1. INTRODUCTION

1.1. Structure, Function, and Mechanics in Healthy Tendon

Tendon is a compositionally complex tissue with a predominantly mechanical function: translating muscular contractions into joint movement by transmitting forces from muscle to bone. Because its stiffness is intermediate between that of muscle and that of bone, tendon acts as a buffer to prevent stress concentrations that would result from a direct muscle-to-bone connection. Moreover, tendon passively stores and releases energy during a joint-loading cycle, resulting in more efficient movement. However, as a result of its viscoelastic nature, tendon also dissipates energy, possibly helping protect bone and muscle from damage. Owing to the critical role of this tissue in body mechanics, injury and degeneration of tendon can be highly debilitating and can result in substantial pain, disability, and health-care costs.

The formation of tendon during development is thought to occur in three distinct steps (1). First, small collagen fibril intermediates nucleate outside fibroblasts in distinct extracellular compartments. Second, these intermediates assemble lengthwise into long, thin collagen structures. Third, these chains fuse laterally to form the thicker fibrils seen in fully developed tissue. In its mature form, tendon is composed predominantly (roughly 70% by dry weight) of type I collagen (also known as collagen I) with small amounts of proteoglycans, glycoproteins, and minor collagens.

In each step of tendon formation, fibrillogenesis is mediated by interactions among these molecules. For example, developing chick tendons express collagen III in regions with collagen fibrils of smaller diameter, suggesting that collagen III plays a role in limiting the lateral growth of collagen I fibrils (2). Conversely, the presence of biglycan, a small leucine-rich proteoglycan with two side chains of either chondroitin or dermatan sulfate attached to its core protein, is associated with thicker, more regularly shaped collagen fibrils (3, 4). Other molecules that have important regulatory roles in tendon development include collagens V, VII, and IX; the proteoglycans fibromodulin, lumican, and decorin; and numerous transcription and growth factors such as scleraxis (5, 6) and transforming growth factor β (TGFβ) (7).

Mature tendon is made up of a hierarchically structured backbone of collagen interspersed with cells (tenocytes) and a network of noncollagenous molecules (Figure 1). On the largest scale is the entire tendon, which is encapsulated by a thin sheet known as the epitenon. Enveloped by the epitenon are fascicles, tube-like structures oriented along the long axis of the tendon that are sometimes visible to the eye after gross dissection (8). Each fascicle is covered by a connective tissue known as the endotenon, which is similar in composition to the epitenon. A tendon's blood supply and nerve supply come in large part from the endotenon and epitenon.

Tendon fascicles, in turn, are bundles of collagen fibers with tenocytes set in between. These fibers are on the order of 10 μm in diameter (9) and are visible at high magnification under an optical microscope (10). Looking within a collagen fiber reveals that the next level of tendon structure is the collagen fibril. These collagen fibrils, which can be imaged with an electron microscope, appear to be periodically “crimped” in the absence of load. On an even smaller scale is the microfibril, an insoluble aggregate of five cross-linked tropocollagen molecules (8). Finally, tropocollagen is composed of three polypeptide chains of collagen wrapped together in a helical pattern. A single tropocollagen molecule is approximately 1.5 nm in diameter (11) and is water soluble.

The intricate fiber-based structure and composition of tendon endow it with nonlinear, viscoelastic, anisotropic, and heterogeneous mechanical properties. As such, its mechanical properties depend on strain, rate of strain, loading direction, and location. When loaded slowly (quasi-statically) under tension, tendon initially stiffens with increasing strain in what is known as the toe region. This stiffening behavior is thought to result from the gradual straightening of initially crimped collagen fibrils. Above strains of approximately 3% and prior to the yield point, tendon maintains a relatively constant stiffness in the linear region of its stress-strain curve (12). The linear-region moduli of human tendons are usually on the order of 1 GPa. Failure in tendon typically occurs at strains near or above 10%.

Owing to its viscoelastic nature, tendon also exhibits a rate-dependent stiffness. In tensile tests of cyclically loaded bovine Achilles tendon, the dynamic modulus |E*| increased by a factor of 4 as the strain rate increased from 3.1 × 10⁻⁴ s⁻¹ to 4.1 × 10⁻² s⁻¹, whereas tanδ, the tangent of the phase angle between stress and strain (a measure of the ability of a material to dissipate energy), was measured to be on the order of 0.1 for loads less than 50 g (13). Even the failure load, failure strain, and failure mode of tendon are rate dependent (14, 15). The viscoelasticity of tendon is further manifested in the phenomena of creep and stress relaxation. In a creep experiment, a constant load is applied to tendon. Unlike an elastic material, which instantaneously deforms when a load is applied, tendons slowly displace until their equilibrium deformation is reached. Similarly, in a stress-relaxation experiment, a constant strain is applied. The stress on the tendon overshoots its stable value before slowly relaxing to equilibrium.

Finally, the mechanics of tendon are sensitive to loading direction and anatomic location. For example, tendon is typically stiffest in the direction of collagen-fiber alignment (i.e., the primary loading direction), and its measured modulus varies with loading angle relative to the natural bone-insertion angle (15). Moreover, mechanical (as well as structural) properties of tendon fluctuate near the tendon-to-bone insertion site (16) and in regions subject to altered loading patterns. For example, the tensile modulus of the human supraspinatus tendon, a rotator cuff tendon that experiences multiaxial loads as a result of its complex surrounding anatomy, varies by more than an order of magnitude from region to region and correlates strongly with local fiber alignment (17).

1.2. Clinical Importance of Tendon Injury

When a tendon is injured, its structure is disrupted, and proper function can be compromised. Unfortunately, tendon injuries are common. For example, 1 of every 10 people and 1 of every 2 runners are afflicted with Achilles tendinopathy (i.e., tendon pain concurrent with possible damage or inflammation) before the age of 45 (18). On one end of the spectrum is chronic tendinopathy, a debilitating injury initiated by biological and physical factors that include aging, oxidative stress, and extreme loading during intensive exercise. Indicators of chronic tendon injury range from pain, inflammation, and increased cross-sectional area to histologically observable changes including increased proteoglycan content, increased cellularity, enhanced vascularity, and disorganization of the collagen-fibril network (10, 19, 20) (Figure 2). On the other end of the spectrum is tendon rupture. Although this type of injury may be spontaneous or induced by direct trauma and/or excessive loading, most tendon tears are preceded by histological changes consistent with chronic tendinopathy, suggesting that tendon rupture is closely associated with degeneration (21).

1.3. Stages of Healing in Tendon

The response to tendon injury can be divided into three overlapping stages (22). In the inflammatory stage, which typically spans a few days, the wound site is infiltrated by red blood cells, white blood cells (leukocytes), and platelets equipped with important growth factors and endothelial chemoattractants. Whereas a fibrin clot is formed to provide temporary stiffness, macrophages digest necrotic debris, and tenocytes are recruited to the wounded area and stimulated to proliferate, particularly in the epitenon (23).

The second stage, known as the proliferative or repair stage, begins roughly two days into the injury response. This phase of healing is characterized by profuse synthetic activity and is directed by macrophages and tenocytes. Macrophages, whose role shifts from phagocytic to reparative a few days after injury, release growth factors and direct cell recruitment (24, 25). Meanwhile, tenocytes deposit a temporary, mechanically inferior matrix composed mostly of collagen III.

In the third and final stage, known as the remodeling phase, collagen I synthesis begins to dominate, and the extracellular matrix (ECM) becomes more aligned. In addition, cell density and general synthetic activity are gradually decreased. This phase begins 1–2 months after injury and can last more than a year. The repaired tissue appears scar-like and never completely regains the biomechanical properties it had prior to injury (25).

2. EXPERIMENTAL INJURY MODELS

Much of our understanding of tendon injury and healing has come through the use of animal and cell-culture models, which are often less expensive and easier to control than clinical trials in humans. Eight successful and widely used models of tendon injury and regeneration are highlighted below.

2.1. Injury and Healing in Rat Tendons

Because the rat has a bony architecture similar to that of humans, it is the most appropriate animal model for human rotator cuff tendon pathology (26). In particular, the rat is the only animal model, other than some nonhuman primates, with a supraspinatus tendon that passes beneath an enclosed structure analogous to the human coracoacromial arch. An intense regimen of running on a declined treadmill has been successfully used to induce tendinopathy in rat supraspinatus tendons (27). After 4 weeks of this regimen, reductions in mechanical properties and increases in cross-sectional area were observed. In addition, collagen alignment became more irregular, and cellularity increased (Figure 2). These macro- and microscale changes are consistent with those seen in human tendinopathy, providing evidence for a direct link between overuse and chronic supraspinatus injury. Other studies utilizing this overuse model have measured an increase in expression of nitric oxide synthases, glutamate signaling proteins, markers of cartilage formation, and markers of apoptosis, demonstrating the role of these molecules in tendon degeneration (28, 29, 30, 31, 32).

The rat is also an important model for understanding healing in torn rotator cuff tendons, a common source of pain and reduced shoulder function in adults. This model has been used to demonstrate an increase in the tension required to repair rotator cuff tendons with time after detachment (33) and the potential benefits of immobilization following detachment of the supraspinatus tendon (34, 35). More recent investigations have shown improved healing in rat rotator cuff tendons biologically augmented with numerous different therapeutic agents (36, 37, 38, 39). These studies demonstrate the utility of the rat model for developing and testing strategies to enhance healing in repaired supraspinatus tendons, for which reported retear rates range from 13% to as high as 94% (40, 41, 42, 43).

Finally, the rat has been used to investigate the etiology of tendinopathy using an in vivo fatigue-loading model (44, 45, 46, 47, 48, 49). In particular, controlled deformations were applied to the patellar tendons of anesthetized rats by gripping the patella with a custom clamp attached to a hydraulic actuator. These studies demonstrated degradative changes in mechanical stiffness, collagen alignment, and molecular activity that depend on the number of applied fatigue-loading cycles, illustrating the progressive nature of tendinopathy.

2.2. Injury and Healing in Dog Tendons

The canine forefoot, whose size and surrounding anatomy are similar to those of the human hand, is an excellent model for understanding healing in flexor tendons of the hand, where the presence of surrounding sheaths and proximity of surrounding bones make repair particularly difficult (50, 51, 52). Notably, this model has been used to test the relative efficacy of suturing techniques (53) and to assess strategies for reducing adhesion formation in healing flexor tendons. For example, addition of a lubricin-containing compound to lacerated canine flexor digitorum profundus tendons following repair inhibits the formation of adhesions at the cost of reduced mechanical properties (54), whereas a regimen of passive-motion rehabilitation can lead to adhesion-free healing in sheathed flexor tendons (55, 56)

2.3. Injury and Healing in Chicken Tendons

Chicken toe flexor tendons are readily available and exhibit anatomical and mechanistic similarities to their analogs in the human hand (57). For example, flexor tendons in the hand of a human and those in the toe of a chicken experience tensile strains and relative motion (sliding) with respect to their encapsulating sheaths. The chicken model has been used to demonstrate that both of these forms of mobilization are critical to improved healing following flexor tendon injury (58). Injury models in chicken tendons include full and partial (58) transection of an interior section of the tendon. Although the latter strategy does not mimic clinically observed tendon injuries, it leaves surrounding fibers intact, precluding the need for sutures that could confound experimental findings.

2.4. Injury and Healing in Mouse Tendons

The primary advantage of the murine model is the availability of transgenic techniques for modifying the mouse genome (59, 60). As a result, tendon healing can be compared in mice with and without the ability to express a particular gene. For example, interleukin 6 (IL-6)-knockout mice demonstrate inferior healing of interior partial-transection patellar tendon injuries compared with controls, suggesting an important role of this molecule in tendon healing (61). In addition, through the use of transgenic mice bred with fluorescent reporters, expression patterns of particular genes can be tracked in time and space throughout the healing process. In a study that used fluorescently tagged collagen I and collagen II genes to investigate murine patellar tendons injured with a central, full-thickness defect, elevated levels of collagen I expression were measured near the insertion sites at 1 week postinjury, whereas elevated expression in the midsubstance was delayed, peaking 1 week later. Collagen II expression, however, was not observed during the healing response (62). The accessibility of murine primers and antibodies for biological assays is yet another advantage of this injury model, as evinced by a recent study measuring increased expression of the tendon marker scleraxis, previously known only for its role in tendon development, 4 weeks after patellar tendon injury in mice (63).

2.5. Injury and Healing in Rabbit Tendons

Owing to its ease of handling and large size compared with the mouse and the rat, the rabbit is a commonly used animal model for understanding tendon injury and healing (64). For example, a model of tendinopathy in the rabbit flexor digitorum profundus tendon was recently employed by way of electrically induced cyclic loading (65, 66). The resulting microstructural damage resembled that seen in pathological human tendon, whereas increased growth factor levels suggested failed regeneration. The rabbit shoulder is also used for understanding healing in rotator cuff injuries (67, 68). Although most studies have focused on the lapine supraspinatus tendon, the lapine subscapularis tendon has recently been used as an analog to the human supraspinatus, where most rotator cuff tears occur (69). The footprint of the lapine subscapularis tendon is similar in aspect ratio to that of the human supraspinatus, and the lapine subscapularis tendon and muscle pass through a bony tunnel similar in geometry to the human coracoacromial arch. Lapine models have also been employed in numerous studies of the repair of both patellar tendon (70, 71, 72) and Achilles tendon (64, 73, 74).

2.6. Injury and Healing in Sheep Tendons

The utility of the sheep model lies in the comparable size of sheep and human tendons (75). In the ovine shoulder, the surrounding anatomy of the infraspinatus tendon is similar to that of the human supraspinatus. Therefore, repairs of surgically torn sheep infraspinatus tendons were used to test the mechanical strength of various tendon-to-bone suturing strategies (76, 77). Similarly, the efficacy of bone anchors was assessed in reconstructed ovine patellar tendons (78). Finally, the sheep is a popular model for studies of healing in fetal tendons. In particular, studies in sheep have demonstrated scarless healing of fetal ovine limb extensor tendons, with a postinjury recovery of mechanical properties no different from that seen in adults (79, 80). Interestingly, transplantation of injured tendons from fetal sheep into adult mice does not impede the scarless healing response (81).

2.7. Injury and Healing in Horse Tendons

Because tendon and ligament injuries are common in athletic and performance horses (82, 83), numerous studies have investigated tendon healing in these animals. Collagen-fibril diameter and collagen-fibril crimp in horse tendons were substantially reduced after injury and remained inferior to controls at 14 months after surgery (84). However, both fibronectin and collagen III were present in the healing horse tendon in the first few months after injury, whereas collagen I fibrils predominated after 6 months (85). These investigations provide a timetable for tendon healing in horses and other large mammals. Many other studies of healing in horse tendons have focused on augmentation (86, 87) and noninvasive diagnostic strategies for assessment of the repair process, including ultrasound and magnetic resonance imaging (82, 88, 89).

2.8. In Vitro Cell-Culture Models of Tendinopathy

In vitro cell-culture models can be used to simulate in vivo environments and offer added controllability over animal models at the expense of less direct applicability to human pathology. For example, an important in vitro injury model was produced by cyclically stretching cells adhered to a flexible membrane in the presence of the cytokine IL-1β (90), suggesting a role of this molecule in tendinopathy. In a separate investigation, an in vitro wound model using gaps generated in fibroblast cell cultures was used to demonstrate accelerated healing in the presence of basic fibroblast growth factor (bFGF). However, another study demonstrated differences in the cellular activities of cultured tendon fibroblasts and fibroblasts removed from a healing rat patellar tendon (91). Although this finding may imply that the latter cell type is preferable as a model for tendon regeneration, in vitro cell-culture studies can provide straightforward, general results that are critical for understanding tendon's elaborate response to injury in vivo.

3. BIOLOGICAL MEDIATION OF TENDON HEALING

The complex cascade of events initiated by a tendon injury is regulated by numerous biological factors with interconnecting pathways. Here, we summarize five classes of biological molecules that play a regulatory role in tendon healing.

3.1. Oxygen and Lactate

Synthetic activity in a healing tendon is strictly regulated by oxygen and lactate levels, particularly in the hypoxic environment of the proliferative stage (25). Although production of collagen decreases when a critically low oxygen level is reached (92), collagen deposition is enhanced with elevated concentrations of lactate (93, 94). Therefore, by releasing high levels of lactate (25), macrophages indirectly contribute to collagen formation. In addition, angiogenesis is stimulated by hypoxic conditions and elevated concentrations of lactate (22). These effects may be in part due to the regulatory effect of oxygen concentration on TGFβ, vascular endothelial growth factor (VEGF), and other growth factors (25).

3.2. Cytokines and Growth Factors

Cytokines and growth factors released by tenocytes and migrating leukocytes after tendon injury are closely involved in the regulation of the repair response (95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106). For example, the cytokine interleukin-1β (IL-1β) promotes inflammation and degradation of the ECM (107). Another proinflammatory cytokine, IL-6, has an inhibitory effect on fibroblast proliferation (108). Nevertheless, IL-6-knockout mice exhibit inferior healing compared with controls, likely the result of an improper immune response (61). IL-1β, tumor necrosis factor α, and IL-10 downregulate collagen I synthesis, whereas the anti-inflammatory cytokine IL-4 promotes deposition of collagen I, collagen III, and fibronectin.

Matrix production is also stimulated by insulin-like growth factor 1 (IGF-1) and TGFβ (109). Other functions of these molecules include mediation of cell proliferation and migration into the wound site as well as, in the case of TGFβ, regulation of proteinases and fibronectin binding (110). On the one hand, biological augmentations of IGF-1 can improve tendon healing, and the absence of IGF-1 leads to an inferior repair response. On the other hand, reductions in levels of TGFβ have been associated with reduced scar formation (111).

IGF-1 and TGFβ are expressed at all stages of healing, but particularly during the inflammatory and proliferative phases (112). In contrast, VEGF, a stimulator of angiogenesis, is most active in later stages of healing. Other growth factors important in tendon healing include bFGF and platelet-derived growth factor (PDGF). Whereas the former stimulates cellular migration and blood vessel formation, the latter has been implicated in regulating DNA synthesis and promoting the production of other growth factors, including IGF-1 (104, 113).

3.3. Matrix Metalloproteinases and Tissue Inhibitors of Matrix Metalloproteinase

Matrix metalloproteinases (MMPs) are a class of zinc-dependent enzymes involved in the repair response of tendon (38, 44, 114). In particular, whereas MMP-9 and MMP-13 help degrade the ECM days after injury, MMP-3, MMP-4, and MMP-14 participate in both matrix degradation and matrix remodeling throughout the healing process (115, 116). Activity of MMPs is inhibited by molecules known as tissue inhibitors of matrix metalloproteinase (TIMPs). Imbalance in the action of MMPs and TIMPs is associated with tendon pathology (117), whereas localized delivery of TIMPs enhances collagen organization in healing tendons (38).

3.4. Proteoglycans and Glycoproteins

Proteoglycans regulate healing in a variety of tissues including skin and annulus fibrosis. Furthermore, their role in mediating tendon development is well established. Although a detailed understanding of their function in tendon healing is lacking, spikes in expression of small leucine-rich proteoglycans measured immediately following tendon injury (118) hint at a potentially important regulatory role for these molecules in tendon regeneration.

The glycoprotein CD44 regulates levels of hyaluronic acid in tendon and acts as a mediator of cell-matrix interactions, cellular migration, and ECM production after injury (119). Postinjury expression levels of this molecule are higher in adult tendons than in fetal tendons, which exhibit enhanced, scarless healing (80, 81, 120). Furthermore, CD44-knockout mice demonstrate improved healing compared with wild-type mice (121).

3.5. Minor Collagens

Minor collagens are believed to play an important role in healing. For example, repaired rat supraspinatus tendons detached at the insertion contained reduced levels of collagen X and increased levels of collagen XII (35). However, the function of these molecules in the repair response is not fully understood.

4. STRUCTURAL AND MECHANICAL CHANGES IN HEALING TENDONS

Throughout the stages of healing, tendons undergo a sequence of structural alterations that significantly modify their mechanical properties. For example, the Young's moduli E in injured rabbit Achilles tendons three weeks after surgery were roughly 5 times lower than in uninjured animals (122). However, E gradually increased to nearly 80% of its uninjured value after 12 weeks. A similar trend was observed with tensile strength, whereas the cross-sectional area and viscoelastic phase angle of the injured tendons were 3 and 1.5 times greater, respectively, than controls at the three-week time point and nearly equivalent to controls at 12 weeks. Collagen became highly disorganized after injury, but collagen alignment and crimp wavelength increased with time after surgery, approaching but not reaching uninjured values. In contrast, a rat supraspinatus tendon-defect injury model demonstrated a recapitulation of less than 10% of the uninjured modulus at 12 weeks postsurgery and an incomplete restoration of collagen organization (123), whereas the ultimate load and stiffness of injured mouse patellar tendons rose to 48% and 63%, respectively, of uninjured values after 8 weeks of healing (62). A longer-term study on healing rabbit medial collateral ligaments found populations of larger-diameter collagen fibrils not seen in uninjured tissue that persist even at 104 weeks postsurgery, suggesting that remodeling may continue for years after an initial injury (124). In contrast, collagen fibrils in equine superficial digital flexor tendons 98 weeks after injury were significantly reduced compared with controls (84). These studies demonstrate that although the extent and timetable of mechanical and structural changes in the healing tendon depend on numerous variables including age, species, repair strategy, injury model, and anatomic location, tendons generally exhibit a substantial decrease in organizational and mechanical parameters immediately after an injury followed by a slow but incomplete recapitulation of initial properties.

Repaired tendons torn at the tendon-to-bone insertion (enthesis) exhibit unique structural and mechanical changes. At 8 weeks following supraspinatus detachment in rats, collagen orientation and levels of collagen I, collagen XII, and aggrecan were elevated, whereas levels of collagen X and decorin were decreased (35). Stiffness and peak load were roughly 40% and 60% lower after injury, respectively, whereas the quasi-linear viscoelastic time constant τ₂ also decreased significantly. In general, animals that were exercised after surgery exhibited inferior healing compared with immobilized animals. Interestingly, progressive healing was even observed in supraspinatus tendons detached without repair (125) (Figure 3). Beginning at 2 weeks postinjury, both tendon stiffness and collagen organization began to increase, surpassing those of the control group after 4 weeks. These improvements in functional properties lasted through 16 weeks after surgery, the final time point studied. This model has led to an improved understanding of the natural healing response of ruptured tendons and the consequences of delayed repair.

5. TENDON HEALING FROM PRENATAL DEVELOPMENT TO SENESCENCE

5.1. Scarless Tendon Healing in Fetal Tendon

Extensive experimental evidence suggests that tissue healing in the fetus—particularly in the early gestational to midgestational period—occurs in a manner fundamentally different from that occurring in the adult. Fetal tissue healing generally occurs at a faster rate and in the absence of scar formation. This type of accelerated scarless healing has been observed in skin (126), articular cartilage (127), nerve (128), and bone (129) injury models. Recent studies have focused on determining whether fetal tendons are also capable of scarless healing. One such investigation tested this hypothesis by examining the healing process of lateral extensor tendons following partial (approximately 50% of the tendon width) tenotomies in adult and fetal sheep (80). Adult tendons and third-trimester fetal tendons healed with disorganized collagen, significant granulation tissue, fiber-bundle discontinuity, increased cross-sectional area, and adhesion to surrounding structures. In contrast, second-trimester fetal tendons healed without gross or histological abnormality or adhesion to surrounding structures (Figure 4). These results suggest that early gestational to midgestational fetal tendon healing occurs at an accelerated pace with less scar formation compared with adult controls. This property of fetal tendons appears to be intrinsic to the tissue itself and not a product of the fetal environment, as demonstrated by studies examining fetal tendons transplanted into an adult environment (81). Therefore, the adult environment itself does not appear to be an impediment to scarless tendon healing.

5.2. Age-Related Decrease in Production of Structural Proteins and Regulatory Biomolecules After Injury

With senescence, tendons undergo numerous biochemical, cellular, and mechanical changes that cause a general decline in the ability of the tendon to recover from injury. There is a decrease in the volume density of tenoblasts as well as in the number of tenoblasts per unit of surface area (130, 131). The plasmalemmal surface density decreases, the tenoblast becomes more slender, the nucleus-to-cytoplasm ratio increases, and the number of organelles responsible for protein synthesis decreases (132). In general, the ability of the tenoblast to synthesize structural proteins and regulatory biomolecules after injury declines with age.

As collagen synthesis and collagenolytic activity diminish with senescence, there is a decrease in collagen turnover (21, 133). This decrease results in an increased diameter of collagen fibers and increased variability in thickness. Furthermore, the collagen-fibril crimp angle decreases with age, and its periodicity increases (134). Owing to alterations in the activity of enzymes involved in the mediation of collagen cross-linking, such as lysol oxidase, there is an increase in nonreducible collagen cross-linking (135), which leads to increased resistance to degradative enzymes, reduced solubility, increased stability to thermal denaturing, increased autofluorescence, and increased birefringence (136). The mechanical result includes decreases in ultimate strain, ultimate load, modulus of elasticity, and tensile strength and an increase in mechanical stiffness. Together, these changes make the aged tendon more likely to tear when subjected to increasing stress (137).

5.3. Healing/Healed Tendon Mechanical Properties Throughout Development and Aging

The biomechanical properties of healed and healing tendons throughout the developmental process have also been an area of focus. Regardless of age, wounded tendons demonstrate less ultimate stress and moduli than do unwounded tendons, and the wounded/unwounded ratios for these values are consistent at all ages (80). Equilibrium/peak stress ratios and the wounded/unwounded ratio for this value are also similar throughout development (80). Across the spectrum of aging, healing tendons exhibit no difference in maximum stress, strain at maximum stress, or modulus at various time points (138). These results suggest that biomechanical recovery is consistent throughout development and aging. Importantly, the minimal loading environment in the womb may delay the speed of tissue remodeling and biomechanical restoration of fetal tendons, and restoration of mechanical integrity may lag behind architectural reconstruction (80).

6. DEVELOPMENT AS A PARADIGM FOR HEALING

6.1. Common Gene Expression Patterns in Developing and Healing Tendons

Tendon healing is a complex and highly regulated process that is initiated, sustained, and terminated by a variety of regulatory molecules. As discussed above, the role of growth factors including IGF-I, TGFβ, VEGF, PDGF, and bFGF in tendon healing has been well characterized in both in vitro and in vivo studies. These molecules are markedly upregulated following tendon injury and are active at multiple stages of the tendon healing process (96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 139). Interestingly, several of the same growth factors are also involved in tendon development. For example, the recruitment and maintenance of tendon progenitors by TGFβ signaling are essential for tendon formation (7). Specifically, TGFβ is a potent inducer of tendon markers in mesenchymal cells, and tendons and ligaments are entirely absent in embryos in which TGFβ signaling is disrupted (7). TGFβ is also involved in distal limb tendon formation during embryonic development (140). In addition, the angiogenic peptide VEGF is present in human fetal tendons and is likely responsible for the differentiation of vascular and avascular zones within tendons (141). As such, there appears to be common gene expression patterns between healing and developing tendons.

6.2. Common Structural Changes in Developing and Healing Tendons

Tendon development and healing are both highly regulated processes that involve certain common structural changes. As described above, the development of mature mechanical properties in the tendon is dependent on the assembly of a tendon-specific extracellular matrix, which is synthesized by tendon fibroblasts and composed of collagen fibrils organized as fibers and fibril-associated proteins (1). Collagen fibrillogenesis occurs in a series of extracellular compartments where fibril intermediates are assembled and fused to form mature fibrils (142, 143). Once the fibril intermediates are incorporated into fibers, both linear and lateral fibril growth occur (143). Tendon fibroblasts regulate this process via interactions between extracellular macromolecules and the collagen fibrils (1).

Tendon healing shares many of the same structural changes that occur during tendon development. In particular, during the proliferative and remodeling stages of tendon healing, tendon fibroblasts synthesize collagens, proteoglycans, and other components of the ECM (144). The collagen is organized into mature fibers that are subsequently reorganized during the remodeling phase (144). Like tendon development, this process is closely regulated by tendon fibroblasts via cytokines, growth factors, and MMPs (144).

7. TENDON HEALING COMPLICATIONS

7.1. Scarring

The remodeling phase of tendon healing can be divided into a consolidation stage and a maturation stage (144). During the consolidation stage, the repair tissue changes from cellular to fibrous in nature with a higher proportion of collagen I synthesis (19). Then, during the maturation stage, this fibrous tissue gradually changes to scar tissue with a decline in tenocyte metabolism and tendon vascularity and an increase in collagen-bundle thickness (19). The resulting scar tissue has biomechanical properties that differ from those of native tendon: decreased strength, increased stiffness, and greater propensity toward adhesion formation. Despite remodeling, the biochemical and mechanical properties of healed tendon tissue never match those of intact tendon (145).

7.2. Adhesions

Adhesion formation after tendon injury poses a major clinical problem (146). Disruption of the synovial sheath at the time of the injury or surgery allows granulation tissue and tenocytes from the surrounding tissue to invade the repair site. Exogenous cells predominate over endogenous tenocytes, allowing the surrounding tissue to attach to the repair site and resulting in adhesion formation.

Tendon healing can occur intrinsically, by proliferation and migration of tenocytes from the epitenon and endotenon into the injury site, or extrinsically, by invasion of cells from the surrounding sheath and synovium (147, 148). The relative balance of each type of healing is determined by tendon location, extent of trauma, and postoperative motion. Intrinsic healing results in superior biomechanics and fewer complications. In particular, a normal gliding mechanism within the tendon sheath is preserved with intrinsic healing, whereas in extrinsic healing, scar tissue results in adhesion formation that disrupts tendon gliding (149). The most important factor implicated in adhesion formation is trauma, induced by either the initial injury or the subsequent surgical repair (150). The actin isoform α-smooth muscle actin has been identified in tendons, and tenocytes that express α-smooth muscle actin are known as myofibroblasts (151). Myofibroblasts play a role in ECM network homeostasis in tendons and ligaments and are likely responsible for the formation of tendon adhesions (152).

The degree and extent of adhesions can be classified into four grades: Grade 0 describes the complete absence of adhesions; Grade I describes adhesions that are thin, avascular, filmy, and easily separable; Grade II describes thick, avascular adhesions that are limited to the site of anastomosis; and Grade III describes thick, vascular, and extensive adhesions (153). These adhesions inevitably result in functional disability due to loss of the gliding mechanism. To prevent or limit adhesion formation, several modalities—such as modulation of the inflammatory response and growth factors that promote scarring by various pharmacological agents, introduction of mechanical barriers between the tendons and the proliferating tissue, use of ultrasound and electromagnetic therapy, and gene therapy—have been explored (154). However, no single method has become widely utilized.

7.3. Failed Repairs

Failure of repair is another notable potential complication following tendon repair (155). Factors that predispose tendon repairs to rupture include inadequate suture material, poor surgical technique, overly aggressive therapy, and early termination of postoperative immobilization.

Patient noncompliance, such as removing the splint, not using a sling, lifting heavy objects, or attempting strong grasp, is another frequent cause of rupture (155). For example, in a study of 526 flexor tendon repairs in 440 patients, 11 of the 23 failed repairs occurred as a result of the patient using his or her hand, in or out of the splint, against medical advice (155).

The location of repair failure is often dictated by anatomic location. Repairs of fresh-frozen digital flexor tendons using modified Kessler technique with running peripheral suture were more likely to fail when subjected to tendon motion arcs with curvatures of small diameter (156). These results suggest that repaired tendons are more likely to fail along curvatures over sheaths, pulleys, or joints (156).

Treatment options for failed tendon repairs include no further surgery, immediate rerepair, one- or two-stage tendon grafting, and tendon transfer. For failed flexor tendon repairs, immediate rerepair with direct suture, whenever possible, has been suggested as the most favorable treatment option (157).

8. STRATEGIES FOR ENHANCEMENT OF TENDON HEALING

8.1. Surgical Tendon Repair

Numerous suturing techniques for the surgical repair of an injured tendon have been described. In flexor tendons, the strength of the repair is proportional to the number of sutures crossing the repair site: Tendons repaired with eight strands have increased ultimate force and rigidity and decreased strain compared with tendons repaired with four strands (158). Thus, whereas classic core suture techniques including the Kessler and the Tajima involve only two suture arms spanning the repair site, more modern repairs attempt to place four to eight sutures across the repair site in addition to a running epitenon stitch (Figure 5). These modern repairs feature a greater number of intratendinous grasping points with an increased number of suture strands extending across the repair site (159). Suture placement in flexor tendon repair is the subject of controversy. One school of thought is that the core sutures should grasp the volar half of the tendon to minimize disruption of blood flow to the tendon, whereas the opposing opinion is that dorsal placement is superior because it exhibits superior pull-out strength (160).

Regarding rotator cuff tendon repair, the merits of single-row versus double-row techniques have been debated. In vitro anatomical studies suggest that a double-row repair produces a significantly larger supraspinatus footprint and a better biomechanical construct than does a single-row repair (161, 162). Furthermore, gap formation during cyclic testing is significantly less for double-row repairs than for single-row repairs, and double-row repairs demonstrate higher ultimate tensile load (163).

The type of suture may also play a critical role in the strength of repair. Studies have established that braided sutures tend to be superior to monofilaments in arthroscopic rotator cuff repairs (164). For these repairs, there has recently been a shift away from the use of simple braided polyester sutures, such as No. 2 Ethibond, and toward hybrid sutures with a core of ultrahigh-molecular-weight polyethylene surrounded by braided polyester, such as No. 2 FiberWire®. Studies comparing FiberWire with Ethibond unanimously agree that the former has approximately 50–80% greater tensile load with at least a factor-of-5 increase in cycles to failure (164, 165, 166). In contrast, for flexor tendon repairs, braided sutures may generate more friction and deform the tendon more than the preferred monofilament sutures would (167).

Tendon autografts may be used to facilitate tendon reconstruction, particularly in cases involving tendon loss or retraction. For example, case reports have described use of a semitendinosus autograft for chronic patellar tendon rupture (168), a hamstring autograft for pectoralis major tendon rupture (169), and a semitendinosus autograft for distal biceps tendon rupture (170). These autografts must be harvested from donor sites, which can potentially result in donor site morbidity. Nevertheless, they remain viable options when primary repair is not feasible.

8.2. Mechanical Stimulation (Mobilization Versus Immobilization)

Extensive studies in animal models have been conducted to investigate the role of mechanical stimulation in tendon healing. Whereas stretching can disrupt tendon healing during the initial inflammatory phase, controlled mobilization of injured tendons after the inflammatory phase (approximately one week following an acute injury) enhances the ultimate strength and excursion properties of the healing tendons (171, 172). Similarly, in humans, early mobilization following flexor tendon injuries helps restore gliding function, increases tensile strength, improves tendon excursion, and stimulates morphological restoration of the injured tendons (173).

The mechanical stimulation from controlled mobilization is suggested to enhance tendon repair and remodeling by stimulating tenoblast activities (such as fibroblast proliferation and collagen synthesis and realignment), leading to increased tensile strength, increased tendon diameter, and fewer adhesions compared with immobilized healing tendons (174, 175). Controlled mobilization promotes healing through fibroblast proliferation and collagen realignment (176). In contrast, immobilization following tendon injury may have a negative effect on tendon healing, as evidenced by lower tensile strength and lower strain at failure compared with control samples (177). Immobilization also reduces the water and proteoglycan content of tendons and increases the number of reducible collagen cross-links (178).

Studies have shown that tenoblasts respond to mechanical loads by altering gene expression, protein synthesis, and cell phenotype. It is through these mechanotransduction mechanisms that tendons are able to respond to mobilization with accelerated healing. Mechanical loading increases ECM protein production by promoting release of growth factors (179) and modulates ECM turnover by regulating the expression and activity of MMPs (90, 180). Mechanical forces applied to cell surfaces also cause changes in cytoskeletal structure, initiating complex signal transduction cascades within the cell through activation of integrins and stimulation of G protein–coupled receptors, receptor tyrosine kinases, and mitogen-activated protein kinases (181).

8.3. Biologics

Biologic solutions to modulate tendon healing are currently being explored. In the past, biologics were utilized to reduce adhesion formation by serving as physical and mechanical barriers between the healing tendon and the surrounding tissues. Agents that have been examined for such use include corticosteroids (182), dimethyl sulfoxide (183), beta-aminopropionitrile (184), hyaluronic acid (185), and 5-fluorouracil (186). As our understanding of the molecular and cellular bases of tendon healing expands, the role of biologic agents will likely expand. For example, the exogenous addition of a cytokine known to limit excessive scar formation or the suppression of a cytokine that promotes excessive scar formation could alter the healing process and result in a more favorable response. Furthermore, through the use of gene therapy, gene expression in the healing tendon may one day be modulated by exogenously delivered genetic material. Researchers have already successfully transduced genetic material into tenoblasts via liposomes (187) and adenoviral vectors (188).

8.4. Tissue Engineering

Tissue engineering has the potential to enhance tendon healing. Mesenchymal stem cells (MSCs) are pluripotent cells that are capable of undergoing differentiation into a variety of specialized mesenchymal tissues, including tendon. These cells can be applied directly to the site of tendon injury or can be delivered on a suitable carrier matrix that functions as a scaffold while tendon repair takes place. Tissue-engineered tendons can also be used to bridge areas of tendon loss or to replace severely degenerated regions (189, 190).

Studies have shown that acellularized allogeneic tendons can be successfully repopulated with epitenon tenocytes, tendon sheath fibroblasts, bone marrow–derived MSCs, or adipose-derived MSCs (191). The resulting recellularized tissue may be used as graft material. Alternatively, collagen sponges or gels may be used to deliver MSCs or other cells of interest (192).

For rotator cuff repairs, tissue engineers have focused on augmenting suture fixation with various biologic scaffolds derived from collagen-rich ECMs, such as dermis (e.g., GraftJacket® Regenerative Tissue Matrix, TissueMend® Soft Tissue Repair Matrix, and Zimmer® Collagen Repair Patch) and small intestinal submucosa (e.g., Restore® Orthobiologic Soft Tissue Implant and CuffPatch™ Soft Tissue Reinforcement) (193). Augmented grafts possess a biochemical composition similar to that of tendon (193) and increase suture fixation strength compared with unaugmented repairs (194).

9. CONCLUSION

Tendon is a compositionally complex tissue with unique structure, function, and mechanics. Injury to this vital connective tissue can result in significant pain and disability. For this reason, tendon healing and repair are topics of considerable research. This review discusses the current understanding of the biological, structural, and mechanical changes that occur during tendon healing, as well as strategies used to repair injured tendons and enhance tendon healing.

Despite the wealth of studies focused on tendon healing, much remains to be elucidated regarding this complex process. The in vitro cell-culture and in vivo animal models that have facilitated tendon research will continue to play a critical role in future experimental endeavors. Through an improved understanding of tendon healing, enhanced techniques for tendon repair and regeneration may be developed.

disclosure statement

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.