthobiologics – minimally invasive approach for tendon healing, mostly anti-inflammatory

Orthobiologics are therapeutic agents, mostly autologous, designed to augment the tissue’s intrinsic regenerative capacity, reduce inflammation and facilitate more effective or accelerated healing [9]. For instance, bone marrow aspirate concentrate, autologous conditioned serum and platelet-rich plasma (PRP) are considered orthobiologics. One of the key advantages of autologous orthobiologics is the ability to be applied directly at the point-of-care, often within a short time of preparation, allowing for rapid therapeutic intervention [10]. Orthobiologics have been reported to carry anti-inflammatory factors, making them a widely applied therapeutic approach for tendonitis and tendinopathy associated with aging or overuse. However, orthobiologics often exhibit significant batch-to-batch variability, provide only short-term symptomatic relief, and, in many cases, fail to demonstrate superiority over placebo treatments [11]. A major limitation in their clinical application is the prevailing “one-size-fits-all” approach, which overlooks key factors such as donor variability, preparation techniques (e.g., leukocyte-rich versus leukocyte-poor platelet-rich plasma), and administration protocols. These factors collectively lead to highly heterogeneous product compositions and inconsistent biological potencies [12].

4. Mesenchymal stromal/stem cells – cell therapy with nonspecific side products and limited expansion capacity

Mesenchymal stromal/stem cells (MSCs) can be found in various tissues, however MSCs derived from bone marrow and adipose tissue have been the most extensively studied MSCs [13,14], have shown potential to differentiate into tenocytes and exhibit immunomodulatory properties that are particularly beneficial in the inflammatory microenvironment of tendon injuries [15]. Notably, their immunomodulation is not constitutive but rather responsive, allowing MSCs to adapt dynamically to injury-induced cues [16]. However, the outcomes remain inconsistent and largely irreproducible. Many in vivo studies have failed to demonstrate meaningful structural regeneration or restoration of biomechanical function in tendon tissue [17]. While MSCs possess immunomodulatory capabilities in vitro, these effects do not consistently translate in vivo. Instead, the fibrotic microenvironment often drives MSCs toward a myofibroblastic fate, inadvertently promoting fibrosis rather than regeneration [18]. Notably, the fibrotic differentiation of α-Smooth muscle actin and Platelet‐derived growth factor receptor‑α positive (αSMA+/PDGFRα+) MSCs into S100 calcium‑binding protein A4 positive (S100A4+) subtype can dominate the wound healing niche and contribute to aberrant remodeling in tendon [19]. Moreover, the clinical translation of MSC-based therapies is challenging due to their limited expansion capacity, which is necessary for therapeutic purposes. Prolonged culture often leads to phenotypic drift and a decline in their regenerative functionality [20].

These limitations are further compounded by the intrinsic heterogeneity within MSC populations, as revealed by single-cell RNA sequencing studies. Only a small subset of MSCs expresses key tendon-associated transcription factors such as scleraxis (SCX), mohawk (MKX), and tenomodulin (TNMD) [21]. As a result, therapeutic preparations often consist of a mixed population in which only a fraction possesses tenogenic potential. Therefore, there is a clinical need for a scalable, tendon-committed cell source that retains tenogenic potential and functional stability during expansion.

5. Induced pluripotent stem cells – regenerative cell therapy

Recent studies have developed iPSC-derived MSCs (iMSCs) engineered to overexpress early tenogenic transcription factors such as Scleraxis and MKX [22–24]. These engineered iMSCs exhibited pronounced tenogenic differentiation, evidenced by elevated expression of late tendon-specific markers including TNMD and TNC, underscoring their potential as a scalable and robust cell source for tendon regeneration.

However, tenocytes derived through directed differentiation by modulation of key developmental signaling pathways have demonstrated superior tenogenic potential compared to iMSC-derived cells. Mimicking tendon developmental cues, particularly through TGF-β/Bone morphogenic protein (BMP) pathway activation, has enabled the generation of iPSC-derived tenocytes (iTenocytes) capable of contributing to functional tendon regeneration in murine injury models [25]. Additionally, induction of self-renewing tendon progenitors via syndetome-like development has facilitated scarless regeneration, offering a developmental blueprint for generating lineage-committed iTenocytes with enhanced regenerative capacity [26]. Building on this, Papalamprou et al. (2023) improved the efficiency and specificity of iTenocyte differentiation by incorporating WNT pathway inhibition during later stages of syndetome induction, thereby minimizing off-target lineage differentiation [27]. While these position iTenocytes as a promising cell source for tendon regeneration, several critical challenges remain. The major drawback is the need to establish definitive temporal dynamics and optimal concentrations of signaling morphogens to further reduce off-target differentiation. Furthermore, the optimal stage of iTenocyte maturation for scalable expansion has yet to be clearly defined, which poses a significant limitation for clinical translation and large-scale production.

To define the true regenerative potential of iPSC-derived tenocytes (iTenocytes), it is imperative to assess their performance in an in vivo tendon‐injury model that faithfully reproduces the hostile, pro-fibrotic microenvironment of tendon defects, where TGF-β signaling is aberrantly elevated [28]. Under these conditions, lineage-stable iTenocytes are expected not only to resist dedifferentiation but also to drive robust, specialized extracellular matrix remodeling, thereby mitigating fibrosis; indeed, sustained TGF-β activity has been shown to be essential for preserving tenogenic identity [29]. Although their potential to restrict fibrosis by the host resident cells is yet to be shown, it is estimated that the availability of regenerative cells in the healing environment will dilute the pro-fibrotic signaling.

Moreover, optimized production protocols for iTenocytes may substantially reduce the risk of tumorigenicity often associated with undifferentiated iPSC derivatives [30,31]. iPSC-derived cells carry a recognized tumorigenicity risk because rare residual pluripotent cells and culture or reprogramminginduced genomic instability can seed teratoma formation [30,31]. To mitigate this, iTenocyte manufacturing may potentially incorporate directed differentiation with quantitative purity gates, targeted depletion of pluripotent contaminants (via Stagespecific embryonic antigen5 (SSEA5) capture or brief selectivekill smallmolecule treatment), genomic surveillance, and an inducible safety switch prior to release added via Crispr/Cas9 technology [32–34]. With these safeguards, iTenocyte preparations are expected to exhibit substantially lower tumor risk than undifferentiated iPSC derivatives. Notably, prolonged expansion of conventional MSCs can also yield chromosomal aberrations and occasional malignant transformation [35,36], underscoring the relative safety advantage of lineage-committed iTenocytes for translational tendon repair. Addressing these gaps will be essential for realizing the full therapeutic potential of iPSC-derived tenogenic progenitors as regenerative cells.

6. Biomaterials – mechanically prominent bioresorbable cell-laden scaffolds

One of the most pressing challenges in tendon tissue engineering is the development of scaffolds that simultaneously support biointegration, bioresorbability, and mechanical competence. A promising solution lies in the design of 3D bioprinted collagen-based scaffolds, which may recapitulate the native tendon microenvironment. These scaffolds are typically constructed from biodegradable hydrogels, allowing encapsulated cells to proliferate and remodel the matrix. In addition, these 3D-printed scaffolds can be tailored to offer enhanced mechanical performance while retaining the pliability and biointegration potential [37,38]. In vivo studies have demonstrated that 3D-printed bioresorbable scaffolds can support tenogenic healing and matrix deposition [39,40], offering encouraging early results. Current scaffolds often fail to provide the mechanical stability required for immediate load-bearing and integrative remodeling. To overcome this, rapid crosslinking chemistries combined with multilayered, anisotropic architectures have been developed to deliver early mechanical support that closely matches native tendon strength [41–43]. In vitro maturation, where we can leverage the specific signaling pathways to stimulate iTenocyte-mediated ECM remodeling, may further enhance the mechanical performance and integrative capacity. Additionally, braiding or twisting 3D-bioprinted scaffold filaments can synergistically optimize both structural strength and cellular accommodation. Given the tendon’s inherently low cellularity and vascularity, direct implantation of lineage-committed iTenocytes is critical for achieving robust regenerative outcomes. Nevertheless, there is still a substantial gap with regard to the collection of robust data from large animal models and clinical trials with cell incorporated scaffolds. Further work is needed to validate the load-bearing capacity, long-term integration, and synergistic effects with regenerative cells such as iTenocytes. Addressing these gaps will be critical for translating 3D-printed, bioresorbable, cell-laden scaffolds into clinically viable solutions for tendon repair.

7. Closing remarks

Based on preclinical evidence supporting the safety but low efficacy of current orthobiologics and stem cell therapies, there is a clear and urgent need for continued investigation into the regenerative efficacy of iPSC-derived technologies. We are nearing a pivotal point where the directed differentiation protocols now allow for the generation of lineage-committed tenogenic progenitors from iPSCs that can be expanded extensively and exhibit minimal immunogenicity when used allogeneically. Although MSCs are readily available and immunologically safe, their use is limited by donor‐site morbidity, low cell yields, and a propensity for dedifferentiation and off‐target lineage commitment, raising safety concerns [44]. Learned from the MSC guidelines, iTenocytes can be harnessed for autologous transplantation in clinical trials in the short term. In the long term, allogeneic approach with hypoimmunogenic engineered iTenocytes will be available off the shelf.

In addition, antifibrotic agents such as metformin to downregulate TGFβ/Smad signaling [45], mRNA therapeutics engineered to elevate PDGFBB to support proliferation [46], and niclosamide to target S100A4driven matrix dysregulation [47] represent tractable pharmacological strategies in cell-free tendon regeneration studies. Therefore, these agents can be employed to augment iTenocyte survival upon implantation, limit scarring, and promote organized matrix remodeling toward scarless tendon repair in a synergistic approach. Furthermore, surface stiffness-conditioning [48] and dynamic mechanical conditioning [49,50] in vitro can prime iTenocytes for improved functional performance, and pairing this with adjunct pharmacologic cues may further enhance postimplantation tendon regeneration.

Recent studies highlight in vitro maturation of iPSC-derived tenocytes as a robust solution, paving the way for plug-and-play cell-based therapies; to realize this potential, concerted efforts must focus on integrating iTenocytes with 3D scaffolds to promote pre‐implantation maturation, aligned matrix deposition, which may bring about organized tissue regeneration. We anticipate the availability of geometrically defined, aligned fibrous scaffolds encapsulating iTenocytes, structures that are not only bioresorbable and suturable but also mechanically robust. Such scaffolds will serve as provisional matrices that bridge defects, support load transmission, and ultimately enable functional regeneration through host integration and remodeling. Then, scarless regeneration and full restoration of tendon injuries will be in sight.

Funding Statement

This work was supported by grant from California Institute for Regenerative Medicine [DISC0–14350 to DS and EDUC4–12751 to AEP].

Author contributions

Conceptualization: AEP, DS

Methodology: AEP, MB, DS,

Writing – original draft: AEP, MB

Writing – review and editing: AEP, MB, DS

Project administration: AEP, DS

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.