Tissue engineering for tendon and ligament repair: insights and advances

Tissue engineering for tendon and ligament repair: insights and advances | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL View This is a preprint and has not been peer reviewed. Data may be preliminary. 7 May 2025 V1 Latest version Share on Tissue engineering for tendon and ligament repair: insights and advances Authors : Francesca Romano , Roberto Di Gesù 0000-0001-6196-643X [email protected] , Francesco Lopresti , and Vincenzo La Carrubba Authors Info & Affiliations https://doi.org/10.22541/au.174660872.21796090/v1 Published VIEW Version of record Peer review timeline 809 views 396 downloads Contents Abstract Melt electrowriting Hybrid fabrication techniques Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Diseases and injuries affecting tendons (Ts) and ligaments (Ls) are among the most frequently diagnosed musculoskeletal disorders. Although many of these are not life-threatening, they can cause severe debilitation, ultimately leading to substantial reductions in the patients’ mobility and quality of life. Even though the relevant advances in surgical and rehabilitative approaches to treat T/Ls diseases, the incidence of complications associated with those approaches is still too high, making T/Ls diseases an unmet clinical need. Currently, there is no consensus on an effective therapeutic strategy able to promote a complete T/Ls healing process. Over the past decade, there has been a growing interest in developing tissue-engineered tendon constructs as viable graft alternatives. In this review, we aim to delve into the latest advancements in the field of tissue engineering for tendon repair. The most advanced technologies such as electrospinning, 3D printing, melt electrowriting and hybrid fabrication techniques have been discussed, adopting critical connections with biological, and biomolecular aspects. Furthermore, this review provides comprehensive insights into current strategies, and future directions, of T/Ls regeneration using tissue engineering approaches, thus paving the way to the development of new effective approaches to T/Ls diseases. Tissue engineering for tendon and ligament repair: insights and advances Francesca Romano 1,2 , Francesco Lopresti 1 , Roberto Di Gesù *2 , Vincenzo La Carrubba 1 1 Department of Engineering, University of Palermo, Palermo, Italy 2 Musculoskeletal Tissue Engineering (MsTE) lab., Ri.MED foundation, Palermo, Italy *Corresponding author - email: [email protected] Highlights Tendon and ligament injuries represent a significant clinical need due to their high incidence, impact on quality of life, and the limitations of current surgical and rehabilitative approaches. Tissue engineering offers promising innovative perspectives for tendon and ligament regeneration by developing biological substitutes that can restore, maintain, or improve tissue function. The design of effective tissue-engineered scaffolds requires careful consideration of biomaterials (natural and synthetic), fabrication techniques (such as 3D printing and electrospinning), and the incorporation of biophysical and structural cues that mimic the native T/L environment. Cell sources like stem cells and tenocytes, along with bioactive components such as growth factors and pharmacological agents, play crucial roles in enhancing scaffold bioactivity and promoting tissue regeneration within the engineered constructs. Despite significant advancements, challenges remain in replicating the complex multiscale structure of native tendons and ligaments, scaling up fabrication for clinical applications, and ensuring long-term efficacy and functional integration of tissue-engineered grafts. CRediT Francesca Romano: Conceptualization, Writing - Original Draft, Writing - Review & Editing Roberto Di Gesù: Conceptualization, Writing - Review & Editing, Visualization, Supervision Francesco Lopresti: Conceptualization, Writing - Review & Editing, Visualization Vincenzo La Carrubba: Visualization Abstract Diseases and injuries affecting tendons (Ts) and ligaments (Ls) are among the most frequently diagnosed musculoskeletal disorders. Although many of these are not life-threatening, they can cause severe debilitation, ultimately leading to substantial reductions in the patients’ mobility and quality of life. Even though the relevant advances in surgical and rehabilitative approaches to treat T/Ls diseases, the incidence of complications associated with those approaches is still too high, making T/Ls diseases an unmet clinical need. Currently, there is no consensus on an effective therapeutic strategy able to promote a complete T/Ls healing process. Over the past decade, there has been a growing interest in developing tissue-engineered tendon constructs as viable graft alternatives. In this review, we aim to delve into the latest advancements in the field of tissue engineering for tendon repair. The most advanced technologies such as electrospinning, 3D printing, melt electrowriting and hybrid fabrication techniques have been discussed, adopting critical connections with biological, and biomolecular aspects. Furthermore, this review provides comprehensive insights into current strategies, and future directions, of T/Ls regeneration using tissue engineering approaches, thus paving the way to the development of new effective approaches to T/Ls diseases. Introduction Every year in the United States, about 17 million people suffer from tendon injuries (TIs) and ligament injuries (LIs), needing medical intervention to be repaired. These kinds of injuries significantly impact patients’ quality of life, drastically limiting their daily activities, and consequently, negatively influencing their social life. Besides this aspect, it is worth highlighting that TI/LI represents a relevant economic burden for medical care, requesting an economic effort that exceeds 40 billion in the United States 1 . TI/LI comprehends a plethora of medical conditions including ruptures, overuse injuries, and inflammatory/degenerative conditions such as tendinopathies 2 . The clinical impact of TI/LIs is intuitively related to the role that tendons and ligaments (T/Ls) play in vivo . They act as influential components of the musculotendinous unit by facilitating the transfer of forces from muscles to bones, ultimately allowing the joint movement 3 . Intuitively, joints represent a high-demanding environment in terms of mechanical solicitation, and this aspect is emphasized in major joints like knee, or hip. For this reason, tendons are constitutively more resistant to tensile forces if compared with muscles, supporting up to 17 times the body weight 4 . Despite their high mechanical resistance, the T/Ls integrity is progressively weakened by the repeated and numerous stresses of daily life, resulting in microtraumas that ultimately lead to ruptures. This process is more evident in structures involved in the most employed joints, such as the Achilles tendon, which is one of the most frequently injured tendons in the body 3 . Regardless of the T/L involved, each damage represents challenging clinical evidence since both T/Ls have a limited self-healing capability, mainly linked to their hypovascular, and hypocellular histological composition 5 . Currently, surgical approaches represent the elective way to repair TI/LIs after a rupture. However, surgery has relevant drawbacks due to postoperative complications that may lead to chronic pain 6 , or hypomotility of the joints 7 . For this reason, different advanced techniques based on tissue engineering have been developed, and successfully applied, either to overcome the postoperative complications or to develop prevention protocols for TI/LI at the early stages. This review aims to provide an overview of the most recent advances in Tissue Engineering (TE) applied to TI/LIs repair, covering a wide range of points of view. We structured the manuscript starting with a general background on T/Ls anatomy, physiology, and biology, as well as on pathologies that often hit T/Ls. Here, we provide information on the importance of T/Ls for the overall musculoskeletal system functionality, underlying how the natural T/Ls self-repair process is unable to regenerate a functional structure after an injury. Next, we examine a comprehensive section focused on the latest TE approaches adopted to treat TI/LIs. In this section, we deeply analyze the criteria followed to choose the right material, as well as the optimal biofabrication technique for each TE application. Furthermore, we provide a clear state-of-the-art on the choice of cells, and bioactive molecules, to obtain a scaffold responding to the different needs in T/Ls reconstruction and regeneration, highlighting strengths, and limitations, with a critical approach. T/Ls structure and composition Extracellular matrix (ECM) Both T/Ls are fibrous and flexible tissues connecting muscles to bones, or bones to bones, respectively. Their primary function is to transfer forces produced by muscles to the adjacent bony structures (Ts), and to stabilize the bone-to-bone interactions (Ls), ultimately allowing complete and cyclic movements around the joints 5 . In terms of both structure and function, tendons T/Ls share many similarities 1,8 . Therefore, throughout this review the terms tendons and ligaments can be broadly interpreted as referring to both structures. The transitional tendon-to-bone tissue is histologically complex and is considered as an independent tissue (enthesis) with its own anatomic structure 9 , which exhibits a zonal organization transitioning from T (or L) to fibrocartilage, mineralized fibrocartilage, and finally bone. The first layer, primarily composed of fibroblasts and type I collagen, resembles T/L tissue characterized by a highly organized longitudinal collagen alignment. Adjacent to this, the uncalcified fibrocartilage layer contains fibrochondrocytes embedded within a proteoglycan-rich matrix, with a composition that includes type I-III collagen. The transition to mineralized fibrocartilage is marked by the tidemark, composed of type II collagen, alongside smaller amounts of types I and X collagen, and houses fibrochondrocytes within a mineralized matrix. Finally, the bone layer consists of osteocytes, osteoblasts, and osteoclasts embedded in a mineralized matrix providing a rigid anchorage for T insertion 10 . This gradient of cell types, extracellular matrix (ECM), and mineralization is essential for transmitting forces during movements 11 , making T/Ls capable of responding to the high mechanically-demanding joints environment. Not only the enthesis, but the whole T/Ls hierarchical architecture contributes to their significant mechanical resistance. The outermost layer of this architecture, the paratenon (figure 1), is composed of an elastic loose connective tissue allowing the tendon’s smooth gliding against surrounding tissues. Contiguously, the underlying epitenon is composed of a network of type I collagen fibers that work as a secondary gliding interface 12 . The epitenon is intimately connected with the endotenon, a loose connective tissue collecting single tendon fibers into fascicles of various sizes. Importantly, the endotenon also has a functional role, since it hosts vascular, lymphatics, and nervous components 13 . Unfortunately, the amount of those components in endotenon is constitutively low, limiting oxygen, and nutrients diffusion, ultimately making T/Ls bradytrophic (slow healing) 14–16 tissues. Collagen is broadly classified into different types based on its structural and functional properties, including fibrillar collagens, fibril-associated collagens with interrupted triple helices (FACITs), and beaded filament-forming collagens .17 . In T/Ls, collagen exhibits a well-defined hierarchical organization, where individual collagen molecules assemble into fibrils, which further aggregate into fibers (primary bundles) and fascicles (secondary bundles) 18 . The ECM of T/Ls is predominantly composed of type I (figure 2), and type III fibrillar collagen, with a constitutive type I/III ratio (I/III ratio) varying between 1.9, and 4.0, as type I collagen represents the 65-80 % of T/L dry weight 19 . T/Ls injuries induce a transient inversion of I/III ratio that spontaneously normalizes during the healing process, making this value a reliable marker for the T/Ls injury recovery 20 . Figure 1 . Schematic illustration of tendon hierarchical structure and its interface with bone. The left panel depicts the microarchitecture of a tendon, while the right panel illustrates the tendon-to-bone enthesis, emphasizing the transition from muscle to bone through distinct regions: the tendon, non-calcified fibrocartilage, and calcified fibrocartilage. Reproduced with permission from Winters et al., (copyright 2019) 21 The T/L’s ECM contains lower amounts of different collagen chains such as type V, VI, XII XIV collagen 5 . Type V collagen is localized at the core of type I collagen fibrils, playing a role in fibrillogenesis and regulating fibril size. FACIT collagens, such as types XII and XIV, serve as molecular bridges between type I collagen and other matrix components. Furthermore, type XII stabilizes collagen fibers, while type XIV regulates fibril diameter 17 . Type VI collagen, abundant in pericellular regions, forms various polymeric structures: beaded micro-filaments, broad-banded structures, and hexagonal lattices. Its absence disrupts fibril organization, leading to a reduction in cross-sectional area. .22 . Beside collagen, non/collagenic molecules complete the T/L ECM composition. Elastin is a fibrillar glycoprotein which is among the most representative non-collagenic molecules in T/Ls since it contributes to 1–2% of the T/L’s dry mass. Elastin provides the characteristic elastic behavior to T/Ls, allowing for the re-establishment of the T/L’s native coil architecture after cyclical mechanical deformations 23,24 . The remaining T/L’s ECM is composed of different biomolecules such as glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins (GPs) (figure 2), which provide a structural integrity to the ECM filling the intra-fibrillar space, ultimately resulting in the characteristic T/L’s viscoelastic properties. GAGs, such as dermatan sulfate (DS), chondroitin sulfate (CS), heparan sulfate, keratan sulfate (KS), and the nonsulfated hyaluronic acid, are highly hydrophilic molecules that regulate tissue hydration. This property influences both fiber organization and tissue’s resistance to transversal compression, contributing to the overall mechanical behavior of T/Ls. Among PGs, Decorin (DCN), and Biglycan (BGN), are the most representative playing a structural role in maintaining the T/L’s fibers architecture. Interestingly, the expression of DCN and BGN genes related are increased during the early stages of tendon regeneration, and remodeling phase 25 respectively, suggesting for an involvement of these PGs in the T/L’s self-healing phenomena. Tenomodulin (TNMD) is one of the most abundant transmembrane GPs in T/Ls and is involved in collagen fibril maturation. Tenascin-C (TNC) is another important GPs as its production increases under mechanical load, ultimately regulating the collagen fibers alignment. Figure 2 . Distribution of cells and matrix components within tendons. Reproduced with permission from Lomas et al. 25 Cells Mature T/Ls are characterized by low density 26 cell population composed for the 90% of tenoblasts, and tenocytes. Tenoblasts are undifferentiated cells histologically characterized by a round shape and a large oval nucleus. They are located in the endotenon and remain in their undifferentiated phenotype until their terminal differentiation toward adult tenocytes during the T/Ls maturation. Tenocytes are characterized by a fibroblast-like morphology showing an elongated cell body, and a thin cytoplasm 20 (figure 2). Notably, these cells show a characteristic arrangement that follows a parallel disposition with collagen fibers in ECM, ultimately resulting in an aligned spatial disposition. The remaining 10% of T/Ls cell compartment is composed of a heterogeneous population, including, synovial cells (SCs), chondrocytes (CHs), vascular cells (VCs), smooth muscle cells (SMCs), and tendon stem/progenitor cells (TSPCs) 13 . Interestingly, two different TSPCs sub phenotypes (type I, and II) 13 have been identified so far, which are regionally distributed in the epitenon and endotenon, respectively 26 . Remarkably, epitenon-derived TSPCs-I exhibit high levels of vascular-, and pericyte-specific markers, whereas endotenon-derived TSPCs (TSPCs-II) are characterized by higher proliferation rates and higher levels of tendon differentiation-related markers, compared to TSPCs-I 27 , suggesting for two slightly-different stages in the TSPCs differentiation path. Intuitively, scientists should be aware of such a dynamic TSPCs-I/II equilibrium and adopt high-standards handling procedures during experimental manipulation to avoid unwanted phenotype switching, and consequential unreliable results. Similarly, a phenotype modification has been noticed for mature tenocytes, making the engineering of T/Ls a challenging task 16 . These cells easily suffer trans-differentiations toward chondrogenic, osteogenic, myogenic, or even adipogenic phenotypes, since they share mesenchymal stem cells MSCs as a common biological precursor 24,28,29 . Luckily, this phenomenon can be easily controlled by assessing the expression of tenocyte-specific markers during the tenocytes handling. Obviously, the optimal experimental setup should maximize the expression of positive markers (e.g. COLI, COLIII, SCX, TNMD), minimizing at the same time the expression of negative markers such as COLII (cartilage-associated collagen), alkaline phosphatase ALP (an enzyme involved in the bone mineralization), RUNX2 (activates and regulates osteogenesis), SOX9 (a transcription factor that participates in sequential events in chondrogenesis) 30 . With this aim, several parameters have been found to avoid cell trans-differentiation such as the O 2 tension, the relative concentration of micro/macronutrients in culture media, the presence of specific growth factors, and the substrate topography 24,31 . Mechanical properties The orientation of collagen fibrils is primarily responsible for the T/Ls high mechanical resistance, and viscoelastic behavior, which are both dependent on the magnitude of the applied stress, and on the stress application rate. The viscoelastic nature of T/Ls at lower strain rates makes those tissues highly deformable, and capable of absorbing a great amount of mechanical energy 32 . Conversely, at high strain rates T/Ls become stiffer and more proficient in transmitting significant muscle-to-bone, and bone-to-bone forces 33 . The T/Ls biomechanics have been established by creating stress-strain curves using different mechanical tests 4 . Regardless of the test adopted, the overall stress-strain curve exhibit a non-linear behavior composed of four distinct regions 34 . When the tissue is stretched, the energy is involved in the system’s entropy reduction, therefore the mechanical stress does not induce any tension response in the tissue. This behavior is described by the first part of the stress-strain graph, where a low slope of the non-linear portion (toe region) (figure 3), and a gradual slope increase are visible until the first part of the linear zone. Specifically, the T/Ls mechanical behavior exhibits a linear stress-strain response for deformations between 2% and 6%, characterized by an elastic modulus ranging from 0.5 to 2 GPa (Table 1). At this stage, deformations are reversible as the tissue returns to its initial length when the load is removed. Deformations exceeding 6% induce an interfiber crosslink breaking-up, allowing fibers to slide over adjacent fibers, and triggering a plastic deformation (microscopic breakage). The slope of the curve decreases due to intense tissue damage (macroscopic breakage) that continues until a complete failure, which is typically reached at deformations greater than 8% 34 . Intuitively, the mechanical behavior varies greatly depending on the anatomical region where T/Ls are inserted to. As an example, muscles responsible for precise movements (e.g.: the flexors of the fingers) rely on thin tendons, while muscles requiring high levels of strength and endurance (e.g.: quadriceps and triceps surae) are connected to bone via larger tendons. A plethora of studies focused on T/Ls mechanical properties at various size scales have been extensively conducted 23 . The wide range of reported mechanical properties in human tendons reflects the technical challenges of characterizing these tissues ex vivo . Factors such as precise dissection and clamping, accurate measurement of tendon’s dimensions (including cross-sectional area), and the need for theoretical models to interpret biological material properties all contribute to this variability. As a result, a single definitive value for a given tendon cannot be determined. Consequently, in vivo measurements relying on non-invasive assessment of human tendon lengthening during voluntary muscle contraction serve as a benchmark. A summary of the key mechanical properties of various human tendons is provided in Table 1 . Figure 3 . Typical response of tendon tissues. This graph illustrates the stress-strain curve of tendons, demonstrating their biomechanical properties across different phases of deformation. The top section visually represents collagen fiber morphology during stretching: (1) crimped fibers at rest, (2) straightened fibers under load, (3) microfailure, and (4) complete rupture. Reproduced with permission from Ruiz-Alonso et al. (copyright 2021) 34 . Table 1 . Mechanical properties of human tendon tissues. Achilles tendon Ex vivo 819 ± 208 5098 ± 1199 79 ± 22 8.8± 2.2 35 Gastrocnemius tendon In vivo 1160 ± 150 875 ± 85 32.4 ± 2.3 4.9 ± 1% 36 Patellar tendon In vivo 597.4 ± 48.5 5452.7 ± 307.3 48 ± 2 4.86 ± 0.5 37 Achilles tendon In vivo 1900 ± 500 1924 ± 229 29 ± 3 4.2 ± 1.1 38 Achilles tendon In vivo 870 ± 200 \(\sim\) 5000 \(\sim\)75 8.3 ± 2.1 39 Tibialis anterior tendon In vivo 1200 ± 150 530 ± 59 25 ± 2.5 2.5 ± 0.4 40 Extensor hallucis longus tendon Ex vivo 448 ± 183 316 ± 88 39 ± 18 11.7 40 Tibialis posterior tendon Ex vivo 187 ± 54 475 ± 60 19 ± 5 16.3 41 Flexor hallucis longus tendon Ex vivo 440 ± 119 525 ± 200 26 ± 10 12.3 41 Long head of the biceps tendon Ex vivo 282.9 ± 144 - 31.7 ± 15.4 11.6 ± 6.6 42 T/L injuries, healing process, and therapeutic approaches T/Ls disorders may be categorized into two macro-groups (acute, and chronic disorders) that overall describe the diseases’ onset time. Specifically, acute injuries occur after sudden traumas following intense physical stresses due to agonistic sport activities, or undesirable non-physiological movements. Chronic injuries are consequential of untreated/mistreated injuries instead and often hit elderly people suffering from recurrent mechanical breakdowns and related inflammatory processes. Both acute, and chronic T/Ls disorders cause well-defined pathological conditions described as tendinopathy, tendinitis, and tendinosis, but their etiology, and physiopathology, are often foggy, and confusing. Tendinopathy is typically used to describe a disorder involving a tendon without knowing the specific pathology, ultimately leading to severe dysfunctionality and localized pain. It is primarily diagnosed through anamnestic investigation supported by semeiotic examination. Despite the involvement of massive phlogistic phenomena, recent findings classify tendinopathies as degenerative processes triggered by a defective self-healing response 43 . Tendinitis refers to a tendon injury accompanied by an inflammatory response and the presence of inflammatory cells caused by an overloading of the musculotendinous unit 44 . In contrast, tendinosis describes the non-inflammatory degeneration of a tendon, as identified histopathologically 43 , which can include changes to its structure or composition. The healing process following tendon injury occurs in three overlapping stages: inflammation, proliferation, and remodeling. The expression profile of cytokines varies throughout tendon healing, with pro-inflammatory cytokines predominating in early phases, while anti-inflammatory and reparative cytokines become more prominent in later stages 45 . The inflammatory phase begins immediately after the injury with a clot formation at the damaged site and lasts approximately one week. Platelets and cells within the clot release key factors, including transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF1), and platelet-derived growth factor (PDGF). These factors help establish a local environment that supports the recruitment of inflammatory cells, such as interleukins (IL), IL-6 and IL-1β. Growth factors recruit neutrophils from the bloodstream, which in turn activate M1 macrophages to clear necrotic debris through phagocytosis. Simultaneously, cytokine-recruited fibroblasts migrate to the injury site and initiate matrix deposition, marking the beginning of the healing process. The proliferative phase begins approximately two days post-injury and is characterized by ECM expansion, fibrous scar formation, and increased cell activity. During this phase TGF- β levels peak, and macrophages gradually shift to M2 phenotype, which later contributes to scar remodeling. TGF-β drives collagen production, recruits TSPCs, and regulates protease activity, whereas IGF promotes ECM production. PDGF is involved in DNA and protein synthesis. As healing progresses, TSPCs proliferate and differentiate into tenocytes which, along with fibroblasts, contribute to tissue regeneration by depositing PGs, GAGs, and collagen at the wound site. The remodeling phase begins two weeks following the injury and is marked by the reorganization of previously synthesized collagen. The whole phase is characterized by a gradual decrease in cellularity alongside an increase in collagen content and density. Tenocytes and collagen fibers align longitudinally to enhance the mechanical strength of the newly forming tissue. Collagenase activity promotes the transition from type III to type I collagen, the latter characterized by larger fiber diameter and higher tensile strength. Moreover, tenocytes express α-smooth muscle actin (αSMA), fundamental in matrix contraction, supporting wound closure. Throughout this phase, tenocytes metabolic activity remains significantly lower compared to the early stages of healing. The overall duration of tendon repair depends on the severity of the injury and the specific tendon affected. Regardless the kind of T/Ls disorder, the inadequate self-healing remains a common challenge, often resulting in the formation of fibrous T/L-like tissue that lacks the native biochemical and biomechanical properties of healthy tissue 1,46 . The treatment of acute and chronic T/Ls injuries relies on conservative methods, surgical intervention, or a combination of both. Conservative approaches refer to non-surgical strategies that typically include rest, physiotherapy, cryotherapy, ultrasound therapy, and laser therapy. Additionally, pharmacological therapies often involving nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids is commonly employed to manage pain and inflammation 47 . However, the inherently limited self-healing capacity of T/Ls frequently reduces the effectiveness of conservative treatments, often leading to partial functional loss and recurrent injuries 48 . A key factor in determining the appropriate treatment strategy is the severity of the injury. Massive injuries like ruptures typically require surgical intervention, while chronic injuries, if left untreated, can lead to progressive tendon weakening, muscle atrophy, contractures, and reduced joint mobility 49 . Although conservative treatment can help avoid the complications of surgery such as infections, scar formation, tendon necrosis, nerve damage, and the risk of retears, surgical intervention remains the primary approach for the treatment of severe injuries 2 . Conventional surgical techniques focus on restoring the biomechanical properties of the tendon through wound suturing or securing the tendon to the bone using sutures, wire loops, and stainless-steel anchors. Both open-air and arthroscopic repair techniques are widely employed, often in combination with postoperative conservative rehabilitation to accelerate functional recovery (known as combined approach). However, while surgery can temporarily restore tendon continuity, it does not fully preserve the structural integrity,resulting in a retear risk ranging from 26% for small (5 cm) tears 46 Tissue grafting is an alternative surgical approach in which a tendon graft is used to bridge the damaged area 50 . Autografts, derived from an autogenous donor tendon, are preferred to minimize the risk of immune rejection but they come with potential complications, including donor site morbidity, and only partial restoration of T/L pre-injury functionality (approximately 50%). 50 . Allografts (from one individual and transplanted into another individual ) and xenografts (from non-human species) serve as alternatives when the autografts are unavailable, such as multi-ligament injuries or revision surgeries. However, they carry their own set of risks including tissue rejection and disease transmission 5,45 , often requiring lifelong immunosuppressive therapy to prevent graft rejection. Moreover, tissue grafting approaches are often less effective in chronic injuries, as they can lead to excessive postoperative tension, with failure rates reaching up to 38% 2 . In the current medical landscape, it is important to recognize that although conservative approaches, traditional surgery, and the use of grafts have played a fundamental role, they have inevitable intrinsic limitations that are prompting an increasing interest in tissue engineering as a promising and innovative perspective. The term ’tissue engineering’ was first introduced by participants at the first meeting sponsored by the National Science Foundation (NSF) in 1988 even though at that time it had a poor connection to the discipline as it is conceived today 51 . In 1993, Langer and Vacanti summarized the early developments in this field by defining tissue engineering as “an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function” 52 . Tissue engineering often begins with cell in vitro expansion under controlled conditions to promote their growth and proliferation. Then, these cells are seeded onto a biocompatible matrix-or scaffold - a structural framework made of synthetic or natural materials that support tissue growth before implantation. Over time, the biodegradable biomaterial undergoes gradual degradation within the body, while the implanted cells proliferate and secrete ECM in vivo , ultimately leading to the repair of damaged tissue. For scaffolds to be effective and minimize side effects, they must meet several criteria, such as non-toxic degradation products, biocompatibility, a degradation rate that matches tissue growth, adequate porosity, and mechanical strength. The role of biomaterials in scaffolds design One of the main design principles in tissue engineering is biomimicry, which is the ability to replicate the native biological environment of the tissue. From a biomimetic perspective, a scaffold designed for tendon/ligament tissue engineering (T/L TE) should ideally exhibit anisotropic mechanical properties, mimicking the natural alignment of collagen fibers 12 that respond differently depending on the direction of the applied force 53 . Additionally, scaffolds should possess a hierarchical structure, reflecting the multiscale organization of tendons, which spans from a molecular to a macroscopic level. Despite the many advantages provided by metallic and ceramic materials, polymers are particularly well-suited for scaffolds biofabrication, thanks to their biocompatibility, and processability 54 . The choice between natural and synthetic polymers depends on the specific application. Natural polymers Natural polymers, such as collagen, chitosan, silk, or hyaluronic acid, offer low immunogenicity, and a biological environment that supports cell adhesion and growth, exhibiting minimal immunogenicity. As intrinsically bioactive, they have been extensively explored in T/L TE. Moreover, different strategies have been employed to overcome their inherent limitations, particularly in terms of mechanical performance and degradation control. One notable approach was proposed by Tawonsawatruk and colleagues 55 , who leveraged the hyperplastic polymer polyhydroxyalkanoate (PHA), produced by microorganisms like Pseudomonas, to fabricate T/L implants. By threading human plantaris through molds with pre-designed holes, they created a composite graft with a tensile strength of 56 MPa, closely resembling that of a human hand tendon. Hydrogel-based scaffolds have gained increasing attention due to their ability to support cell functions while replicating the hydrated environment of native tissues. For example, Diaz and colleagues 56 developed a scaffold for supraspinatus tendon repair using a chitosan-gelatin matrix with varying cellulose concentrations. Crosslinking with genipin improved the scaffold’s stability, while cellulose incorporation enhanced cell metabolic activity and proliferation. A different strategy was explored by Ruiz-Alonso and coworkers 57 who engineered a hydrogel-based scaffold composed of alginate, hyaluronic acid, gelatin, and fibrinogen. This formulation exhibited remarkable biocompatibility, facilitating tenocytes recovery from bioprinting-induced stress and promoting rapid cell proliferation, while preserving their phenotype. Further advancing hydrogel-based strategies, Li and colleagues 58 proposed a 3D hydrogel-based sandwich model in which TSPCs were cultured on a grooved 2D hydrogel substrate before an additional hydrogel layer was added to form a 3D construct. In vivo implantation in a rat Achilles tendon defect model demonstrated not only enhanced tendon regeneration but also a reduction in heterotopic ossification. Despite these promising advantages, natural polymers still pose significant challenges, including uncontrolled degradation rates and mechanical properties that do not fully match those of tendons 46 . Moreover, batch-to-batch variability in molecular weight and purity can compromise reproducibility, thereby limiting their clinical applicability. Synthetic polymers Unlike natural polymers, synthetic polymers such as poly(lactic-co-glycolic acid) (PLGA), polylactide (PLA), polycaprolactone (PCL), and polyurethane (PU), offer superior mechanical properties and tunable degradation rates. They enable precise control over scaffold architecture, porosity, and degradation kinetics. Moreover, synthetic polymers can be engineered to meet the mechanical properties of native tendon tissue, providing temporary support during healing 59 . One approach to improving the bioactivity of synthetic scaffolds was demonstrated by Wang’s team, who developed a PLGA scaffold functionalized with PDGF-AA (PLGA-PDGF-AA) onto the surface of PLGA fibers. Functionalization significantly enhanced tendon repair, as in vitro experiments confirmed the scaffold’s biocompatibility and lack of cytotoxic effects. In a mouse hind limb tendon injury model, PLGA-PDGF-AA scaffold significantly improved tendon repair and collagen synthesis compared to PLGA alone. Beyond biochemical modifications, mechanical stimulation has also been explored as a key factor in optimizing synthetic scaffolds for tendon repair. Wang and colleagues 60 demonstrated that mechanical loading on a cell-free polymeric composite scaffold significantly enhanced the regeneration of a fully functional Achilles tendon in a rabbit model. The scaffold, composed of a combination of PGA and PLA filament fibers, promoted host cell infiltration, matrix production, and tissue remodeling. Histological analysis showed the formation of parallel-aligned collagen fibers and tenocytes similar to those on native tendons. A different strategy was pursued by Bahrami and colleagues 61 , who used PU to create multiscale nanofibrous scaffolds designed to replicate the architecture and mechanical characteristics of T/Ls fascicles. The bundles closely replicated the mechanical properties of other PU-based grafts, such as Artelon (SportMesh), as well as native tendon tissues like patellar tendon. The work by Turgut and coworkers 62 explored the use of fibroblast growth factor-2 (FGF-2) PCL/PU nanofibrous scaffold. In vivo results on rat Achilles tendon injury model demonstrated that the use of FGF-2 effectively supported tendon healing with a better collagen fibril organization, enhanced vascularity, and reduced inflammatory responses. Challenges related to biocompatibility, inflammatory responses, and the potential cytotoxicity of degradation byproducts remain significant obstacles in the clinical translation of synthetic polymers. The breakdown of polymeric chains into acidic byproducts can lead to pH changes, subsequently triggering inflammatory reactions 63 . To address these limitations, researchers are actively investigating strategies to improve the biological performance of synthetic scaffolds. For instance, surface modifications such as plasma treatment, bioactive molecule coatings, and functionalization with anti-inflammatory agents have shown promise in reducing immune responses and promoting cell interactions 64 . Furthermore, hybrid approaches combining synthetic polymers with natural polymers, such as collagen or gelatin, can leverage the mechanical advantages of synthetic scaffolds while minimizing adverse reactions 65 . Optimizing biophysical and structural cues T/Ls are mechanosensitive tissues that experience continuous mechanical stretching, a process essential to the maintenance of T/L cell morphology and phenotype 66 . Besides promoting tenogenic differentiation of stem cells by augmentation of tendon-specific markers, mechanical loading improves the mechanical properties of the engineered tissues through ECM remodeling and regulation of cell behavior. A strategy of replicating this native mechanical milieu is the application of mechanical stretching, which can be classified as static and dynamic. Among these types, dynamic stretching is the most employed, whose outcome influenced by three parameters: strain, frequency, and rest intervals. Each of these factors independently impacts tenogenic differentiation, thus their interdependence complicates the determination of optimal conditions. To address this challenge, advanced bioreactors have become essential 66 . Such systems regulate multiple variables simultaneously, thereby closely mimicking the biomechanical and biochemical environment of native T/Ls, which consequently delivers the required stimuli to the engineered constructs. For instance, Xu and team 67 investigated the effects of mechanical stimulation on TSPCs cultured on poly(L-lactide-co-ε-caprolactone)/collagen (P(LLA-CL)/Col) scaffolds using a custom-made 3D bioreactor. The results of their research demonstrated that cyclic tensile strain at different frequencies (0.3 Hz, 0.5 Hz, and 1.0 Hz) and amplitudes (2%, 4%, and 8%) did not compromise cell viability. However, differences in cell proliferation and tendon-specific marker expression were observed, and the optimal response was achieved at 0,5 Hz and 4% strain. Apart from mechanical stimuli, scaffolds design is a key player in T/Ls repair, with porosity being one big factor. An optimized pore structure allows the scaffold to function as a cytoskeletal substitute during the initial stages of healing, where cell elongation and migration are promoted. Moreover, deeper pore architectures provide the necessary space for cell proliferation and extracellular matrix deposition. Beyond porosity, the recreation of hierarchical organization of T/L ECM is another fundamental prerequisite in scaffold design. To this end, biomaterials with aligned topographies have been developed, as they resemble the native T/L microenvironment, promoting aligned collagen fiber deposition 68 , and tenogenic differentiation 69 . Scaffold manufacturing techniques for T/L scaffolds The choice of the biofabrication approach greatly influences the properties of the scaffold, and is driven by the severity of the injury, and the type of tendon involved 5 . Several fabrication techniques have emerged as prominent strategies in T/L TE including 3D printing, electrospinning, melt electrowriting, and textile technologies. Each of these techniques possess distinct benefits and drawbacks (Table 2). 3D printing provides accurate control over scaffold geometry, allowing the design of customized architectures tailored to specific functional requirements. Electrospinning enables the production of nanofibrous structures with high porosity and surface area, facilitating cell attachment and proliferation. Melt electrowriting, a variation of electrospinning, uses a heated polymer to generate well-organized and customizable fiber arrangements. Textile-based fibrous scaffolds offer significant advantages of scalability and versatility, and they can be engineered to mimic the architecture of T/Ls 16 . Decellularization is another widely used technique aimed at removing cell components while preserving the ECM’s structural integrity. Moreover, new technologies grouped as “hybrid fabrication techniques” have gained attention for their ability to integrate multiple materials and architectures into a single construct, offering new possibilities for scaffold optimization. To make easier the comprehension of the section 4, we included a schematic overview of the different fabrication techniques adopted for T/Ls scaffolds, highlighting their respective advantages and drawbacks (table 2). 3D Printing 3D printing is an additive manufacturing method based on computer-aided design (CAD) that creates 3D structures in a layer-by-layer fashion starting from a 2D pattern and using a liquid binder that is printed onto a powder bed. The technique is characterized by a precise control over biomaterial deposition at the micrometer level, with the possibility to achieve controllable porosity (both in geometry and size) and an accurate replication of the macroscopic architecture of native tissues 70 . Through 3D printing, multiple materials can be combined in the same scaffold, with a broad design space, ranging from plain structures (like patches, or sheaths) 71–75 to more complex tubular shapes 76–79 . . Different 3D printing techniques have been developed, including stereolithography, bioprinting, inkjet printing, fused deposition modeling (FDM), selective laser sintering (SLS), and laser beam melting. While each has its technical realization, the common principle among them is the sequential addition of material in a layer-by-layer manner to achieve the final structure 70 . Current research has demonstrated the application of 3D printing for tendon regeneration through the utilization of different combinations of materials. Li and colleagues 76 fabricated a Gelatin Methacrylate (GelMa)/PCL scaffold. In the study, the PCL shell was used to enhance the mechanical properties of the hydrogel core. Functional assessments in a rat Achilles tendon defect model showed that the scaffold can promote tendon regeneration while preventing heterotopic ossification. Similarly, Kempfert and colleagues 71 developed a PCL patch coated with Collagen I and Fibronectin to increase the overall biocompatibility. Additionally, authors performed plasma modifications that further enhanced the hydrophilicity of the construct, as showed by experiments with human bone marrow stem cells (BMSCs). Innovative structural designs have also been explored. Rodriguez-Reinoso and coworkers 72 explored a PLA-based device composed of two superposed thin patches featuring barbs inspired by rose thorns and limpet teeth. Their findings indicate that the device provides superior load distribution compared to conventional suture-based methods. Jiang and team 73 enhanced stem cell function and proliferation by incorporating Collagen I and Fibrin hydrogels into a PLGA scaffolds (figure 4). In vivo , subcutaneous implantation in mice revealed excellent biocompatibility and degradation properties. Likewise, Zhang and colleagues 77 employed electrohydrodynamic 3D printing to fabricate a PCL scaffold, incorporating Pluronic F127 at different concentrations. In vitro tests showed that PCL scaffolds with 5% F127 exhibited optimal cell adhesion and growth, whereas in vivo data obtained from rats’ Achilles demonstrated enhanced collagen deposition and organization. While there has been enormous advancement in 3D printing, there are still some problems. Among the primary limitations is achieving native T/L biomechanical strength, which requires oriented fibers that mimic natural collagen fibers. However, obtaining aligned fibers with 3D printing technology is challenging. While electrospinning excels in producing aligned nanoscale fibers, conventional 3D printing methods struggle to achieve the same level of organization. Post printing processes may offer a potential solution to improve scaffold mechanical properties. Moreover, the issue of reproducibility is a significant concern, especially for clinical application. However, ongoing optimization of printing parameters and material selection has already shown promise in improving consistency, suggesting that these limitations may be progressively overcome. Figure 4 . Schematic representation and close-up photographs of 3D-printed PLGA composite scaffolds with different configurations. Reproduced with permission from Jiang et al. 73 Electrospinning Electrospinning is acknowledged as a versatile technique in used materials science and biomedical engineering which involves the creation of ultrafine fibers through the application of an electric field to a polymer solution. The standard experimental setup for electrospinning relies on a syringe, a spinneret (usually a needle), a syringe pump, a high-voltage power supply, and a collector, which can either be planar or rotary 80 . During the process, the polymeric solution is extruded from the syringe, forming a droplet at the needle tip. Upon application of a high voltage, the droplet elongates into a conical shape known as the “Taylor cone”, and a thin liquid jet emerges once the electrical field overcomes the solution’s surface tension. As the jet travels towards the collector, it undergoes rapid whipping motion, during which solvent evaporation occurs, leading to fiber deposition. The resulting fibers possess unique properties such as high surface area-to-volume ratio, tunable diameters, flexibility in surface functionalization, and interconnected porous structure 81 . Beyond its material flexibility, electrospinning offers a wide range of adjustable parameters that influence fiber morphology and orientation. This adaptability makes it a promising approach to address challenges like low cellularization efficiency, one of the critical hurdles in the development of 3D-engineered tissues, particularly when pore sizes are either excessively small or large 82 . Nowadays, several \soutInizio moduloelectrospinning variants have been developed, such as coaxial electrospinning 83,84 , wet electrospinning 85 , in-line blending 86 , and emulsion electrospinning 87 each offering specific advantages. In a study conducted by Wu and coworkers 88 a nanofibrous Poly(p-dioxanone)/Silk Fibroin (PPDO/SF) scaffold was fabricated by electrospinning, followed by a thermal ethanol treatment to induce a wavy fibrous morphology (figure 5). Biological tests on tenocytes showed that the incorporation of SF improved cell adhesion and proliferation, while in vivo subcutaneous implantation in rats reduced the inflammatory response, highlighting the scaffold’s biocompatibility. Expanding on the role of biochemical modifications, Tu and their team 79 fabricated Poly(L-lactide-co-ε-caprolactone) (PLCL)/Collagen I nanoyarn scaffolds using an electrospinning setup equipped with a basin and a water vortex. By crosslinking Fibromodulin (Fmod) with collagen, they successfully promoted the tenogenic differentiation of human adipose-derived stem cells (hASCs), underscoring the potential of biochemical modifications in guiding cellular behavior. In a related study, Tu’s group 83 employed co-axial electrospinning to fabricate a PLGA/Chitosan tubular scaffold modified with soluble tendon extracellular matrix (sTECM). PLGA served as the core structure, while PLGA and sTECM formed the outer shell. Biomechanical testing at 12 weeks post-implantation in rat Achilles tendons showed a Young’s modulus of approximately 600 MPa, suggesting that both biochemical and structural modifications synergistically contribute to scaffold function. However, a direct comparison between these approaches is needed to fully assess their relative advantages. Similarly, Sheng and colleagues 75 focused on fiber alignment by developing a biomimetic electrospun sheath composed of PCL/Gelatin to encase the patellar tendon in a rabbit model. Histological examination at 8 weeks post-implantation demonstrated favorable orientation of collagen fibrils and spindle nuclei of fibroblasts, further corroborating that the alignment of the fibers is an important factor in tissue regeneration. Although these results align with those of Tu’s nanoyarn scaffolds, the different materials and fabrication techniques employed raise questions about the optimal approach for achieving a functional regeneration. Another key aspect in electrospinning-based scaffold design is the effect of fiber diameter on cell responses. Baldwin and colleagues 89 investigated the effect PDO electrospun scaffold topography on the transcriptional response of healthy and diseased tendon fibroblasts. Scaffolds with aligned fibers exceeding 2000 nm in diameter led to an upregulation of genes involved with DNA and cell replication and downregulated those linked with inflammation. These results are consistent with previous studies highlighting the influence of fiber alignment, but they also introduce fiber diameter as an additional parameter for modulating cell behavior. In another innovative approach, Song’s team 90 developed an immunoregulatory PCL scaffold functionalized with a layer-by-layer coating of SF linked with mechano-growth factor (MGF) via click chemistry. In a rat Achilles tendon injury model, inflammation was undetectable 28 days post-implantation, demonstrating the scaffold’s ability to mitigate foreign body reactions. Seeking to enhance mechanical properties, Li and their group 91 sought to enhance the mechanical properties of electrospun scaffolds by fabricating a nanofibrous PCL/Methacrylated poly(trimethylene carbonate) (PTMC-MA) composite. Their results showed that increasing PTMC-MA content significantly improved mechanical performance, with scaffolds containing a 1:3 ratio of PCL/PTMC-MA exhibiting a stress at break of 23.80 ± 3.44 MPa, comparable to native tendons. Figure 5 . Schematic of the electrospinning process and subsequent ethanol and thermal treatment, resulting in aligned fibers with a wavy pattern. Reproduced with permission from Wu et al. 88 Despite its widespread use, electrospinning suffers several limitations, among which the cytotoxicity 92 due to solvents residual within the electrospun fibers is the most frequent. This risk can be mitigated by using low-boiling points or low-toxicity solvents. Alternatively, the investigation of water-based solvents systems is a viable path for environmentally friendly manufacturing processes. Additionally, integrating advanced drying systems such as heated airflows, or vacuum-assisted collection chambers could further enhance solvent removal. Another main obstacle is the inherently low yield of production of electrospinning, making it difficult to scale up to industrial applications. A multineedle electrospinning system is a valid alternative to address this issue by increasing the throughput while maintaining a high fiber quality. Melt electrowriting Melt electrowriting (MEW) is an emerging fabrication technique that like electrospinning uses an electrical field through which molten polymers can deposit into fibers in a layer-by-layer manner 93 , with diameters ranging from 2 to 50 μm 94 . The standard setup includes a heated polymer delivery system, a three-axis positioning mechanism for an accurate material deposition, and a high-voltage power supply for generating a potential between the delivery system and the collector. The electric field created between the printhead and the collector charges the molten droplet 95 , and when these charges exceed the surface tension of the material, a Taylor cone is generated, propelling the molten jet toward the collector 96 . Several studies have explored the potential of MEW in T/L TE by optimizing fiber architectures and mechanical properties. Von Witzleben and colleagues 97 fabricated a rectangular PCL scaffold via the MEW technique, followed by a type I collagen coating extracted from rat tail (figure 6). Their comparative analysis of fiber architecture demonstrated that rhombohedral pore geometries enhanced cell alignment of hASCs more effectively than traditional grid structures. This shows the important role of pore geometry in regulating cell behavior, with certain designs having the ability to promote cell alignment and function. To approximate the biomechanical behavior of human T/Ls, Hochleitner and their team 98 fabricated poly(e-caprolactone-co-acryloyl carbonate) (p(e-CL-AC)) scaffolds with a sinusoidal architecture by MEW at speeds below the critical translation threshold, ensuring the fibers retained their sinusoidal morphology. These scaffolds demonstrated cytocompatibility with murine L929 cells and mimic the characteristic toe region seen in tendon stress-strain curves. Despite its potential, MEW faces significant challenges related to ink formability. A crucial problem concerns the viscosity control of the ink. Low-viscosity inks are easy to extrude and to manipulate but collapse when deposited, while high-viscosity inks are difficult to extrude and may not respond adequately to the electric field 99 . Therefore, finding an optimal balance between these extremes is essential for improving MEW accuracy and reliability. One potential solution to this problem involves shape memory polymers that dynamically adjust their viscosity in response to external stimuli 96,100 . This approach would enable more precise control over the extrusion process thereby enhancing overall accuracy and reliability. Figure 6 . Schematic representation of the scaffold fabrication process, combining melt electrowriting (MEW) to create embroidery structures with a superficial PCL pattern and collagen coating (A). Quantitative analysis of fiber diameter (B), fiber spacing (C), and fiber alignment angle (D) for different scaffold designs. Reproduced with permission from von Witzleben et al. 97 Hybrid fabrication techniques The integration of different fabrication techniques allows researchers to retain the advantages of each individual methodology while mitigating their respective limitations. In this way, the fabrication of scaffolds is custom designed for either single or multiple tissue types 101 . For instance, while textile techniques allow for the fabrication of macroscopic structures with defined patterns and porosities, 102 the incorporation of electrospinning enables the integration of nano-scale fibers within such constructs, enhancing their functional properties 78,103–106 . Cai and their group 103 explored this synergy by combining electrospinning with traditional textile weaving. They employed SF/PLLA nanofiber yarns as weft and commercial PLLA microfiber yarns as warp producing a nanofibrous scaffold with a plain-weaving structure. These scaffolds promoted cell adhesion, alignment, proliferation, and the preservation of tenocyte phenotype in vitro . Biomechanical tests performed six months post-surgery in rats Achilles tendon defect model revealed a failure load of 44.2 ± 6.9 N, surpassing that of native tendons (39.9 ± 4.3 N). Similarly, Xie and fellow researchers 104 introduced an innovative micro-nano hierarchical scaffold designed mimic the natural organization of collagen fibrils. Using electrospinning, they created aligned PLGA nanoyarns loaded with Fibrin which were then braided onto a triple-helix structure. This design promoted tendon cells alignment and proliferation in vivo . Shalumon and co-authors 78 fabricated a scaffold by collecting aligned PCL electrospun fibers around a commercial suture. Three of these single yarns were braided together and surface modified with heparin grafting. Retrieved samples of rabbit extensor digitorum tendon at 6 weeks post-implantation exhibited a failure load of 16.58 ± 3.05 N, demonstrating its mechanical viability as an alternative to autologous tendon grafts. Another example of the combination of technologies is the assay conducted by Pensa’s group 107 in which hybrid patches were obtained by printing a PLA mesh onto PCL/Gelatin electrospun mats (figure 7A). In vivo functional tests in a rat bone defect model showed no inflammatory response 20-weeks post implantation. Taken together, the findings of several studies indicate a promising potential for combining electrospinning with other fabrication approaches to bridge scaffolds toward the native T/L’s structural and mechanical properties. A recurring theme among them is the employment of aligned nanofibers, whether woven, braided, or incorporated into hierarchical structures, to guide cell alignment and improve mechanical performance. This alignment appears to be an important part of enhancing tenocyte proliferation and phenotype maintenance. In the near future, it can be expected that more techniques of this kind will be applied. Figure 7 . Examples of scaffolds developed through advanced hybrid fabrication techniques. (A) 3D-printed reinforced electrospun scaffold. Reproduced with permission from Pensa e al., licensed under Creative Commons Attribution 4.0 International 107 . (B) Scaffold created by braiding three yarns, which were twisted from electrospun fibers. Scale bar: 200 μm. Readapted from Laranjeira et al. 106 Decellularization is a process that removes cell components from the ECM without disrupting its inherent structure and mechanical integrity. It begins with the extraction of tissues from an animal donor, followed by treatment with specific reagents and wash solutions to eliminate cell material, resulting in a protein-based matrix. The resulting ECM can be seeded with cells for transplantation as a regenerative medicine application. 108 Tendons 109–111 and other connective tissues 112 are common sources for decellularization-derived scaffolds, which possess several advantages including reduced immunogenicity and enhanced biocompatibility 113 . One of the primary benefits of decellularization over synthetic fabrication methods, such as electrospinning and 3D printing, is the ability to retain the native ECM architecture (figure 8). This biomimetic structure provides a biologically relevant environment that can facilitate cell attachment, proliferation, and differentiation, features that are difficult to fully replicate using synthetic approaches. Figure 8. Schematic illustration of the fabrication process of decellularized tendon scaffolds (DTSs). Reproduced with permission from Dede Eren et al. 113 Several studies have demonstrated the regenerative potential of decellularized tendon scaffolds. In a study undertaken by Niveditha and co-researchers 111 decellularization of rat Achilles tendon was accomplished using tri (n-butyl) phosphate (TnBP). The tendon was used a as platform for further seeding rat ASCs and for the delivery of Tinospora cordifolia extract , used in many ayurvedic preparations. Cell viability and proliferation assays demonstrated increased proliferation rates, with stem cells adopting a spindle-shape morphology typical of tenocytes. Similarly, Cui and colleagues 109 employed bovine tendon sheets functionalized with collagen-binding domain extracellular vesicles (CBD-EVs) from TSPCs. Biomechanical tests on regenerated rat Achilles tendons 12 weeks post-implantation revealed a failure load of 110 N, denoting the mechanical strength of the implant. Further innovations have been aimed at improving the mechanical properties of decellularized scaffolds. Wang and colleagues 114 applied a double cross-linked chitosan modification to enhance the mechanical properties of decellularized mussel adductor muscle scaffolds. Histological evaluations performed after implantation on rats’ Achilles tendon defect model indicated that the engineered tendon may be beneficial towards tissue remodeling and regeneration. However, it remains unclear how the chitosan modification would fare in long-term in vivo applications, particularly in terms of maintaining scaffold integrity under dynamic loading conditions typical of tendons. The research team led by Chen 112 fabricated a Bioactive Collagen I scaffold (BCS) from porcine connective tissue seeded with hTSCs exposed to cyclic uniaxial loading. The work progressed to a pilot human safety study, confirming the feasibility in mini-open or arthroscopic rotator cuff repair (figure 9). Figure 9 . Schematic and intra-operative acquisitions of on-lay Bioactive collagen scaffold (BCS) repair of human supraspinatus tendon. Reproduced with permission from Chen et al. 112 The limited availability of donor tissue, along with the biological variability of native T/Ls restrict shape tailoring and scalability for clinical applications. Moreover, the decellularization process is capable of compromise ECM integrity, leading to the loss of essential components, including GAGs and growth factors. Such limitations can lead to the reduction of the scaffold’s bioactivity, which can affect its long-term functionality. To deal with these challenges the development of cutting-edge strategies is fundamental. Decellularized ECM has been investigated as bio-ink for 3D bioprinting 115 . Additionally, the integration of decellularized ECM with other biofabrication techniques offers the potential to deliver bioactive molecules and cells, which enhances the regenerative potential of scaffolds. Table 2 . Overview of advantages and disadvantages of the main fabrication techniques employed in T/L TE. 3D Printing Rapid prototyping Design freedom Limited resolution Post-processing required GelMa/PCL In vitro and in vivo (rat) Achilles tendon 76 PCL In vitro - 71 PLA In vitro - 72 PLGA/Collagen/Fibrin In vitro and in vivo (mouse) Back subcutaneous implantation 73 . PCL/Pluronic F127 In vitro and in vivo (rat) Achilles tendon 77 Electrospinning High surface area to volume ratio Submicron dimensions High pores interconnections Tunable properties Challenges in collecting and handling nanofibers Trapped solvents require removal from electrospun nanofibers Low production yield PPDO/SF In vitro and in vivo (rat) Subcutaneous implant 88 PLCL/Collagen I In vitro and in vivo (mouse) Back subcutaneous implantation 79 PLGA/Chitosan/sTECM In vitro and in vivo (mouse) Achilles Tendon 83 PCL/Gelatin In vitro and in vivo (rabbit) Patellar tendon 75 PDO In vitro - 89 PCL/SF In vitro and in vivo (rat) Achilles Tendon 90 PCL/PTMC-MA In vitro - 91 Melt electrowriting Highly organized structures Highly controlled pore size and interconnectivity Careful control of temperature to avoid material degradation Low production speed PCL/Collagen I In vitro - 93 p(e-CL-AC) In vitro - 98 Electrospinning/textile technologies High surface area Fabrication of complex structures Enhanced mechanical properties Production costs Limited scalability Need for specialized expertise PLLA/SF In vitro and in vivo (rat) Achilles Tendon 103 PLGA In vitro and in vivo (rat) Achilles Tendon 104 PCL In vitro and in vivo (rabbit) Extensor digitorum tendon 78 Decellularization Biocompatibility Pre-formed structure Inferior mechanical properties Low reproducibility Complex process ECM from rat Achilles tendon In vitro - 111 ECM from bovine Achilles tendon In vitro and in vivo (rat) Achilles Tendon 109 ECM from mussel adductor muscle In vitro and in vivo (rat) Achilles Tendon 114 ECM from pig connective tissue In vitro and vivo (human) Supraspinatus tendon 112 GelMA= Gelatin Methacrylate; PCL = Polycaprolactone; PLA = Poly(Lactic acid); PLGA=Poly(lactic-co-glycolic acid); PPDO = Poly(p-dioxanone); SF = Silk Fibroin; PLCL = Poly(L-lactide-co-ε-caprolactone); sTECM = Soluble tendon extracellular matrix; PDO = Polydioxanone; PTMC-MA = Methacrylated poly(trimethylene carbonate); p(e-CL-AC) = poly(e-caprolactone-co-acryloyl carbonate); PLLA= Poly (L-lactic acid); ECM= extracellular matrix. Cell sources Cells, as basic units of all living tissues, represent a central element in TE since they constitute the biological framework in which ECM components are synthesized. Cells in scaffolds create dynamic microenvironments during repair and promote cell retention at the injury site. This aspect prevents undesired migration to surrounding tissues, which could potentially result in unintended side effects. Advances in cell manipulation and differentiation have significantly broadened the range of available options, allowing more specific and effective regenerative approaches 116 (table 3). However, the clinical translation of cell-based approaches remains in its early stages. Currently, a limited number of phase 1 and 2 clinical trials are either in progress or have been completed for the cell treatment of T/Ls disorders 117 . Inizio moduloAmong the most commonly used cell types in T/L TE are tenocytes 118 , fibroblasts, and stem cells that possess the ability to differentiate into tendon-like cells 119 . Stem cells Stem cells offer a highly versatile solution in T/L TE, owing to their ability for self-renewal and differentiation into multiple mesodermal lineages, including osteoblasts (bone), chondrocytes (cartilage), tenocytes (tendon) 13 when exposed to specific biochemical and mechanical cues. Of special interests are mesenchymal stem cells (MSCs), which have attracted significant interest based on their high proliferation rates and high biosynthetic activity. The use of either autologous or allogeneic MSCs presents this great trade-off: with allogeneic MSCs, off-the-shelf availability and large-scale expansion can be achieved but with risks of pathogen transmission and immune rejection. Several studies have explored the use of MSCs in combination with advanced biomaterials and mechanical stimulation. Li’s research team 120 , for example, fabricated a PCL tubular scaffold via e-jetting and coated it with alginate before seeding it with hMSCs. Subjecting the construct to cyclic uniaxial strain (0,5 Hz, 3%) significantly upregulated tendon-specific protein expression and promoted cell alignment along the scaffold’s longitudinal axis. Similarly, Chae and colleagues 115 employed 3D bioprinting techniques, embedding hBMSCs in a bio-ink composed of decellularized T/Ls tissues. PCL was incorporated to provide mechanical stability, and live/dead staining showed high cell viability with an elongated morphology indicative of tenogenic differentiation. Yang and team 121 developed collagen tubular scaffolds through counter-rotating extrusion and seeded them with BMSCs. The aligned collagen fibers not only replicated the structural organization of native tendons but also promoted tenogenic differentiation by guiding cell elongation along the fiber direction. Nevertheless, BMSCs are not without limitations. Ectopic bone formation in has been reported 122 , along with inferior strength compared to uninjured tendons. In addition, the aspiration procedure involving the harvest of MSCs is invasive and often painful, thus limiting its clinical applicability 123 . With these limitations TSPCs have emerged as an attractive alternative since, unlike BMSCs, TSPCs are naturally predisposed toward tenogenesis. The existence of a pool of stem cells in tendons can be confirmed in multiple tendons and ligaments from different species 26,124,125 . Ning and coworkers 110 demonstrated the regenerative potential of TSPCs by first seeding them onto a decellularized tendon substrate (DTS) and then taking the construct through a second decellularization process. The resulting composite was then used to assess its ability in providing support for BMSC migration, proliferation, and tenogenic differentiation, with favorable outcomes. Meanwhile, Kim and colleagues 126 investigated the influence of nanotopographical cues on TSPC behavior by fabricating a PCL patch via capillary force lithography. Immunofluorescence analysis revealed that TSPCs underwent alignment along the patterned surface, forming organized cell-cell interactions indicative of tenogenic commitment. However, TSPCs present limitations that hinder their use on a large-scale for treating T/Ls injuries. The limited quantity of cells present in tendon tissue requires a significant volume of source material to secure an adequate number of cells. Fibroblasts Fibroblasts are the predominant cell type in connective tissues and are responsible in the synthesis of ECM proteins such as type I and III collagen 127 , fibronectin, and proteoglycans and are typically characterized by their spindle- or stellate-shaped morphology, which is contingent upon the specific tissue in which they are located. Dermal fibroblasts and tenocytes share a common mesodermal origin, which makes fibroblasts an attractive alternative for tendon repair 75,77,117,119 . Their ease of extraction (which requires only a small skin biopsy) minimizes donor site morbidity compared to tendon-derived cells. Clinical trials have shown promising results, as injections of autologous dermal fibroblast have reduced pain in patients with refractory patellar tendinopathy 128 . Xuan and team 87 fabricated a PLLA scaffold with a core-shell structure by emulsion electrospinning, incorporating L-Arginine (L-Arg) and hyaluronic acid (HA) was to support different phases of tendon healing. Mouse fibroblasts exhibited significantly higher proliferation rates on the Arg/HA-modified scaffold. One of the major concerns in the use of fibroblasts is the potential fibroproliferative response, which could lead to the deposition of excessive scar tissue formation rather than a functional T/L regeneration 129 . Tenocytes As resident tendon cells, tenocytes hold particular advantages in T/L TE due to their specialized function in ECM maintenance. Tenocytes already express the genes and proteins required for tendon homeostasis, unlike stem cells, eliminating the need for directed differentiation 130 . Furthermore, they do not pose the risk of teratoma formation, a concern associated with pluripotent stem cells 131 . Maghdouri-White and colleagues 132 evaluated the potential of tenocyte-seeded scaffolds by developing an electrospun PDLLA-collagen I scaffold coated with platelet-rich plasma (PRP). In vitro testing confirmed tenocyte alignment along the scaffold’s fibers, and in vivo implantation in a rabbit Achilles tendon injury model revealed early remodeling at 16 weeks and complete integration at 52 weeks, suggesting the long-term viability of tenocyte-based constructs. However, tenocyte-based therapies face several barriers to clinical translation. Isolation of tenocytes requires a tendon biopsy, an invasive procedure with the risk of donor site morbidity. Moreover, tenocytes exhibit limited proliferative capacity which requires extended culture periods to obtain therapeutically relevant cell numbers. This limitation, when compared with more expandable cell sources such as MSCs or fibroblasts, represents a substantial constraint in terms of mass clinical implementation. A key takeaway from the studies discussed here is the widespread appreciation that cell behavior is greatly influenced by scaffold architecture and mechanical stimulation. Throughout multiple studies, aligned topographical cues and dynamic mechanical loading allow enhanced tenogenic differentiation, regardless of the cell type used. Table 3 . Summary of the main cell types used in scaffolds for tendon repair. PCL MSCs Human - - Increased production of COL1, DCN, TNC, TNMD 120 PCL BMSCs Human Mouse Subcutaneous implantation Increased expression in SCX, TNMD, COL1A1, COL3A1 115 Collagen BMSCs Rat Rat Achilles tendon Increased expression in Col1a1, Bgn, Tnc, Scx, Tnmd, Dcn 121 Decellularized tendon BMSCs Rat - - Increased expression in Scx, Tnmd, Tnc, Col1, Col3, Thbs4 110 PCL TSCs (supraspoinatus tendon) Human Rabbit Supraspinatus tendon Increased production of COL1, OCN, FN 126 PLLA Fibroblasts (adipose tissue) Mouse Rat Achilles tendon Enhanced proliferation 87 PDLLA/Collagen I Tenocytes Human Rabbit Achilles tendon Cell elongation along the direction of fibers 132 PCL = Polycaprolactone; MSCs = mesenchymal stem cells; COL1= Collagen type I; DCN=Decorin; TNC= Tenascin C; TNMD; Tenomodulin; BMSCs = Bone marrow mesenchymal stem cells; SCX = Scleraxis; COL1A1= Collagen type I Alpha 1 Chain; COL3A1 = Collagen type III alpha 1 chain; BGN= Biglycan; COL3= Collagen type III; THBS4 = Thrombospondin 4; TSCs = Tendon-derived stem cells; OCN= Osteocalcin; FN= Fibronectin; PLLA = Poly (L-lactic acid); PDLLA= Poly(D,L-lactide). Bioactive components Over the last decade, there has been a growing body of research exploring the functionality of bioactive compounds in TE 133 . To this end, multiple strategies have been developed to enhance T/L regeneration by delivering bioactive molecules including hormones 134,135 , nanoparticles 136,137 , drugs or growth factors all of which play a crucial role in the modulation of the immune response at the injury site 85 (Table 4). The incorporation of bioactive components into scaffolds isn’t limited to a single method: researchers have explored different techniques, such as surface coating, encapsulation within the scaffold itself, direct chemical bonding with the scaffold material 74 , and loading within micro-or-nanoparticles as carriers for the delivery bioactive molecules at the target site. Growth factors Growth factors are signaling molecules, typically proteins or steroid hormones, involved in the regulation of cell processes such as proliferation, differentiation, and growth 138 . Among these, transforming growth factor beta (TFG-β) is a protein superfamily that exhibits a multifaceted function in wound healing, through promotion of inflammation during initial stages and enabling ECM deposition as healing occurs 139,140 . Donderwinkel and team 141 investigated the effects of TGF-β3 supplementation on tenogenic differentiation of hBMSCs cultured on 3D printed poly(D,L-lactic acid ethylene glycol-D,L-lactic acid) P(LA-EG-LA) and GelMA hydrogel scaffolds, widely used in musculoskeletal TE 142 . Their findings indicated that samples supplemented with 5 ng/ml TGF-β3 and subjected to 3% of intermittent cyclic uniaxial strain exhibited optimal tenogenic differentiation, with increased SCX and COL1A1 expression and improved ECM organization. Similarly, the fibroblast growth factor (FGF) family regulates a broad spectrum of biological processes, including proliferation, inflammation, angiogenesis, and collagen synthesis 86,105,143 . Inizio modulo Building on the bioaffinity with heparin, Darshan and coworkers 86 encapsulated FGF-2 within a gelatin/PCL/heparin electrospun scaffold. The scaffold effectively maintained the tenocyte phenotype, promoting enhanced expression of key tenogenic markers during in vitro culture. Figure 10. Nuclear and actin cytoskeleton staining of tenocyte-seeded gelatin-PCL-heparin (GPH) and GPH-bFGF scaffolds. Scale bar = 250 μm. Reproduced with permission from T.G. et al. 86 Bone morphogenic proteins (BMPs), also known as Growth differentiating factors (GDFs) belong to the TGF-β superfamily and regulate chemotaxis, proliferation, ECM synthesis, and tenogenic differentiation during tendon healing 144 . BMP levels are elevated during the early phases of tendon healing and gradually decline over time 145 . In the context of embryogenic development, BMP-12 and BMP-13 have been observed to promote the expression of elastin and collagen I, thereby contributing to the development of mechanically robust tendons 146 . Rinoldi and coworkers 137 fabricated electrospun meshes using a combination of PCL, Polyamide 6 (PA6), and mesoporous silica particles, subsequently functionalizing them with BMP-12. Results demonstrated that BMSCs cultured on BMP-12-enriched scaffolds exhibited enhanced TNMD expression, indicating improved tenogenic differentiation. This aligns with findings from Güner and team 85 , who investigated BMP-14’s role in the tenogenic differentiation of hADSCs seeded on a dual-phase PCL (shell)/PCL-Gelatin (core) scaffold fabricated via rotary jet electrospinning and wet electrospinning (figure 10). Supplementation with 100 ng ml⁻¹ proved to be most effective on the expression of TNMD, total collagen deposition, COLIII deposition, and cell migration. Beyond BMPs and FGFs, the use of combined mechanical and biochemical stimulation has been explored to optimize T/L regeneration. Jayasree and their lab 105 developed a multi-scale fibrous scaffold by integrating electrospinning with textile techniques. They loaded FGF-2 into collagen-poly(vinyl alcohol) (PVA) nanofibers, which were further blended with electrospun PCL microfibers to form braided scaffolds. Their work demonstrated a synergistic effect between mechanical stimulation (applied at 0.5 Hz for 3 hours/day with 5% elongation) and growth factor delivery, leading to increased expression of COLI, COLIII, TNC, BGN, and FN, as confirmed by qPCR analysis. Platelet-derived growth factors (PDGFs) is another growth factor group relevant in T/L TE. PDGFs are dimeric growth factors held together by disulfide bonds. These factors have several isoforms, each being a dimer of two polypeptide chains, either AA, BB, or a combination of the two. Furthermore, PDGFs stimulate the migration and proliferation of several cell populations involved in T/Ls regeneration such as fibroblasts, tenocytes, and mesenchymal stem cells 147 . Evrova and colleagues 84 investigated the use of the polyesterurethane DegraPol® for the sustained delivery of PDGF-BB. They employed two electrospinning techniques, coaxial and emulsion electrospinning to incorporate the growth factor within the polymeric solution, enabling a controlled release over 30 days. In vivo studies using a rat Achilles tendon defect model demonstrated the potential of these scaffolds, with tubular scaffolds obtained via coaxial electrospinning leading to a two-fold increase in the maximum load of healed tendons after three weeks post-surgery. Although these studies highlight the potential of growth factor incorporation into scaffolds, significant challenges exist. The clinical application of growth factors is limited due to high costs, short half-life, and uncertainties regarding optimal dosages 148 . Given the involvement of multiple growth factors in T/Ls regeneration as described above, their simultaneous use is a strategic solution to better replicate the natural healing cascade. Although this strategy holds some promise in maximizing the regenerative outcome, it also significantly increases treatment costs and complexity. Pharmacological agents Systemic or local administration are the routes for delivery of pharmacological agents which help to modulate inflammation, pain, and ECM remodeling. Systemic administration methods, such as intravenous, intramuscular, and oral routes, ensures widespread drug distribution but could lead off-target effects 149 . To mitigate side effects and improve drug effectiveness, it is essential to develop carriers that preserve the drug’s functions, protect it from decomposition or denaturation, and deliver it to the target site 150 . Conversely, local delivery through biomaterials provides precise control over drug release, minimizing systemic exposure while enhancing therapeutic outcomes 151 . Local delivery strategies include hydrogels, films, sponges, fibers, and injectable carriers like cross-linkable hydrogels and nanoparticles 149,152 . Adel and coworkers 149 explored the use of a bi-polymeric hydrogel scaffold for the localized delivery of rosuvastatin calcium (RSV) as an inhibitor of farnesyl pyrophosphate (FPP), a known wound healing inhibitor. RSV was incorporated into a scaffold made of Gellan gum (GG) and a secondary polymer (either κ-Carrageenan, Carboxymethyl cellulose, or PVA). Functional tests on rat Achilles tendons showed enhanced vasculature formation and high rates of collagen expression. A common approach among recent studies involves the incorporation of bioactive agents directly into the polymeric solution before electrospinning, ensuring homogeneous drug distribution within the scaffold and allowing for controlled, sustained release profiles. Yu and fellow researchers 153 developed an electrospun scaffold incorporating Celecoxib, a widely used analgesic, blended with collagen, bupivacaine (a potent local anesthetic), and PLGA . In vitro release profiles indicated a sustained Celecoxib release over 30 days, while in vivo studies on an Achilles tendon rat model guaranteed a release of 28 days. The prolonged drug delivery profile suggests a viable strategy for managing inflammation and pain following injury. Likewise, Pien and colleagues 154 designed a tubular scaffold composed of an Acrylate-endcapped urethane-based polymer (AUP)/PCL blend loaded with Naproxen, an anti-inflammatory agent, and hyaluronic acid (HA), specifically designed to reduce scar tissue formation. In vitro biocompatibility assays employing human fibroblasts confirmed the scaffold non-cytotoxic nature, whereas ex vivo biomechanical testing on a cadaveric sheep flexor digitorum profundus tendon model revealed substantial improvements of the mechanical properties with the reinforced AUP530:PCL construct. Zhour and coworkers 155 incorporated Methylprednisolone (MP), known for its anti-inflammatory, antifibrotic, and immunosuppressant properties, into a PLGA polymeric solution before electrospinning. Drug release analysis showed an initial burst release within the first 24 hours, followed by a sustained release profile that ensured gradual drug delivery with most of the drug released by the 14th day . Notably, the MP/PLGA scaffold exhibited antibacterial activity against E. coli and S. aureus . Implantation of the 25% MP-loaded PLGA scaffold resulted in reduced adhesion formation compared to control tendons, highlighting the potential advantages of integrating anti-inflammatory and antimicrobial properties into tendon repair scaffolds. One common challenge in many applications discussed in this paragraph is the need for accurate control over the drug release rate. Furthermore, residual empty carriers post-drug depletion could potentially trigger inflammation, making it essential to eliminate them alongside the drug release. To address these challenges, personalized design of drug release modifiers that can be combined with a drug and assist in regulation of the drug release independently of polymer degradation kinetics offer a promising solution. Fillers Polymer composites are advanced materials designed to improve mechanical, biological, and functional properties by blending a reinforcement phase within a polymeric matrix 156 . The matrix maintains cohesion between interfaces and facilitates the transfer of forces experienced by the composite. The resulting material is characterized by high heterogeneity and often exhibits anisotropic properties 157 . Several factors affect these features, including the type of matrix and filler, the shape and proportion of the filler, the quality of the interface, and the manufacturing processes employed. Various fillers like titanium dioxide, hydroxyapatite, and bioactive glass, can be incorporated into scaffolds to improve their functional properties 158 . To confer antibacterial properties Silva and coworkers 136 integrated 0.5 wt.% of functionalized graphene nanoplatelets decorated with silver nanoparticles ((f-EG)+Ag) into a medical-grade PLA matrix. The resulting filaments were used to fabricate cylindrical scaffolds via 3D printing, further seeded with human tendon stem cells (hTSCs). As expected, silver nanoparticles exhibited antibacterial activity against S. aureus and E. coli . In addition, the presence of (f-EG)+Ag significantly upregulated the expression of the tenogenic markers SCX , TNMD , and COL1 , highlighting the scaffold’s potential in promoting tendon regeneration. Bioactive glass (BG) represents a class of reactive materials recognized for their biocompatibility and ability to bond with mineralized bone tissue 159,160 . It is generally made up of Na 2 O, CaO, P 2 O 5 , and SiO 2 , with SiO 2 content kept below 55%. The properties of bioactive glass can be customized by modifying the concentrations of its components to trigger specific biological responses. Touré and team 143 incorporated BG into a PCL/ Poly(glycerol sebacate)(PGS) blend, using a combination of 3D printing and electrospinning techniques. The electrospun mats provided a reinforcement to the 3D-printed patches due to strong interfacial adhesion between the layers, resulting in a in Young’s modulus of 311 ± 20 MPa for scaffolds containing 10% w/w bioactive glass. In vitro assays with mouse fibroblasts demonstrated the highest cell viability at day 7 in scaffolds containing 10% BG. Strontium-hardystonite (Sr-Ca2ZnSi2O7, Sr-HT) is a bioactive calcium silicate with potential applications in musculoskeletal TE. Although native T/Ls contain less than 0.2% inorganic material 161 , trace amounts of metal ions such as calcium, silicon, zinc, and strontium have been found to be essential in the growth, repair, and maintenance of musculoskeletal tissues, including T/Ls 162 . No and team 163 investigated the incorporation of Sr-HT into a hydrogel, which was injected into an ultra-high molecular weight polyethylene (UHMWPE) fibrous scaffold and subsequently pultruded it through a circular channel to fabricate a fiber-reinforced hydrogel construct. In vitro studies using TSPCs suggested that Sr-HT might inhibit tenogenic differentiation, while in vivo implantation in a rat patellar tendon model demonstrated substantial collagenous tissue ingrowth within the scaffold six weeks post-implantation. The use of micro- and nanoparticles for the controlled release of microRNAs (miRNAs) has emerged as a promising strategy in T/L TE 164 . miRNAs are small non-coding RNA molecules that regulate gene expression post-transcriptionally, influencing biological processes of major relevance such as cell differentiation, proliferation, and apoptosis. Integrating miRNA-loaded nanoparticles into scaffolds enables targeted and sustained release, addressing challenges related to miRNA stability and bioavailability. Studies have shown that polymer-based miRNA nanoparticles not only enhance cell viability and tendon-specific differentiation but also mitigate fibrosis. Important miRNAs for T/Ls repair include miR-21-5p, which promotes tendon cell proliferation and differentiation while modulating fibrosis, and miR-210, known to modulate angiogenesis 165 . This cutting-edge strategy holds significant potential for improving T/L regeneration through precise molecular modulation. Figure 11 . SEM images at different magnifications depicting: (a, b) 3D-printed scaffolds with BG microspheres either embedded within the polymer matrix or exposed on the surface (see insets); (c, d) the composite scaffold coated with electrospun PCL-PGS mats; (e, f) the composite scaffold surface without the electrospun fiber layer. Reproduced with permission from Tourè et al. 143 Table 4 . Overview of the key bioactive components used in scaffolds for tendon repair. TGF-β3 Growth factor (P(LA-EG-LA))-GelMa 3D Printing Human BMSCs Increased expression in SCX and COL1A1 141 FGF-2 Growth factor Gelatin/PCL/Heparin Electrospinning Rabbit tenocytes Increased expression in COL1, BGN, TNC 86 BMP-12 Growth factor PCL/PA6 Electrospinning Human BMSCs Increased production of TNMD 137 BMP-14 Growth factor PCL/Gelatin Wet electrospinning/Rotary jet electrospinning Human ASCs Increased production of TNMD, COL3 85 FGF-2 Growth factor PCL/Collagen Electrospinning/Textile technologies Rabbit tenocytes Incresead expression in COL1, COL3, TNC, BGN, FN 105 PDGF-BB Growth factor DegraPol®(polyester urethane) Coaxial electrospinning Rabbit tenocytes Increased production of COL1, COL3 84 RSV Drug GG Hydrogel preparation - Enhanced vasculature formation and collagen deposition 149 Celecoxib Drug CCBP Electrospinning - Local stimulation of growth factors around the wound site 153 Naproxen Drug AUP/PCL Electrospinning Human fibroblasts High cell viability after 7 days 154 Methylprednisolone Drug PLGA Electrospinning - Antibacterial activity against E. coli and S. aureus Reduction in the formation of adhesions (for 25% Methylprednisolone-loaded PLGA scaffolds) 155 AgNPs Filler PLA 3D Printing Human TSCs Antibacterial activity against E. coli and S. aureus Increased expression in SCX, COL1, TNMD 136 Bioactive glass Filler PU/Silk fibroin Electrospinning BMSCs Increased production of ALP 166 Bioactive glass Filler PCL/PGS Electrospinning/3D Printing Mouse fibroblasts High cell viability after 7 days for scaffolds with 10% of Bioactive glass 143 Sr-HT Filler UHMWPE/PVA/Gelatin Extrusion Rat TSCs Hindered expression in Col1, Col3, Scx,Tnmd, Tnc Good collagenous tissue ingrowth in vivo 163 TGF-β3= Transforming growth factor-β3; (P(LA-EG-LA)) = poly(D,L-lactic acid ethylene glycol-D,l-lactic acid); GelMA = Gelatin Methacrylate; SCX= Scleraxis; COL1A1= Collagen type I Alpha 1 Chain; FGF-2 = fibroblast growth factor-2; PCL = Polycaprolactone; COL1= Collagen type I; BGN = Biglycan; TNC= Tenascin C; BMP = Bone morphogenic protein; PA6 = Polyamide 6; BMSCs= Bone marrow mesenchymal stem cells;TNMD= Tenomodulin; ASC= Adipose-derived stem cells; COL3 = Collagen type III; FN= Fibronectin; PDGF-BB = Platelet-derived growth factor-BB; RSV = Rosuvastatin calcium; GG= Gellan gum; CCBP = Celecoxib, collagen,bupivacaine, and PLGA; AUP = Acrylate-endcapped urethane-based polymer; AgNPs = Silver nanoparticles; PLA = Poly(Lactic acid);TSCs = Tendon-derived stem cells; PU = Polyurethane; ALP = Alkaline Phosphatase; PGS = Poly (glycerol sebacate); Sr-HT = Strontium-doped hardystonite(Sr-Ca2ZnSi2O7); UHMWPE = Ultra high molecular weight polyethylene; PVA = Poly(vinyl alcohol). Animal models The successful clinical translation of T/L TE strategies depends on the selection of relevant preclinical models. The appropriate selection of an animal model in proof-of-concept studies includes a careful evaluation of significant characteristics: anatomical site, surgical accessibility, biomechanical properties, and overall clinical relevance 167 . Rodents, particularly rats and mice, are among the most employed animal species 149,153 due to their small dimensions and ease of handling that facilitate reproducible experimental techniques. Additionally, their cost-effectiveness compared to larger animal species makes them an attractive option for preclinical research. However, the advantages they provide are somewhat limited by their small tendon sizes and differences in biomechanical loading that present a significant barrier toward direct clinical translatability. As a result, larger animals such as rabbits 75 , dogs, sheep, pigs, and horses have gained increasing relevance due to their closer resemblance to human tendon anatomy 167,168 . Among these species, rabbits offer a practical compromise between small and large animal models, having tendons large enough for surgical manipulation and biomechanical analysis. Rabbit flexor tendons are very close to human tendons in terms of diameter and the presence of a synovial sheath, thus of extremely high relevance in both flexor and Achilles tendon research. Nonetheless, species differences must be considered; for example, rabbits typically maintain greater knee flexion in ventral recumbency, and their posterior knee structures exhibit distinct biomechanical properties 169 . Dogs are also a useful model due to their anatomical and functional similarities to human flexor tendons. Furthermore, their ability to participate in post-operative mobilization exercises adds translational relevance. However, ethical concerns and their status as companion animals have led to a decline in their use for experimental research 170 . Sheep, particularly skeletally mature ewes are widely used in rotator cuff injuries, given the anatomical and functional similarities between the ovine infraspinatus tendon and the human supraspinatus tendon 171 . Their size and shape adds value of being compatible with a variety of non-invasive imaging such as ultrasound. However, due to the high cost of procurement and maintenance, and breed-dependent variability, makes standardizing outcomes a challenge. Pigs represent a relevant large-animal model, particularly for anterior cruciate ligament (ACL) research (figure 12). The porcine ACL exhibits mechanical properties similar to its human counterpart, including comparable load-to-failure and stiffness, which are significantly lower in small animal models. However, anatomical differences need to be considered, such as the larger cross-sectional area and distinct insertion sites of the porcine ACL. Nevertheless, structural measurements, including overall ACL length and tibial plateau width, are comparable between pigs and humans, and therefore suitable for translational studies 172 . The equine model has a distinct set of advantages that maximizes the utility when studying high-load tendons such as the flexor digitorum superficialis tendon, which shares functional similarities with the human Achilles tendon 173 . Like humans, horses rely on energy-storing tendons for high-speed locomotion. However, its application is hampered by significant logistical challenges, including high maintenance costs, specialized housing requirements, and ethical considerations. Ultimately, the choice of an animal model should be carefully aligned with the specific research objectives, taking into consideration the biological relevance as well as the practical feasibility. In many cases, a strategic approach that integrates multiple models may be necessary to bridge the gap between basic research and clinical translation. Figure 12. Comparative in situ anatomy of the human (top row) and porcine (bottom row) knee joints, viewed from anterior (left) and posterior (right) perspectives. Adapted from Little et al. 170 Conclusions and future challenges Further improvements in T/L TE will not be without challenges. The main drawback in this regard is the ability to replicate the complex multiscale structure of T/L tissue. In terms of microstructure, their complex hierarchical organization extends across multiple scales, ranging from the nanoscale to the macroscale. With such complexity, no single biomaterial can fully replicate the structural and functional characteristics of native T/Ls tissues. Hence, composite scaffolds that include multiple scale-specific components would provide a mechanism to guide the cell behavior toward a desired end and hence facilitate regeneration. In addition, one of the main barriers in widespread clinical applications is the scaling up of the scaffold fabrication. Demand for tissue-engineered T/L constructs is now growing, and most solutions are still in an experimental stage; therefore, establishing high-throughput processes for scaffold biofabrication will be crucial. Addressing this issue requires interdisciplinary collaboration among bioengineers, clinicians, and material scientists to drive technological advancements and optimize translational potential. Lastly, the other major limitation lies in the predominant reliance on small animal models in preclinical studies. 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Collection View Keywords bioengineering biofabrication tendons and ligaments tissue engineering Authors Affiliations Francesca Romano Ri.MED Foundation View all articles by this author Roberto Di Gesù 0000-0001-6196-643X [email protected] Ri.MED Foundation View all articles by this author Francesco Lopresti University of Palermo View all articles by this author Vincenzo La Carrubba University of Palermo View all articles by this author Metrics & Citations Metrics Article Usage 809 views 396 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Francesca Romano, Roberto Di Gesù, Francesco Lopresti, et al. Tissue engineering for tendon and ligament repair: insights and advances. Authorea . 07 May 2025. DOI: https://doi.org/10.22541/au.174660872.21796090/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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