Exploring the Frontier of 3D Bioprinting for Tendon Regeneration: A Review

Abstract

The technology of 3D bioprinting has sparked interest in improving tendon repair and regeneration, promoting quality of life. To perform this procedure, surgical intervention is often necessary to restore functional capacity. In this way, 3D bioprinting offers a scaffold design, producing tendons with precise microarchitectures, promoting the growth of new tissues. Furthermore, it may incorporate bioactive compounds that can further stimulate repair. This review elucidates how 3D bioprinting holds promise for tendon repair and regeneration, detailing the steps involved and the various approaches employed. They demonstrate future challenges and perspectives and provide valuable information on the concept, bioprinting design, and 3D bioprinting techniques for the repair of tendon injuries.

1. Introduction

Tendons are an integral component of the musculoskeletal system, serving as a critical medium in the transmission of force from muscle to bone during joint movement and maintenance of posture. The structure of tendons can vary depending on their specific function but can be generally described as a collection of tightly packed, longitudinally arranged, collagen fibrils embedded within a highly hydrated matrix of proteoglycans and glycoproteins. The collagen fibrils are organized in hierarchical manner, first bundling as fibers, which further bundle as fascicles [1].

Each fascicle is wrapped in a connective tissue layer called the endotenon, which binds adjacent fascicles to form the full tendon unit, which is itself wrapped by the epitenon. These connective tissue sheaths provide structural integrity, facilitate the sliding of tendon fibres, and serve as vascular and neural conduits [2]. The primary cell population found in tendons are tenocytes, a class of specialized fibroblast cells which are responsible for the synthesis and turnover of the extracellular matrix [1].

At the termini of the tendon structure, specialized junctions can be found. The myotendinous junction (MTJ) represents the interface between the tendon and muscle. Here, the collagen fibres of the tendon interdigitate with the muscle’s sarcolemma through finger-like projections, enhancing the interface surface area. At the other end of the tendon, there is the osteotendinous junction (OTJ), which connects the structure to bone. This insertion point forms a complex and specialized interface that can be either fibrous, where the tendon inserts directly into the bone, or the fibrocartilage. Both the MTJ and OTJ are biomechanically optimized to minimize stress concentration and facilitate efficient force transfer [1].

Due to their high mechanical strength, tendons exhibit viscoelastic characteristics. This means that their mechanical behaviour varies with the rate of strain applied. At lower strain rates, tendons become more deformable, allowing them to absorb more mechanical energy but also causing them to become less effective at bearing mechanical loads. Conversely, at higher strain rates, tendons stiffen, enhancing their ability to transmit substantial muscular loads to bones [3].

Additionally, tendons demonstrate a phenomenon known as “creep”, where they elongate over time under a constant load, and “stress relaxation”, where the stress within a tendon decreases under a constant strain [4]. Though most commonly occurring during sports participation, tendon ruptures can occur spontaneously during routine activities as well, usually as a consequence of underlying degenerative changes within the tendon matrix [5]. Although such severe acute tendon injuries have the potential for functional recovery, the affected tendon seldom returns to its original pre-injury state [6]. Chronic tendinopathies, on the other hand, are degenerative in nature and develop over an extended period as a result of prolonged overuse or repetitive mechanical stress.

Current treatment strategies for tendon injuries involve a combination of non-surgical and surgical approaches, each with its inherent limitations and challenges. Non-surgical treatment often serves as the first line of management, especially for less severe tendon injuries. For some tendinopathies, extracorporeal shock wave therapy (ESWT) or platelet-rich plasma (PRP) injections may also be used to help stimulate healing [7,8]. While non-surgical treatments can be effective for many patients, they may not always promote complete recovery, especially in chronic cases or with significant tendon damage.

In severe cases, such as significant tears or complete ruptures, surgical tendon repair is often necessary for re-establishing the structural integrity and functionality of the tendon. In cases where the tendon has been severely damaged, a graft may also be required to replace the injured tissue. However, despite advancements in surgical methods, patients often face a long and challenging recovery process. Furthermore, current surgical treatments are often unable to fully restore the tendon to its pre-injury state, leading to a higher risk of re-injury or chronic disability [8]. This gap in treatment efficacy highlights the need for innovative solutions that can more closely mimic the natural architecture and function of healthy tendons.

Emerging techniques, using tissue engineering, offer promising avenues for enhancing tendon repair and regeneration. Among these, tendon-specific stem/progenitor cell (TSPC) therapy and 3D scaffolding stand out for their innovative approaches to facilitate the natural healing process. This method leverages the inherent ability of TSPCs to differentiate into tendon cells, and other supporting cells, to promote the regeneration of damaged tendon structures [9]. Advances in 3D scaffolding techniques, such as electrospinning and soft lithography, have opened new avenues for creating tendon scaffolds with precise microarchitectures [8].

3D printing offers unparalleled control over scaffold design, enabling the fabrication of structures that precisely match the patient’s anatomy and injury specifics [10]. This personalized approach not only supports the integration and growth of new tissue but also opens the door to incorporating bioactive compounds that can further stimulate healing. The versatility and precision of 3D printing hold significant promise for overcoming the limitations of current tendon repair methods, marking a new frontier in the field of regenerative medicine.

In this review, we elucidate the main advances, challenges, and future directions in tendon repair. Furthermore, several bioprinting techniques that can be used have been described. Therefore, this review aims to address the concept of 3D bioprinting, bioprinting design, and techniques for tendon injury repair.

2. 3D Printing

The additive manufacturing technique of 3D bioprinting is based on depositing biomaterials, or bioink, on a microscale to create structures that can imitate natural tissues. As opposed to traditional printing of tissue engineering scaffolds, bioprinting involves live cells mixed with biomaterials being directly deposed. In cell-free printing, the cells are seeded onto the scaffold afterwards [11]. The bioprinting process begins with a digital design of the desired structure, which is then divided into thin layers for printing. It typically involves an extruder being controlled by a three-axis mechanical platform. The movement of the platform is determined by coordinates related to the digital design. Many of the steps remain the same as conventional 3D printing, with different methods for bioprinting including extrusion-based, inkjet, and light-based techniques [12].

2.1. Inkjet Bioprinting

Inkjet bioprinting is a technique that involves the precise ejection of small droplets of bioink, which is a mixture of cells and hydrogel, stored in an ink cartridge. The print head, controlled by a mechanical lifting platform, releases these droplets through electrostatic or piezoelectric stimulation. This method allows for high-resolution printing and maintains high cell viability, making it particularly valuable in academic research. While not as widely commercialized as other bioprinting techniques, inkjet bioprinting is favoured in research settings due to its ability to produce detailed and accurate tissue constructs. The high resolution of this technique enables the creation of complex tissue structures with precise cell placement, which is critical for replicating the intricate architecture of natural tissues. Inkjet bioprinting also allows for the simultaneous deposition of multiple cell types and biomaterials, facilitating the creation of heterogeneous tissue constructs that better mimic the natural cellular environment. This capability is essential for developing functional tissues that can integrate seamlessly with the patient’s existing biological structures. Despite its advantages, inkjet bioprinting faces challenges such as the need for bioinks with suitable viscosity and the potential for nozzle clogging. However, ongoing research and technological advancements continue to address these issues, further enhancing the feasibility and effectiveness of inkjet bioprinting for tissue engineering applications [13]. While not as widely commercialized, inkjet bioprinting is prevalent in academic research due to its high resolution and cell viability [14].

2.2. Extrusion-Based Bioprinting

Extrusion-based bioprinting is a widely used technique in tissue engineering, particularly for tendon regeneration, due to its ability to print continuous filaments of bioink. This method involves the deposition of bioink through a nozzle, using either pneumatic, mechanical, or solenoid forces to extrude the material layer by layer onto a substrate. The bioink typically consists of a mixture of cells and biocompatible hydrogels that provide the necessary support for cell growth and differentiation. One of the significant advantages of extrusion-based bioprinting is its versatility in handling a wide range of bioink viscosities, allowing for the use of more complex and mechanically robust materials. This is particularly beneficial for tendon regeneration, where the printed constructs need to mimic the high tensile strength and elasticity of natural tendons.

The technique also allows for the incorporation of gradient materials and the creation of anisotropic structures, which are essential for replicating the hierarchical organization of tendon tissues. By adjusting the printing parameters, such as nozzle diameter, extrusion speed, and layer thickness, researchers can fine-tune the mechanical properties and microarchitecture of the printed tendons to closely match those of native tissues [13]. However, extrusion-based bioprinting faces challenges, including maintaining cell viability during the extrusion process and ensuring uniform cell distribution within the printed construct. High shear forces during extrusion can lead to cell damage, while uneven cell distribution can affect the overall functionality of the printed tendon. To mitigate these issues, advancements in nozzle design, bioink formulation, and printing protocols are being continually developed.

Despite these challenges, extrusion-based bioprinting remains a promising approach for tendon regeneration. Its ability to produce large-scale, mechanically stable constructs with precise control over the spatial distribution of cells and biomaterials makes it a valuable tool in the field of regenerative medicine. Ongoing research and technological innovations are expected to further enhance the capabilities of extrusion-based bioprinting, paving the way for more effective and reliable tendon repair solutions [14]. This method is compatible with a variety of hydrogels and supports high cell densities, making it suitable for musculoskeletal tissue engineering [15].

2.3. Stereolithography

Stereolithography (SLA) is a sophisticated bioprinting technique that utilizes light to cure liquid photopolymer resins into solid structures with high precision. This process involves the use of a laser or digital light projector to selectively polymerize the bioink layer by layer, creating highly detailed and complex tissue constructs. SLA is particularly advantageous for tendon regeneration due to its ability to produce constructs with fine resolution and intricate geometries.

One of the primary benefits of SLA is its exceptional accuracy and surface finish, which are crucial for replicating the detailed microarchitecture of tendon tissues. The high resolution of SLA enables the fabrication of constructs that closely mimic the natural extracellular matrix, providing an optimal environment for cell attachment, proliferation, and differentiation. This precision is essential for developing functional tendons that can effectively integrate with the surrounding tissues.

SLA also allows for the incorporation of various bioactive molecules and growth factors within the printed constructs, enhancing the regenerative potential of the engineered tendons. By embedding these bioactive compounds within the scaffold, SLA can promote cellular activities such as migration, proliferation, and matrix synthesis, which are vital for tendon healing and regeneration.

Despite its advantages, SLA faces several challenges in tendon bioprinting. One of the main limitations is the need for photopolymerizable bioinks that are both biocompatible and capable of supporting cell viability. The exposure to light and the photoinitiators used in the process can potentially affect cell health and functionality. Additionally, the mechanical properties of the printed constructs must be optimized to match those of native tendons, which requires the careful selection and formulation of the bioinks.

To address these challenges, ongoing research is focused on developing new photopolymerizable bioinks that are tailored for tendon regeneration. Innovations in bioink composition, such as the incorporation of natural polymers and hybrid materials, are being explored to enhance the biocompatibility and mechanical strength of the printed constructs. Furthermore, advancements in SLA technology, including multi-material printing and dynamic light projection, are expanding the capabilities of this technique for more complex and functional tendon tissue engineering.

In summary, stereolithography holds significant promise for the bioprinting of tendons, offering unparalleled precision and the ability to create highly detailed and functional tissue constructs. Continued research and technological developments are expected to overcome current limitations, paving the way for more effective and reliable tendon repair and regeneration strategies [12].

2.4. Laser-Assisted Bioprinting

Laser-assisted bioprinting (LAB) is a cutting-edge technique that leverages laser energy to precisely deposit bioink onto a substrate. This method involves the use of a focused laser beam to generate high-pressure bubbles that propel droplets of bioink from a donor slide to the target area. LAB offers several advantages for tendon regeneration, including high resolution, precise cell placement, and minimal shear stress, which collectively enhance cell viability and functionality.

One of the standout features of LAB is its ability to achieve high spatial resolution, enabling the creation of intricate and finely detailed tendon constructs. This precision is vital for replicating the complex hierarchical structure of tendons, which includes aligned collagen fibres and specialized cellular arrangements. By accurately positioning cells and biomaterials, LAB can produce constructs that closely mimic the native tendon architecture, promoting effective integration and function. LAB also supports the use of a wide range of bioinks, including those with varying viscosities and compositions. This flexibility allows researchers to incorporate different cell types, growth factors, and extracellular matrix components into the printed constructs, enhancing their biological and mechanical properties. Such versatility is particularly beneficial for tendon regeneration, where the engineered tissues must withstand significant mechanical loads while supporting cellular activities essential for healing [16].

Despite its advantages, LAB faces several challenges in the context of tendon bioprinting. One of the primary concerns is the potential for laser-induced damage to cells and biomaterials. The high energy required for the printing process can generate localized heat, which may affect cell viability and the integrity of the bioink. To mitigate these effects, ongoing research is focused on optimizing laser parameters and developing more resilient bioinks that can withstand the laser-assisted printing process. Another challenge is the scalability of LAB for producing larger tissue constructs. While LAB excels in precision and detail, scaling up the process to create larger, clinically relevant tendon grafts remains a complex task. Efforts are being made to improve the throughput and efficiency of LAB, including the development of multi-laser systems and automated printing platforms [17].

2.5. Fused Deposition Modeling

Fused Deposition Modeling (FDM) is a widely utilized extrusion-based bioprinting technique that involves the layer-by-layer deposition of thermoplastic materials to create three-dimensional structures. In the context of tendon regeneration, FDM offers a robust and versatile approach to fabricating scaffolds that can support cell growth and tissue development. This method uses a heated nozzle to extrude bioink, typically composed of biodegradable polymers and cells, onto a build platform, where it solidifies to form the desired structure. One of the primary advantages of FDM is its ability to produce mechanically strong and stable scaffolds, which are essential for mimicking the load-bearing properties of natural tendons. The technique allows for precise control over the scaffold’s architecture, including pore size, shape, and distribution, which are critical parameters for facilitating nutrient diffusion, waste removal, and cellular infiltration. By optimizing these parameters, FDM can create scaffolds that promote effective tissue integration and regeneration [10,17].

FDM is also highly adaptable, capable of processing a wide range of thermoplastic materials, including polylactic acid (PLA), polycaprolactone (PCL), and other biocompatible polymers. These materials can be tailored to achieve specific mechanical properties and degradation rates, ensuring that the scaffold provides adequate support during the initial stages of tendon healing and gradually degrades as the new tissue forms. Additionally, FDM can incorporate bioactive molecules and growth factors into the bioink, enhancing the scaffold’s regenerative potential. However, FDM faces several challenges in tendon bioprinting. One significant limitation is the relatively high processing temperatures required to melt the thermoplastic materials, which can affect cell viability. To address this issue, researchers are exploring the use of temperature-sensitive bioinks and developing hybrid printing techniques that combine FDM with other bioprinting methods to preserve cell viability. Another challenge is achieving the fine resolution needed to replicate the intricate microarchitecture of tendons. Advances in nozzle design and printing technology are being pursued to enhance the precision and detail of FDM-printed constructs.

Despite these challenges, FDM remains a promising technique for tendon regeneration. Its ability to produce durable, customizable scaffolds with controlled architecture makes it a valuable tool in the field of tissue engineering. Ongoing research and technological innovations are expected to further improve the capabilities of FDM, enabling the creation of more effective and reliable tendon repair solutions [10].

3. Bioinks

Bioinks are a cornerstone in the field of bioprinting, providing the essential medium that supports cells during the printing process and ultimately forms the scaffold for tissue regeneration. For tendon bioprinting, the development of suitable bioinks is critical, as they must not only support cell viability and proliferation but also replicate the mechanical properties and biological functions of natural tendon tissue [18].

3.1. Composition and Properties

The ideal bioink for tendon bioprinting should exhibit several key properties. Biocompatibility is paramount to ensure that the bioink does not provoke an adverse immune response and supports cell adhesion, growth, and differentiation [17]. Additionally, the bioink must have appropriate rheological properties, such as viscosity and shear-thinning behaviour, to facilitate smooth extrusion and precise deposition during the printing process. Mechanical strength and elasticity are also crucial, as the bioink must withstand the mechanical demands of the tendon while maintaining its structural integrity.

3.2. Natural and Synthetic Polymers

Bioinks can be derived from both natural and synthetic polymers, each offering distinct advantages. Natural polymers, such as collagen, gelatin, and hyaluronic acid, are favoured for their inherent biocompatibility and their ability to promote cell–matrix interactions. These materials closely mimic the extracellular matrix (ECM) of native tissues, providing a familiar environment for cells. However, natural polymers often lack the mechanical strength required for tendon applications and may degrade too quickly.

Synthetic polymers, such as polycaprolactone (PCL), polylactic acid (PLA), and polyethylene glycol (PEG), offer greater control over mechanical properties and degradation rates. These materials can be engineered to provide the necessary support during the initial stages of tendon healing and gradually degrade as the new tissue forms. Combining natural and synthetic polymers in hybrid bioinks can leverage the strengths of both, creating a balanced environment that supports both mechanical integrity and biological functionality [19]. Table 1 displays the biomaterials currently in use for tendon bioprinting, highlighting their properties and applications.

3.3. Functionalization and Bioactive Additives

To enhance the regenerative potential of bioinks, they can be functionalized with bioactive molecules, such as growth factors, peptides, and cytokines. These additives can promote cellular activities essential for tendon healing, including migration, proliferation, and matrix synthesis. For instance, incorporating transforming growth factor-beta (TGF-β) or bone morphogenetic proteins (BMPs) into bioinks can stimulate tenogenic differentiation and improve the overall quality of the regenerated tissue [17,18,19].

3.4. Challenges and Future Directions of Bioinks

Despite significant advancements, several challenges remain in the development of bioinks for tendon regeneration. Achieving the optimal balance between printability, mechanical strength, and biological functionality is a complex task. Additionally, ensuring uniform cell distribution and maintaining cell viability during the printing process are critical considerations. To address these challenges, ongoing research is focused on developing novel bioink formulations and optimizing printing parameters.

Future directions in bioink development include the exploration of smart bioinks that can respond to environmental cues, such as temperature, pH, or mechanical stress, to enhance tissue regeneration. Advances in nanotechnology and biomaterials science are also expected to contribute to the creation of more sophisticated bioinks that can better replicate the dynamic and hierarchical nature of tendon tissues [10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29].

Table 1. Biomaterials and their properties and application in tendon bioprinting.

Biomaterial	Properties	Applications in Tendon Bioprinting
Collagen [26,27,28]	Natural polymer, high biocompatibility, promotes cell adhesion and proliferation	Mimics native extracellular matrix (ECM), supports cellular alignment and growth, used in scaffold fabrication
Polylactic Acid (PLA) [19]	Biodegradable, good mechanical strength, tunable degradation rate	Used in FDM printing, provides structural support, and can be combined with other materials for enhanced properties
Polycaprolactone (PCL) [25]	Biodegradable, flexible, slow degradation rate	Provides long-term mechanical support, used in combination with bioactive molecules for enhanced regeneration
Gelatin Methacrylate (GelMA) [10]	Photocrosslinkable, good cell compatibility, adjustable mechanical properties	Used in Digital Light Processing (DLP) printing, supports cell encapsulation and tissue formation, and can be modified for improved properties
Silk Fibroin [10]	High tensile strength, biocompatible, promotes cell attachment	Used for creating mechanically robust scaffolds, supports tendon-like mechanical properties and tissue regeneration
Alginate [11]	Biocompatible, easy to process, forms hydrogels upon crosslinking	Used as a bioink component, provides a hydrated environment for cells, often combined with other materials for improved stability
Hyaluronic Acid [11,19]	Natural polymer promotes cell migration and proliferation, and hydrophilic	Enhances scaffold hydration and cell migration, used in combination with other materials for improved mechanical properties
Decellularized Extracellular Matrix (dECM) [11]	Contains native ECM components, promotes cell attachment and differentiation	Used to create bioactive scaffolds that closely mimic the native tendon environment, support tissue-specific regeneration

Table 1. Biomaterials and their properties and application in tendon bioprinting.

Biomaterial	Properties	Applications in Tendon Bioprinting
Collagen [26,27,28]	Natural polymer, high biocompatibility, promotes cell adhesion and proliferation	Mimics native extracellular matrix (ECM), supports cellular alignment and growth, used in scaffold fabrication
Polylactic Acid (PLA) [19]	Biodegradable, good mechanical strength, tunable degradation rate	Used in FDM printing, provides structural support, and can be combined with other materials for enhanced properties
Polycaprolactone (PCL) [25]	Biodegradable, flexible, slow degradation rate	Provides long-term mechanical support, used in combination with bioactive molecules for enhanced regeneration
Gelatin Methacrylate (GelMA) [10]	Photocrosslinkable, good cell compatibility, adjustable mechanical properties	Used in Digital Light Processing (DLP) printing, supports cell encapsulation and tissue formation, and can be modified for improved properties
Silk Fibroin [10]	High tensile strength, biocompatible, promotes cell attachment	Used for creating mechanically robust scaffolds, supports tendon-like mechanical properties and tissue regeneration
Alginate [11]	Biocompatible, easy to process, forms hydrogels upon crosslinking	Used as a bioink component, provides a hydrated environment for cells, often combined with other materials for improved stability
Hyaluronic Acid [11,19]	Natural polymer promotes cell migration and proliferation, and hydrophilic	Enhances scaffold hydration and cell migration, used in combination with other materials for improved mechanical properties
Decellularized Extracellular Matrix (dECM) [11]	Contains native ECM components, promotes cell attachment and differentiation	Used to create bioactive scaffolds that closely mimic the native tendon environment, support tissue-specific regeneration

4. Tendon 3D Bioprinting

4.1. Functional Properties of Healthy Tendons

While all tendons act as tensile support, also known as the positional role, some tendons can also perform the role of energy storage [21]. As a result, the functional properties of tendons can differ based on their classification, anatomical location, physical training, age, and other factors [22]. In general, the Young Modulus of healthy tendons can range from 800 to 1500 MPa. The stress at failure can range from 50 MPa to 200 MPa and the strain failure can range from 12 to 22% [21,23].

4.2. Functional Properties of 3D Bioprinting Tendons

To fully replace a healthy tendon, a 3D bio-printed tendon must exhibit similar mechanical strength. Most attempts at 3D bioprinting fall under two categories; organic and semi-synthetic [24]. The synthetic materials are used to support the mechanical strength of the tendon or serve as scaffolding for the cell structures. A general overview of 3D bioprinting is depicted in Figure 1. The process of 3D bioprinting begins with acquiring images of the organ or segment of interest through CT scan or MRI. These images are then used to create a segmented 3D model, which is converted into a suitable format. Following this, the appropriate bioprinter and bioink are selected for the construction of the 3D tissues. This process allows for the precise and customized fabrication of complex biological structures with potential applications in tissue engineering and regenerative medicine. Unfortunately, even in the best cases, neither organic nor semi-synthetic 3D bio-printed tendons exhibit similar mechanical characteristics to healthy tendons [25,26,27]. One study found that in optimal conditions, Electronically Aligned Collagen (ELAC), had a wet Young’s modulus of 900 MPa, a wet ultimate stress of 105 MPa and a wet ultimate strain of 10% [28]. Unfortunately, the ideal conditions of the ELAC are not biocompatible, making such a solution unviable as a tendon replacement. Due to this the current gold standard for tendon treatment are either tendon autografts or allografts.

In recent years, tough and stretchable hydrogels (e.g., GelMA, Silk Fibroin) have garnered considerable interest for their potential applications in tendon repair, owing to their ability to withstand high mechanical loads and their flexibility, both of which are crucial characteristics of natural tendons [29]. These hydrogels are engineered to maintain their structural integrity under stress, providing a robust scaffold for cell attachment and growth. Koo et al. (2023) [30] made noteworthy contributions to this field by developing a novel hydrogel that combines toughness and stretchability, positioning it as an ideal candidate for tendon repair. Their research demonstrated the hydrogel’s ability to endure repeated mechanical loading without significant degradation, closely mimicking the behaviour of natural tendons. The composition of the hydrogel included a combination of synthetic and natural polymers, striking a balance between mechanical strength and biocompatibility.

The key features of Koo et al.’s hydrogel include high tensile strength, crucial for supporting the mechanical loads experienced by tendons, as well as elasticity that allows the material to stretch and return to its original shape, akin to natural tendon tissue. Moreover, the hydrogel supports cell viability and proliferation, essential for tissue regeneration, and exhibits self-healing properties, which could be beneficial for repairing micro-damages occurring during normal tendon function [30].

Furthermore, other researchers [11,12,13,14,15,16,17,18,19,20,21,22,23,31,32] have also made significant progress in developing tough and stretchable hydrogels for tendon repair. These studies have focused on enhancing the mechanical properties and biological functionality of hydrogels through various approaches, such as composite hydrogels, double-network hydrogels, and bioactive hydrogels. Composite hydrogels involve combining natural and synthetic polymers to create materials with improved mechanical properties and biocompatibility, while double-network hydrogels consist of two interpenetrating polymer networks, one providing elasticity and the other offering toughness to achieve a balance between flexibility and strength. Additionally, bioactive hydrogels incorporate growth factors or peptides to promote cell differentiation, tissue regeneration, adhesion, and proliferation, facilitating better tissue integration.

In

Table 2. Correlation between bioprinting techniques and biomaterials for tendon repair: applications and advantages.

Bioprinting Technique and References	Example of Application	Advantages
Inkjet Bioprinting [23]	Creating cell-laden constructs for tendon repair by precisely depositing droplets of bioink containing tendon-derived cells and growth factors.	High resolution and precision, ability to print multiple cell types and bioactive molecules simultaneously, relatively low cost, and rapid printing speed.
Extrusion-Based Bioprinting [33]	Fabricating PCL scaffolds that support tenocyte proliferation and alignment, enhancing tendon regeneration.	Ability to print a wide range of biomaterials, high mechanical strength of printed constructs, suitability for creating large and complex structures, incorporation of cells and growth factors within the bioink.
Stereolithography (SLA) [34]	Creating high-resolution GelMA-based scaffolds with intricate microarchitectures that mimic the native tendon structure, promoting cell viability and alignment.	High resolution and precision, ability to create complex and detailed structures, suitability for printing photocrosslinkable hydrogels.
Laser-Assisted Bioprinting (LAB) [35]	Depositing cells and biomaterials with high precision to create constructs that promote tendon regeneration, such as patterning tenocytes and ECM components.	High precision and resolution, ability to print cells and biomaterials without direct contact, minimal thermal damage to cells, creation of highly detailed and organized tissue constructs.
Fused Deposition Modeling (FDM) [36]	Fabricating PCL scaffolds that mimic the mechanical properties of native tendons, supporting cell attachment, proliferation, and alignment.	High mechanical strength and stability of printed constructs, ability to print a wide range of thermoplastic materials, suitability for creating large and complex structures, relatively cost-effective and widely accessible.

, each technique is paired with examples of how it has been applied in tendon repair, showcasing the practical use cases and the benefits they offer.

While these bioprinting techniques offer numerous advantages for tendon repair applications, they also face several limitations. Inkjet bioprinting, despite its high precision, often struggles with viscous bioinks and may cause cell damage due to shear stress during droplet formation [23]. Extrusion-based bioprinting, while versatile, can compromise cell viability due to the pressure required for extrusion and may have lower resolution compared to other methods [33]. Stereolithography (SLA) is limited by the availability of biocompatible, photocrosslinkable materials and potential cytotoxicity from residual photoinitiators [34]. Laser-assisted bioprinting (LAB), though highly precise, faces challenges in scaling up for larger constructs and can be prohibitively expensive [35]. Fused Deposition Modeling (FDM), while cost-effective, is primarily limited to thermoplastic materials and often lacks the resolution needed for intricate cellular-level structures [36]. Additionally, all these techniques face common challenges in recreating the complex hierarchical structure and mechanical properties of native tendons, ensuring the long-term stability of the printed constructs in vivo, and achieving proper vascularization and innervation of the engineered tissue. Furthermore, the translation of these technologies from laboratory settings to clinical applications is hindered by regulatory hurdles, scalability issues, and the need for standardization in bioink formulations and printing protocols.

5. Challenges and Future Perspectives

Tendons are subject to tensile loads exerted by the body’s muscles but require more recovery time. Thus, tendon injuries can occur even when the surrounding muscle suffers little to no injuries. Since the issue of tendinopathy is not limited to tendon damage, a more holistic approach must be taken to completely resolve the issue both in the short and long term. A multi-pronged strategy must be followed to stop the degeneration of the cells, promote proper function of the support cells, and stop inflammation of the cells in the area.

One of the challenges involves using a material that is strong enough to withstand the tensile loads that tendons are subject to, while also serving as a good environment for future cell development [28]. Most tendon material development focuses on creating collagen-based tissue mimics. While there have been some improvements in the strength of tissue mimics, the overarching results demonstrate that they are not strong enough to withstand the tensile loads that tendons are normally subject to [24,28]. It is probable that emerging technologies such as Digital Light Processing (DLP) and volume printing will bring gains in the field of bioprinting. DLP uses a digital light projector to cure photopolymer resins layer by layer, allowing for high-resolution and rapid fabrication of complex structures. Its precision and speed make it a promising technique for creating detailed tissue constructs. Volume printing, including techniques such as volumetric bioprinting, enables the simultaneous solidification of entire 3D structures within a single step. This approach significantly reduces printing time and can produce highly complex geometries with excellent cell viability [13,31,32].

Another challenge has to do with the difficulty of replicating the organized matrix of the tendons. The extracellular matrix (ECM) of the tendon is one of the characteristics of healthy tendon tissue. It is primarily composed of collagen organized in a unique pattern, allowing the tendon to perform its function as medium of force transfer [21]. When tendinopathy occurs, the organized matrix changes, making the tendon tissue weaker and thicker.

Replacement tendon tissue must replicate both the tensile strength and the unique matrix of the tendon to restore the full function of the tendon. This replication of the ECM requires fine 3D bioprinting. Recent studies have shown that electrohydrodynamic jet 3D printing or E-jetting is capable of printing tendon scaffolding with enough precision to mimic the unique order of the ECM from fibre to fascicle level. Unfortunately, such collagen-based tissue biomimics are currently unable to match the tensile strength of healthy tendons [25]. The mechanical properties of tendons, such as tensile strength, elasticity, and viscoelasticity, are crucial for their function. Biomimetic scaffolds aim to replicate these properties to provide adequate support during the healing process and to withstand physiological loads. Materials such as polylactic acid (PLA), polycaprolactone (PCL), and other biocompatible polymers are often used in bioprinting due to their tunable mechanical properties. By adjusting the composition and structure of the bioink, researchers can create scaffolds with mechanical properties that match those of native tendons. Additionally, incorporating reinforcing elements like nanofibers or microfibers can enhance the scaffold’s strength and durability. Also, successful tendon repair requires the scaffold to support cellular activities such as proliferation, differentiation, and ECM production. By mimicking the biochemical cues present in native tendons, biomimetic scaffolds can promote the recruitment and differentiation of tendon-derived stem cells or other progenitor cells. The use of growth factors like transforming growth factor-beta (TGF-β) and bone morphogenetic proteins (BMPs) can further stimulate tendon regeneration. Additionally, the scaffold should facilitate vascularization to ensure adequate nutrient and oxygen supply, which is essential for tissue survival and integration [25].

Restoring the tendon repair process is also an obstacle, as one of the important functions of the tendons is the ability to repair damage it sustains due to excessive loads. This repair process is part of the normal function of the tendon and if performed sparingly it can cause the tendon to strengthen [29]. However, when the tendons are exposed to an excessive load it can damage the tendon and change its response to future loads. Tendons that are tendinopathic are not only weak, they also are less able to repair future damage to the tendon. This starts a process of the tendon gradually losing tensile strength due to its inability to reinforce itself when exposed to a load. This makes the tendon predisposed to injury in the future. The most common cause of tendinopathy is the exposure of the tendon to high-volume repetitive loads [23]. To restore proper function of the tendon, the oxidative and apoptotic process must be stopped. This prevents the further deterioration of the tendon in the case that a 3D bio-printed tendon is grafted in.

6. Conclusions

While the main function of tendons is the transfer and/or storage of tensile force produced by muscles, the fulfillment of that function is dependent on many mechanical and biological properties of tendons. The goal of 3D bioprinting design is to fulfil both those requirements. While there are design options that maximize either the mechanical or biological requirements of a tendon there is often a trade-off to maximizing one or the other. Modern 3D bio-printed tendons currently are not significantly comparable to healthy tendons or even tendon autographs and allographs. Further research will need to be performed holistically to design a 3D bio-printed tendon that is both mechanically and biologically viable.