Chapter 5: Summary and Future prospects
2.2 Scaffolds in Neural Tissue Engineering
2.2.3 Electrical stimulation and electrically conductive scaffolds in Neural Tissue Engineering
2.2.3 Electrical stimulation and electrically conductive scaffolds in Neural
electrical stimulation’ (Kitchen et al, 2002). A variety of cellular responses to electric stimulation of different cell types, including fibroblasts, osteoblasts, myoblasts, chick embryo dorsal root ganglia and neural crest cells, have been reported (Schmidt et al., 1997; Kimura et al., 1998; Wong et al., 1994; Li et al., 2006; Bidez et al., 2006;Wood et al., 2006). Several theories have been suggested to explain the effect of electric stimulation on nerve regeneration. Patel et al. (1982) suggested three possible ways by which electrical stimulation could act directly on a neuron, including the redistribution of cytoplasmic materials, the activation of growth-controlling transport processes across the plasma membrane due to change in cell membrane potential, and the electrophoretic accumulation of surface molecules responsible for neurite growth or cell–substratum adhesion. Changes in ionic currents around the growing fibre tips induced by electric fields have been suggested by Freeman et al.
(1985) as one possible mechanism through which electrical stimulation can affect nerve cells. Sisken et al. (1989) suggested that electrical stimulation affects protein synthesis in transected sciatic nerve segments and stimulates neurite outgrowth in vitro. Kimura et al. (1998) postulated that gene expression for nerve growth factor (NGF) is electrically activated for rat neuronal pheochromocytoma cells (PC12 cells) by alternative potential, while Kotwal et al. (2001) showed that fibronectin adsorption increased with immediate electrical stimulation and explained enhanced neurite extension on electrically stimulated PPy films. Initial studies investigating the effect of electrical stimulation on neurons were performed on Xenopus neurons after exposing them to a steady direct current field. Extracellular electric fields (0.1–10 V/cm) applied in solution reversibly influenced the direction of neurite growth and increased the neurite initiation and length in Xenopus (Li and Hoffman-Kim, 2008).
Applied electrical fields have been shown to influence the rate and orientation of neurite outgrowth from cultured neurons in vitro (Valentini et al., 1992). For example, applied electric fields influenced the extension and direction of neurite outgrowth from neurons cultured in vitro and pulsed electromagnetic fields stimulated sciatic nerve regeneration in vivo (Wang et al., 2004). A separate study demonstrated that 7 days of electric field imposed within a damaged adult guinea-pig spinal cord can both induce the regeneration of axons and guide their growth into the ends of a hollow silicone rubber tube inserted into the dorsal half of the cord (Borgens et al,1999) (Fig 2.6).
Conductive polymer based scaffolds in neural tissue engineering
The importance of conductive polymeric composites is based on the hypothesis that such composites can be used to host the growth of cells, so that electrical stimulation can be applied directly to the cells through the composite, proved to be beneficial in many regenerative medicine strategies, including neural and cardiac tissue engineering (Bettinger et al., 2009). Conductive polymers show great promise in biomedicine and are stimulus-responsive polymers that can be synthesized to form composites that could serve as ‘smart’ biomaterials (Skotheim and Reynolds, 2007). The biggest limitation of conductive polymers for in vivo applications is their inherent inability to degrade, which may induce chronic inflammation and require surgical removal (Huang et al., 2007). To address the drawbacks of existing conductive polymers, attempts to blend them with suitable biodegradable polymers have been carried out. Zhang et al. (2010) synthesized a novel, electrically conductive and biodegradable polyphosphazene polymer containing aniline pentamer with glycine ethyl ester as side chains and evaluated its biocompatibility using Schwann cells. Their results showed that the polymer exhibited no cytotoxicity, indicating suitability of this polymer as a scaffold material for peripheral nerve regeneration or other biomedical devices that require electro-activity.
Polypyrrole
Polypyrrole (PPy) is a conductive synthetic polymer that has numerous applications in drug delivery and nerve regeneration and it has also been used in biosensors and coatings for neural probes (Sanghvi et al., 2005). Figure 2.7 (i) shows the chemical structure of PPy before and after doping. The high degree of conjugation in the molecular backbone of PPy makes it very rigid, insoluble and poorly processable. It is therefore very difficult to be used alone as a structural material and must be optimized and transformed into a mechanically manageable and processable form (Shi et al., 2004). Wang et al. (2004) showed that PPy has good biocompatibility with rat peripheral nerve tissue and showed it to be a suitable substrate for bridging the peripheral nerve gap. They reported that PPy extraction solution showed no evidence of acute and subacute toxicity, pyretogens, haemolysis, allergens and mutagenesis, and the migration of Schwann cells and the neurite extension from dorsal root ganglia on the surface of PPy membrane-coated glass were found to be
better than those on bare glass (Wang et al., 2004). George et al(2009) also fabricated conductive PPy tubes using the electrodeposition of PPy onto wire templates and subsequent separation from the template after electrochemical reduction. Conduits made of this material did not show any acute or active chronic inflammatory infiltrate or tissue damage in the surrounding tissues, for at least 8 weeks of in vivo implantation as sciatic nerve guides.
Polyaniline
Polyaniline (PANI) is the oxidative polymeric product of aniline under acidic conditions and is commonly known as aniline black (Nalwa, 1997). Depending on the oxidation level, PANI can be synthesized in various insulating forms, such as the fully reduced leucoemeraldine base, half-oxidized emeraldine base (PANI- emeraldine base) and the fully oxidized pernigraniline base. PANI emeraldine base is the most stable and widely investigated form of PANI (Le´on,2001). Fig 2.7 (ii) shows the reduced, oxidized and half oxidized forms of PANI (MacDiarmid and Epstein, 1994). Wang et al. (1999) investigated the in vivo tissue response to PANI and found no characteristic features resulting from tissue incompatibility after PANI implantation. In general, no significant inflammation at the implant site and no signs of abnormality of muscle and adipose tissues in the vicinity of the implants were observed (Guimarda et al., 2007).
Poly (3,4-ethylenedioxythiophene)
Poly (3,4-ethylenedioxythiophene) (PEDOT) is considered the most successful polythiophene derivative, with interesting properties.Its chemical structure is represented in Fig 2.7 (iii) Luo et al. (2008) synthesized PEDOT films and investigated their biocompatibility by seeding NIH3T3 fibroblasts on PEDOT films, and carried out subcutaneous implantation of PEDOT films by in vivo study. Their results showed that PEDOT films exhibit very low intrinsic cytotoxicity and that their inflammatory response upon implantation was good, making them ideal for bio- sensing and bioengineering applications. Implantable electrodes can be used for the treatment of different disabilities and neurological disorders and can be used either to electrically elicit neural impulses or to record neuron signalling (Asplund et al., 2009). Asplund et al. (2009) coated platinum electrodes with PEDOT and investigated the biocompatibility of resultant electrode. Further in vivo study using
polymer-coated implants in rodent cortex indicated that platinum electrodes coated with PEDOT were non-cytotoxic and showed no marked differences in immunological response in cortical tissue compared to pure platinum controls. Green et al. (2009) used anionically modified laminin peptides, such as DEDEDYFQRYLI and DCDPGYIGSR, to dope PEDOT electrodeposited on platinum
Fig 2.7: Chemical structure of conducting polymers. (i) Chemical structure of PPy: (A) before doping; (B) after doping, (ii) Polyaniline (PANI): (A) reduced; (B) oxidized; and (C) half-oxidized forms, (iii) The structure of PEDOT. (i) and (ii) Adapted with permission from Ghasemi-Mobarakeh et al, 2011, (iii) Adapted with permission from Balint et al, 2014).
electrodes and assessed the bioactivity of incorporated peptides and their effect upon nerve cell growth (PC12 cells). Longer neurite outgrowth was observed on PEDOT films doped with synthetic anionic laminin peptides than that on PEDOT films doped with conventional paratoluene sulphonate dopant. The interactions between neural cells and PEDOT for the development of electrically conductive biomaterials intended for direct and functional contact with electrically active tissues, such as the nervous system, heart and skeletal muscle, was also studied by Richardson-Burns et al. (2007).
Carbon nanotubes and Carbon nanofibers
Carbon nanotubes (CNTs) are another group of conducting polymers incorporated into non-conducting polymers to provide structural reinforcement and impart novel
properties, such as electrical conductivity, into the scaffolds and to direct cell growth (Harrison and Anthony,2007). CNTs are shown to be toxic to cells when used as a suspension in cell culture media in any given experiment, while they appear to be non-toxic if immobilized to a matrix or to a culture dish (Hussain et al., 2009).
Potential cytotoxic effects associated with carbon nanotubes may be mitigated by chemically functionalizing their surfaces (Harrison and Anthony, 2007). Chemically functionalized CNTs have been used successfully as potential devices to improve neural signal transfer while supporting dendrite elongation and cell adhesion. These results strongly suggest that the growth of neuronal circuits on a CNT grid is accompanied by a significant increase in network activity. In a study dissociated embryonic rat hippocampal neurons on a mat of multi-walled CNTs (MWNTs) were deposited onto a polyethyleneimine (PEI) covered glass. In this condition, neurons could grow and elongate their neurites in all directions. In fact, CNTs represent a scaffold composed of small fibres or tubes that have diameters similar to those of neural processes such as dendrites (Lovat et al., 2005). Interest in carbon nanofibres has also been growing exponentially due to their unique electrical, mechanical and surface properties. Webster et al. (2004) developed a carbon nanofibre-reinforced polycarbonate urethane composite in an attempt to determine the possibility of using carbon nanofibres as either neural or orthopaedic prosthetic devices. Their results showed that this composite supports neural cell function and has the ability to tailor electrical properties for polyurethane composites containing carbon nanofibers.
However, there are several limitations to the application of carbon nanotubes being non-degradable (Harrison and Anthony, 2007).