1.4 SILK FIBROIN IN TISSUE ENGINEERING
1.4.1 Bone tissue engineering
Bone is a rigid, dynamic, highly vascularised, connective tissue with a unique capacity to heal and remodel without leaving a scar. The major constituent of bone is mineralized osseous tissue which includes osteoblasts, osteocytes, osteoclasts, inorganic matter (hydroxyapatite) and organic matter (collagen). Other types of tissue found in bones include marrow, endosteum and periosteum, nerves, blood vessels and cartilage. The key function of the bone is to provide structural support and protect various internal organs of the body. Besides, they also serve as a mineral reservoir, support muscular contraction resulting in motion and withstand load bearing (Salgado et al, 2004). Hence, any major alterations in its structure due to injury or disease can severely alter one’s body equilibrium and quality of life. In spite of the progress made in bone regenerative medicine filed, current therapies, such as bone grafts, still suffer from serious limitations.
Consequently, alternative therapeutic strategies involving wide range of biomaterials to engineer the bone tissue are continuously being perused since last few years (Salgado et al, 2004; Sharma and Elisseeff 2004).
In this context, owing to its remarkable mechanical properties and proven biocompatibility, SF has received a significant attention as a biomaterial for bone TE (Weska et al, 2009; Xu et al, 2008; Jones et al, 2009; Kim et al, 2005; Kim et al, 2007;
Kim et al, 2008; Jiang et al, 2006; Meinel et al, 2006a; Hofmann et al, 2007; Zhang et al, 2010; Uebersax et al, 2006). For example, the implantation of a porous BmSF scaffold based tissue engineered bone implants (grown in bioreactors for 5 weeks prior to implantation) into calvarial critical size defects in mice demonstrated the capacity of these systems to induce advanced bone formation within 5 weeks (Meinel et al, 2005). The in vitro osteogenic ability of BmSF scaffolds seeded with human mesenchymal stem cells (hMSCs) and its in vivo ability to heal size femoral segmental defects in nude rats was also demonstrated successfully (Meinel et al, 2006b). The apatite-coated SF scaffolds combined with bone marrow stromal cells (bmSCs) were successfully used to repair mandibular critical size border defects and the premineralization of these porous SF protein scaffolds provided an increased osteoconductive environment for bmSCs to regenerate sufficient new bone tissue (Figure 1.8) (Zhao et al, 2009).
Besides using BmSF alone, blends of BmSF and Hydroxyapatite (HAp) are explored to a great extent because of their stoichiometric similarity to the organic and
inorganic parts of native bone, respectively (Leukers et al., 2005; Cui et al, 2007; Liu et al, 2008; Wang et al, 2008; Wang et al, 2009). For example, the preparation of HAp/SF composite and its ability to support the growth of mesenchymal cells towards bone regeneration was successfully demonstrated (Hirose et al, 2006). A composite of needle- like nano-Hydroxyapatite (n-HAp)/SF with good homogeneity, strong interfacial bonding and preferential orientation along c-axis was successfully prepared by Wang et al (2007).
SF-chitosan/n-HAp (SF-CS/n-HAp) based porous scaffolds fabricated through freeze drying technique were characterized by Wen et al (2007). Subsequently, the fabrication of n-HAp/SF sheets and their compatibility with rat bone marrow mesenchymal cells were also demonstrated (Tanaka et al, 2007). It was suggested that the surface of the n-HAp/SF sheets was covered with appropriate HAp crystal for mesenchymal cells adhesion/proliferation and that the sheets effectively support the osteogenic differentiation of mesenchymal cells.
Recently, the use of SF/HAp composite co-cultured with rabbit bmSCs in the healing of a segmental bone defect was evaluated. The subcutaneous implantation of SF/HAp scaffold combined with bmSCs into Sprague-Dawley rats with segmental bone defects demonstrated the capacity of these systems to repair the defect completely after 12 weeks of implantation, while the repair was incomplete by SF/HAp without bmSCs (Wang et al, 2010). Similarly, HAp/SF based composite scaffold was designed to induce and support the formation of mineralized bone matrix by hMSCs in the absence of osteogenic growth factors, where it was found that the incorporated HAp enhances the formation of tissue engineered bone through osteoconductivity of the material and by providing nucleation sites for new mineral (Bhumiratana et al, 2011). Besides synthetic HAp/SF, eggshell derived n-HAp blended with SF was also studied for its use in bone regeneration and found that the n-HAp from eggshells exerted successful bone formation in the rabbit calvarial bony defect model (Kweon et al, 2011).
Bone Morphogenetic Protein 2 (BMP-2) is a member of the transforming growth factor (TGF) super-family. It plays an important role in stimulating osteoblast differentiation and bone formation, and has been widely utilized in clinical bone repairing by implantation (Karageorgiou et al, 2004; Bessa et al, 2008). Recombinant human BMP- 2 (rhBMP-2) loaded BmSF scaffold were fabricated and studied in association with hMSCs for its feasibility in bone regeneration, where, it was found that in comparison to rhBMP-2 free BmSF scaffold, the loaded scaffold had efficiently produced more bone
formation (Kirker-Head et al, 2007). Lately, rhBMP-2, loaded n-HAp/SF porous scaffold, was found to promote the osteoblasts adhesion and proliferation and stimulated a significant increase in alkaline phosphatase activity of osteoblasts in vitro on the n- HAp/SF scaffolds (Zhang et al, 2011).
The microsphere-mediated growth factor (rhBMP-2 and recombinant human insulin-like growth factor (rhIGF-I)) delivery in polymer scaffolds and its impact on osteochondral differentiation of hMSCs was demonstrated (Wang et al, 2009). It was found that the silk microspheres were more efficient in delivering rhBMP-2 than rhIGF-I for hMSCs osteochondrogenesis. Thus, the silk microsphere/scaffold system offers a new option for the delivery of multiple growth factors with spatial control in a 3D culture environment for both understanding natural tissue growth process and in vitro engineering complex tissue constructs. The efficiency of SF microparticles as a delivery carrier for BMP-2 was also evaluated successfully in vitro and in vivo (Bessa et al, 2010a, 2010b). The premineralized silk scaffolds combined with BMP-2 modified bmSCs were found to be efficient in repairing mandibular bony defects in a rat model (Jiang et al, 2009). It was demonstrated that the presence of BMP-2 gene enhanced tissue-engineered bone in terms of the most new bone formed and the highest local bone mineral densities found. Thus, BMP-2 gene therapy and TE techniques could be used in mandibular repair and bone regeneration.
Electro spinning (e-spinning) is a versatile technique that enables the development of nanofiber-based biomaterial scaffolds. BmSF based e-spun nanofibrous scaffolds were widely studied to evaluate their efficiency in bone TE (Li et al, 2006; Ki et al, 2008; Park et al, 2010; Wei et al, 2011). The BmSF based scaffolds containing BMP-2 and/or nanoparticles of HAp prepared via e-spinning were found to help in bone formation from hMSCs (Li et al, 2006). It was found that the coexistence of BMP-2 and nanoparticles of HAp in the e-spun BmSF fibers resulted in the highest calcium deposition and upregulation of BMP-2 transcript levels when compared with the control systems. The in vitro and in vivo ability of e-spun BmSF scaffolds for bone regeneration in comparison to a commercially available porous 3D poly(lactic acid) (PLA) scaffold was also evaluated, where, the proliferation and alkaline phosphatase activity of osteoblasts was found to be higher on BmSF scaffolds than on PLA. Also, upon implantation at critical bone defect in rat calvaria, the bone regeneration was nearly 78.30% with BmSF while it was about 49.31% using PLA scaffolds, thus, the BmSF scaffold may be a good bone substitute for
bone regeneration in comparison to the commercially available PLA scaffold (Park et al, 2010). Lately, e-spun SF/n-HAp biocomposite was prepared by an effective calcium and phosphate alternate soaking method and successfully evaluated for its use in bone regeneration using osteoblaste-like MC3T3-E1 cell line (Wei et al, 2011).
The survival and functioning of a bone biomaterial requires a rapid and stable vascularization after implantation. The coculture of endothelial cells and osteoblasts on porous 3D SF scaffolds gradually lead to tissue-like self-assembly and formation of microcapillary-like structures and these microcapillary-like structures were intertwined between cell layers of osteoblasts (Unger et al, 2007). Thus, such coculture systems may be explored as a prevascularization strategy for biomaterials prior to implantation. Later, the formation of pre-vascular structures by human outgrowth endothelial cells from progenitors in the peripheral blood and the osteogenic differentiation of primary human osteoblasts on micrometric SF scaffolds were assessed by Fuchs et al (2009). The rationale was to gain more insight into the dynamic processes involved in the differentiation and functionality of both cell types depending on in vitro culture time. The study suggested a progressing maturation of the tissue construct with culture time which seemed to be not effected by culture conditions mainly designed for outgrowth endothelial cells. However, whether such in vitro pre-formed microvasculature persists and functions in vivo in an immune-deficient mice and how the host responds to the cell- containing scaffolds were also studied in great detail, where the in vitro pre-formed microcapillaries in a coculture system survive and anastomose with the host vasculature to become functional microcapillaries after implantation (Unger et al, 2010). Also, the coculture stimulates the host capillaries to rapidly grow into the scaffold to vascularize the implanted material. Thus, such coculture-based pre-vascularization of a biomaterial implant may have great potential in the clinical setting to treat large bone defects.
Figure 1.8. Apatite-coated silk fibroin scaffold seeded with bone marrow stromal cells was found to treat a bilateral inferior mandibular border full-thickness defect measuring 2 × 1 cm after 12 months post-operation. (a) Scaffold filled defect and (b) radiograph of implantation site after 12 months. (Reproduced with permission, Zhao et al, 2009).
Figure 1.9. Knitted silk-collagen sponge scaffold combined with hESC-derived mesenchymal stem cells were implanted into the dorsum of nude mice (a), and gross morphology of repaired tendons after 4 weeks post-operation (b). (Reproduced with permission, Chen et al., 2010).
Figure 1.10. (a) Macroscopic view of ACL reconstruction in pig knee joint (the arrow points to implant); Inset shows MSCs-seeded scaffold. (b) Macroscopic view of regenerated ACL in experiment group at 24 weeks postoperatively. (Reproduced with permission, Fan et al., 2009).