Connective Tissue: Tendon and Ligament
Connective tissue is one of the basic organizations of people and higher animals and consists of cells, fibers, and ECM. Connective tissue is widely dispersed with diverse forms, including tendons, ligaments, fascia and dermis, and has multiple functions of support, connection, nutrition, protection, etc. Connective tissue injury is very common in the clinical and surgical care .
Ligaments and tendons are dense connective tissues, composed of polysaccharide phases, proteoglycans, and protein phases, collagen and elastin, whose function is mainly to maintain the stability and normal movement of joints in the musculoskeletal system. Each year more than 800,000 people require medical care due to these structures damage, which can lead to significant joint instability and the development of other tissue injuries or degenerative joint disease [118-120]. Surgical repair is a commonly used clinical procedure, but it does not maintain normal activity and has a certain failure rate [121, 122]. Autologous and allografts have also become increasingly common for tendon and ligament repair [1, 123]. The structural properties of grafts should mimic native tendons and ligaments with sufficient mechanical properties and reasonable structure morphology [118, 120, 124, 125]. Although some scaffolds derived from natural and synthetic polymers have been investigated for repairing tendons and ligaments, the success of these materials as substitutes is limited due to the lack of mechanical properties, such as stiffness, strength, creep, ductility, axial splitting, elasticity and stress-shielding. There are many problems that most biomechanical properties of the scaffolds can’t simulate or sustainably maintain the biomechanical properties of the original ligaments and tendons [120, 125, 126]. Centered on these design goals of overcoming the limitations, a new generation of the synthetic composites in ligament and tendon tissue engineering tends to be heterogeneous from two or more materials through one or more manufacturing techniques. Many studies have found that the use of fiber- or tube- reinforcement is a viable method to obtain the ideal scaffold for the tendon and ligament repair.
Numerous groups have reinforced scaffolds with fibers to develop biomechanically functional tissue constructs for tendon and ligaments repair. As far as more methods are available for producing reinforced scaffolds for the repair of tendons and ligaments, Shepherd et al.  designed lyophilized structures of collagen- reinforced collagen-chondroitin-6-sulfate (C6S) material to produce bioactivities and biomechanical scaffolds for repairing and regenerating soft tissues such as tendons, ligaments as well as cartilage. The pore structures of the collagen-based composite were produced by axially aligned collagen fibers derived from acidic swollen gel type I collagen, and generally considered to be conducive to cell migration, proliferation and attachment. Although collagen-glycosaminoglycan has biological activities and porous structures favorable for cell infiltration and attachment, the poor mechanical properties reduce their range of applications in tissue engineering, particularly for the applications requiring tensile strength. Considering the carbodiimide-based cross-linking method, the collagen fiber bundles were extruded and then crosslinked in the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide solution. Next, they were mixed with the collagen C6S matrix slurry to be lyophilized for two times. The collagen C6S matrix reinforced by crosslinked fibers remained a pore structure and good interaction between fiber and matrix, and they bear the maximum load of about 60N before failure. Compared to the matrix that was not crosslinked, the collagen C6S matrix reinforced by cross- linked fibers had higher mechanical properties and more pore channels, which enhanced cell migration and provided the potential to guide aligned tissue regeneration, like tendons and ligaments.
De Santis et al.  designed a continuous fiber-reinforced polymer scaffold with mechanical properties like the native connective tissue that could be used as substitutes of tendons and ligaments. Many soft tissue stress-strain curves are similar in shape, and the Fig. 8.12 shows this nonlinear relationship. HydroThane™, Hydrophilic Thermoplastic Polyurethane by Cardio-Tech International, has excellent mechanical properties, but native connective tissues have higher the mechanical properties, as Fig. 8.12 shown. The polyethylenterephtalate (PET) fibers were used to reinforce HydroThane™ polyurethane matrix by filament winding to simulate the mechanical properties of ligaments and tendons (Fig. 8.13). The mechanical behavior of the composite could be seen as a function of the winding angle, so the value of the elastic modulus can be easily controlled by the angle of the different
Fig. 8.12 Stress-strain behavior of connective tissues and Hydrotane. The mechanical properties of natural connective tissue are higher than that of hydrogels, thus a composite structure must be designed to match the mechanical properties of tendons and ligaments (Adapted with permission from Ref. . Copyright 2004 Elsevier Ltd)
Fig. 8.13 HydroThaneTM reinforced PET as ligament prostheses. The composite structures was achieved by using the filament winding technology in order to simulate the morphology and mechanical properties of natural ligaments (Adapted with permission from Ref. . Copyright 2004 Elsevier Ltd)
fiber winding. In fact, the reinforcing effect of PET fibers in the HydroThane™ matrix with 35 °C winding angle was a better than that of 20 °C and 15 °C. The wide range of the fibers aligned along the load direction increased the modulus and strength of the scaffolds. The structure of the reinforced composite had a static stress-strain behavior close to the native ligament.
Webb et al.  investigated a poly(3-hydroxybutyrate-co-3 -hydroxyhexano- ate) (PHBHHx) scaffold with a PHBHHx fiber-reinforced collagen core used in a rat Achilles tendon repair mode, which had a good delayed biodegradability and
Fig. 8.14 Histological staining of explanted scaffolds. A microscopic assessment of the damage to the tendon site using Haematoxylin and Eosin confirmed evidence of cellular infiltration and partial structure regeneration along the fiber-tissue interface (indicated by the arrows). Different levels of sinusoidal morphology are observed at the interface with control (C), PHBHHx scaffold (S), PHBHHx scaffold-collagen (SC) and PHBHHx scaffold-collagen-tenocyte (SCT), where the last two present the greater degree of sinusoidal morphology (Adapted with permission from Ref. . Copyright 2013 Elsevier Ltd)
biocompatibility. The mechanical properties PHBHHx scaffolds with maximal loads at 23.73 ± 1.08 N in the mechanical testing could be compared with health rat tendons with maximal loads at 17.35 ± 1.76 N. Therefore, PHBHHx fiber-reinforced collagen gels were implanted into the lumen of the PHBHHx tube to produe a PHBHHx-based composite scaffold. The composite scaffolds encapsulated with tenocytes were implanted in rat model to verify that PHBHHx could be used to promote the repair of tendon tissue damage without inducing immune response or prolonging inflammation. During 40 days of culture in vivo, prolonged immunological or inflammatory responses did not occur in the rat model, and the functional recovery of the Achilles tendon was accelerated. The tenocytes not only migrated to the PHBHHx-collagen core but also formed new tendon tissue along the fiber surface of the core. Thus, the core promoted a regenerative cellular response based on the contribution of the mechanical properties of the scaffold and facilitated subsequent tissue reconstruction (Fig. 8.14). The PHBHHx-based scaffold with PHBHHx fiber-reinforced collagen core has a 3D structure morphology, which contributes to the successful applications in an in vivo tendon repair model.
Yang et al.  introduced a new aligned nanoyarn-reinforced nanofibers (NRS) scaffold by electrospinning with improved mechanical strength and cell infiltration for tendon tissue regeneration. Three different types of scaffolds consisting of the hybridization of poly (L-lactide-co-caprolactone) (P (LLA-CL)) and silk fibroin were prepared, that were aligned nanofibers scaffold, random nanofibers scaffold and NRS scaffold. The aligned and randomly NRS scaffolds had relatively high porosity and large pore sizes, which contributed to cell adhesion and migration, oxygen and nutrient delivery and metabolite discharge. The results of the three scaffolds cultured with marrow-derived mesenchymal stem cells (MSCs) in the bio-
Fig. 8.15 HE-stained histology images of MSCs interactions with the random nanofibrous scaffolds (a-c), aligned nanofibrous scaffolds (d-f) and NRS (g—i) on day 7 (a, d and g), 14 (b, e and h) and 28 (c, f and i). The MSCs (black arrow) are infiltrated from the surface of NRS at a depth of about 150 pm on day 7 (g), approximately 300 pm on day 14 (h) and approximately 500 pm on day 28 (i). Scale bars 100 pm for all images (Adapted with permission from Ref. . Copyright 2014 Elsevier Ltd)
compatibility analysis demonstrated that the reinforced nanoyarn nanofibers had the ability to enable cell infiltration and improve cell proliferation (Fig. 8.15). The MSCs on the NRS scaffolds showed an elongated and aligned growth pattern along the nanoyarn, but their distribution was not uniform in the yarns (Fig. 8.16). Furthermore, the tensile properties of the composite were enhanced in parallel to the direction of nanoyarns, which was required for the repair of tendons.
Hakimi et al.  developed a layered surgical scaffold by electrospinning and braiding for the repair of endogenous tendon. Electrospinning submicron materials with excellent cellular and tissue responses have promising results to be used as a scaffold material for tendon repair, however, whose mechanical properties are too poor to be surgically implanted or support the damage tendons during the period of recovery. In the published literature on tendon repair, several technologies only
Fig. 8.16 Confocal microscopy fluorescence images of the actin filaments (red) and nuclei (blue) of MSCs on the random nanofibrous scaffold (a), aligned nanofibous scaffold (b) and NRS (c) after 7-day culture. Magnifications of all images are 400x (Adapted with permission from Ref. . Copyright 2014 Elsevier Ltd)
improve marginal strength of the scaffold, which does not have close mechanical properties to tendon tissue. In this study, a bonding technology with biocompatible and non-destructive was developed to create a first prototype of the scaffold, where a multi-layered woven fabric was used to enhance an aligned electrospun sheet (Fig. 8.17). Both layers were made up of polydioxanone (PDO) with excellent compatibility, and they could be detected by ultrasonography, which could be used to locally inject cells or growth factors postoperatively . As expected, the weaving and electrospinning layer resulted in the scaffold strength of at least 20 times stronger than that of the electrospun layer in the tensile tests. The scaffold with the twill woven fabrics and aligned electrospun sheets exhibited a maximum suture trength at 167 N similar to that of human tendon. The multi-layered scaffold implanted in an in vivo rat model was gradually more embedded in dense tissue, and PDO fiber pattern was less visible by weeks 6 and 12 (Fig. 8.18). Moreover, the scaffold with a multi-layered reinforced structure maintained integrity during the duration of culture, and there was no separation or delamination of layers. For the manufacturing process is non-destructive to the aligned electrospun layers, the porosity and thickness of the electrospinning layer can be controlled, which provides the potential to design various scaffolds with reinforced electrospun sheets for more medical applications. The use of woven medical fabrics to enhance aligned electrospun layers has resulted in a promising implantable scaffold, which has the potential to improve the endogenous repair of the rotator cuff tendon.