The limited regeneration capacity of AT is usually attributed to their low cellular content (Ruiz-Alonso et al., 2021). These cells are ultimately responsible for the synthesis of ECM components as well as the production of biologically active molecules that regulate a variety of physiological processes. Therefore, cell infiltration of the damaged area is regarded as a significant estimation for AT injury (Schneider et al., 2018). However, cell infiltration at the injured AT is not as efficient as postulated, owing to the low persistence and survival rate of cells at the original location (Geburek et al., 2016). Thus, tissue engineering is advanced in this area because cells can be integrated into scaffolds. This represents a major advantage of this technique, which allows cells to remain where damage occurs, minimizing cell migration to other tissues and possibly even detrimental effects (Niveditha et al., 2021).

Cell source is an important consideration because poor cell source selection can lead to tendon regeneration and phenotype drift toward osteogenesis, resulting in ectopic bone formation. Tenocytes and tenoblasts are two of the most commonly used cell types of tendon tissue engineering. However, due to the limited number of autologous cells available and the risk of donor site disease, getting autologous tenocytes is difficult (Wu et al., 2018). To be effectively used for clinical purposes, cells should be readily available, expandable in vitro, and gross in culture and implantation. They must be morally and ethically acceptable, functionally integrated with recipient tissue, and safe and non-immunogenic; that is, they must not be contaminated by any pathogen or tumorigenic (Almela et al., 2016). Stem cells are characterized as non-committable, self-renewing, long-dividing cells, which can differentiate in all cell lines. Current evidence supports that stem cells can promote regeneration during tendon healing (Migliorini et al., 2020). Since they are stem cells, it is crucial to provide them with the proper signals so that they differentiate toward tendinocyte lines, and they do not differentiate toward osteogenic lines resulting in ECM characteristics of bone tissue rather than tendon tissue (Loebel and Burdick, 2018). The most commonly used stem cells in tendon tissue engineering are MSCs (Kim et al., 2021), TSPCs (Zhang et al., 2019), and IPSCs (Tsutsumi et al., 2022), which can also give rise to tendinocytes or fibroblasts after differentiation.

The TSPCs are pluripotent adult stem cells involved in tendon healing. The TSPCs can clone, differentiate, and express specific stem cell surface markers (Bi et al., 2007). Tendon healing is poor and may deteriorate in the elderly, due to a decrease in endogenous TSPCs with age, coupled with impaired cellular activity (Yin et al., 2020). In a recent study, rat TSPCs were implanted on asymmetric CS scaffolds. In vitro studies revealed that TSPCs transplanted on CS scaffold showed higher levels of protein production and tenogenic-specific gene expression. At 4 and 6 weeks after implantation of all AT abnormalities in rats, the TSPCs group produced more COL1, COL3, and TNMD proteins as well as better arrangement of collagen fibers with elongated spindle cells (Chen et al., 2018). However, TSPCs account for approximately 4% of all mature tendinocytes. Given this low number, in vitro amplification of TSPCs is required prior to treatment to achieve therapeutic effect (Song et al., 2018; Migliorini et al., 2020). One of the major challenges is that the phenotype of TSPCs is gradually lost during in vitro culture, and the expression of tendinocyte-associated gene markers is significantly reduced (Zhang Y. et al., 2021).

The MSCs are pluripotent non-hematopoietic adult stem cells generated from the germinal layer of mesoderm (Pillai et al., 2017). The MSCs can be found in bone marrow, adipose, periosteum, skin, blood vessel walls, tendons, muscles, peripheral circulation, umbilical cord blood, periodontal ligament, and tooth tissue (Wang et al., 2013; Chen J. et al., 2021; El Khatib et al., 2021). A range of active molecules (e.g., BMP-12/7 and growth factor) can promote further expansion and tendinogenic differentiation (Tang and Lane, 2012; Zhou et al., 2019a; Perucca Orfei et al., 2019). The expression of tendon surface proteins (such as tendinomodulins) demonstrates a commitment to tendinogenesis. Longer dilation can induce differentiation and growth of osteoblast cell lines, therefore this stage must be strictly regulated (Chamberlain et al., 2007; Veronesi et al., 2017).

The BMSCs and ADSCs are more readily available in large quantities than TSPCs and other MSCs and have been investigated in various preclinical trials for tendon tissue healing (Leong and Sun, 2016). The BMSCs are the most often used source of stem cells for tendon tissue engineering and regeneration. Nevertheless, due to their higher proliferative capacity and easier accessibility for tissue harvesting, ADSCs are also increasingly being used to facilitate tendon regeneration, increasing the number of cells available for clinical usage (Torres-Torrillas et al., 2019). In a recent study, scaffolds loaded with and without autologous BMSCs were implanted into adult rabbit models of AT injury (Chailakhyan et al., 2021). The BMSC-loaded scaffold group developed spindle-shaped tenocytes, neovascularization, and parallel collagen fibers 6 months after surgery. The lesion area in the cell-free control group was filled with non-specific fibrotic tissue, with a small number of incompletely regenerated lesions. The rigidity of the BMSC-loaded scaffold repaired AT improved 98% of the value for the entire sample, according to biomechanical measurements. In other studies, autologous ADSC-loaded scaffolds cultured with GDF-5 were implanted in the rat AT models, and at 4 weeks after surgery showed higher cell density and more uniform COLIII distribution in the ADSC-loaded scaffolds group than the non-ADSC-loaded scaffolds groups (Aynardi et al., 2018).

Many studies have revealed that the therapeutic effect of non-tendon MSCs is mainly due to the ability of resident cells to be endowed by paracrine mechanisms or intercellular communication (Daneshmandi et al., 2020; Chen et al., 2021b; Shah et al., 2022). The MSCs regulate inflammation by secreting cytokines and stimulate AT tissue growth by secreting exosomes (Shi et al., 2019; Yu et al., 2020). Gissi et al. (2020) discovered that both high and low-dose rBMSCs-EVs could stimulate the migration of AT cells in vitro. They stimulated AT cell proliferation and enhanced COLI expression at higher concentrations. In the rat AT injury model, rBMSCs-EVs accelerated the remodeling phase of AT repair in a dose-dependent manner. High-dose rBMSCs-EVs resulted in better recovery of AT structure, optimal AT fiber alignment, and lower vascular density in histomorphological assessment and histology. The higher the EV concentration, the greater the expression of the COLI and the lesser the expression of the COLIII.

The IPSCs induced by mature cells under specific cell culture conditions have the potential to be used in cell therapy (Takahashi and Yamanaka, 2006; Nakajima and Ikeya, 2021). An approach based on IPSCs differentiation was used because it avoids autologous tissue harvesting and ethical concerns, as well as its high proliferative capability and minimal risk of heterotopic tissue development, this technique may be used in tissue engineering therapy (Nakajima et al., 2021; Tsutsumi et al., 2022). Komura et al. (2020) generated mouse IPSCs with tenocyte-like characteristics after tendon differentiation and facilited AT regeneration in mice.

Although tenocytes have been generated using mice MSCs or IPSCs, the use of human IPSC derived in cell therapy would be revolutionary (Czaplewski et al., 2014; Machova Urdzikova et al., 2014; Zhang et al., 2015a). Nakajima’s team has been developing an IPSC-based transplantation therapy procedure, and they recently published the first method to induce differentiation of IPSC-derived tenocytes (IPSC–tenocytes) (Nakajima et al., 2021). They recently established a novel approach to efficiently differentiate human IPSCs into tenocytes. Furthermore, IPSC–tenocyte transplantation can contribute to the recovery of motor function after AT injury in rats via implantation and paracrine action. The biomechanical strength of the regenerated AT was comparable to that of the healthy AT.

Bioactive molecules are molecules or compounds (including growth factors, angiogenic factors, cytokines, siRNA, DNA, hormones, and others) that are synthesized and secreted by cells, as controllers of cell proliferation, differentiation, and matrix deposition and play an important role in the development and repair of AT tissue (Raghavan et al., 2012; Ruiz-Alonso et al., 2021; Chen et al., 2022a). Bioactive compounds, particularly recombinant growth factors, are effective, readily available, dose-controlled, and stable. Specific bioactive chemical formulations can effectively induce tendon development (Zhang Y. J. et al., 2018a). Binding these bioactive molecules into scaffolds is simple. There are different approaches to this. They can be incorporated into biomaterials and then manufactured to form scaffolds with the bioactive molecules already bound or scaffolds that have been synthesized (Elsner and Zilberman, 2009). One specific approach involves preparing recombinant bioactive molecules containing scaffold material binding domain and controlling the release of the recombinant bioactive molecule by tethering the recombinant bioactive molecule to the scaffold (Sun et al., 2018).

Different types, concentrations, or release gradients of the bioactive molecule are appropriate for the proliferation and differentiation of different types of cells (Teh et al., 2015; Shiroud Heidari et al., 2021). The significant growth factors for tendon tissue engineering are IGF-1 (Olesen et al., 2021), PDGF-BB (Evrova et al., 2020), BMP-7 (Zhang P. et al., 2020), TGF-β1 (Rajpar and Barrett, 2019), VEGF (Zhou Y. L. et al., 2019; Yuan et al., 2022), BMP-12 (Rinoldi et al., 2019), SDF-1α (Chen C. et al., 2020), bFGF (Liu et al., 2013; Zhao et al., 2019), and TGF-β3 (Chen et al., 2022a). Raghavan et al. (2012) discovered that IGF-1, bFGF, and PDGF-BB increased the proliferation of all cell types (hADSCs, fibroblasts, and tenocytes) in in vitro scaffold experiments. The most effective proliferation stimulator among single growth factors was 50 ng/ml PDGF-BB. The ideal concentration of 50 ng/ml IGF-1, 5 ng/ml bFGF, and 50 ng/ml PDGF-BB showed the best regeneration effect of the injured tendon with numerous active molecules.

The FGF-2 belongs to the FGF family, which promotes not only cell proliferation but also angiogenesis, collagen production, and tissue remodeling (Benington et al., 2020; Zhang J. et al., 2020a). Low FGF-2 levels may be the main cause of poor tendon healing, with FGF-2 downregulated during the tendon healing process as per the studies (Zhang J. et al., 2020b). Increased FGF-2 concentration of repair site may stimulate AT regeneration (Zhang J. et al., 2020b). Guo et al. (2020) transfected FGF-2 into hTPSCs and FGF2 overexpressed hTPSCs significantly increased the expression of SCXA and COL3α1 in vitro. In the rat AT defect model, COLI/III expression was significantly increased in the FGF-2 group 4 weeks after surgery compared to the control group. Biomechanical tests revealed that the failure load of the FGF-2 group was higher than that of the control group 4 and 8 weeks after surgery.

Several studies have demonstrated the relevance of the combined role of the MAPK pathway and TGF-β in tenogenic differentiation (Zhang Y. J. et al., 2018a). Havis et al. (2014) performed transcriptome analysis on mouse tendinocytes isolated at various stages of development showing that TGF-β and MAPK were the two most strongly modified signaling pathways. TGF-β signaling induces the differentiation of mouse mesodermal stem cells into tendines via Smad2/3 in vitro and in vivo. The MAPK signaling inhibition was sufficient to activate Scx expression in mouse limb MSCs and mesodermal progenitors.

Platelet concentrates, such as PRF, PRP, and concentrated growth factor, contain high superphysiological concentrations of platelet-derived chemokines, growth factors, cytokines, and high concentrations of platelets (Chen et al., 2022b). The preparation procedure is simple and quick, and it is possible to evaluate the quality of PRP products by calculating the relative proportion of leukocytes, platelets, and growth factors. To date, the FDA has approved several commercial preparation systems for PRP and expanding, mainly in cosmetic and plastic surgery (Sharara et al., 2021), orthopedic medicine (McCarrel et al., 2014; Fang et al., 2020), sports and musculoskeletal medicine (Jiang et al., 2020), dermatology (Gentile and Garcovich, 2020), reproductive medicine (Sharara et al., 2021), and oral and maxillofacial surgery (Anitua et al., 2021). PRP can be delivered to the target by intradermal injection or topical administration. The therapy premise is that PRP injection can activate the repair mechanism and speed up tendon healing (Chen et al., 2022b). The injection in the rabbit AT injury model revealed that LP-PRP and LR-PRP improved AT recovery more than LP-PRP (Yan et al., 2017). Maghdouri-white et al. developed a novel type of TEND consisting of PDLLA and type I collagen (Maghdouri-White et al., 2021). For 2 weeks, annealed TEND rapidly adsorbs PRP and gradually releases PDGF-BB and TGF-β1. After implantation of the scaffold into a rabbit AT injury model, new AT tissue was generated, and mature, dense, regular, and directed connective tissue remodeling was observed in vivo.

However, a large number of randomized controlled trials have found that PRP injections do not improve objective tendon function or self-reported function or quality of life in patients with acute and chronic Achilles tendinopathy, and there is no evidence that they have any benefit on Achilles tendinopathy (de Vos et al., 2010; de Vos et al., 2011; Keene et al., 2019; Kearney et al., 2021). Because each PRP preparation method leads to different products with different biological functions, the regulation of PRP applications, both in clinical research and basic science, is confusing. Although the FDA has approved several commercial preparation systems for PRP preparation, uniform quality assessment and standardized preparation procedures have not been agreed upon worldwide (Zhang Y. J. et al., 2018b; Everts et al., 2021).

Because of the scarcity and high cost of growth factors, in vivo efficacy is inadequate. Chemically manufactured or naturally occurring small molecules, on the other hand, provide numerous advantages over growth factors in regulating cell behavior, including quantity control, cell permeability, cost efficiency, and time efficiency (Zhang C. et al., 2018). MLT (Yao et al., 2022), RGD (Yin et al., 2020), and simvastatin (Weng et al., 2022)-loaded nanofiber scaffolds have been proven to enhance AT regeneration in vitro and in vivo.

Although the therapeutic benefits of the bioactive molecule for tendon regeneration have been proven, there are still some challenges in using bioactive molecules, such as the potentially detrimental effects of uncontrolled release in vivo (de Girolamo et al., 2019).

The scaffold’s primary role in AT tissue creation is to offer biomechanical support, a bionic matrix for cell proliferation, differentiation, and migration, and to promote AT integration and growth (Paredes and Andarawis-Puri, 2016). Scaffolds are developed and created to mimic the shape and function of natural tissue ECM and to generate ideal cell interactions (adhesion, differentiation, and proliferation) that contribute to the formation of new functional tissues (Lin et al., 2018; Ruiz-Alonso et al., 2021). Several scaffold manufacturing approaches have been reported over the past decade, including collagen matrix–based creation (Dewle et al., 2020; Uday Chandrika et al., 2021), particle leaching (Ye et al., 2018), rapid prototyping (Billiet et al., 2012; Gu et al., 2016), solvent casting (Yeo et al., 2015), freeze drying (Shahbazarab et al., 2018), and fibrous structures (Tiwari et al., 2016; Reid et al., 2021; Uribe-Gomez et al., 2021). Sun et al. (2018) used a rat model SDF-1α loaded collagen scaffolds manufactured from bovine type I and type III collagen for AT regeneration. The findings showed that collagen scaffolds can enhance the diameter of collagen fiber, facilitate the expression of type I collagen of AT, and improve the mechanical characteristics of regenerated AT 4 and 12 weeks after surgery. Li et al. (2019a) implanted a parallel PCL microfiber bundles template subcutaneously in rats, resulting in collagen-rich ECM deposition and cellularization in the template. The polymer template and cells were then removed to form autologous ECM scaffolds with aligned and hollow microchannel structures. Three months after surgery, the mechanical strength of the regenerated AT filled with tenocyte was comparable to that of the pre-injury state AT.

Natural organizational microarchitecture is widely acknowledged as being important in functional organizational behavior. Cells in natural tendons also align along the direction of collagen fibers. This cell arrangement stimulates the formation of collagen matrix (e.g., COLI and COLIII) and key tendinins proteins (Wu Y. et al., 2017). Therefore, the use of fibrous scaffolds in tissue engineering is beneficial to its microstructure, which is similar to that of ECM. Aligned fibrous scaffolds, in particular, can be used to guide cell alignment and create tissues with anisotropic microstructure (Wu et al., 2019; Nagiah et al., 2021). The tendon regeneration fibrous structures can be divided into three types: nanofiber structure, microfiber structure, and nano/microfiber structure. The nanoscale properties of the nanofibers lead to effective cell attachment, proliferation, and differentiation in nano/microfibrous scaffolds, whereas coarser fibers in micron diameters can be chosen for mechanical strength (Yongcong et al., 2019; O'Brien et al., 2016; Tan et al., 2021). The aligned nanofibers are postulated to be suitable for providing topographic cues for tendon seed cells to guide cell-oriented growth and enhance cell proliferation and differentiation to generate functional tendon tissue. The anisotropic structure also enhances the tensile strength of the scaffold while responding to directional stimuli such as uniaxial mechanical loads. Therefore, the directional fiber terrain scaffold is expected to provide appropriate biophysical cues to help cells elongate along the direction of the directional fiber, thereby increasing cell proliferation rate and function (Chen et al., 2021c; Sarıkaya and Gümüşderelioğlu, 2021).

The insertion can be separated into four discrete but structurally continuous zones within a few hundred microns of the tendine-bone insertion location. The collagen fibers are parallel and aligned in the tendon region. Before adhering to the bone, the tendon fibers break into thinner, smoother interface fibers and spread symmetrically along the insertione (Lei et al., 2021). The implanted tissue had varied mineral content, collagen orientation, and protein type gradients at the interface at the micron scale (Calejo et al., 2019). Even after surgical repair, the injured tendon-bone interface does not regenerate the well-aligned collagen fibers found at natural attachments during healing. The healed scar tissue is an order of magnitude weaker than natural tissue and does not integrate well into the bone (Lei et al., 2021; Shiroud Heidari et al., 2021; Zhu et al., 2021). Therefore, an enhanced tendon-to-bone repair can be achieved by a combination of strategies: I) inducing the establishment of a grading interface; II) promoting bone formation; and III) promoting deposition of aligned collagen, all of which will result in higher adhesion strength and better integration (Zhu et al., 2021). Typical anisotropic scaffolds typically consist of three zones: aligned fibers, random fibers, and interfaces. These three zones are morphologically distinct but structurally continuous, resembling changes in the direction of collagen fibers at the tendon-bone insertion site.

Nanofiber-based scaffolds are prepared using various techniques. The best-known nanofiber manufacturing techniques include molecular self-assembly, phase separation, and electrospinning (Eskandari et al., 2017; Manoukian et al., 2017; Ko, 2020).

Self-assembly often involves intermolecular bonding (Barnes et al., 2007). Using non-covalent forces such as electrostatic, van der Waals forces, hydrogen bonds, hydrophobicity, and π–π stacking interactions, simple molecules and macromolecules assemble rapidly into stable, organized, and well-defined structures (Lyu et al., 2020) (Figure 3A). These bonds are often weak, but when they combine to form a single unit during assembly, they affect the structural stability and conformation of the assembly and have a significant impact on the interactions between supramolecular structures and other tissues, cells, and molecules (Nemati et al., 2019; Yao et al., 2020). Natural biopolymer-based materials, such as proteins, DNA, and polysaccharides, offer excellent alternatives to tissue-engineered scaffolds. Due to a wide range of non-covalent interactions (ionic, hydrogen bonding, and hydrophobicity) between molecular chains, they exhibit superior self-assembly capabilities (Zhang R. et al., 2021). Since the late 1980s, interest in peptides derived from natural proteins with self-assembly properties has increased. Consequently, numerous engineered peptides that mimic the conformation of self-assembled peptides of protein molecules have been developed (Chen and Zou, 2019; Park, 2020). In response to external stimuli, secondary structures such as α-helices and β-folds are responsible for assembly. The pH, temperature, and ionic strength are the most critical factors in supramolecular nanofiber formation (Koutsopoulos, 2016; Vedhanayagam et al., 2017; Delfi et al., 2021). Polysaccharides such as cellulose, chitosan, and chitin have highly similar structures and are rich in hydrogen bonds, which makes it easy for them to form nanofibers through parallel self-assembly (Zhang R. et al., 2021). By adding molecules to nanofibers via step by step self-assembly, thin and dense functional molecular layers are formed on the nanofibers’ surface, resulting in the formation of multifunctional nanomaterials (Ma et al., 2021).

The phase separation process can be initiated by a non-solvent or by heat, and it has been widely used to make foams or porous membranes for separation and filtration methods (Asadian et al., 2020). Typically, non-solvent-induced phase separation results in a matrix with an uneven pore structure, which is undesirable for tissue scaffolds, which require a homogeneous pore structure (Yao et al., 2020; Asadian et al., 2020). Thermal-induced phase separation occurs when a homogeneous multi-component system (polymer, filler, solvent, drug, etc.) becomes thermodynamically unstable under specific conditions, resulting in the formation of two distinct phases, a polymer-poor phase and a polymer-rich phase (Figure 3B). After solvent removal by sublimation, extraction, or evaporation, the polymer phase is transformed into the skeleton of the porous scaffold, while the removed solvent is responsible for the final porosity (Zeinali et al., 2021). Changing formulation variables, such as solvent polymer concentration and mixture composition, can significantly affect the morphology of the resultant scaffold (Sabzi et al., 2021).

Electrospinning is a simple and widely used technique for producing nanofibers for application in tissue engineering (Han et al., 2021; Sensini et al., 2021a; Song et al., 2021). The electrospinning device is so simple that it can be used in almost any laboratory. The primary components include an injection pump, spinneret (usually a hypodermic needle with a blunt tip), conductive collector, and high-voltage power supply (direct current or alternating current) (Xue et al., 2019; Xue et al., 2017). In electrospinning, a high electric field is applied to the polymeric solution drawn using a capillary, and the jet begins when the electrostatic charge exceeds the surface tension (Figure 3C). Finally, by stretching the droplets of the polymer solution, the surface charge density increases as the solvent evaporates, and the fine fibers are collected on a grounded collector (Sundaramurthi et al., 2012; Sundaramurthi et al., 2013; Jayasree et al., 2019; Zhang C. et al., 2020).

Changes can be made to the polymer, its concentration in a liquid solution, polymer molecular weight, feed rate, and electric field voltage to change the diameter, composition, and direction of the nanofiber (Kalantari et al., 2019; Wu et al., 2020a).

AT reconstruction (Sankar et al., 2017) employs natural polymers such as silk fibroin protein (Yi et al., 2018), collagen (Sandri et al., 2016), chitosan (Chen et al., 2018), sodium alginate, and gelatin, as well as degradable synthetic polymers such as PCL (Fotticchia et al., 2018), PLA (Wu et al., 2020b), PLGA (Hasmad et al., 2018), PET (Cai et al., 2018a), and PEO (Table1). Different materials used as scaffolds for tendon tissue engineering have their advantages and disadvantages (Chen et al., 2021a). The advantage of synthetic polymer scaffold polymers is that their sizes may be easily modified to meet requirements and they degrade slowly. In general, the use of synthetic polymer scaffolds constrained by the adverse effects of their degradation products in vivo (such as inflammatory reactions) and their relatively poor integration with host tissues (Chen et al., 2021a). PGA, PLA, and PLGA are considered biocompatible and cause only mild or minor foreign body reactions because their hydrolysis degradation product (glycolate lactate) is typically present in the metabolic pathway of the human body (Bianchi et al., 2021). However, their extensive degradation occasionally causes local inflammation due to the accumulation of acidic degradation products that are difficult to eliminate (Hafeez et al., 2021). PCL is also biocompatible and degrades significantly more slowly than PGA, PLA, and PLGA, making it a desirable material for long-term scaffolds such as tendons (Silva et al., 2020; Zhu et al., 2021). Because of its excellent biocompatibility and biodegradability, the synthetic polymer PLGA is a copolymer used in the majority of FDA-approved therapeutic devices. PLGA significantly decreased peritendinous adhesions, an effect that may be associated with histopathological findings of inflammation, vascular density, and fibrosis (Russo et al., 2020; Uyanik et al., 2022).

The advantage of using natural polymer-based fibers is that they are comparable to natural tendon ECM (Salvatore et al., 2021), which is beneficial for cell adhesion, proliferation, and differentiation (Zha et al., 2020). However, natural fibers have low mechanical strength. Silk, on the other hand, is readily available and inexpensive, has a relatively slow controlled degradation rate, and possesses excellent mechanical properties. Numerous previous studies have demonstrated that silk is an ideal scaffold material for AT tissue engineering and has a promising clinical application (Chen et al., 2021a). Insoluble collagen fibers are more suitable for scaffold preparation than soluble collagen since their mechanical properties are closer to those of natural tendon ECM (Yang et al., 2019).

Moreover, natural polymers blended with synthetic polymers increase their mechanical strength and promote tissue regeneration (Jayasree et al., 2019; Lim et al., 2021; Liu et al., 2022). The combination of natural and synthetic polymers can maximize the benefits of each biomaterial, allowing the composite scaffold to possess significant properties (Nikolova and Chavali, 2019). Therefore, different natural/synthetic polymers, such as PCL/nCol (Jayasree et al., 2019), PCL/chitosan (Almeida et al., 2019), Col/PDS (Moshiri et al., 2013; Moshiri et al., 2015), Col/BDDGE (Sandri et al., 2016), SF/PEO (Yi et al., 2018), and SF/PCL (Yuan et al., 2016) have been developed for Achilles tendon tissue engineering (Jayasree et al., 2019; Yi et al., 2018; Moshiri et al., 2015; Yuan et al., 2016; Li et al., 2020a) (Table 1). The FDA has approved drug delivery systems and sutures for PCL. However, PCL is extremely hydrophobic, resulting in reduced cell-scaffold interactions. Blends of PCL with hydrophilic natural polymers such as cellulose acetate (CA) improve its hydrophilic, biocompatibility, and matrix erosion profile (Ramos et al., 2019).

In addition, some special materials are also used in nanoscaffolds. MOFs assembled by metal ions and organic connectors possess the advantages of large pore volume, high specific surface area, and hydrophilicity, which can enhance the performance of tissue scaffolds. In addition, these types of MOFs have great potential for tissue repair and regeneration (Chen J. et al., 2020; Luo et al., 2021; Yang et al., 2022). It has been established that motion-driven electromechanical stimulation of tendon tissue by ferroelectric nanofibers can modulate ion channels and specific tissue regeneration signaling pathways in vitro (Fernandez‐Yague et al., 2021).

Due to their excellent bionic properties, numerous studies over the past decade have established the beneficial effects of nanofibrous scaffolds as cell delivery scaffolds for tendon regeneration (Moffat et al., 2009). In addition, several studies have investigated nanofibrous scaffolds with alignment and/or porosity gradient (Li et al., 2020b), metal-organic framework (Yang et al., 2022), mineral gradient (Grue et al., 2020; Zhou et al., 2020), and growth factor gradient (Madhurakkat Perikamana et al., 2018) for tendine-bone interface healing (Lowen and Leach, 2020; He et al., 2021). Table 1 shows a variety of nanofibrous scaffolds for regeneration of the Achilles tendon and tendine-bone region.

Wang et al. (2016) demonstrated the potential of nanofiber as scaffolds for AT regeneration. Random and aligned electrospun nanofibers of PCL/PEO/gelatin were inoculated with hDFs to analyze the in vitro efficiency of the scaffold, and cell-free scaffolds were implanted in rat AT fractional defects to study the effects of AT regeneration. In vitro experiments revealed elongated cell morphology and tenogenic differentiation of seeded hDFs on aligned nanofibers and irregular morphology on the random nanofibers. In addition, elongated cells produced more parallel aligned collagen fibers ECM than irregularly shaped cells. In vivo experiments revealed that endogenous cells were able to infiltrate aligned nanofiber scaffolds more efficiently than random nanofiber scaffolds. In the aligned group, the regeneration tissue structure of parallel collagen fibers was superior to that of the random group, and the cell density was relatively lower. It has been demonstrated that well-aligned nanofibers can induce tendon differentiation in host fibroblasts; consequently, they could be used for de novo AT regeneration via cell-free methods together with tenogenic inductive nanofiber aligned scaffold (Wang et al., 2016). Chen et al. (2017) studied the effect of aligned PCL/SF (APSF) nanofiber scaffolds on AT regeneration in vivo and in vitro. By binding rDFBs to APSF, the gene expression of tendon marker proteins (fibronectin, Col I, and biglycan) of rDFBs was upregulated, and there was no statistical difference between the maximum load of regenerated AT tissue and that of normal AT.

Cai et al. (2018b) successfully prepared a random and ordered double-layer P (LLA-Cl) /SF nanofiber scaffold using the electrostatic spinning method to study tendon-bone regeneration in AT. After 12 weeks, the maximum breaking strength and stiffness of AT in the double-layer scaffold group were significantly higher than in the random nanofiber group and the control group. Aligned and random double-layer P (LLA-Cl) /SF nanofiber scaffolds can enhance gradient microstructure and tendine-bone bonding by improving collagen tissue maturity and organization, inducing new bone formation, and expanding fibrous cartilage area (Cai et al., 2018b).

Numerous research has investigated various strategies for guiding stem cell phenotypic expression and manipulating tendon matrix properties using different fibrous scaffolds (Guner et al., 2020; Sarıkaya and Gümüşderelioğlu, 2021; Zhu et al., 2021). The stem cells formed more mature tendinous tissue on aligned fibers and displayed greater osteogenic differentiation on random fibers (Yin et al., 2015a; Orr et al., 2015; Guner et al., 2020). The mechanical properties and self-assembly of insoluble collagen fibers are more similar to natural collagen ECM than those of soluble collagen. Yang et al. (2019) fabricated aligned and random nanofiber scaffolds of insoluble collagen for in vitro and in vivo Achilles tendon regeneration. The tensile strength of the nanofiber scaffold was comparable to that of natural rat AT. In vitro assays of tendon-related genes and proteins indicate that oriented scaffolds induce much more tenogenic differentiation of rBMSCs than random scaffolds by producing an elongated cell shape morphology on fibers. The rat AT repair study revealed that rBMSCs on orientated scaffolds can produce healing properties comparable to those of autologous AT. Wu et al. (2021) prepared wavy nanofiber scaffolds by adjusting the PPDO/SF ratio. In vitro, the combination of mechanical stimulation and GDF-5 enhanced the tenogenic differentiation of hADMSCs and maintained the viability and phenotype of hTCs. Furthermore, piezoelectric descendent electric fields generated during physiological exercise provide additional bioelectrical cues to activate tendon-specific regeneration pathways (Gouveia et al., 2017). Several studies have demonstrated that the motion-driven electromechanical stimulation of tendon tissue by piezoelectric bioelectric devices can regulate ion channels and affect specific tissue regeneration signaling pathways in vitro (Cheah et al., 2021; Lee et al., 2021). Fernandez‐Yague et al. (2021) studied the piezoelectric collagen nanofibrous scaffold made of arrays of nanofibers constructed from a ferroelectric material known as poly (vinylidene fluoride-co-tri-fluoroethylene) for rat AT regeneration. It is a self-powered piezoelectric material that represents a paradigm shift in biomedicine, eliminating the need for external power or batteries and complementing existing mechanical therapies to accelerate the repair process. In vitro experiments of mechanical and bioelectric stimulation of hTDCs revealed that BMP, MAPK, FAK, and Wnt/β-catenin form a complex signal network that controls tenogenic differentiation. Using a rat model of mechanical acute tendon rupture, the researchers demonstrated the efficacy of EMS in regulating ion channel expression (PIEZO1/2, TRPA1, and KCNK3/4) and the disrupted regeneration process associated with BMP signal overactivation. This discovery lays the engineering foundation for a variety of synthetic piezoelectric bioelectric nanofibrous scaffolds that enable bioelectrical control of the AT tissue regeneration process (Fernandez-Yague et al., 2021).

The tenogenic differentiation of stem cells in functional tendon tissue engineering relies not only on biophysical clues of scaffolds but also on biochemical clues (Theiss et al., 2015). Yuan et al. (2021) prepared highly oriented PLLA nanofiber scaffolds by electrostatic spinning; the surfaces of these scaffolds were modified with two key tendon ECM components, CS and COL1. In vitro tenogenic differentiation of hBMSCs using PLLA (core) /CS-COL1 (shell) fibers, revealed higher cell spreading and proliferation rates on aligned PLLA/CS-COL1 fibers compared to plain PLLA fibers. The expression of tendon-related genes COL1, SCX, and TNMD was significantly increased. Introducing mechanical stimulation had a synergistic effect on the tenogenic differentiation of hBMSCs. Higher expression of Smad3, TGF-βR2, and TGF-β2 was observed in cells on the PLLA/CS-COL1 fiber matrix, indicating that PLLA/CS-COL1 nanofibers induce tenogenic differentiation of hBMSCs by activating the TGF-β signaling pathway. The role of PLLA/CS-COL1 in promoting tissue regeneration was confirmed in a rat AT repair model (Yuan et al., 2021). Wang et al. (2021) used transcriptional profiling to compare changes in gene expression associated with tendon development in rats and discovered that Postn is a secreted ECM protein that regulates tenogenic differentiation potential and TSPCs stemness. Further, parallel ACF loaded with rPOSTN was implanted into the damaged AT of rats. The ACF-rP group was found to have a well-organized collagenous fibrous structure and a large number of spindle tendine-like cells. After 8 weeks of remodeling and healing, there was a significant reduction in inflammation. In comparison to the ACF group, the ACF-rP group achieved more satisfactory regeneration outcomes, as shown by the tighter and ordered arrangement of ECM deposits, which resembled natural tendons. In addition, it was demonstrated that TSPCs cultured with parallel collagen fibers loaded with rPOSTN had excellent spheroidization ability and the ability to regenerate tendon ECM structure. Currently, organoid and spheroid models are extensively used in stem cell culture. Therefore, this discovery may aid in the optimization of TSPCs culture techniques, and the discovery of additional genes that promote TSPCs function, AT regeneration, and repair (Wang et al., 2021). TSA is reported to explain the mechanism by which MSCs differentiate into functional hepatocellular, adipocyte, and cardiomyocyte cells (Yin et al., 2015b; Li et al., 2020c). Given the role of these epigenetic mechanisms in regulating MSCs differentiation and the observation of decreased HDACs expression in cells cultured on aligned fibers, Zhang et al. (2018) proposed a strategy to enhance the tenogenesis effect of aligned fibers using a small molecule of TSA, an HDACs inhibitor. Tendon-related gene and protein expression demonstrated that TSA-containing targeted scaffolds promoted TSPCs tendon differentiation more effectively than targeted fibers alone. Importantly, random fibers induced the tenogenic differentiation of TSPCs with extremely low efficiency, but the TSPCs tenogenic differentiation was significantly enhanced when random fibers bind to TSA. This study implies that TSA combined with the aligned nanofiber scaffolds may be a more effective strategy for promoting stem cells to tenogenic differentiation and promoting AT defect repair.

To overcome the limitations of nanofibrous scaffolds, integrating nanofibers with different structures is an attractive research area (Liu et al., 2020; Jin et al., 2018; Chen Y. et al., 2020). It has been demonstrated that textile structures have enhanced mechanical properties and can mimic natural structures by providing a wide range of fiber forms and pore sizes (Saveh-Shemshaki et al., 2019; Ouyang et al., 2002; Chen et al., 2012). Textile-based scaffolds, such as knit, braid, and woven (Jiang et al., 2021) (Figure 4), have the potential to mimic the mechanical properties of natural tendons, overcoming the limitations of nanofibrous scaffolds, and are used in many tendon regeneration studies (Zhang et al., 2015b; Wu et al., 2017b; Cai et al., 2018a; Araque-Monrós et al., 2020; Han et al., 2021).

One strategy is to create multiscale nanofibrous textile-based scaffolds using strands or yarns of nanofibers (Almeida et al., 2019; Chen et al., 2021d; Ma et al., 2022). Another method is to coat the fabric with nanofibers (Chen L. et al., 2020; Olkhov et al., 2018). Nanofibrous strands or yarns have the potential to reconstruct the structure of natural collagen fibers (Figure1) in tendon nanofibrous to microfibrous construction (Mouthuy et al., 2015; Cai et al., 2020). Nanofibrous strands or yarns can be braided/twisted to replicate fiber bundles, or knitted/woven to simulate 3D tendon macrostructures (Li et al., 2019b; Silva et al., 2020). Studies on multiscale nanofibrous textile-based scaffolds for tendon regeneration are listed in Table 2.

Rashid et al. (2020), examined the biological potential of woven and electrospun PDO/PCL patches to induce repair responses as shown in Figure 5. The electrospinning assembly of the patch consists of seven layers of aligned electrospun PDO fibers sandwiched between six layers of thin PCL random electrospun mesh (used as a binder) (Figure 5A). A single layer of aligned electrospun PCL fibers connects the electrospinning components with a braided PDO monofilament layer (Figure 5B). All tendon defects had healed 3 months following the implantation of patch scaffolds. No residual polymer of the electrospun scaffold was found on the tendon surface. HE staining revealed various key characteristics. Electrospinning scaffolds exist in the area of cell proliferation and vascularization. The deepest layer of the section corresponds to the native lamb tendon below the patch. These areas resemble normal tendons. The serology and hematology of inflammatory markers were normal 3 months after scaffold implantation, with no indications of increased local or systemic inflammation.

Morais et al. (2020) developed various AT regeneration structures based on PET or PP multifilament yarns. These structures are based on a dual shell/core system in which the core is comprised of numerous sub-components (braided fabric) and the shell is based on the braided yarn that surrounds the core. The PET-based yarn structure exhibited a non-linear force and strain curve comparable to that of typical natural AT, showing optimal load, strain-failure, and stiffness levels for AT replacement. In addition, the PET rope also demonstrated adequate creep resistance and very promising fatigue properties in its final application, which is consistent with the native AT’s report. Jayasree et al. (2019) fabricated a braided multiscale fiber scaffold consisting of neatly arranged PCL microfibers/collagen bFGF nanofibers (mPCL-nCol-bFGF) and coated with sodium alginate to inhibit perinetendinous adhesions. Rabbit Tenocytes exhibited the highest levels of tenogenic marker expression and cellular proliferation in response to In vitro dynamic stimulation. AT tissue regenerated 12 weeks following surgical implantation, and the collagen morphology was neatly arranged.

The effects of multiscale structure and polymer type on cell function (after implantation of rBMSCs) and AT mechanical properties were investigated (Sankar et al., 2021; Yin et al., 2022). Zhao et al. (2019) added an exogenous BFGF-loaded fibrin gel to a porous network of knitted PLGA scaffolds. The scaffold stimulated rBMSCs proliferation and tenocytes differentiation, which synergistically enhanced the reconstruction of damaged Achilles tendons. The biomechanical test 8 weeks following transplantation revealed that the elastic modulus of the regenerated AT was comparable to that of an intact tendon (43.65 ± 5.09 MPa, 45.73 ± 11.64 MPa, respectively). Cai et al. used braided PET scaffolds to induce rBMSCs tenocytes differentiation in the absence of a bioactive molecule; the mechanical properties of regenerative AT with cellular scaffolds were significantly better than those with cell-free scaffolds.

The promising outcomes of these experiments on tendon repair scaffolds suggest a new direction for AT regeneration. Further research using multiscale nanofibrous scaffolds may contribute to the regeneration and integration of the AT structure.