Trend of Bioactive Molecules and Biomaterial Coating in Promoting Tendon—Bone Healing

Abstract

The tendon-bone junction (TBJ) is a graded structure consisting of tendons, nonmineralised, and mineralised fibrocartilage and bone. Given the complex gradient of the TBJ structure, TBJ healing is particularly challenging. Injuries to the TBJ such as anterior cruciate ligament (ACL) tears and rotator cuff injuries are common and serious sports injuries, affecting more than 250,000 patients annually in the United States, particularly people older than 50 years. ACL reconstruction and rotator cuff repair are the commonly performed TBJ repair surgeries. However, the re-tear rate is high post-operation. In recent years, studies on improving TBJ healing have focused on promoting tendon-bone integration at tendon sites. This process includes the use of periosteum, hydrogels, scaffolds, growth factors, stem cells or other reconstruction materials that promote bone growth or ligament attachment. In this study, we will highlight the utilisation of the unique properties of biomaterial coating in promoting tendon-bone healing and discuss recent advances in understanding their role in TBJ healing. Furthermore, we aim to provide a systematic and comprehensive review of approaches to promoting TBJ healing.

1. Introduction

Injury to the tendon-bone junction (TBJ) has become a common sports injury. Anterior cruciate ligament (ACL) injury and rotator cuff injury are the common tendon injuries [1]. About 30 million cases of TBJ injuries are reported worldwide every year [2], and about 200,000 tendon or ligament repair operations are reported in the United States every year [3]. The incidence of rotator cuff injury is about 2%–3.8%, and its incidence amongst the elderly is higher, which is about 5%–7%; moreover, approximately 75,000–250,000 cases of shoulder arthroscopic surgery are performed every year [4]. Statistics from the Centers for Disease Control and Prevention of the United States show that the annual incidence of ACL injury is as high as 3.09%, and more than 100,000 people undergo ACL reconstruction operations each year [5]. Although China does not have specific statistics, more than 100,000 people suffer ACL injuries every year. From the perspective of medical economics, TBJ injuries are costly for patients, families and society [6].

The TBJ can steadily transmit the traction force generated by the muscle from the tendon to the bone [7,8]. Given the long-term stress concentration, the TBJ is prone to acute or chronic rupture [9,10]. The TBJ can be inserted directly and indirectly: The indirect point refers to the penetrating collagen fibre connection between the tendon-bone interface tissues, namely, the Sharpey fibre. Direct insertion indicates that the soft tissue is connected to the bone tissue through the typical fibrocartilage tissue. It has a complex and typical four-layer structure: tendon, uncalcified fibrocartilage, calcified fibrocartilage and bone. The first tendon (or ligament) layer is composed of type-I collagen fibres arranged in parallel and a small amount of proteoglycan-modified protein, which has the mechanical properties of tendons (or ligaments). The second uncalcified fibrocartilage layer is composed of fibrochondrocytes. The beginning of the transition from tendon tissue to bone tissue is composed of type-II and type-III collagen, with a small amount of type-II and type-X collagen, protein polysaccharides and core proteins. In the third calcified fibrocartilage layer, an evident transition to bone tissue can be observed, which is primarily composed of type-II collagen fibres, and it has a large amount of type-X collagen and aggrecan. The fourth layer of bone tissue is primarily composed of bone tissue, and it has relatively high mineral content. The type-I collagen matrix contains osteoblasts, bone cells and osteoclasts [7]. The four-layer structure is continuous, and it has evident gradient changes and a ‘tide line’ separation between the calcified and non-calcified fibrocartilage areas [11,12]. Thus, its mechanical force characteristics are gradually changing, from hard bone tissue to soft tendon tissue, to avoid stress concentration during the transmission of force [13]. The four zones at the TBJ are shown in Figure 1.

Anterior cruciate ligament reconstruction and rotator cuff repair are the common tendon-bone repair operations to reduce pain and functional limitations caused by TBJ damage [14]. Insertions formed by endochondral ossification include the use of anchors, interface screws, cortical suspension devices and cross pins to directly fix tendons or ligaments to bones [15,16,17]. The site is regenerated through fibrovascular scar tissue rather than graded fibrocartilage, accompanied by fatty infiltration. Thus, mechanical properties cannot reach a normal level. Poor healing after ACL reconstruction may cause the graft to slip and then increase joint laxity, resulting in a graft failure rate ranging from 3% to 25% [18]. After rotator cuff reconstruction, the normal tendon-bone interface tissue structure is also well reconstructed. The cartilage layer where the tendon is inserted into the bone cannot be regenerated, and about 20%–94% of the repair of rotator cuff tears fails during scar healing between the tendons and bones [19]. Harryman et al. [20] followed up 105 patients undergoing rotator cuff tendon repair surgery. Amongst the patients who only underwent supraspinatus repair surgery, 20% experienced postoperative re-tear, and the infraspinatus and supraspinatus muscles were repaired. A total of 43% of patients undergoing surgery experienced full-thickness re-tear postoperatively, and 68% of patients who underwent three tendon repair surgeries had full-thickness re-tear postoperatively. Therefore, the poor healing of the TBJ primarily causes unsatisfactory treatment results [21].

The healing of the TBJ is divided into three stages. In the inflammatory reaction stage, a blood clot forms and releases growth factors in the injured area, whereupon inflammatory cells accumulate. Inflammatory cells (macrophages and neutrophils) accompany tendon-derived cells to release mediators that initiate repair. The cell proliferation stage includes angiogenesis, proliferation, fibroblast-driven activities, and collagen and extracellular matrix deposition [22]. The third stage includes reconstruction: during reconstruction, the blood vessels and cells in the tissue are reduced, and collagen is arranged in order. The healing between soft tissue and bone is completed by the re-establishment of the collagen bridge between them. The reconstructed interface completes osseointegration through the gradual ossification of the extracellular matrix [23,24]. Inflammation is widely involved in the regeneration and repair of various tissues and organs in the body, which is necessary for tissue repair. However, excessive and continuous inflammation can result in the formation of a large amount of scar tissue at the tendon-bone interface instead of the normal typical four-layer structure. Studies in animal models have shown that the healing of soft tissue to bone is due to the formation of fibrovascular scar tissue, rather than the regeneration of a gradient structure containing fibrocartilage. Killian et al. [25] used the rat rotator cuff healing model to analyse biological strength after natural healing. The results showed that although the TBJ reached healing at 8 weeks after the operation, the structure reached two-thirds of the normal structure, and biological strength had an order of magnitude lower than that of the control group [9,26]. Tomita et al. [27] used a flexor tendon autograft for ACL reconstruction in a dog model and found that no fibrocartilage formation occurred between the graft and bone tunnel for up to 12 weeks, resulting in a final strength of 42% of the normal ACL. In a similar study, Newsham-West et al., conducted a long-term healing study of the TBJ after rebuilding the patellar tendon in the sheep model. After the second year, the tendon and bone were observed with the naked eye. The organisation conducted fusion without boundaries. Under microscopic observation, no layer of fibrocartilage tissue was observed, and the cells in this area were round fibroblasts. Therefore, we hypothesised that the current surgical methods cannot form the four areas of natural fibrocartilage attachment points, which leads to reduced mechanical performance and increased risk of re-injury [28,29]. Thus, the hope is that the tendon-bone will heal to its original four-layer structure and return to normal mechanical strength after repair. Compared with simple bone or tendon healing, the tendon-bone interface requires two materials of different properties to heal each other. The process is more complicated and affected by many factors, such as different graft materials and surgical procedures, the choice of formula, and the type of internal fixation. However, many mechanisms have not been clarified, and further investigation of factors such as inflammation, fibrocartilage or bone tissue formation, capillary growth, and other factors involved in the healing of transplanted tendons and bone canals is necessary. At present, many scholars have focused on these factors and tried to improve tendon healing through them, and certain progress has been made. In recent years, studies on the use of stem cell therapy, cytokine therapy, exosomes and biomaterials to promote tendon bone healing have been reviewed.

2. Stem Cell Therapy

Stem cells (SCs) can be found in the mesenchyme and connective tissues of organs all over the body. They are derived from the mesoderm, and they have high multidirectional differentiation potential. They can be differentiated into a variety of mesoderm sources under specific conditions. Mesenchymal SCs are a common type of SC therapy. The International Society for Cellular Therapy has defined mesenchymal stem cells (MSCs) as cells that: (1) adhere to plastic in vitro cell cultures; (2) have a certain surface marker profile (CD105+, CD73+, CD90+ and CD45−; CD34−, CD14−, CD11b−, CD79−, CD19− and HLA-DR−); and (3) have a trilineage ability to differentiate into osteoblasts, adipocytes and chondroblasts [30]. Apart from their ability to regenerate mesenchymal tissues, SCs have a wide range of immunomodulatory properties [31]. The earliest mesenchymal SCs were isolated from the bone marrow and then extracted from adipose tissue, tendons, dental pulp, muscle, synovium and other tissues to obtain mesenchymal SCs. Animal transplantation studies have confirmed that mesenchymal SCs can enhance tissue repair and promote the regeneration of bone, articular cartilage, intervertebral discs and tendons. Various sources of mesenchymal SCs and cartilage-related cells can promote the formation of fibrocartilage at the tendon-bone interface, thereby improving the biomechanical strength of tendon-bone joints. Stem cells can be differentiated into multiple types under stimulation by endogenous and exogenous factors. Stem cell technology has been widely used in orthopaedic disease research of tissues, including tendons, cartilage and bones, amongst which BMSCs are the most used type of SCs [32]. Therefore, some scholars have tried to use SC technology to enhance cartilage regeneration at the tendon-bone interface after rotator cuff repair and form a normal tendon-bone connection structure. Shi, Z et al. [33] compared the scar-like healing of rats in the fibrin and control groups and found that BMSC-EVs promote tendon healing by suppressing inflammation and apoptotic cell accumulation and increasing the proportion of tendon-resident SCs. Nourissat et al. [34] found that MSC-injected rats showed a uniform type-II collagen distribution at the bone-tendon junction and chondrocyte columns 45 days after surgery (Figure 2).

(Figure 2A) Native enthesis contained chondrocyte columns (arrows) at the bone-tendon junction, and positive collagen II immunostaining was observed at the junction. (Figure 2B) Control bone-tendon junction 45 days after surgery showing the absence of cells and type-II collagen. By contrast, the bone-tendon junction of repairs with cell injection was intensely stained for collagen II (Figure 2C,D). However, the type-II collagen distribution was not uniform, and no chondrocyte columns were observed in chondrocyte-injected rats (Figure 2C), whereas the repair of MSC-injected rats 45 days after surgery showed a uniform type-II collagen distribution at the bone-tendon junction and chondrocyte columns (arrows) (Figure 2D). Scale = 200 μm. T, tendon; Bo, bone. Dotted lines delimitate bone and tendon.

In addition, Tan, Z. et al. [35] found that tendon-derived stem cells (TDSCs) exhibit a higher population compared with BMSCs, indicating that more primitive SCs can be recruited in TDSC culture, and the proliferation rate of TDSCs is also faster than that of BMSCs. Bi et al. [36] compared TDSCs with BMSCs and found that TDSCs expressed higher levels of tenomodulin (Tnmd) and tendon extracellular matrix components (Col1A1, Col1A1/Cl3A1 ratio and decorin [Dcn]), which indicate that tendon SCs may be a good cell source for tendon repair. Moreover, adipose-derived stem cells (ASC) promoted tendon injury. The horse model has been used to study the regenerative potential of ASCs in the treatment of this trauma [37,38,39,40]. Some studies have shown that a single injection of ASCs can promote the organisation of collagen fibres, reduce inflammation in injured tissues, stimulate neovascularisation and limit the risk of tendinopathy progression in horses receiving treatment. Meanwhile, the application of ASCs reduces the migration and proliferation of inflammatory cells, promotes the expression of anti-inflammatory cells and downregulates the synthesis of pro-inflammatory cytokines [41,42]. Based on observations in the rotator cuff repair model, the topical application of ASCs may reduce inflammation in the tissue, increase bone mineral density and improve its biomechanical function [43,44,45].

Table 1. A search of the literature, performed as described previously, allowed us to retrieve 11 studies on stem cells for tendon repair.

Objective of the Study	Author	Tendon	Type of Study	Model
BMSCs	Shi et al. [32]	Achilles tendon	In vivo	Mouse
BMSCs	Shi et al. [33]	Patellar tendon	In vivo	Mouse
TDSCs	Tan et al. [35]	Patellar tendon	In vitro	Rat
TSPCs	Bi et al. [36]	Patellar tendon and human hamstring tendon	In vivo and in vitro	Human and Mouse
AT-MSCs	Geburek et al. [37]	Superficial digital flexor tendon	In vivo	Horse
adMSC	Carvalho et al. [38]	Superficial digital flexor tendon	In vivo	Horse
adMSC	Ahrberg et al. [39]	Superficial digital flexor tendon	In vivo	House
BM-MSCs and AT-MSCs	Romero et al. [40]	Superficial digital flexor tendon	In vivo	House
ASCs	Mora et al. [43]	Supraspinatus tendon	In vivo	Rat
ASCs	Kaizawa et al. [44]	Supraspinatus tendon	In vivo	Rat
ADSCs	Rothrauff et al. [45]	Supraspinatus and infraspinatus tendons	In vivo	Rat

BMSCs: bone marrow stromal cells. TDSCs: tendon-derived stem cells. TSPCs: tendon stem/progenitor cells. AT-MSCs: adipose tissue-derived mesenchymal stromal cells. adMSC: mesenchymal stem cells derived from adipose tissue. BM-MSCs: bone marrow-derived mesenchymal stromal cells. ASCs: adipose tissue-derived stem cells. ADSCs: adipose-derived stem cells.

shows various SCs on TBJ healing.

3. Cytokine Therapy

Cytokines are small molecular proteins with biological activity secreted by immune cells and certain non-immune cells (such as endothelial cells and epidermal cells), which regulate cell proliferation and differentiation, as well as affecting growth and development. A number of studies have shown that cytokines can promote tissue regeneration and repair. In tendon-bone healing, studies have shown that transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), bone morphogenetic protein-2 (BMP-2) and other activity substances can promote tendon-bone healing by promoting osteogenesis, cartilage or angiogenesis [46,47,48,49,50,51].

Wang et al. [47] found that BMSCs could promote TGF-β expression by injecting them into the bone tunnel and transplanted tendon cavity after ACL reconstruction in a rabbit model, thereby promoting the proliferation of fibroblasts, chondrocyte-like cells, fibrochondrocytes and collagen fibres; tendon-bone healing was thus accelerated and their biomechanical properties improved. Setiawati and his colleagues found that BMSCs and VEGF injected into a rabbit model after ACL reconstruction can promote early healing of tendon grafts and increase reconstructed ligament type-III collagen fibres, and they have shown good biomechanical analysis results [49]. PDGF is produced shortly after tendon injury to stimulate the production of other growth factors (including IGF-I), and it plays a role in tissue remodelling. It helps the proliferation and migration of fibroblasts, thereby increasing collagen production [50]. However, cytokines are unstable, and they require low-temperature storage, which limits their application to a certain extent.

In the past decade, researchers have become interested in using biologically active adjuvants to treat sports injuries. Platelet-rich plasma (PRP) is a common therapy, which is an autologous blood product that is collected from peripheral venous blood and centrifuged to remove red blood cells, leaving behind concentrated platelets and plasma [50]. PRP is rich in many growth factors, including but not limited to PDGF, VEGF, TGF-β, EGF, FGF and IGF. In the past 20 years, many basic science and in vivo animal studies have supported the use of PRP in various orthopaedic diseases. In clinical settings, many isolated studies have shown the beneficial effects of PRP. Although the evidence for PRP continues to evolve, no clear, unbiased, high-level clinical research has provided clear evidence for the efficacy of PRP therapy. This problem is multifaceted. Firstly, the pathogenesis of human tendinopathy may be different from artificial tendinopathy induced under highly simulated conditions or Achilles tendon rupture in rats or rabbits. Secondly, PRP preparations are not standardised. Third, human growth factors and animal PRP compounds may be different [52]. At present, PRP has been used for various tendon and ligament injuries, such as rotator cuff tendinopathy, patellar tendinopathy, Achilles tendinopathy, lateral epicondylitis, partial tear of the ulnar collateral ligament and partial tear of ACL [53].

Chen et al. [54] found that injecting PRP into patients with ACL reconstruction could promote ligament repair and improve knee function. Figure 3 shows a schematic diagram of the experimental operation. Recent studies have shown that certain PRP preparations have a positive effect on the healing of injured skeletal muscles, particularly those with enhanced anti-fibrotic healing ability [55]. Kia et al., recently conducted a randomised controlled trial on the use of PRP to increase ACL reconstruction of autogenous patellar tendon grafts [56]. Despite the lack of high-level clinical evidence, PRP remains a viable conservative treatment, and the risk of complications or adverse reactions is low. The main disadvantage of clinical use is the patient’s out-of-pocket expenses because PRP is rarely covered by insurance. The summary of clinical studies that use cytokines for tendon repair is shown in

Table 2. Summary of clinical studies using cytokines for tendon repair.

Objective of the Study	Author	Tendon	Type of Study	Model
TGF-β	Wang et al. [47]	Anterior cruciate ligament	In vivo	Rabbit
VEGF	Setiawati et al. [49]	Anterior cruciate ligament	In vivo	Rabbit
PDGF	Petersen et al. [50]	Achilles tendons	In vivo	Rat
PRP	Abate et al. [53]	Supraspinatus tendon	In vivo	Human

TGF-β: transforming growth factor-β. VEGF: vascular endothelial growth factor. PDGF: platelet-derived growth factor. PRP: platelet-rich plasma.

.

4. Exosomes

Exosomes (Exos) are an important part of the extracellular microenvironment. They are released into the surrounding body fluid after the fusion of multivesicular bodies and plasma membrane. These types of extracellular vesicles are approximately 30–100 nm in diameter [57,58]. After exosomes are recognised by recipient cells, the exosomes can regulate the biological functions of recipient cells by carrying information molecules. Intercellular communication mediated by exosomes is primarily carried out in the following three ways. Firstly, exosomal membrane proteins can bind to receptor proteins on the target cell membrane, and then activate the signalling pathways in the target cells to affect the biology of the recipient cells. Secondly, the exosomal membrane can directly fuse with the target cell membrane, non-selectively releasing its protein, mRNA and microRNA (microRNA). Thirdly, exosomes can deliver activated receptors to target cells [59]. Studies have shown that exosomes have a variety of biological functions, such as inhibiting cell apoptosis, immune regulation, promoting collagen and angiogenesis, and they can be used in the repair and regeneration of tissue damage [60]. Xu et al. [61] found that exosomes derived from tendon cells can induce MSCs to be differentiated into tendon cells in a TGF-β-dependent manner. MSCs have powerful paracrine, anti-inflammatory, immunomodulatory and angiogenic potential MSCs, which can achieve many regulatory functions through exosomes [62]. Lange-Consiglio et al., found that exosomes secreted by equine amniotic mesenchymal SCs can regulate the expression of pro-inflammatory cytokines by downregulating the expression of matrix metalloproteinases and tumour necrosis factor-α, which promotes tendon healing [63]. Zhang et al., found that TSC-Exos can promote the proliferation and migration of tendon cells via the PI3K/Akt and MAPK/ERK1/2 pathways; they can inhibit inflammation, apoptotic cell aggregation and scar formation, and improve high-quality healing of tendon injuries. Therefore, TSC-Exos have a potential clinical application in tendon injury repair [64].

Macrophages will be polarised into two functional phenotypes, namely, the classically activated pro-inflammatory (M1) phenotype or the alternately activated anti-inflammatory (M2) phenotype, based on different microenvironmental signals [65,66]. M1-type macrophages participate in the positive immune response by secreting pro-inflammatory cytokines and chemokines, whereas M2-type macrophages downregulate the immune response by secreting inhibitory cytokines, which have anti-inflammatory properties in immune regulation. Based on the study of BMSC-Exos in tendon-bone healing, Huag et al., found that BMSC-Exos inhibited the polarisation of M2 macrophages, and secreted pro-inflammatory factors by regulating the angiogenic signalling pathway, thereby promoting the proliferation and migration of vascular endothelial cells and forming new blood vessels. BMSC-Exos can induce angiogenesis around the rotator cuff and promote healing after rotator cuff reconstruction in rats [66]. Wang et al., injected ASC-Exos in the chronic rotator cuff tear model and found that such exosomes can hinder fatty infiltration and promote tendon-bone healing with improved biomechanical properties, thereby indicating that an injection of ASC-Exos can be used as a cell-free adjuvant therapy to inhibit fat penetration and promote rotator cuff healing [67]. The combined application of BMSCs-Exos and hydrogel also promotes tendon-bone healing. Shi et al., found that hydrogel combined with BMSC-Exos inhibited group M1-type macrophages and related pro-inflammatory factors, reducing the apoptosis rate [32]. During tendon-bone healing, the topical application of exosomes can induce the polarisation of M2 macrophages, improve the microenvironment, promote the regeneration of fibrocartilage at the tendon-bone interface and improve the biological performance. Therefore, exosomes have excellent circulation stability, biocompatibility, low immunogenicity and toxicity, and they are an effective therapeutic drug delivery system. However, only few clinical studies have been conducted on the treatment of tendon-bone healing using exosomes at present (

Table 3. Summary of studies using exosomes for tendon repair.

Objective of the Study	Author	Tendon	Type of Study	Model
Tenocyte-derived exosomes	Xu et al. [61]	Achilles tendon	In vitro	Rat
Bone marrow mesenchymal stem cell-derived exosomes	Huang et al. [62]	Supraspinatus tendon	In vivo	Rat
Tendon stem cell-derived exosomes	Zhang et al. [64]	Achilles tendon	In vivo	Rat
Adipose stem cell-derived exosomes	Wang et al. [67]	Supraspinatus tendon	In vivo	Rabbit
Bone marrow stromal cell-derived exosomes	Shi et al. [32]	Achilles tendon	In vivo	Mouse

).

5. Scaffolds Based on Biomaterial Coating

The scaffold for tissue engineering should have good biocompatibility, biodegradability, three-dimensional structure, plasticity and considerable mechanical strength. In addition, the scaffold should have a good microenvironment for seed cell adhesion and reproduction. The seed cells on the scaffold can be differentiated into different types of cells, in which the hierarchical structure of the natural tendon will be built [68]. The main materials used as scaffolds for tissue engineering include poly(ε-caprolactone) (PCL), poly-L lactic acid, polyglycolic acid (PGA), polylactic acid-glycolide (PLGA), calcium phosphate ceramics (hydroxyapatite [HA], β-calcium triphosphate and bioactive glass [primarily 45S5 bioglass based on silicate]) and decellularised tendon scaffold (DTS). Differentiated stem cells are collected from the human body, cultured, proliferated and adhered to the designed scaffold. The structural characteristics of the scaffold are used to induce and differentiate them into tissues similar to the TBJ, which are implanted in the site to be repaired. Finally, such tissues form the ideal tendon-bone tissue (Figure 4) [69]. The summary of clinical studies using biomaterial scaffolds for tendon repair is shown in

Table 4. Summary of clinical studies using biomaterial scaffolds for tendon repair.

Objective of the Study	Author	Tendon	Type of Study	Model
PCL and CS/HA	Han et al. [70]	Anterior cruciate ligament	In vitro	Rabbit
Decellularised tendon scaffold	Schulze-Tanzil et al. [71]	Achilles tendon	In vitro	Rabbit
Decellularised tendon scaffold	de Lima Santos et al. [72]	Gastrocnemius muscle tendons	In vitro	Rabbit
Decellularised tendon scaffold	Liu et al. [73]	Infraspinatus tendons	In vitro	Rabbit
Decellularised tendon scaffold	Zhou et al. [74]	Patella-patellar tendon	In vitro	Rabbit
Hydrogels (chitosan/gelatin/β-glycerol phosphate)	Huang et al. [75]	Anterior cruciate ligament	In vitro	Rabbit
Hydrogels (an injectable, thermoresponsive, type-I collagen-rich, decellularised human tendon-derived)	Kaizawa et al. [76]	Supraspinatus tendon	In vitro	Rabbit

PCL: polycaprolactone; CS/HA: chitosan/hyaluronic acid.

.

5.1. Polymer Materials

As a synthetic material, polycaprolactone (PCL) can also be used as a scaffold to repair tendon and bone defects. PCL and polylactic acid (PLA) are synthetic biodegradable polymers that are non-toxic, rich in sources and biocompatible [69,77,78]. PCL is a thermoplastic crystalline polyester obtained by ring-opening polymerisation of caprolactone with diol as initiator. It contains many methyl groups, making it hydrophobic. Jiao et al., found that the use of hydroxyphosphate lime (HA) and PCL as raw materials for melt blending to make tissue-engineering scaffolds can form a three-dimensional porous and high-porosity structure, which can promote cell adhesion, proliferation and nutrient transport [79]. It has a great potential application in soft tissue engineering. In addition, the PCL scaffold can be loaded with biological interventions such as BMP-2 and SDF-1α to form fibrous scar tissue and a new bone for tendon-bone healing [70].

PLA and PGA have three main structural forms (fibre scaffold, porous foam and tubular structure). The degradation products of PLA and PGA are lactic acid and glycolic acid, respectively, which are intermediate metabolites of the tricarboxylic acid cycle. They have good biodegradability and compatibility, which can cause low inflammation, immune response and cytotoxicity.

The polymer matrix is composed of synthetic biodegradable aliphatic polyesters reinforced with nano-hydroxyapatite (nHA), or graphene oxide (GO) nanofillers for bone tissue engineering applications. Considering that nHA or GO can enhance their biological activity and biocompatibility by promoting biomineralisation, bone cell adhesion, proliferation and differentiation, these bio-nanocomposites have been explored for the manufacture of 3D porous scaffolds for bone defect repair, thereby promoting the formation of new bone tissue after implantation. The mechanical strength of aliphatic polyester scaffolds can be greatly improved with the incorporation of nHA or GO, particularly the hybrid GO/nHA nanofiller. Nanocomposite scaffolds with high mechanical strength can support and enhance cell attachment to promote tissue growth. Porous scaffolds made from traditional porogen leaching and thermally-induced phase separation have many shortcomings, resulting in the use of organic solvents, poor control of pore shape and pore interconnectivity, whereas electrospinning pads can limit cell infiltration and tissue grown in small holes [80]. In addition, John M Moran et al., coated the nonwoven mesh of PGA with PLA and produced PLA composites containing different proportions through solvent evaporation. They found that PGA/PLA composites can increase the adhesion and proliferation of bovine articular cartilage cells [81]. Cao, Y et al., repaired a 4 cm tendon defect with autologous tendon cells combined with PGA and biofilm [82]. The implanted tissue-engineered tendon is similar to a normal tendon only in general morphology and histology, and its biomechanical performance is 83% higher than that of a normal tendon.

PLGA has excellent biocompatibility and adjustable mechanical and degradation properties. It is a copolymer of PLA and PGA, which is commonly used in tendon tissue engineering. PLGA has good biocompatibility, and it can induce the upregulation of certain genes. The degradation rate can also be controlled by changing the ratio of PLA to PGA, combining the high degradation rate of PGA and the high strength of PLA. Therefore, PLGA can also be used as a cell scaffold for artificial tendons [81]. Guo, J et al., used electron beam evaporation to coat a new bioactive calcium phosphate silicate (CPS) ceramic on the surface of the PLGA membrane, to prepare a customisable composite with a layered chemical composition similar to the tendon-bone interface membrane [83]. They found that CPS-modified samples could promote the attachment and proliferation of rBMSCs and NIH3T3 cells and improve osteogenic activity. Therefore, the CPS-modified PLGA film has great potential application for tendon-bone healing. However, the biological safety and biological activity of these stents made of polymer materials remain unclear, which partially limits their application.

These materials have good cytocompatibility, standardised and regulated production and degradability, but sometimes local inflammation of materials will affect the treatment, and the effect is not ideal [84]. Given the inherent hydrophobicity of most synthetic polymers, the adhesion of cells in the scaffold is reduced, which is not conducive to cell differentiation and the formation of new tissues [85]. In addressing this problem, physical or chemical modifications may be performed to the surface of polymer molecules to change their hydrophobicity, which can enhance the adhesion of cells after being fabricated into bioscaffolds. We look for new biomaterials and combine the excellent properties of two or more biomaterials to synthesize optimal bioscaffolds. The corrosion resistance and biocompatibility of titanium alloy materials can even be used to create bioscaffolds [86].

5.2. Hydrogels

Hydrogel is a three-dimensional network formed by cross-linking water-soluble polymers. It has good biocompatibility, and it can absorb a large amount of water. Hydrogel has been widely studied as a scaffold material for tissue engineering, and it is considered as an ideal articular cartilage repair material [87,88,89].

Hydrogel can better simulate the extracellular matrix environment of cartilage cells, and its high water content is conducive to the entry of nutrients and the discharge of cell metabolic wastes, which facilitates the repair of cartilage. In addition, this high water content environment can maintain the natural form of chondrocytes, and help chondrocytes to secrete the cartilage extracellular matrix [90]. Based on different sources of raw materials, hydrogels can be divided into three categories: natural polymer hydrogels, synthetic hydrogels and composite hydrogels. Some common hydrogels are composed of collagen, gelatin, fibrin, chitosan (CS), alginate, hyaluronic acid (HA), polyethylene glycol diacrylate (PEGDA), methacryloyloxy preparation of ethyl phosphocholine (MPC) polymer and polyethylene glycol [90]. Huang added the gelatin molecule to heat-sensitive chitosan/β-glycerophosphate disodium salt gel to form a chitosan/gelatin/β-glycerophosphate (C/G/GP) hydrogel, which was injected into the TBJ of a rabbit model. The results showed that the strength of the tendon-bone healing in the rabbit model increased by 66% after injection [75]. Wang et al., found that injecting BMP-2/type-I collagen hydrogel into the tibial tunnel of a rabbit ACL reconstruct model can lead to the formation of the fibre-cartilage-bone tissue at the tendon-bone interface, and the fibrous and cartilage tissues are tight. Kaizawa et al., found that the injection of acellular human tendon-derived hydrogel (tHG) rich in type-I collagen at the RTC repair site can enhance the biomechanical properties of the tendon and the formation of fibrocartilage at the TBJ [76].

The hydrogel material can well simulate the extracellular matrix environment and provide growth space for seed cells. In the future, we can load other drugs on the hydrogel to achieve better anti-inflammatory and anti-extracellular matrix degradation effects.

5.3. Decellularised Tendon Scaffold

Decellularised tendon scaffold is a tissue engineering material, in which all cells in tissues were removed with chemical, mechanical or perfusion methods, and only the extracellular matrix and its three-dimensional structure were retained [71,89,91,92]. Considering that most of the cells are removed and immune rejection is greatly reduced, it is more biocompatible than artificial tissues or organs. The ideal decellularised tendon bio-scaffold not only has a good three-dimensional structure, but also has a cell. Certain anti-degradation ability, biomechanics and good biocompatibility can maintain the original shape after transplanting into the body. The scaffold can repair and reconstruct damaged tissue and promote the growth and proliferation of seed cells, and it can provide sufficient space support and mechanical support. The decellularised tendon has been proven to be a good source of alternative biomaterials for tissue engineering [93,94].

DTS materials usually contain cell-removed natural tendon ECM, and such materials can promote better repair results. The retained tendon ECM shows striking similarities to a natural tendon with regard to its bioactive components, collagen arrangement and biomechanical characteristics. In addition, DTSs retain many active growth factors, and they have good biocompatibility, which can promote cell growth and differentiation [72]. Therefore, DTS has a similar ultimate tensile load, high porosity and disorganised core material to normal tendons, and it shows tissue integration in the rotator cuff model [95,96]. Guo, M et al., bridged the extracellular matrix scaffold of multi-layer decellularised tendon slices to the defect of the rotator cuff in a rabbit model. They found that the graft promoted the ingrowth of host cells and enhanced the weight of the regenerated tendon. Plastic promotes the formation of fibrocartilage, thereby improving the biomechanical properties of repairing tendons [73]. Zhou, Y et al., cultured bone marrow mesenchymal stem cell sheets (MSCS) on acellular tendon scaffolds (ATS) and implanted them into a rabbit’s patella defect. They found that regeneration was formed at the rabbit’s patella-patella tendon interface, bone and fibrocartilage. Moreover, the cell tendon scaffold has high resistance strength [74]. Some scholars have studied the effect of a BMP-decellularised tendon complex on adhesion after allogeneic tendon transplantation. However, decellularised scaffolds derived from dense tissues have low porosity; thus, they are inconvenient for cell infiltration, which partially limits their application [97,98]. Woon, C et al., also reported that peracetic acid increases the porosity of the scaffold and improves cell penetration and migration [99]. Whitlock and his colleagues observed that peracetic acid does not damage the tensile properties of the scaffold, and it neither makes the scaffold cytotoxic nor causes inflammatory reactions in vitro [100].

The possibility of DTSs has been previously demonstrated, and the decellularised extracellular matrix can replant cells. However, process methods have disadvantages such as the time-consuming, unclean removal of cell components and incomplete biological scaffolds. Therefore, the preparation methods of decellularised scaffolds need further research.

5.4. Composite Material

Natural materials such as collagen have good biocompatibility, but they also have defects such as poor mechanical properties, fast degradation and poor processing properties. Composite materials and other synthetic materials have defects such as a low degradation rate, and acid degradation products can cause inflammation and poor mechanical properties. Therefore, two or more biological materials with complementary characteristics can be combined in a certain ratio to construct a biological material that meets the requirements [68]. Using collagen and HA, the collagen matrix is calcified by calcium phosphate impregnation to precipitate HA, which simulates the four-layer structure of a natural tendon and bone. The physical and chemical structure formed by the scaffold and the mechanical environment are suitable for the adhesion and proliferation of human fibroblasts, chondrocytes and osteoblasts to each corresponding matrix. The research team of Li and Erisken obtained similar results when analysing the mechanical properties of nonwoven mats of electrospun poly(ε-caprolactone) nanofibers covered with gradient β calcium triphosphate [101,102]. However, regarding tensile stress deformation, the Young’s modulus of the stent gradually increases with the increase of the calcification content, but the elongation ability decreases. In addition, cell adhesion and migration analysis indicate that MC3T3 pre-osteoblasts have different adhesion behaviour and morphologies along the scaffold. Graphene is an inorganic material with electrical conductivity. Some studies have shown that it is not toxic to cells, and it has a biocompatible biological surface that can enhance cell adhesion and proliferation because of its electrical conductivity.

If graphene is fully incorporated, then it can significantly improve the physical properties of composites even at small loads, which has been shown to increase the probability of TBJ healing [103]. In the future, more multiple chemical gradient embeddings will be developed in the scaffold design.

6. Conclusions

Mao et al. [68], Q et al. [89] and Xu et al. [91] have published reviews related to coatings in tendon healing in recent years. They focus on polymers, biogenic materials, functionally graded scaffolds and stem cell therapy, respectively. There is a lot of research to support the effectiveness of these methods. Stem cell therapy, cytokine therapy and exosome therapy have been studied to promote tendon-bone healing, but these methods cannot form the complex structure of the TBJ. Although scaffolds based on biomaterial coating can form similar natural structures, all these studies were performed on animal models, and there was not enough clinical evidence to show that the methods worked. Moreover, no breakthrough, effective treatment methods for the biomechanical properties have been reported after healing. In addition, existing research strategies are still far from clinical application and clinical requirements, because the structure of the TBJ is complex, and healing after injury takes place between two different tissues (soft and hard), making it difficult to study. Moreover, the mechanism of healing remains unclear. Therefore, future research must start with exploring the most basic healing mechanisms, including the causes of scar tissue formation, the source of scar cells, the causes of restricted fibrocartilage regeneration, the regulation mechanism of SC differentiation at the healing site and the inflammatory microenvironment. Furthermore, studying the mechanism of mechanical stimulation affecting healing is important to explore effective treatment methods.

Finding a composite material that is resistant to degradation, wear and immune rejection in the human body is a future challenge. We also need to investigate more precision and controllability methods to generate gradients at appropriate length scales. Such materials would adapt to different environments based on the gradient changes of their own properties and functions, and achieve particular functions. At present, most of the clinical experiments are carried out on animals, and further research on humans is needed in the future.