**3. Natural materials**

#### **3.1. Collagen**

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

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

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

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

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

of different levels of exercise—and therefore cannot only be attributed to gender [39].

lineages like osteoblasts, chondrocytes, or adipocytes [33, 34].

Achilles tendons have ultimate stresses of 79 ± 22 MPa [36].

other physical activities should be considered too.

**2.2. Biomechanical baseline values**

164 Tissue Regeneration

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


**Table 1.** Selected commercially available collagen scaffolds sold as patches for tendon or ligament augmentation in the order of decreasing strength according to [48].

Depending on the processing technique and the final architecture and structure of the collagen scaffold, ultimate stress and elastic modulus vary over a range of six orders of magnitudes, with the following increasing order:

considerably enhanced by the presence of silk fibroin in this blend compared to mere PCL [63] because silk is a very stress-resistant material and can be tuned so well in order to cover a wide range of mechanical properties; it has not only been considered for tissue engineering

Tissue Engineering of Tendons

167

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

Chitin and its derivative chitosan are getting more and more attractive as a suitable natural biomaterial for tissue engineering purposes [68], especially for tendons [69]. In a combination with poly acrylic acid, composite films were fabricated by a layer-by-layer technique. These films exhibited elastic moduli of 27–420 kPa suitable for tissue engineering of tendons exhibiting low elastic moduli [70]. Other composites like chitosan-hyaluronic acid were used to close defects of *infraspinatus* in a rabbit model. The result was that ultimate stress and elastic modulus were significantly increased as compared to defects closed without this scaffold [71]. Moreover, the same composite material was also used for *medial collateral ligament* reconstruction in a rabbit model and it was found to be a promising substitute in case cells were seeded

Chitosan in combination with collagen has also been investigated to serve as a material for tissue engineering: addition of chitosan to bovine and salmon collagen scaffolds improved the mechanical properties by increasing the compressive strength and the swelling ratio [73]. Moreover, a rat Achilles tendon study, where a scaffold based on chitosan-β-glycerophosphate-collagen was

If synthetic polymers are used for (tendon) tissue engineering, the fabrication process highly decides upon its biocompatibility and its effectiveness as graft. As nicely shown by Prof. Ratner, the same polymer, once applied as a porous foam and once as a dense block, can evoke quite different reactions of the body: while the porous material is penetrated by ingrowing cells as well as vasculature and there is practically no foreign body reaction, the dense block is encapsulated as a foreign body going along with an inflammation reaction [75]—*in vivo veritas* [76]. Hence, the processing of a synthetic material, mostly polymers in tendon tissue engineering, has to be optimized in order to get a biocompatible material that fulfills the requirements

Many polymers have been synthesized and modified in order to get suitable materials in terms of implants for tendon repair and regeneration; polyglycolic acid (PGA) [75, 77], polylactic-co-glycolic acid (PLGA) [78], PLGA/alginate composite [6], polylactic acid (PLA) [79–81], poly-l-lactic acid (PLLA) [82–84], polycaprolactone (PCL) [85], polycaprolactone/ polyethylene oxide (PCL/PEO) [7], polyurethane (PU) [86, 87], polyethylene terephthalate (PET) [88, 89], DegraPol® [90, 91] (**Figure 2**), nanocarbon fiber [92], and polyurea [93], among others. The architecture of the synthetic materials has to be chosen carefully, as gene expression of (stem) cells may be significantly influenced by the microenvironment that the cells

used, demonstrated the effectiveness of this composite material for this purpose [74].

of tendons but also for applications in bone tissue engineering [64–67].

**3.3. Chitosan**

on the chitosan-hyaluronan [72].

**4. Synthetic materials**

encounter [94].

encountered in tendon tissue engineering.

sponges < gels < yarns, mats < cross-linked mats < cross-linked yarns <3D extruded fibers

As shown by Kato and colleagues, extruded collagen fibers highly resemble mechanical characteristics of rat tail tendon tissue [49], with elastic moduli >1000 MPa and ultimate stress >600 MPa. However, these mechanical assessments were made under dry conditions. As tendons are hydrated tissues, wet conditions should rather be taken into account. For that reason, Zeugolis et al. compared extruded collagen fibers under wet and dry conditions and found that wet extruded fibers were swelling (increase in CSA), while ultimate stress values decreased by factors up to 2000 [50]. Therefore, other optimization strategies like blending collagen with PEG (polyethylene glycol) were undertaken in order to achieve not only the desired fiber thickness but also envisioned mechanical properties [51]. Moreover, crosslinking of extruded collagen fibers with different chemical agents like aldehydes and isocyanates, biologically by microbial transglutaminase or physically by photo-oxidation was compared in terms of fiber diameter and mechanical properties [52]. A total of 16 different ways for crosslinking were compared and the high variability in characteristics was summarized [52].

#### **3.2. Silk**

Silk is derived from silkworm cocoons named *Bombyx mori* (mulberry silk) consisting of two fibroin proteins, and has been approved by the Food and Drug Administration [53]. The physical properties of silk fibroin (which is achieved after sericin is removed) are ideal for tendon grafts. Moreover, silk fibroin is biodegradable and compatible and can also be structurally changed and adapted for different purposes [54]. Silk fibroin exhibits ultimate stress values up to 4800 MPa, which is far beyond maximum ultimate stress limits of human tendons (approximately 80 MPa) and animal tendons (around 120 MPa). Physical properties can be tuned by giving the silk fibroin different architectures. Li and Snedeker showed that wired, braided, and straight silk fibroin fibers behaved differently in biomechanical fatigue tests [55]. They found that a wired structure best fitted their final target which was an anterior cruciate ligament. In addition, also knitted silk fibroin gained from a non-mulberry silk intended at tendon tissue engineering has been tested *in vitro*, and Musson and co-workers found that cell attachment and growth was satisfactory [56]. Finally, biphasic silk fibroin scaffolds with different pore alignments (anisotropic and isotropic) mimicking the tendon-bone interface are very promising TECs based on this natural material [57].

Often, silk is combined with other materials like collagen [58, 59], PDLLA [60], or PLGA [61, 62] in order to manipulate and adapt the TEC under view. As a promising example, silk fibroin was combined with PCL and electrospun nanofibers of this blend were seeded with rabbit dermal fibroblasts, with the result that silk fibroin favored and supported cell proliferation compared to blank PCL and tendon-specific proteins like collagen and tenascin-C were increased and deposited to a higher amount in an *in vivo* experiment using New Zealand White rabbits and an Achilles tendon partial defect [63]. Moreover, also biomechanics were considerably enhanced by the presence of silk fibroin in this blend compared to mere PCL [63] because silk is a very stress-resistant material and can be tuned so well in order to cover a wide range of mechanical properties; it has not only been considered for tissue engineering of tendons but also for applications in bone tissue engineering [64–67].

#### **3.3. Chitosan**

Depending on the processing technique and the final architecture and structure of the collagen scaffold, ultimate stress and elastic modulus vary over a range of six orders of magni-

As shown by Kato and colleagues, extruded collagen fibers highly resemble mechanical characteristics of rat tail tendon tissue [49], with elastic moduli >1000 MPa and ultimate stress >600 MPa. However, these mechanical assessments were made under dry conditions. As tendons are hydrated tissues, wet conditions should rather be taken into account. For that reason, Zeugolis et al. compared extruded collagen fibers under wet and dry conditions and found that wet extruded fibers were swelling (increase in CSA), while ultimate stress values decreased by factors up to 2000 [50]. Therefore, other optimization strategies like blending collagen with PEG (polyethylene glycol) were undertaken in order to achieve not only the desired fiber thickness but also envisioned mechanical properties [51]. Moreover, crosslinking of extruded collagen fibers with different chemical agents like aldehydes and isocyanates, biologically by microbial transglutaminase or physically by photo-oxidation was compared in terms of fiber diameter and mechanical properties [52]. A total of 16 different ways for crosslinking were compared and the high variability in characteristics was summarized [52].

sponges < gels < yarns, mats < cross-linked mats < cross-linked yarns <3D extruded fibers

Silk is derived from silkworm cocoons named *Bombyx mori* (mulberry silk) consisting of two fibroin proteins, and has been approved by the Food and Drug Administration [53]. The physical properties of silk fibroin (which is achieved after sericin is removed) are ideal for tendon grafts. Moreover, silk fibroin is biodegradable and compatible and can also be structurally changed and adapted for different purposes [54]. Silk fibroin exhibits ultimate stress values up to 4800 MPa, which is far beyond maximum ultimate stress limits of human tendons (approximately 80 MPa) and animal tendons (around 120 MPa). Physical properties can be tuned by giving the silk fibroin different architectures. Li and Snedeker showed that wired, braided, and straight silk fibroin fibers behaved differently in biomechanical fatigue tests [55]. They found that a wired structure best fitted their final target which was an anterior cruciate ligament. In addition, also knitted silk fibroin gained from a non-mulberry silk intended at tendon tissue engineering has been tested *in vitro*, and Musson and co-workers found that cell attachment and growth was satisfactory [56]. Finally, biphasic silk fibroin scaffolds with different pore alignments (anisotropic and isotropic) mimicking the tendon-bone interface are

Often, silk is combined with other materials like collagen [58, 59], PDLLA [60], or PLGA [61, 62] in order to manipulate and adapt the TEC under view. As a promising example, silk fibroin was combined with PCL and electrospun nanofibers of this blend were seeded with rabbit dermal fibroblasts, with the result that silk fibroin favored and supported cell proliferation compared to blank PCL and tendon-specific proteins like collagen and tenascin-C were increased and deposited to a higher amount in an *in vivo* experiment using New Zealand White rabbits and an Achilles tendon partial defect [63]. Moreover, also biomechanics were

very promising TECs based on this natural material [57].

tudes, with the following increasing order:

**3.2. Silk**

166 Tissue Regeneration

Chitin and its derivative chitosan are getting more and more attractive as a suitable natural biomaterial for tissue engineering purposes [68], especially for tendons [69]. In a combination with poly acrylic acid, composite films were fabricated by a layer-by-layer technique. These films exhibited elastic moduli of 27–420 kPa suitable for tissue engineering of tendons exhibiting low elastic moduli [70]. Other composites like chitosan-hyaluronic acid were used to close defects of *infraspinatus* in a rabbit model. The result was that ultimate stress and elastic modulus were significantly increased as compared to defects closed without this scaffold [71]. Moreover, the same composite material was also used for *medial collateral ligament* reconstruction in a rabbit model and it was found to be a promising substitute in case cells were seeded on the chitosan-hyaluronan [72].

Chitosan in combination with collagen has also been investigated to serve as a material for tissue engineering: addition of chitosan to bovine and salmon collagen scaffolds improved the mechanical properties by increasing the compressive strength and the swelling ratio [73]. Moreover, a rat Achilles tendon study, where a scaffold based on chitosan-β-glycerophosphate-collagen was used, demonstrated the effectiveness of this composite material for this purpose [74].
