Current progress in growth factors and extracellular vesicles in tendon healing

Abstract

Tendon injury healing is a complex process that involves the participation of a significant number of molecules and cells, including growth factors molecules in a key role. Numerous studies have demonstrated the function of growth factors in tendon healing, and the recent emergence of EV has also provided a new visual field for promoting tendon healing. This review examines the tendon structure, growth, and development, as well as the physiological process of its healing after injury. The review assesses the role of six substances in tendon healing: insulin‐like growth factor‐I (IGF‐I), transforming growth factor β (TGFβ), vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and EV. Different growth factors are active at various stages of healing and exhibit separate physiological activities. IGF‐1 is expressed immediately after injury and stimulates the mitosis of various cells while suppressing the response to inflammation. VEGF, which is also active immediately after injury, accelerates local metabolism by promoting vascular network formation and positively impacts the activities of other growth factors. However, VEGF's protracted action could be harmful to tendon healing. PDGF, the earliest discovered cytokine to influence tendon healing, has a powerful cell chemotaxis and promotes cell proliferation, but it can equally accelerate the response to inflammation and relieve local adhesions. Also useful for relieving tendon adhesion is TGF‐ β, which is active almost during the entire phase of tendon healing. As a powerful active substance, in addition to its participation in the field of cardiovascular and cerebrovascular vessels, tumour and chronic wounds, TGF‐ β reportedly plays a role in promoting cell proliferation, activating growth factors, and inhibiting inflammatory response during tendon healing.

1. INTRODUCTION

The tendon is a highly organised structure that allows the transmission of strength between muscles and bones. With the economic development，Tendon injury is one of the most prevalent disorders, affecting one in 2700 people each year in America. ¹ Tendon injuries, in addition to the significant financial burden associated with handling them, have a deleterious effect on patients' quality of life. Knowing how to treat patients with tendon injuries effectively, reducing tendon adhesions, enable patients to exercise as early as possible, reducing disability and secondary surgical injury caused by tendon adhesions and joint stiffness is key to facilitating postoperative healing and to restoring tendon function to the greatest extent feasible. More research on the mechanism of tendon healing is required to enhance patient treatment experience and achieve better therapeutic results.

2. STRUCTURE OF TENDON

2.1. Tendon cells

Mature tendons are usually characterised by low cellular density. Reportedly, the low metabolic rate of mature tendons reduces the risk of ischaemia and necrosis upon an increase in the local tension of the tendon. However, this feature is a disadvantage for tendon recovery and healing (Table 1). ²

TABLE 1.

Differences and connections between tennocytes and tenoblasts.

Differences	Tenocytes	Tenoblasts
	Spindle‐shaped, fibroblast‐like cells with elongated nuclei and thin cytoplasm	Round cells with larger ovoid nuclei
	Lower apoptosis index	Higher apoptosis index
	Limited proliferative capacity	Higher proliferative capacity
	Easy accumulation	Difficult accumulation
	Scattered evenly on the collagen fibres in tendons	Clusters are distributed on the collagen fibres
	Mainly distributed on mature and aging tendons	Mainly distributed on young tendons
Connections	Tenoblasts are precursor cells for tenocyte differentiation. Tenoblasts transform into tenocytes during maturation and aging. Tenoblasts are generated from the activation of tenocytes in response to tendon injury.

It is generally believed that the main resident cells of the tendon are fibroblast‐like cells‐termed tenocytes. Using immunohistochemical analysis of healthy patellar tendons from 14 individuals, the researchers further subdivided the tendon cells into tendon cells and tendon cells according to the phenotype and activity of the cells. Tenocytes and tenoblasts are the main resident cells of tendons and are located on collagen fibres in tendons, which can synthesise most of the ECM. ³ Approximately 90%–95% of tendon cells are composed of tenocytes and tenoblasts. Chuen et al.'s analysis of the immunohistochemical properties of the tendon revealed that tenoblasts often appeared in clusters with a localised pericellular region devoid of collagen fibre anchorage. In contrast to tenoblasts, tenocytes were scattered evenly on the collagen fibres in tendons. ⁴ In terms of cell proliferation capacity, tenoblasts are traditionally regarded as having a stronger proliferative capacity. In the experiment by Chuen et al, it is proved that tenoblasts had a stronger proliferative capacity, which is consistent with the traditional view. ⁵

Benjamin et al. suggested that tenocytes could actively regulate collagen synthesis in response to changes in mechanical loading. ⁶ In addition to the effects on collagen synthesis, mechanical loading influences the production of pro‐inflammatory growth factors and the gene expression of Cox‐2, pge2, MMP‐1, and other mediators. ⁷ , ⁸ Notably, tendon cells themselves can produce growth factors (eg, IL‐1β), especially if they are located near the site of tendon injury. ⁹

Tenoblasts are relatively round cells with larger ovoid nuclei of tendon cells that are sometimes regarded as an activated form of the tenocytes of tendon injuries. ¹⁰ Tenoblasts are terminally differentiated cells, which, when subjected to various stimuli, such as exercise and trauma, will transform into tenocytes to attain higher proliferation rates and matrix remodelling in response to these stimuli. ⁴ Tenoblasts are more active in matrix remodelling ⁴ ; however, because they can be converted into tenocytes under certain conditions, a debate on whether the two kinds of cells can be regarded as the same group rumbles on. For the convenience of presentation, tenocytes are used as a general term for tendon cells.

Besides tenoblasts and tenocytes, Yanming Bi et al isolated another cell population from both human and mouse tendons using clonogenicity, self‐renewal and multipotent differentiation capacities and named these cells tendon stem/progenitor cells (TSPCs). ¹¹ Examining TSPCs should help the subsequent exploration of promoting tendon healing and even reconstructing tendons. Our speculation is that the activity of stem cells in a tendon is linked to the function associated with the healing potential of tendon injuries. However, because research in this area is sparse, extracting and utilising such cells remains a limitation.

Yu et al. found that Postn, a secreted ECM protein, is involved in regulating cell–cell and cell‐matrix interactions, promoting TSPC stemness and tendon‐derived differentiation potential, and plays an important role in promoting tendon healing. ¹² , ¹³ However, due to the lack of understanding of specific regulatory factors during postnatal tendon development, long‐term in vitro culture of TSPC and functional regeneration of tendon have not yet been achieved.

2.2. Extracellular matrix of tendon (ECM)

Tendon ECM comprises collagen fibres, elastic fibres, ground substance, and inorganic components. Collagen fibres' primary role is to resist tension. In the ECM, collagen fibres are arranged orderly and can be divided further into fascicles, fibrils, subfibrils, microfibrils, and tropocollagen components. ¹⁴

Collagen fibres are the smallest structural units of collagens. Each fibril is built from soluble tropocollagen molecules forming cross‐links to create insoluble collagen molecules, which then aggregate progressively into microfibrils, fibrils and finally into fibres. Bundles of fibres are bound together by thin layers of loose connective tissues known as the epi‐ and endotenon, allowing the fibre group to slide with almost no friction; they also transport blood vessels, nerves and lymphatic vessels deep into the tendons. ⁶ The complex three‐dimensional structure endows the tendon with high tension and elasticity and prevents the damage and separation of the fibre under mechanical stress. ¹⁵

Elastic fibres have flexibility and extensibility to reduce the energy input of long‐range deformability and passive recoil. ¹⁶ This is greatly significant in protecting tissues from sports injuries. Furthermore, elastic fibres are thought to be involved in the recovery of the crimp pattern of the collagen fibres after tendon stretching. ¹⁷

Collagens in the tendons include types I, III, V, VI, XII, XIV and XV. Collagen type I is the most abundant molecule in the ECM, accounting for almost 60% of dry tissue mass and approximately 95% of total collagen. ¹⁵ Type III, which is bountiful in pathological tendons and is also produced in large amounts during tendon healing the first type of collagen, ¹⁵ is the next most plentiful collagen. ¹⁸ Because various collagen types have different properties, for example, type I collagen is relatively hard collagen and type III collagen is elastic collagen, the different proportions of collagen involved in repair will inevitably affect the mechanical properties of the tendon after repair. Finding an appropriate proportion of collagen and applying drugs or biological agents to guide the proportion could lead to accelerated patient recovery and mitigate the risk of tendon rupture.

3. TENDON INJURY AND REPAIR

Natural tendon healing is considered a recapitulation of the developmental processes of a tendon. For optimal treatment, an enhanced understanding of tendon physiology and the process of ruptured tendon healing facilitates the development of treatment after tendon injury. Tendon healing is believed to encompass three phases: inflammatory, proliferation, and remodelling phases. In general, these phases can overlap, and their duration depends on the location and severity of the disease.

Until approximately 3 to 7 days after injury, blood collects and clots at the injured site and releases pro‐inflammatory growth factors to attract neutrophils, monocytes, macrophages and other inflammatory cells to the injured site. ¹⁴ At the same time, platelets degranulate and secrete a considerable amount of growth factors and growth factors, ¹⁹ thus, activating fibroblastic cells that upregulate the production of ECM elements. ²⁰ Extrinsic cells from the peritendinous soft tissue, such as tendon sheath, fascia, periosteum, and subcutaneous tissue, and intrinsic cells from the epitenon and endotenon then migrate and proliferate in the area of tendon injury. These cellular activities are the basis of granulation during the proliferation phase. Starting from the fifth day of tendon healing, this immature tissue synthesises mainly type III collagen. ¹⁹ At this stage, the strength of the tendon is almost entirely dependent on the blood clot. Surgical sutures from surgical repair—if performed—provides most of a tendon's mechanical strength.

Proliferation occurs a few days later. Its principal features include the synthesis of abundant ECM components, proteoglycans and collagen (mostly type III collagen), accompanied by an increase in cell numbers and large water uptake. Infiltration by fibroblast‐like cells, which are thought to originate from endogenous tenocytes and/or tendon progenitor cells or extrinsically from paratenon and epitenon, dominate this phase. ²¹ These cells are responsible for the deposition of ECM in the repair tissue. ²² Proteoglycans, which account for less than 1% of the dry weight of tendons and ligaments, are a noteworthy component, but their presence contributes to proper tendon assembly and tissue. For example, decorin and biglycan are known to regulate the formation of collagen fibres. Decorin restricts the diameter of collagen fibres during fibre formation, with the aim of limiting the diameter of fibrils and changing the rate of the process. Meanwhile，Decorin may inhibit TGFβ activity by isolating growth factors, thus affecting tendon healing. ²³ In the fourth week, tenocytes that is from endotenon begin to proliferate gradually and play the most active role in tendon healing, aggressively resorbing collagen, while producing new collagen. ¹⁹ Although collagen fibres are still not oriented in parallel at this time, they provide partial tensile strength to the tendon. Up until the fifth week, the amount of collagen increases steadily, and the repair callus attains its largest size. ²⁴

The tendon tissue matures, and the fibres become more longitudinally oriented during a formation phase that lasts approximately 2 months. ¹⁹ According to one investigation, the directional arrangement of collagen not only refers to the longitudinal arrangement in the same direction but also includes a crossover between horizontal, spiral and collagen fibres, forming a complex tendon ultrastructure that provides a good buffer capacity to resist vertical, horizontal and rotational forces. ¹⁸ Still, some questions with significant implications remain unanswered. Is growth factor‐instigated collagen rearrangement involved in the restoration of the original arrangement of collagen fibres after an injury? Or is this handled solely by the longitudinal arrangement of collagen? Is this related to the decrease in mechanical properties after tendon injury repair?

The remodelling phase begins 6–8 weeks after injury and takes around 1–2 years depending on the age and condition of the patient. ² During the remodelling phase, ECM components are assembled into deposits of random tissues that reorganise longitudinally. However, despite this reorganisation, the strength of repaired tendons remains inferior to that of healthy tendons. ²⁵ In this phase, the emergence of physiological load promotes the recovery of the biomechanical strength of the tendon. ¹⁹ Under the action of an appropriate load, collagen fibres become more organised and more cross‐linked. ¹⁹ Moreover, the type III collagen produced during the formative phase is replaced by the mechanically more resistant type I collagen. ¹⁹ During the entire reconstruction phase, a prolonged exposure of tenocytes to inflammatory signals could lead to the overproduction of ECM components and excessive proliferation of tendon scarring, resulting in tendon adhesions (see Table 2). ²⁶

TABLE 2.

Summary of the healing process in tendons.

Time (day)	Phase	Process
0–7	Inflammatory stage	Blood cells accumulate, and blood clots form Pro‐inflammatory growth factors, which are released; Inflammatory cells are attracted, and fibroblasts are activated; The synthesis of the extracellular matrix begins; The migration and proliferation of the peripheral and intrinsic cells of the tendon begin; The synthesis of Collagen III collagen begins.
8–42	Multiplicative stage	More extracellular matrix, proteoglycans, and type III collagen are secreted; Tenocytes proliferate further; Water is absorbed in large amounts; the shift by Collagen from disorder to longitudinal direction begins.
42+	Remodelling stage	Extracellular matrix components are assembled into deposits of random tissues; Collagen is further arranged and cross‐linked; Type I collagen replaces Type III collagen; Cell numbers and the matrix begin to approach normal levels.

The tendon healing process is orchestrated by a variety of secreted molecules in a complex manner. Several growth factors and growth factors released by tenocytes and migrating leukocytes after tendon injury closely participate in the regulation of the repair response (Table 3). IL‐1β, transforming growth factor‐α (TGF‐α), and IL‐10 downregulate collagen I synthesis, whereas the anti‐inflammatory cytokine IL‐4 promotes the deposition of collagen I, collagen III, and fibronectin. Insulin‐like growth factor‐1 (IGF‐1) and transforming growth factor‐beta (TGF‐β), ²⁷ which are expressed in all phases of healing, especially in the inflammatory and proliferative phases, also stimulate matrix production. ²⁸ Other fundamental growth factors to tendon healing include b‐fibroblast growth factors (bFGF) and platelet‐derived growth factor (PDGF). The former rouses cell migration and angiogenesis, while the latter is involved in regulating DNA synthesis and promoting the production of other growth factors. ²⁹ , ³⁰

TABLE 3.

Summary of the roles of factors during tendon healing.

Factors	Roles
IGF‐1	Triggers the migration of functional fibroblasts.
	Inhibits local inflammation.
	Increases the mechanical strength of healing tendons.
	Collaborates with PDGF and PDGF‐BB to promote cell proliferation.
	Stimulates mitogenic responses in a variety of cells, particularly fibroblasts.
	Increases the synthesis of related proteins, especially collagen and proteoglycan synthesis.
VEGF	Initiates the formation of a vascular network.
	Promotes fibroblast proliferation.
	Promotes inflammatory cell chemotaxis.
	Initiates the production of TGF‐β.
PDGF	Promotes the chemotaxis and mitosis of inflammatory cells.
	Promotes tenocytes that enter the wound site to regenerate damaged tissues.
	Attracts tenocytes and fibroblasts that migrate to the wound site and begin synthesising extracellular matrix components.
	Stimulates tenocyte and tenoblast proliferation, collagen production, and collagen crosslinks, leading to better tissue organisation and improved biomechanical properties.
	Accelerates the inflammatory stage process in tendon healing.
	Alleviates local adhesion.
FGF	Has greater angiogenic capacity than VEGF or PDGF.
	Accelerates the response to inflammation.
	Stimulates fibroblasts to secrete type III collagen.
	Promotes the conversion of type III collagen to type I collagen at the reconstruction stage.
	Increases healing strength, but not adhesion formation.
	Combines with TGF‐β1 to amplify the synthesis of type I collagen in fibroblasts.
TGF‐β	Modulates the response to inflammation; a suppressor of excessive inflammation.
	Promotes the secretion of fibronectin and collagen by epithelial and mesenchymal cells.
	Promotes collagen accumulation and fibrotic tissue responses.
	TGF‐β3 has Antifibrotic properties.
	TGF‐β3 Promotes scar‐free healing.
	TGF‐β2 and TGF‐β3 are potent inducers of tendon progenitors.
	Upregulates tenogenic gene expression in MSCs.
	Stimulates extrinsic cell migration, regulates proteinases, enhances fibronectin binding interactions.
	High levels of TGF‐β1 have been implicated in tendon adhesion formation.
Exosome	Induces the activation of the VEGF signalling pathway and promotes angiogenesis around the injury site.
	MSC‐derived EV enhance the expression of tenomodulin and type I collagen, as well as the mechanical properties of neotendon.
	MSC‐derived EV promote the proliferation of local TSPCs in vivo.
	EV from bone marrow‐derived multipotent mesenchymal stromal cells increase the proportion of tendon‐resident stem cells.
	Inhibits the expression of pro‐inflammatory factors.
	Significantly reduces tendon adhesion.

Abbreviations: bFGF, basic fibroblast growth factor; IGF‐I, insulin‐like growth factor‐I; PDGF, platelet‐derived growth factor; TGFβ, transforming growth factor; VEGF, vascular endothelial growth factor.

Several investigations on the mechanism of growth factors during tendon injury healing have attempted to enhance tendon healing by delivering various growth factors known to play significant roles in the healing and development of tendons .

3.1. Role of growth factors in tendon healingInsulin‐like growth factor‐1 (IGF‐1)

IGF was first isolated from human serum by Lindkenett et al. in 1976 and named after its structural similarity to proinsulin. ³¹ As a powerful growth factor, IGF‐1 plays a key role in the whole process of tendon healing, including maintaining the increase in the number of tendon cells and the migration of functional fibroblasts, increasing the production of collagen and extracellular matrix at the remodelling stage, inhibiting local inflammation, increasing the mechanical strength of the healing tendon, and liaising with other growth factors to promote tendon healing.

In Chuan et al.'s research on the gene expression of several growth factors at several time points during the early healing phase in a chicken model, they found that IGF‐1 became active almost immediately after tissue injury and remained active throughout most phases of tendon healing. The expression of growth factor genes, except the bFGF gene, was upregulated on day 3, with IGF‐1 gene expression seeing the most substantial increase. ³² IGF seems to play a role during most stages of tendon healing, and this role appears to somewhat correlate with the expression of the IGF gene.

Studies have shown that during the first two weeks of tendon injury, IGF‐I is released mainly by inflammatory cells, endothelial cells, granulocytes, and macrophages, ³³ which is consistent with the findings of Chuan et al. IGF‐1 is expressed extensively in the early phase of inflammation. In an equine collagenase‐induced SDF tendinitis model, IGF‐I treatment alleviated soft tissue swelling, increased DNA and collagen synthesis, and improved the mechanical properties of a healing tendon. Linda A. Dahlgren et al. also demonstrated that IGF‐I had anti‐inflammatory properties in local use. ³⁴ Although the exact mechanism by which IGF‐1 inhibits the inflammatory response is unclear, inflammatory cells are, reportedly, not significantly reduced in wounds treated with IGF‐1. According to speculations, IGF‐1 does not act by reducing inflammatory cell migration, but rather by inhibiting the early inflammatory cascade with a negative feedback on increasing the concentration of IGF‐1. ²⁷

By week 4, IGF‐I is expressed primarily in tendon cells. ³³ Research proved that IGF‐1 has a potent synthetic metabolic effect on cells and stimulates mitogenic responses in a variety of cells, particularly fibroblasts. IGF‐1 can also inhibit cell death. During tendon injury and healing, it increases the synthesis of related proteins by promoting fibroblast proliferation, especially collagen and proteoglycan synthesis. ³⁵

As with many other growth factors, synergy with other molecules is vital to their stimulatory activity. Conceivably, IGF‐I can synergize with PDGF and PDGF‐BB to promote cell proliferation. Tsuzaki et al. demonstrated this assertion in an in vitro study in which both growth factors were most potent when applied together during mitogenesis and the subsequent cell division of tendon fibroblasts and tendon surface cells compared with their individual applications. ²⁷

At present, some researchers have tried to apply IGF‐1 to the treatment of tendon injury through local injection or hydrogel, although IGF‐1 has clearly promoted the proliferation of tendon cells in vitro. ³⁶ However, it is disappointing that IGF‐1 has not shown a very significant role in promoting tendon healing in clinical trials. We still need a large number of in vivo experiments to fully stimulate the potential of IGF‐1 in tendon healing to use it in clinical treatment.

3.2. Vascular endothelial growth factor (VEGF)

As with most tissues, angiogenesis is crucial to facilitating tendon healing via delivering oxygen and nutrients, removing waste products, and controlling immune responses. Angiogenesis is one of the earliest events to occur during tendon healing, and VEGF is one of the most critical angiogenic factors that regulate angiogenesis during tendon healing. ³⁷ VEGF‐mediated angiogenesis traverses the epithelial cell surface to the normal avascular area, bringing with it external cells, nutrients, and growth factors to the site of injury. ²⁷ As the tendon heals, oxygen consumption at the site of injury, response to inflammation, and VEGF expression all diminish. ³⁸ , ³⁹

VEGF becomes active immediately after inflammation, most notably during the proliferative and remodelling phases. ²⁷ After a tendon injury, α‐granules secreted by platelets at the site of trauma can release large amounts of growth factors, such as VEGF, TGF‐β, FGF, IGF and PDGF. ⁴⁰ In addition, inflammatory growth factors (IL‐1β, IL‐6, and IL‐8) accompany inflammatory infiltration at the injury site after acute tendon injury, promoting the upregulation of VEGF. ³⁷ This upregulation both accelerates the progression of inflammation and promotes local growth factor infiltration, stimulating angiogenesis, and combines with other growth factors to foster tendon healing. Furthermore, VEGF increases TGF‐β expression during the early stages of tendon repair ²² and furthers collagen accumulation and tendon healing; however, excess TGF‐β can also exacerbate local tissue adhesion.

VEGF may have different effects during different tendon healing periods. Studies have shown that an early postoperative application of exogenous VEGF promotes neovascularization and increases the mechanical strength of tendons. ³⁹ However, the prolonged presence of blood vessels will lead to an adverse healing microenvironment. ³⁷ This side effect could be more pronounced if high levels of VEGF persist during the remodelling phase, causing persistent pain and decreased biomechanical performance. ⁴¹ In addition, high VEGF levels culminating in high vascular inward growth have been shown to sustain inflammation and increase tissue scarring. ⁴²

During tendon healing, secreted angiogenic factors initiate the formation of a vascular network that is responsible for the survival of the newly formed fibrous tissue at the injury site. ⁴³ VEGF also boosts fibroblast proliferation, chemotaxis for macrophages and granulocytes, and initiates the production of other growth factors. ⁴⁴

The VEGF protein family includes VEGF‐A, VEGF‐B, VEGF‐C, VEGF‐D, placental growth factor (PlGF), virus‐encoded VEGF‐E and snake venom‐derived VEGF‐F. ⁴⁵ Herbert Tempfer et al.proved that VEGF‐D‐mediated signal transduction promotes tendon cell proliferation, but it is also related to tendon degeneration specimens, such as matrix destruction and cell migration. ⁴⁶

Based on the research of antiangiogenic therapy in cancer and other fields, some scholars have proposed that antiangiogenic therapy may be beneficial to tendon healing. ⁴⁷ According to the findings of Dallaudière et al., intratendinous injection of bevacizumab, a VEGF binding inhibitor, in Achilles tendon and patellar tendinopathy (induced by chemical collagenase) may accelerate tendon healing, reduce collagen fibre disorders and reduce new angiogenesis. ⁴⁸

3.3. Platelet‐derived growth factor (PDGF)

Platelet‐derived growth factors (PDGFs) were discovered more than two decades ago. ⁴⁹ They were first isolated from platelets. ¹⁹

At the beginning of the inflammatory and proliferative phases of the tendon healing process, different isomers of PDGF are released from the platelets. ⁵⁰ These factors have chemotactic and mitogenic effects on neutrophils, macrophages and phagocytes, which are responsible for the breakdown and cleaning of tissue debris, as well as tenocytes that enter the wound site to regenerate the damaged tissue. ⁴⁰ In addition, PDGF attracts tenocytes and fibroblasts that migrate to the wound site and start synthesising extracellular matrix components. ⁵¹

PDGF‐bb is one of four isoforms of the PDGF growth family (A, B, C and D). It is considered a promising candidate for tendon repair because of its ability to promote cell mitosis, chemotaxis and angiogenesis. ⁴⁰ Stavros et al revealed that PDGF‐BB stimulates the cell activity of canine flexor tendon fibroblasts in vitro. ⁵¹ They also proposed that PDGF‐BB delivered via a fibrin matrix further increased fibroblast proliferation, matrix synthesis, and matrix remodelling at the repair site. ⁵⁰ Researchers have concluded that PDGF‐bb's ability to further tenocyte and tenoblast proliferation, collagen production, and collagen crosslinks can aid the tendon healing process during the initial phase and lead to better tissue organisation and improved biomechanical properties. ⁴⁰ PDGF‐bb accelerates the inflammatory stage process in tendon healing and plays a role in alleviating local adhesion.

Unlike VEGF, PDGF not only attracts inflammatory cells at the early stage of tendon injury, decomposes local necrotic tissue fragments, and accelerates the process of inflammatory response, but it also acts as a chemo‐attractant and mitogen for fibroblasts and endothelial cells. ⁵² In addition, PDGF‐BB promotes the mitosis of tendon cells and fibroblasts at the late stage of tendon healing, as well as the synthesis of extracellular matrix components, accelerates tendon healing, and improves the mechanical properties of tendon healing, without enhancing adhesion formation at the time of sacrifice and dissection. ⁵⁰ PDGF has a longer lasting and more potent impact with fewer adverse effects than VEGF.

Although a moderate increase in collagen synthesis and repair site stiffness was demonstrated, its effectiveness may be limited because of the rapid clearance of growth factors from the wound site. ⁵³ Therefore, how PDGF‐BB is delivered to the wound site in a controlled and sustained manner is important for tendon rupture repair.

3.4. Fibroblast growth factors (FGFs)

FGFs, first identified in pituitary extracts in 1973, are peptide growth factors that are widely expressed in developmental and adult tissues. ⁵⁴ FGFs have a variety of biological functions both in vivo and in vitro, including roles in mitogenesis, cellular migration, differentiation, angiogenesis, and wound healing. ⁵⁴ Typical FGF activates the FGF receptor (FGFR) through paracrine or autocrine mechanisms, which requires cooperation with heparan sulfate proteoglycans. ⁵⁵ One study showed that particular FGFRs can direct the development of new tissues during tendon healing, particularly through an increase in the expression of bFGFR that has been implicated in fibroblast cellular proliferation. ⁵⁶

Basic fibroblast growth factor (bFGF) belongs to the FGF family and is expressed in almost every tissue. ⁵⁷ In a rabbit model of synovial tendon injury, bFGF mRNA was found to be upregulated in tendon cells and tendon sheath fibroblasts. ⁵⁸ And in Chan et al.'s investigation of the effects of a single injection of bFGF on type III collagen expression and content at the initial stages of healing in the rat patellar tendon, ⁵⁹ type III collagen expression and cell proliferation increased after 7 days of bFGF injection, suggesting that b‐FGF possibly plays a significant role in tendon repair by promoting tendon cell mitogenesis and increasing local collagen involvement. ²⁰

One of the early events in tendon healing is angiogenesis, in which neovascularization promotes the delivery of inflammatory cells and fibroblasts to the wound site. FGFs are more potent angiogenic factors than VEGF or PDGF. Therefore, FGFs can also stimulate tendon healing by boosting angiogenesis. Furthermore, bFGF enhances the production of pro‐inflammatory factors during the early phase of tendon healing, thereby accelerating the inflammatory response. Moreover, the cell proliferation phase is accompanied by the synthesis of many extracellular matrix components. bFGF speeds up tendon healing by stimulating fibroblasts to secrete type III collagen, and it can advance the conversion of type III collagen to type I collagen at the reconstruction stage. ⁶⁰

In a recent in vitro study, Tang et al. established that bFGF amplified the strength of healing, but not adhesion formation, during the critical tendon healing phase. ⁶¹ Another research also revealed that combining bFGF with TGFβ‐1 intensified the synthesis of type I collagen in fibroblasts. ⁶² However, FGFs tend to rapidly lose their biological functional activity when injected as free solutions in vivo, limiting the application of bFGF in the field of tendon healing. ⁵⁴

In conclusion, if FGFs are adsorbed or encapsulated in materials, their biological activity would be largely protected from their risk of degradation. Therefore, appropriate materials and substrates must be developed to accommodate and deliver them to defective areas and then release them at a controlled and sustainable rate. If performed properly, much of the potential of these factors will be realised.

3.5. Transforming growth factor‐beta (TGF‐β)

The TGF‐β family of peptides was first isolated in the late 1970seconds by de Larco and Todaro before being divided further into TGF‐α and TGF‐β. ⁶³ , ⁶⁴ Chuan et al.'s scrutiny of the gene expression of some growth factors at several time points during the early healing phase in a chicken model showed that the gene expression and production of TGF were far higher than those of all other growth factors. TGF‐β has been studied extensively as therapeutic candidates to promote tendon repair following tendon injury.

Currently, the TGF‐β family comprises three isoforms: TGF‐β1, TGF‐β2, and TGF‐β3. ⁶⁵ The structural prototype of TGF‐β1 is a protein first isolated from human platelets called TGF‐β, cloned from a human cDNA library, and later named TGF‐β1. ⁶⁶ Platelets are its main storage sites. TGF‐β1 is involved in the regulation of inflammation. It is released immediately after tendon injury, with this immediate release after injury playing a key role in the chemotaxis of macrophages and fibroblasts to the wound. ⁶⁷ TGF‐β could be a suppressor of excessive inflammation. ⁶⁸

A potential role for TGF‐β1 in physiological repair and collagen accumulation has been established in mammals. TGF‐β1 allegedly promotes the secretion of fibronectin and collagen by epithelial and mesenchymal cells through the transcriptional activation of related genes during culture. ⁶⁹ In addition, the subcutaneous injection of TGF‐β1 has been shown to strongly boost collagen accumulation and fibrotic tissue responses. ⁷⁰ These observations consolidate the role of TGF‐β1 as a master regulator of ECM accumulation and, consequently, a potential key driver of fibrosis.

Although there is evidence that the disruption of TGF‐β1 reduces the extent of scar formation, the mechanical strength of the tendon and the site of repair are lessened as a result of this disruption, ⁷¹ , ⁷² , ⁷³ suggesting that the complete blockade of TGF‐β1 does not provide optimal treatment. Therefore, determining the appropriate dose and isoform combination is critical to the successful application of TGFβ to promote tendon healing.

TGF‐β3 and TGF‐β1 are homologous, and recent data suggest that TGF‐β3 may have antifibrotic effects in wound healing and different tissues compared with TGF‐β1. ⁷⁴ Several pieces of preclinical evidence suggest that the excessive production of TGF‐β1 enhances scarring, while TGF‐β3 promotes scar‐free healing. ⁷⁵

TGF‐β2 appears to be tenogenic and was found in an embryonic tendon. ⁷⁶ It upregulates tenogenic gene expression in MSCs, ⁷⁷ , ⁷⁸ maintains the tenogenic commitment of embryonic tendon cells, ⁷⁹ and induces tenogenic markers in vivo. ⁸⁰ Scx is a transcription factor containing basic helix–loop–helix (bHLH). It can be observed that the expression is up‐regulated in the early stage of embryonic development and the healing of tendon injury, and has a regulatory effect on the maturation process of tendon, that can drive MSC commitment to tenogenesis. ⁸¹ , ⁸² One study showed that TGF‐β2 upregulates Scleraxis expression in embryonic murine limbs in vivo. ⁷⁹ Havis et al noted the involvement of TGF‐β2 molecules in the commitment of undifferentiated chick limb mesodermal cells towards the tendon lineage and their action downstream of mechanical forces to regulate tendon differentiation during chick limb development. ⁸⁰ These two experiments once again validated the tenogenicity of TGF‐β2, that is, its ability to promote embryonic tendon development. But its role in tendon morphogenesis is largely unknown. Perhaps it can heal the damaged tendon? Extensive experiments will have to be conducted to explore that possibility and many others.

Growth factors of the TGF‐β family are ubiquitous, multifunctional, and essential for survival. They play an important roles in growth and development, repair, and host immunity. TGF‐β especially stimulates the production of the ECM, including the increased secretion of collagen types I and III through all three isoforms; TGF‐β1, TGF‐β2, and TGF‐β3. ⁸³ TGF‐β1 is expressed by tenocytes, infiltrating fibroblasts, and inflammatory cells ⁷¹ , ⁸⁴ and is thought to be associated with the pathogenesis of excessive scar tissue formation. At all stages of in vitro development, TGF‐β2 is a tendonogenic cell of tendon progenitors. ⁸⁵ TGF‐β2 and TGF‐β3 are regarded as essential for tendon formation and are potent inducers of tendon progenitors. ⁷⁹ , ⁸⁵

In summary, TGFβ is active in almost all stages of tendon healing ⁸⁴ and has multiple roles, such as extrinsic cell migration stimulation, proteinase regulation, ⁸⁶ and fibronectin binding interactions. ⁸⁷ However, for many growth factors, excessive doses can be detrimental. High levels of TGFβ‐1, for example, have been implicated in tendon adhesion formation, which can significantly decrease the range of motion of a tendon. ⁸⁸

4. ROLE OF EXTRACELLULAR VESICLE (EV) IN TENDON HEALING

The emergence of stem cells once opened a new field of vision for the promotion of tendon regeneration and repair. However, given the risks of tumour formation and teratoma, attention has turned to an extracellular component—EV. EV, first identified by Johnstone et al. in 1983 in the supernatant of sheep reticulocyte as a small membrane‐structured vesicle that delivers protein between cells, got its name EV in 1987. In 2007, Valadi et al. were the first to demonstrate the existence of RNA as well as mRNA and miRNA in mouse and human mast cells. With the discovery of functional proteins and mRNA and miRNA, EV have become a research hotspot in the past decade. ⁸⁹

EV are extracellular nanovesicles, 30–100 nm in size, which are now known to be released by many different cell types and can be found in most bodily fluids acting as a mode of communication between cells. EV participate in multiple biological activities, including in age‐related tissue degeneration, the modulation of microbial infection, autoimmune and inflammatory disease pathology, and tumour initiation and progression. ⁹⁰

Like VEGF, studies have shown that Mesenchymal stem cells (MSCs)‐derived EV induce the activation of the VEGF signalling pathway and promote angiogenesis around the injury site. ⁹¹ Yu et al. revealed that MSCs‐derived EV enhanced the expression of tenomodulin and type I collagen, as well as the mechanical properties of neotendons, and stimulated the proliferation of local TSPCs in vivo. ⁹² Per Shi et al.'s finding, EVs from bone marrow‐derived multipotent mesenchymal stromal cells boosted tendon healing by increasing the proportion of tendon‐resident stem/progenitor cells. ⁹² This finding is consistent with the aforementioned conjecture about the TSPC.

EV have a wide range of sources. Studies have shown that exosomes derived from adipose mesenchymal stem cells and BMSCs. Similar to most growth factors, EV have some anti‐inflammatory properties. Four studies have shown that EV also inhibit the expression of pro‐inflammatory factors when promoting the expression of anti‐inflammatory factors, such as IL‐10 and IL‐β1. ⁹¹ , ⁹² , ⁹³ , ⁹⁴ This effect can also significantly diminish the thorny problem of tendon adhesion. Chamberlai et al. observed Exosomes derived from exosome‐educated macrophages (EEMs) and mesenchymal stromal/stem cells (MSCs) improved ligament healing, now with increased collagen type I and III, improved collagen tissue, and reduced scar size 14 days after injury. ⁹⁵ A reduction in scar formation has both advantages and disadvantages for tendon healing. Its advantage is that the appropriate scar tissue not only provides support for the broken tendon, but it also reduces the friction resistance caused by excessive scarring. On the other hand, reduced scarring increases the risk of tendon rerupture. Extensive experimentation is required to find a suitable balance.

In the context of biomechanics, Yu et al. noticed that stress at the failure of the regenerated tendons and Young's modulus were higher in the exosome treated group than in controls in a murine patellar tendon injury model. ⁹² Wang et al. identified significantly increased ultimate stress and maximum loading in the exosome‐treated group compared with the injury group. ⁹⁶ This makes up for the problem of reduced strength after tendon repair to a certain extent. If tendon repairs rely solely on promoting the formation of more scar tissues to increase the strength of the repair, this will only result in improved structural, but not material, properties of the tendon. These two experiments show precisely that EV can, in the process of promoting tendon healing, improve the structure and performance of a repaired tendon, reduce the risk of postoperative re‐rupture to a certain extent, and lay a solid foundation for early postoperative exercise for patients. EV can effectively reduce the risk of postoperative adhesion, secondary surgery, and joint stiffness.

In addition to the direct promotion of tendon healing, EV also opens up new horizons for tendon repair as a carrier. In the field of tumour treatment, chemotherapeutic drugs, biological agents and natural products have been carried through EV for anti‐cancer treatment. At present, research teams have promoted the proliferation of rat tendon stem/progenitor cells by loading platelet‐derived exosomes of recombinant Yap1 for functional tendon regeneration. ⁹⁷

In summary, EV are significantly likely to promote tendon healing by attenuating the initial inflammatory response and accelerating tendon matrix regeneration, providing a basis for potential clinical use in tendon repair. However, most investigations on EV in tendon injuries have follow‐up for less than 4 weeks, which is a considerably short follow‐up period, and lack of assessment of long‐term treatment efficacy. In addition, transitioning from small animal experiments to clinical application and promoting the safety and effectiveness of testing of products have proven challenging to clinical progress.

5. CONCLUSIONS

Understanding the role of growth factors opens a new window for tendon healing. Some success has already been achieved utilising growth factors as therapeutic agents applying a variety of delivery techniques. In most of these studies, growth factor utilisation has shown some degree of enhanced healing; however, in general, this pure application of a single factor has a limited effect on the outcome. Combining growth factors with surgery to promote healing of injured tendons has become an important and potentially very fruitful area of research. The clinical application of each material and cytokine must address a few important challenges, including determining cytokine dosage, the type of cytokine, the types of materials, the manner of delivery, and the optimum time point of delivery. Regarding the timing of the complete procedure, the following factors should be considered: (1) how to control the rate and dose of cytokine release; (2) the need for changes in cytokine type and dose at different stages of healing; (3) how to monitor local and long‐term treatment effects of growth factors; (4) treatment of postoperative adverse reactions (eg, infection, prolapse). Having a better understanding of the various materials and growth factors would address some of the limitations in the tissue engineering of tendons.

The emergence of EV has provided new horizons for promoting tendon healing and created new routes for their administration. There may be only a handful of studies on using EV in tendon healing, but there is no denying that EV have great potential. Moreover, EV derived from different cells appear to have different effects on tendon healing, and we speculate that EV acquired from tendon stem cells have a more potent effect on tendon healing.

Wang Y, Li J. Current progress in growth factors and extracellular vesicles in tendon healing. Int Wound J. 2023;20(9):3871‐3883. doi: 10.1111/iwj.14261

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.