**2. Native tendons**

transplant rejections. Only autografts are easily transplanted—but the donor site morbidity may cause a lot of pain and go along with impaired function. In addition, other disadvantages of autografts are reported to be insufficient strength [2] because other tendons than the one to

Hence, an excellent alternative to decellularized tendons is the tissue engineered construct (TEC) aimed to be attached to tendon stumps [4] (**Figure 1**). In this field, tissue engineering has covered natural materials like collagen constructs [5], combinations of natural and synthetic components as realized in PLGA and alginate [6] or entirely synthetic polymers such as PCL ± PEO [7]. Many reports on seeding cells onto the corresponding materials have determined their impact, including extracellular matrix deposition and inherently going along changes in stability [8]. Other strategies include growth factors implemented in the graft material [9, 10] with the ultimate aim to be released sustainably to the repair site in order

**Figure 1.** Fabrication of a tissue engineered construct. As a first step, a scaffold material is used and processed as exemplified by electrospinning. Then, cells may be seeded onto the construct. After that, the cell-seeded construct may be cultivated under static conditions or under perfusion in a bioreactor before being implanted into an appropriate

animal model. As a final step, performance of the TEC is assessed in a clinical trial.

be replaced might be different in strength, cellularity as well as gliding capacity [3].

to support and accelerate the innate healing process [11].

162 Tissue Regeneration

#### **2.1. Structure and composition of tendons**

Before we turn our interest toward tissue engineering of tendons, a brief summary of what native healthy tendon tissue is composed of and of the characteristics of human and selected animal tendons is given here. The hierarchical structure of the tendon tissue is very well-known and has been characterized by multiple imaging and analysis techniques [14]. Starting with the smallest molecular entity, tropocollagen molecules assemble to form microfibrils. Covalently connected, these microfibrils form sub-fibrils and fibrils of the collagen which is typically seen in histological sections of tendon tissue as slightly waved "crimps" [15, 16]. Fibrils form bundles resulting in fascicles. Between the fascicles, there are cell-rich layers called endotenons that can be very well seen in histological sections, as the tenocytes form "lines," one behind each other connected by gap junctions that are important for mechanotransduction [17–19].

In addition, there are some tendons that have a peritenon around the whole tendon. The peritenon is a thin sheath around the tendon, but should not be confused with the tendon sheath on intrasynovial tendons [20]. More information on the organization of the tendon tissue are found in several articles [14, 21, 22], with a special emphasis on the extracellular matrix (ECM) [23].

In the ECM, the main component is collagen I (around 95% of the dry weight). The noncollagenous part of tendons is composed of proteoglycans like lubricin, decorin or biglycan, glycosaminoglycans (GAGs; typically encountered as chondroitin sulfate, dermatan sulfate or heparan sulfate) and glycoproteins such as fibronectin. Proteoglycans are important for tissue hydration (especially decorin) and it was found that they are essential for limiting the viscoelastic behavior by preventing tissue fatigue [24]. When GAGs of an extracted fascicle were enzymatically digested, they exhibited higher reductions in failure stress and more stress relaxation, supporting the regulation of viscoelasticity [24]. Noteworthy, water makes up 60–80 wt% of the entire tendon tissue and is—together with the GAGs—a highly important component regulating viscoelasticity [25]. Moreover, elastin has not to be neglected although it makes up only 2% of the tendon dry weight. Elastin fibers are found closely to the tenocytes the cells in the tendon tissue [26].

Tenocytes are the mature tendon cells, while tenoblasts are the immature ones. Tenoblasts build up the ECM components. They are spindle-shaped, very similar to fibroblasts—and their morphology changes upon aging [27] and mechanical loading [28]. Surface marker characterization of tenocytes and tenoblasts includes tenomodulin [29], which is induced by scleraxis (Scx), a transcription factor identifying tendon cells during development [30]. Other cell types occurring in tendon are synovial cells, typically found in the tendon sheath and synovial lining cells. One subtype of synovial lining cells produces hyaluronic acid, an important lubricant facilitating the gliding of the tendon in the sheath [31]. In addition, tendons do also have stem cells, primarily residing in a niche composed of biglycan and fibromodulin [32]. Like other adult stem cells, tendon stem cells are able to self-renew, form colonies, and differentiate into lineages like osteoblasts, chondrocytes, or adipocytes [33, 34].

only have ultimate stresses of 16 ± 6 MPa [42]. In terms of elasticity, the range for animals is also larger than for humans. While for human tendons it is up to around 800 MPa, for animals, values 1.5 times as high are found like for the rabbit *flexor digitorum profundus* which has an elastic modulus of 1166 ± 281 [43] or the horse *flexor digitorum superficialis* with a modulus of 1189 ± 63 MPa [44]. Xenografts, although rejection problems may arise, might nevertheless be useful starting points if refinements by cell seeding or other cues manipulating the graft are applied too. Otherwise, tendon tissue engineering intended at veterinary clinical application

Tissue Engineering of Tendons

165

http://dx.doi.org/10.5772/intechopen.73507

Tendon tissue basically consists of type I collagen [14, 45]. Therefore, many approaches in tendon tissue engineering take collagen as a material in order to fabricate appropriate TECs [46]. It has to be noted that mechanical properties of collagen greatly depend on the processing. Kumar and co-workers produced robust planar collagen fiber constructs by drying collagen gels to form dense collagen mats that were layered [47]. With this approach, they were able to tune ultimate stress values between 0.6 and 1.8 MPa; if they used an additional crosslinking step, the range of ultimate stress increased to 4.7 up to 10.5 MPa [47]. As for the elastic modulus, not cross-linked collagen mats exhibited elastic moduli of 2.0–6.3 MPa; with crosslinking, however, such fabricated mats had moduli of 52–114 MPa [47]. Obviously, with only one processing step (crosslinking), mechanical characteristics could be changed by an order of magnitude, enabling the tissue engineer to adapt his material to the mechanics envisioned.

Also, commercially available collagen scaffolds show a wide range of mechanical properties and may be chosen upon those selection criteria [48]. Generally, such scaffolds are patches that are used as augmentations in order to increase the primary repair strength after operation. The following commercially available collagen patches are described further in [48]. They are

**Table 1.** Selected commercially available collagen scaffolds sold as patches for tendon or ligament augmentation in the

**Trade name Tissue Ultimate load (N)**

GraftJacket® extr. 2.0 Human dermis 229 ± 72 MaxForce® 1. 4 Human dermis 182 ± 50 GraftJacket® 1.0 Human dermis 157 ± 38 Permacol® 1.0 Porcine dermis 128 ± 26 TissueMend® 1.1 Fetal bovine dermis 76 ± 22 TissueMend® 1.2 Fetal bovine dermis 70 ± 13 Restore® 1.0 Porcine small intestinal submucosa 38 ± 3 CuffPatch® 1.0 Porcine small intestinal submucosa 32 ± 4

should include such baseline values when planning to fabricate appropriate TECs.

**3. Natural materials**

presented with decreasing strength (**Table 1**).

order of decreasing strength according to [48].

**3.1. Collagen**

#### **2.2. Biomechanical baseline values**

For successful tissue engineering of tendons, it is essential to know the basic mechanical properties of the tendons that have to be reconstructed in order to plan processing steps accordingly. Hence, *ex vivo* determined biomechanical properties of target tendons are crucial and should always be taken as background information to compare (a) *in vitro* mechanical properties of TECs and (b) *in vivo* mechanical properties of TECs [13]. Tendon ultimate stress values of all human tendons are in a range of approximately 5–80 MPa. *Supraspinati* of the shoulder exhibit quite weak tendon tissue in the posterior portion with only 4 ± 1 MPa [35], while Achilles tendons have ultimate stresses of 79 ± 22 MPa [36].

The age influences the stability of the tendon tissue; while Achilles tendons of old people aged 79–100 years were reported to have ultimate stresses of 48 ± 16 MPa, younger people (36– 50 years old) had corresponding values of 73 ± 8 MPa; interestingly, an age group in between 52 and 67 years had the strongest Achilles tendons with 81 ± 14 MPa [37]. Besides age, also gender plays a significant role when mechanical properties of tendons are assessed and compared; female donors usually have weaker tendons and ligaments than male donors [38], however, weaker and softer Achilles tendons of women compared to men might also be a consequence of different levels of exercise—and therefore cannot only be attributed to gender [39].

Besides these intrinsic factors (age and gender), physical activity also plays an important role and has a major impact on tendon strength and elasticity. As a consequence, surgical intervention at a ruptured tendon of an athlete might need a different graft material compared to a ruptured tendon of a person that does not do any exercise beyond daily low-impact activities. Interestingly, also exercise in elderly people has a massive impact on tendon strength and elasticity. In a study performed with two groups of elderly people [aged 74 ± 5 years (n = 9) for group 1 and group 2 had and age of 68 ± 6 years (n = 8)], significant impact on the stiffness and elastic modulus of the patellar tendon was found when assessed by ultrasound measurements. Group 1 did one lesson of exercise per week going only to 40% of their maximum capacity, while group 2 had two lessons weekly and went to 80% of their highest capacity. As a result 12 weeks later, the elasticity of the tendons in group one was not changed, while group 2 had 1.6-fold increased stiffness and 1.5-fold increased elastic modulus [40]. Hence, intrinsic factors should not be interpreted alone; however, extrinsic factors like exercise and other physical activities should be considered too.

Compared to humans, the animal realm covers a wider range of mechanical properties; from rat tendons to horse tendons, there is a span of one order of magnitude in ultimate stress; with horse *flexor digitorum superficialis* having values of 109 ± 8 MPa [41], while rat Achilles tendons only have ultimate stresses of 16 ± 6 MPa [42]. In terms of elasticity, the range for animals is also larger than for humans. While for human tendons it is up to around 800 MPa, for animals, values 1.5 times as high are found like for the rabbit *flexor digitorum profundus* which has an elastic modulus of 1166 ± 281 [43] or the horse *flexor digitorum superficialis* with a modulus of 1189 ± 63 MPa [44]. Xenografts, although rejection problems may arise, might nevertheless be useful starting points if refinements by cell seeding or other cues manipulating the graft are applied too. Otherwise, tendon tissue engineering intended at veterinary clinical application should include such baseline values when planning to fabricate appropriate TECs.
