JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE REVIEW ARTICLE J Tissue Eng Regen Med 2011;5: e17–e35. Published online 10 January 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.383
Application of conductive polymers, scaffolds and electrical stimulation for nerve tissue engineering Laleh Ghasemi-Mobarakeh1 , Molamma P Prabhakaran2∗ , Mohammad Morshed3 , Mohammad Hossein Nasr-Esfahani4 , Hossein Baharvand5 , Sahar Kiani5 , Salem S Al-Deyab6 and Seeram Ramakrishna2,6∗ 1 Islamic
Azad University, Najafabad Branch, Isfahan, Iran Care & Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore 117576 3 Department of Textile Engineering, Isfahan University of Technology, Isfahan, Iran 4 Department of Cell and Molecular Biology, Royan Institute for Animal Biotechnology, Isfahan, Iran 5 Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 6 Department of Chemistry, Faculty of Science, King Saud University, Riyadh, Saudi Arabia 2 Health
Abstract Among the numerous attempts to integrate tissue engineering concepts into strategies to repair nearly all parts of the body, neuronal repair stands out. This is partially due to the complexity of the nervous anatomical system, its functioning and the inefficiency of conventional repair approaches, which are based on single components of either biomaterials or cells alone. Electrical stimulation has been shown to enhance the nerve regeneration process and this consequently makes the use of electrically conductive polymers very attractive for the construction of scaffolds for nerve tissue engineering. In this review, by taking into consideration the electrical properties of nerve cells and the effect of electrical stimulation on nerve cells, we discuss the most commonly utilized conductive polymers, polypyrrole (PPy) and polyaniline (PANI), along with their design and modifications, thus making them suitable scaffolds for nerve tissue engineering. Other electrospun, composite, conductive scaffolds, such as PANI/gelatin and PPy/poly(ε-caprolactone), with or without electrical stimulation, are also discussed. Different procedures of electrical stimulation which have been used in tissue engineering, with examples on their specific applications in tissue engineering, are also discussed. Copyright 2011 John Wiley & Sons, Ltd. Received 5 March 2010; Accepted 12 October 2010
Keywords tissue engineering; electrical stimulation; conductive polymers; nanofibres; nerve cells; modification
1. Introduction The nervous system plays a central and complex role in human biological processes interacting in physiological processes, such as cognition and individual cell function. Damage to the nerve may impose tremendous *Correspondence to: Molamma P Prabhakaran or Seeram Ramakrishna, Health Care and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore 117576. E-mail:
[email protected] or
[email protected] Copyright 2011 John Wiley & Sons, Ltd.
consequences and its recovery is difficult. Moreover, malfunctions in other parts of the body might also occur because mature neurons do not undergo cell division (Huang et al., 2006). Neurodegenerative disorders of the spinal cord and brain after injury, stroke or multiple sclerosis have increased over the past few years (Prabhakaran et al., 2009). Peripheral nerve lesions are also common, with serious injuries affecting 2.8% of trauma patients annually, leading to lifelong disability (Ciardelli and Chiono, 2006). In the USA, 360 000 people suffer from upper extremity paralytic syndromes on an annual basis and approximately 253 000 people in the USA live with the after-effects of spinal cord injury. Moreover, each
e18
year this number grows by an estimated 11 000 people in the USA (Willerth and Sakiyama-Elbert, 2007) and in Europe more than 300 000 cases of peripheral nerve injury are reported annually (Ciardelli and Chiono, 2006; Bueno and Shah, 2008). Numerous strategies have been applied for the repair of peripheral nerve lesions, with the common goals of directing the regenerating nerve fibres into the proper distal endoneural tubes and improving the prospects of axonal regeneration and functional recovery (Schmidt et al., 1997). Implantation of autografts, allografts and xenografts, providing grafts from the patient, cadavers and animals, respectively, are a few strategies applied in this field. But the loss of function at the donor nerve graft site and mismatch of damaged nerve and graft dimensions are major disadvantages of using autograft nerve repair system (Schmidt et al., 1997; Schmidt and Leach, 2003). On the other hand, allogenic and xenogeneic tissues have the advantages of their availability, along with the benefit of not requiring harvesting from patients. However, their disadvantages include disease transmission and problems of immunogenicity (Schmidt and Leach, 2003). Tissue engineering provides a new medical therapy as an alternative to conventional transplantation methods, which regulates the cell behaviour and tissue progression through the development and design of synthetic extracellular matrix (ECM) analogues of novel biomaterials to support three-dimensional (3D) cell culture and tissue regeneration (Yang et al., 2004a; Subramanian et al., 2009). The fundamental approach in neural tissue engineering involves the fabrication of polymeric scaffolds seeded with nerve cells to produce a 3D functional tissue suitable for implantation (Yang et al., 2004). Historically, tissue-engineering strategies have been used in an effort to develop therapies for peripheral nerve and spinal cord injury, combining biomaterials, cell therapy and drug delivery approaches (Li and Hoffman-Kim, 2008). Successful nerve regeneration requires tissueengineered scaffolds that provide not only mechanical support for growing neurites and prevention of ingrowth of fibrous scar tissue, but also biological signals to direct the axonal growth cone to the distal stump (Huang et al., 2006). Recently, a synthetic nerve guidance channel has provided surgeons with an interesting option to bridge severed nerves instead of conventional methods including autografts, allografts and xenografts (Zhang et al., 2007), and numerous efforts have been devoted towards the development of synthetic guidance conduits for the repair of peripheral nerve defects (Hadlock et al.,2000; Kijima et al., 2009; Rooney et al., 2008a; Ellis and Chaudhuri, 2008; Jiang et al., 2008; Rooney et al., 2008; Patel et al., 2009; Houchin-Ray et al., 2009; Park et al., 2007). Nerve guidance channels are biomaterials-based devices that are designed to be transplanted for nerve repair, experimentally studied as a possible alternative to nerve grafts. Nerve guidance channels aim to provide a conduit through which regenerating axons can grow and connect to their appropriate targets (Li and Hoffman-Kim, 2008). An appropriate synthetic material for fabricating Copyright 2011 John Wiley & Sons, Ltd.
L. Ghasemi-Mobarakeh et al.
nerve guidance channels must readily shape to a conduit with the desired dimensions, must be sterilizable, tear-resistant, easy to handle and suture, biodegradable, and should maintain its shape and resist collapse during implantation over the course of nerve regeneration (Blacher et al., 2003). The physical, chemical and electrical properties of synthetic conduit affect the outcome of nerve regeneration. The inherent nature of neurons is to transmit electrochemical signals throughout the nervous system and, as a result, they are highly influenced by electrical stimuli (Yu et al., 2008b). Previous studies have shown that electrical stimulation is an effective cue in stimulating either the proliferation or differentiation of various cell types (Zhang et al., 2007; McCaig and Zhao, 1997; Sun et al., 2006; Ciombor and Aaron, 1993; Aaron and Ciombor, 1993; Goldman and Pollack, 1996; Dust and Bawornluck, 2006; Shi et al., 2008; Zhao et al.,1996, 1999; Yaoita et al.,1990; Guimarda et al., 2007; Rivers et al., 2002; Jeong et al., 2008; Whitehead et al., 2007; Ateh et al., 2006; Schmidt et al., 2003; Valentini et al., 1992). In this review we discuss the electrical properties of nerve cells, the most commonly utilized conductive polymers, namely polypyrrole (PPy) and polyaniline (PANI), the principle of electrical stimulation and the application of electrical stimulation through conductive scaffolds to nerve cells. A brief evaluation of the methods of electrical stimulation, electrospinning of conductive polymers and modifications carried out to conductive scaffolds, thus making them suitable substrates for tissue engineering, are also discussed.
2. Electrical properties of nerve cells Recent interest in electrical stimulation arises from a growing knowledge of the electrical properties of tissues and cells (Snyder-Mackler, 1987). Living cells employ many of the properties of electrical systems. For example, they generate electromotive force, maintain a required difference in potential, increase or decrease the difference in potential as necessary, use varying resistances in series or parallel, switch current on and off, control current flow, rectify current flow and store charge, which is even more crucial (Kitchen, 2002). An electrical voltage exists across the plasma membrane, while the inside of the cell remains more negative than the outside. By convention, the potential outside the cell is called zero; therefore, the typical value of the membrane potential is −60 to −100 mV (Matthews, 2003). This potential difference is maintained at a steady level when excitable cells are inactive and is called the resting potential (Paul, 1975). Regarding the electrical properties of cells, electrical signals strongly affect cell behaviour, affecting ion influx across the cell membrane, altering the membrane potential and conditioning the intracellular signal transduction pathways (Mattioli-Belmonte et al., 2003). The transition of information from one place to another in the nervous system takes place along the axon. It is known that the activity in axons is accompanied by J Tissue Eng Regen Med 2011;5: e17–e35. DOI: 10.1002/term
Conductive polymers, scaffolds and electrical stimulation for nerve tissue engineering
e19
polarity for a brief period before re-establishing the resting potential.
3. Electrical stimulation and its effects
Figure 1. Various stages (resting state, depolarization and repolarization) of nerve cells during electrical stimulation
electrical changes in and around them and the specific electrical event that occurs is called the action potential, which is an active response generated by the neuron that appears on an oscilloscope as a brief (∼1 ms) change from negative to positive in the transmembrane potential. The action potential represents transient changes in the resting membrane potential. One way to elicit an action potential is to pass an electrical current across the membrane of the neuron. Several steps are involved during this process, the first being a stimulus received by the dendrite of a nerve cell. This causes the Na+ channels to open and, if the opening is sufficient to drive the interior potential from −70 mV to −55 mV, it reaches the action threshold. In this step, more Na+ channels are opened and the Na+ influx drives the interior of the cell membrane up to about +30 mV. The process at this point is called depolarization. Further, the Na+ channels close and the K+ channels are opened, and since the K+ channels are much slower to open, the depolarization process is completed. The membrane begins to repolarize back towards its resting potential as the K+ channels open. The repolarization typically overshoots the resting potential to about −90 mV, when it is termed hyperpolarization and prevents the neuron from receiving another stimulus, or at least raises the threshold for a new stimulus. Hyperpolarization also assists in preventing any stimulus that has already been sent up an axon from triggering another action potential in the opposite direction. In other words, hyperpolarization assures that the signal is always proceeding in one direction. After hyperpolarization, the Na+ /K+ pump eventually brings the membrane back to its resting state of −70 mV (Kandel, 2000). Typically, the membrane must be depolarized by about 10–20 mV in order to trigger an action potential. Figure 1 shows a schematic illustration of the resting state, depolarization, action potential and repolarization of nerve cells. As can be observed in this figure, in response to the appropriate stimulus, the cell membrane of a nerve cell goes through a sequence of depolarization from its resting state, followed by repolarization to that resting state. In the sequence, it actually reverses its normal Copyright 2011 John Wiley & Sons, Ltd.
An action potential can be elicited artificially by changing the electrical potential of a nerve cell by inducing an electrical charge to the cells, and the process is termed ‘electrical stimulation’ (Kitchen, 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). The proposition related to electrical stimulation is based on the fact that bioelectricity present in the human body plays an integral role in maintaining normal biological functions, such as signalling of the nervous system, muscle contraction and wound healing (Shi et al., 2008). McCaig et al. (1997) reported the generation of electrical fields during major cellular events such as cell division, development and migration. They also found that the presence of a steady weak direct current (DC) electrical field in some biological systems affects cellular activities such as cell division, differentiation, migration and the extension of motile processes (Zhao et al., 1999). Endogenous electric fields in the form of voltage gradients have been observed to polarize the nervous system along the rostral–caudal axis (5–18 mV/mm) and to direct nerve growth (Li and Hoffman-Kim, 2008). 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). Borgens (1999) 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 (1999) concluded that this was due to production of a DC voltage gradient within the injured spinal cord, with the cathode located within the experimental tubes. Electrical stimulation has been shown to influence the differentiation of stem cells. Yamada et al. (2007) showed that mild electrical stimulation strongly influences embryonic stem cells to assume a neuronal fate. Although the resulting neuronal cells showed no sign of specific terminal differentiation in culture, they showed potential to differentiate into various types of J Tissue Eng Regen Med 2011;5: e17–e35. DOI: 10.1002/term
e20
neurons in vivo, and contributed to the injured spinal cord as neuronal cells. The induction of calcium ion influx is significant in this differentiation system. 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.
3.1. Method of electrical stimulation in the tissue engineering context Electrical stimulation is a relatively simple, flexible and feasible method carried out for both in vivo and in vitro two-dimensional (2D) and 3D cultured cells (Sun et al., 2006). The behaviour of an excitable cell such as nerve cells can be modified by application of electrical current through two external electrodes. The current passing between the electrodes can cause depolarization of the membrane (Paul, 1975). Surface electrodes, usually made of silver plates, available in different sizes in the range 0.5–1.0 cm in diameter, are commonly used for clinical applications. Gold and platinum electrodes have also been used for electrical stimulation procedures (Kimura, 2001). Some researchers applied voltage to conductive materials, considering it as one of the electrodes, where another electrode was used separately as anode or cathode. For example, Schmidt and co-workers (1997) utilized PPy film as the anode and a gold wire as the cathode for the electrical stimulation of PC12 cells. In other cases, voltage was applied between two electrodes through conductive scaffolds seeded with cells (Shi et al., 2007, 2008; Ghasemi-Mobarakeh et al., 2009). Shi et al. (2007) studied the effect of electrical stimulation in culture media and did not detect any measurable variations in pH or temperature during the period of cell culture. Moreover, no biologically significant ionic current was observed in the electrical cell culture system. To avoid cultures from potentially toxic electrode Copyright 2011 John Wiley & Sons, Ltd.
L. Ghasemi-Mobarakeh et al.
products, agar-gelled salt bridges have also been applied for connecting the metal electrodes within the culture medium, where one end of a salt bridge is connected to a scaffold placed in the culture medium, and the other end of the salt bridge is placed in a beaker of electrodes, along with their relevant salt (e.g. Ag/AgCl) and the electrodes in each beaker are attached to a DC power supply (Mccaig et al., 2005). Salt bridges are prepared from flexible plastic tubing filled with 2% agarose in PBS. This provides a conducting pathway for applied current and prevents electrolysis products from contaminating the chamber by providing a pathway for current transfer from each media reservoir to the Ag–AgCl electrode (Tandon et al.,2009). The amplitude of the stimulus is also very important and a direct relationship exists between stimulus amplitude and response amplitude within a small range, which is quite enough to induce the depolarization of nerve cells (Paul, 1975). If the amplitude of the electrical stimulus is too weak to produce a threshold depolarization, an action potential will not take place. If the applied current depolarizes the membrane to threshold, an action potential will result. Steady DC voltage has been applied in many previous studies for electrical stimulation, mainly due to the existence of DC electrical gradients of voltage within tissues (endogenous electrical fields) (Mccaig et al., 2005). Wood et al. (2006) investigated the influence of brief DC electric stimulation on neurite outgrowth and outgrowth rates after application. Their results showed that the presence of a 25 V/m electrical field for 10 min increased overall neurite outgrowth over controls for up to 48 h after stimulation. In the literature, both electrical field and current have been reported to be effective in modulating cell behaviour. There are a few reports indicating more effectiveness of DC electrical fields than DC currents in electrical stimulation. Shi et al. (2007) applied both electrical field (100 mV/mm) and current (2.5–250 µA/mm) for electrical stimulation and concluded that a wide range of surface current density (2.5–250 µA/mm) had no significant effect on cell adhesion or cell viability, while a constant electrical field of 100 mV/mm upregulated the mitochondrial activity of human cutaneous fibroblasts and enhanced their adhesion. Their results showed that electrical field plays a more substantial role than electrical current in modulating the activity of cells cultured on conductive polymeric scaffolds compared to the non-stimulated cell cultures. However, these researchers did not rule out the importance of current as potential gradient reaches a certain critical level. Some studies also showed that, on exposure to an electrical field (
