1.4 SILK FIBROIN IN TISSUE ENGINEERING
1.4.3 Ligament / tendon tissue engineering
(38.9%) and GAG content (54.3%) at day 21, in addition to facilitating the maturation of cartilage tissue. In the clinical feasibility studies, large defects on rabbit knee joints were repaired with regenerated cartilage, which resembled hyaline cartilage at 12 weeks after operation. Recently, the effects of perfusion bioreactors on the chondrogenic potential of engineered constructs prepared from porous SF scaffolds seeded with human embryonic stem cell derived MSCs was investigated, where, after four weeks of incubation, in comparison to static culture, constructs cultured in perfusion bioreactors showed significantly higher amounts of GAG, DNA, total collagen and collagen II, along with cartilage-related gene expression (Tigli et al, 2011). Also, the mechanical stiffness of constructs increased 3.7-fold under dynamic culture conditions and distinct differences were noted in tissue morphology, including polygonal extracellular matrix structure of engineered constructs in thin superficial zones and an inner zone under static and dynamic conditions, respectively. Thus, the dynamic culture conditions in bioreactors modulate the growth of tissue-engineered cartilage and enhance tissue growth in vitro, and such mechanically stimulated scaffold/cell constructs have great potential to support chondrogenesis in vivo.
methods. Application of cell-matrix composites with an appropriate combination of cells and biochemical cues have shown to affect the process of ligament and tendon healing.
Besides, it will also include mechanical manipulation of tissue environments to accelerate cell differentiation and to improve matrix formation (Woo et al, 1999; Kuo et al, 2010).
Besides providing unique benefits in terms of mechanical properties as well as biocompatibility and slow degradability, silkworm fiber matrices were found to provide support for the attachment, expansion of adult human progenitor bmSCs and its differentiation toward ligament lineages for in vitro engineering of anterior cruciate ligaments (ACL) (Altman et al, 2002a). A computer controlled bench-top bioreactor system with a capability to apply complex concurrent mechanical strains and its potential use towards the development of an engineered ACL to 3D matrices was demonstrated (Altman et al, 2002b). The system supported cell spreading and growth on the silk fiber matrices, as well as the differentiation of the cells into ligament-like cells and tissue.
The adhesion, spreading, proliferation and collagen matrix production of human bmSCs and ACL fibroblasts was found to be significantly higher on Arg-Gly-Asp (RGD)-modified silk fibers substrate than on the nonmodified group (Chen et al, 2003).
Similarly, in the rabbit medial collateral ligament (MCL) defect model, MCLs treated with knitted silk scaffold combined with collagen matrix deposited more collagen, had better mechanical properties, and showed more native microstructure with larger diameter collagen fibrils and stronger scaffold-ligament interface healing than untreated MCLs and those treated with silk scaffolds (Chen et al, 2008). Thus, the knitted silk/collagen sponge scaffold improves structural and functional ligament repair by regulating ligament matrix gene expression and collagen fibril assembly.
The use of nonwoven / braided / knitted SF based scaffolds for use in ligament / tendon TE was evaluated successfully (Dal Pra et al, 2005; Horan et al, 2009; Chen et al., 2010) (Figure 1.9). For example, the use of a silk-based scaffold with knitted architecture and its strengths as compared to previous poly-lactide-co-glycolide (PLGA)-based knitted scaffolds investigated (Toh et al, 2006). It was found that the e-spun nanofiber surface on knitted microfiber architecture is adopted and was found to have better composite- material integrity, in vitro degradation resistance, and encourages cell adhesion and proliferation. Seo et al estimated the mechanical properties and evaluated the biocompatibility of braided / knitted silk scaffold as an artificial ligament to an ACL
reconstruction (Seo et al, 2007). The mechanical strength and human ACL cell growth were significantly higher for silk matrices than PGA control scaffold. Notably, in vitro studies with T lymphocyte and mononuclear cell culture and in vivo studies in rats revealed that the immunogenic / inflammatory reactions were more conspicuous with PGA scaffold compared with silk matrices.
Fan et al (2008a, 2009) performed the in vivo experiments of in vitro grown MSCs/ silk scaffold constructs, where it was found that the direct ligament-bone insertion with typical four zones (bone, mineralized fibrocartilage, fibrocartilage, ligament) was reconstructed that resembled the native structure of ACL-bone insertion (Figure 1.10). In comparison to ACL fibroblasts the cell growth, proliferation, phenotype and ligament specific ECM production was found to be significantly higher by bmSCs as demonstrated by Liu et al (2008a). On the other hand, the adhesion, growth and cellular functions of hMSCs cultured on combined silk scaffolds were found to be more active in comparison with the knitted silk scaffolds seeded with MSCs in SF gel (Liu et al., 2008b). Thus, while the knitted structure holds the microporous silk sponges together and provides the structural strength of the combined silk scaffold, the microporous structure of the silk sponges mimic the ECM and promotes cell proliferation, function, and differentiation.
The application of a combination of sequential in vitro biochemical and mechanical stimuli to developing bmSCs/silk cultures offers the potential to create biomimetic ligament tissue. For example, the sequential administration of growth factors (epidermal growth factor, bFGF - basic fibroblast growth factor and then TGF- β) was found to first proliferate and then differentiate bmSCs cultured on RGD-modified silk fiber matrices (Moreau et al, 2005). The sequential application of growth factors through extended culture, to reinforce the effectiveness of bFGF/TGF-β as the optimal g rowth factor regimen, revealed the positive effects of sequential biochemical and mechanical stimulation on the development of optimized ligament tissue (Moreau et al, 2006; 2008).
Additionally, a unique correlation between innate MSCs development processes on a surface-modified silk matrix and dynamic environmental signaling was identified, where, an appropriate time frame for applying mechanical stimuli to induce bmSCs differentiation for ligament TE (Chen et al, 2006).
The use of blend scaffolds composed of SF and other natural materials, including natural polymers, for use in ligament / tendon TE have been investigated (Fan et al,
2008b). For example, Takezawa et al evaluated an "On vitrigel" system - 3D floating culture system, to culture the fibroblasts on a pressed silk sheet and type-I collagen based scaffold to reconstruct hard connective tissue such as ligaments, tendons, and other connective tissues (Takezawa et al, 2007). Liu et al demonstrated that gelatin coated silk fibers (prepared using using nordihydroguaiaretic acid as a cross-linking agent), unlike native silk fibers, are biocompatible with little or no inflammatory reaction after 4 weeks of subcutaneous implantation in rats (Liu et al, 2007). A composite scaffold composed of SF fibers wrapped with pig SIS and fabricated and characterized by Cui et al, where, the biomechanical properties of this scaffold were found to match with those of natural ligament (Cui et al, 2009). The in vitro and in vivo studies using silk/collagen-hyaluronan composite scaffold revealed that the scaffold is biocompatible and supports cell migration and new blood vessel formation (Seo et al, 2009).
Similarly, the use of blend scaffolds composed of SF and other synthetic polymers for use in ligament / tendon TE have been investigated. For example, Bosetti et al fabricated a composite scaffold composed of SF fibers and polyelectrolyte modified HEMA hydrogel (HEMA-co-METAC) and revealed that the scaffold possess acceptable biomechanical behavior and successfully supported growth and differentiation of hMSCs (Bosetti et al, 2008). Sahoo et al demonstrated the fabrication of biocompatible and mechanically robust hybrid nano-microscaffolds by coating the silk fibers with an intervening adhesive layer of silk solution followed by e-spun (PLGA) nanofibers (Sahoo et al, 2010a). Similarly, a biohybrid fibrous scaffold system by coating bioactive bFGF- releasing ultrafine PLGA fibers over knitted microfibrous silk scaffolds was developed and its ability to stimulate mesenchymal progenitor cell proliferation, subsequent tenogeneic differentiation and ligament/tendon-specific ECM production was successfully evaluated (Sahoo et al, 2010b).
Hairfield-Stein et al studied the preparation and characterization of scaffold-free ligament analogs from a clinically relevant cell source (Hairfield-Stein et al, 2007).
Porcine bmSCs were seeded on laminin-coated substrates with silk suture segments as anchor points. Cells developed into monolayers that subsequently delaminated and self- organized into cohesive rod-like tissues that were held in tension above the substrate. It was found that, mechanically and histologically, engineered ligament resembled native embryonic connective tissue and had an ultimate stress approximately 15% of native adult mouse tissue. Fan et al investigated the feasibility of using co-culture system to induce
the differentiation of MSCs on silk cable-reinforced gelatin/SF hybrid scaffold for constructing the tissue-engineered ligament in vitro (Fan et al, 2008c). Notably, the MSCs in co-culture system were found to differentiate into ligament fibroblasts by expressing ligament ECM specific genes including collagen I, collagen III and tenascin- C, and thus the specific regulatory signals released from fibroblasts in 3D co-culture system can enhance the differentiation of MSCs for ligament TE. Recently, Sell et al (2011) evaluated the suitability of nanofibrous scaffold by airgap e-spinning for ligament TE and found that the morphological, physical and mechanical properties of air gap e- spun scaffolds were superior to traditionally e-spun scaffolds. Also, the in vitro cell culture experiments using human dermal fibroblasts revealed that the degree of cellular orientation and penetration on airgap e-spun structures was significantly increased in contrast to their traditional e-spun counterparts.