1.4 SILK FIBROIN IN TISSUE ENGINEERING
1.4.6 Neural tissue engineering
Nerve system is a complex biological network, which is broadly categorized into peripheral nervous system (PNS) and central nervous system (CNS). The PNS consists of the cranial nerves arising from the brain, the spinal nerves arising from the spinal cord and sensory nerve cell bodies (dorsal root ganglia) and their processes. Peripheral nerves innervate muscle tissue, transmitting sensory and excitatory input to and from the spinal column (Schmidt and Leach, 2003). While the CNS, which includes the brain, spinal cord, optic, olfactory and auditory systems, conducts and interprets signals as well as provides excitatory stimuli to the PNS. In the PNS, nerves can regenerate on their own if injuries are small. Larger injuries must be surgically treated, typically with nerve grafts
harvested from elsewhere in the body. Spinal cord injury is more complicated, as there are factors in the body that inhibit repair. Unfortunately, a solution to completely repair spinal cord injury has not been found. Thus, bioengineering strategies for the peripheral nervous system are focused on alternatives to the nerve graft, whereas efforts for spinal cord injury are focused on creating a permissive environment for regeneration.
Fortunately, recent advances in neurobiology and biomaterials science provide optimism for new treatments for nerve injuries.
Yang et al (2007a) studied the biocompatibility of SF material with peripheral nerve cells and tissues, where, it was found that the SF extract fluid showed no significant cytotoxicity on rat sciatic nerve Schwann cells, and the substrate made up of SF fibers showed good biocompatibility with rat dorsal root ganglia (DRG). Later, Chen et al (2007) and Yang et al (2007b) demonstrated that the SF-based nerve graft, composed of a SF-NGC (nerve guidance conduit) inserted with oriented SF filaments, could promote peripheral nerve regeneration in rats over 6 months of implantation. Similarly, Tang et al (2009), investigated the suitability of SF as a candidate biomaterial for CNS therapy. The substrates made up of SF fibers demonstrated good biocompatibility with primarily cultured hippocampal neurons without any significant cytotoxic effects on their cell phenotype and functions, and the results were comparable with that of cells cultured in plain neuronal culture medium. Recently, Xu et al (2011) successfully demonstrated the fabrication of e-spun SF mats and evaluated its biocompatibility with SCs in vitro.
The fabrication of blend scaffolds composed of SF and other naturally derived polymers was investigated by Ren et al (2009). The SF-based scaffolds with 3-6% of hyaluronic acid showed improved affinity to primary neural cells. Additionally, in 6%
blend scaffolds, neurosphere-forming cell migrated from their original aggregate and adhered tightly to the surface of scaffolds. Tan et al evaluated the feasibility of using SF/CS as nerve conduit for facial nerve regeneration, where, it was found that 2, 4, 6 and 8 weeks of post-operative observations revealed the successful regeneration of facial nerve of rabbit (Tan et al, 2009).
Kim et al (2011) demonstrated the fabrication of natural/synthetic hybrid films using 0, 10, 20, 40 and 80 wt% of SF and PLGA, and found that the PLGA/SF hybrid film containing 40% and 80% of SF interrupted adhesion and proliferation of Schwann cells (SCs), while the films containing 10% and 20% of silk provided suitable
environment for growth and proliferation of SCs. Similarly, Wang et al (2011a) investiagetd the regeneration of 10 mm defect in the sciatic nerve of rats using SF/P(LLA-CL) (poly(l-lactic acid-co – caprolactone)) e-spun nanofibrous NGC.
The preparation of composite scaffold composed of a mixture of more than one natural and synthetic polymer has been studied by Wu et al (2009). The SCs viability, growth and proliferation were relatively higher in PLGA-SF-collagen e-spun scaffold than in e-spun PLGA nanofibrous scaffold alone. In a similar study, Wang et al (2011b) demonstrated the fabrication of e-spun nanofibrous scaffolds composed of different weight ratio PLGA-SF- collagen (50:25:25, 30:35:35), and evaluated their use in nerve TE. The biocompatibility assays using Schwann cells confirmed that PLGA-SF- collagen scaffolds particularly the one that contains 50% PLGA, 25% SF and 25% collagen is more suitable for nerve TE compared to PLGA nanofibrous scaffolds.
Madduri et al (2010) developed SF nerve conduit that were loaded with glial cell line-derived neurotrophic factor and nerve growth factor, and topographically functionalized with aligned and non-aligned SF nanofibers. The DRG sensory neurons and spinal cord (SpC) motor neurons, both from chicken embryos, exhibited an augmented length and rate of axonal outgrowth parallel to the aligned nanofibers. In addition, glial cells from DRG proliferated and migrated in close association and even slightly ahead of the outgrowing axons (Figure 1.12). On the contrary, axonal and glial growth was slower and randomly oriented on non-aligned nanofibers. The DRG and SpC explants were also inserted into the lumen of the finished SF nerve conduit. The unidirectional orientation of axo-glial outgrowth from the explants evidenced the preservation of the trophic and topographical functionalities in the SF nerve conduit.
The degradation behaviors of NGCs made up of SF in vitro and in vivo was investigated by Yang et al (2009). It was found that, after incubation in the protease XIV solution or subcutaneous implantation in rabbits, the SF-NGCs and SF fibers were able to degrade at a significantly increasing rate as compared to SF fibers, thus meeting the requirements of peripheral nerve regeneration. Furthermore, based on the possible involvement pathway in the in vivo degradation of SF-NGCs, the time-dependent changes in the mRNA level of lysosome-related genes (Hip1R, cathepsin D, and tPA) in subcutaneous implantation site within 24-week period post-implantation was determined
by real-time RT-PCR, and the resulting data might contribute to our understanding of the molecular aspects that affect in vivo degradation and absorption of SF-NGCs.