Electroconductive Nanocomposite Scaffolds: A New Strategy Into Tissue Engineering and Regenerative Medicine

Nanocomposites are a combination of a matrix and a filler, where at least one dimension of the system is on the nanoscale being less than or equal to 100 nm. Much work has focused on the construction of nanocomposites due to the structural enhancements in physico-chem‐ ical properties, and functionality for any given system [1-6]. The physico-chemical enhance‐ ments result from the interaction between the elements being near the molecular scale. Nanocomposite materials have also received interest for tissue engineering scaffolds by be‐ ing able to replicate the extracellular matrix found in vivo. Currently, researchers have creat‐ ed composite materials for scaffold formation which incorporate two or more materials. Some of these materials consist of minerals for bone tissue engineering including calcium, hydroxyapatite, phosphate, or combinations of different polymers, such as poly (lactic acid), poly (ε-caprolactone), collagen and chitosan, and many other different combinations [7-9]. Other work has focused on doping the polymer scaffolds with specific growth hormones or adhesion sequences to influence how cells attach to the scaffold and cause the scaffold to be‐ come a drug delivery vehicle for different kind of tissue engineering applications [10]. Among different materials used in preparation of nanocomposits, conducting polymers are one of the effective materials that can be employed to facilitate communication with neural system for regenerative purposes.


Introduction
Nanocomposites are a combination of a matrix and a filler, where at least one dimension of the system is on the nanoscale being less than or equal to 100 nm. Much work has focused on the construction of nanocomposites due to the structural enhancements in physico-chemical properties, and functionality for any given system [1][2][3][4][5][6]. The physico-chemical enhancements result from the interaction between the elements being near the molecular scale. Nanocomposite materials have also received interest for tissue engineering scaffolds by being able to replicate the extracellular matrix found in vivo. Currently, researchers have created composite materials for scaffold formation which incorporate two or more materials. Some of these materials consist of minerals for bone tissue engineering including calcium, hydroxyapatite, phosphate, or combinations of different polymers, such as poly (lactic acid), poly (ε-caprolactone), collagen and chitosan, and many other different combinations [7][8][9]. Other work has focused on doping the polymer scaffolds with specific growth hormones or adhesion sequences to influence how cells attach to the scaffold and cause the scaffold to become a drug delivery vehicle for different kind of tissue engineering applications [10]. Among different materials used in preparation of nanocomposits, conducting polymers are one of the effective materials that can be employed to facilitate communication with neural system for regenerative purposes.
However, the major obstacle concerning the electrically conducting polymers has been the difficulty associated with the processing of them [11]. To overcome this problem, most researchers have electrospun conducting polymers by blending them with other spinnable

Conductive polymers 2.1. General approaches and considerations
Conducting polymers are a special class of materials with electronic and ionic conductivity [22]. The structures of the widely used conducting polymers are depicted in Fig. 1 [23]. These polymers have immense applications in the fields of drug delivery, neuroprosthetic devices, cardiovascular applications, bioactuators, biosensors, the food industry and etc.
One of the first electrically conducting polymers, polypyrrole (PPy) was introduced in the 1960s, but little was understood about this polymer at that time [24]. In 1977, a research team reported a 10 million-fold increase in the conductivity of polyacetylene doped with iodine as the first inherently conducting polymer [25,26]. Unlike polyacetylene, polyphenylenes, are known to be thermally stable as a result of their aromaticity [27]. Polyheterocycles, such as PPy, polythiophene (PT), polyaniline (PANI), and poly(3,4-ethylenedioxythiophene) (PEDOT), developed in the 1980s, have since emerged as another class of aromatic conducting polymers that exhibit good stabilities, conductivities, and ease of synthesis [28]. Table 1 shows a list of different conducting polymers and their conductivities [29]. Conducting polymers have an inherently unstable backbone, resulting from the formation of alternate single and double bonds along with the monomer units during polymerization. The delocalized π bonding electrons, produced across the conjugated backbone, provide an electrical pathway for mobile charge carriers which are introduced through doping. Consequently, the electronic properties, as well as many other physicochemical properties, are determined by the structure of the polymer backbone and the nature and the concentration of the dopant ion [30].   [30] Conjugated aliphatics, including polyacetylene, and benzene derivatives such as PANI, have been largely ruled out for biomedical applications due to their oxidative degradation in air and the cytotoxic nature of their by-products. Although recent research has shown that the emeraldine salt of PANI (EPANI) can be successfully fabricated in a biocompatible form [31,32], modern biomedical conducting polymers are typically composed of heterocyclic aromatics, such as derivatives of thiophene and pyrrole [33,34]. Specifically, PEDOT and PPy have been widely studied for their superior environmental and electrochemical stability [35][36][37]. Fig. 2 shows the chemical structure of various thiophene derivatives including EDOT, EDOT-MeOH and 3-alkylthiophene [30].

Surface modification of conducting polymers
For biomedical approaches, sometimes we need to modify the outer surface of the materials to induce special features. Conducting polymers can be also modified to enhance the functionality of nanocomposites. The surface modifications of conducting polymers have some concerns including: • Enhancement of the charge transport of carriers between the implant and tissue • Mediating the large difference in mechanical modulus

• Improvement of biodegradation
• Decreasing the impedance to enhance the sensitivity of the recording site • Cell response enhancement

• Bioactivity enhancement
Surface modification and functionalization of conducting polymers with different biomolecules or dopants has allowed us to modify them with biological sensing elements, and to turn on and off different signalling pathways required for cellular processes. In this way, conducting polymers can show significant enhancement in cell proliferation and differentiation. Thus, conducting polymers provides an excellent opportunity for fabrication of highly selective, biocompatible, specific and stable nanocomposite scaffolds for tissue engineering of different organs [39,40].

General use of conducting polymers
A range of applications for conducting polymers are currently being considered, such as the development of tissue-engineered organs [41], controlled drug release [42], repaire of nerve chanels [43], and the stimulation of nerve regeneration [44]. In addition, electrically active tissues (such as brain, heart and skeletal muscle) provide opportunities to couple electronic devices and computers with human or animal tissues to create therapeutic body-machine interfaces [45]. The conducting and semiconducting properties of this class of polymers make them important for a wide range of applications. The important properties of various conducting polymers and their potential applications are discussed in

Conductivity mechanism
Generally, polymers with loosely held electrons in their backbones can be called conducting polymers. Each atom on the backbone has connection with a π bond, which is much weaker than the σ bonds in the backbone. These atoms have allways a conjugated backbone with a high degree of π-orbital overlap [46]. It is known that the neutral polymer chain can be oxidized or reduced to become either positively or negatively charged through doping process [47]. It is also known that conducting polymers could not be perfectly conductive without using dopants, and doping of π-conjugated polymers results in high conductivity [24]. The doping process is influenced by different factors such as polaron length, chain length, charge transfer to adjacent molecules and conjugation length [46]. There have been different dopants for the addition of H + (protonation) to the polymers. For example, strong inorganic hydrochloric acid (HCl), organic and aromatic acids containing different aromatic substitution have been used as dopants for PANI. It is also reported that the surface energies of the doped conducting polymers vary greatly, depending on the choice of the dopants and doping level. Recently, PPy doped with nonbiologically active dopants (tosylate) and it has been characterized for biological interactions as they can trigger cellular responses in biological applications. However, the incorporation of more biologically active dopants can significantly modify PPy-based nanocomposites for biomedical applications [48].
One of the most important challenges of nanocomposite scaffolds based on conducting polymers is their inherent inability to degrade in the body, which may induce chronic inflammation [49]. Hence, belending of conducting polymers with biodegradable polymers seems to solve the problem. PPy and PANI are the most importan conducting polymers for tissue engineering, and they are important in terms of their biocompatibility and cell signaling especially for nerve tissue engineering [24].

Polypyrrole
PPy is among one of the first conducting polymers that studied a lot for its effect on the behaviour of cells. This material has been reported to support cell adhesion and growth of different cells [50]. This conducting synthetic polymer has numerous applications in tissue engineering and drug delivery. Recently, Moroder et al. [51] studied the properties of polycaprolactone fumarate-polypyrrole (PCLF-PPy) nanocomposite scaffolds under physiological conditions for application as conductive nerve conduits. In their study, PC12 cells cultured on PCLF-PPy nanocomposite scaffolds were stimulated with regimens of 10 µA of either a constant or a 20 Hz frequency current passed through the scaffolds for 1 h per day. The surface resistivity of the scaffolds was 2 kΩ and the nanocomposite scaffolds were electrically stable during the application of electrical stimulation. As can be seen in Fig. 3, in vitro studies showed significant increases in the percentage of neurite bearing cells, number of neurites per cell and neurite length in the presence of electrical stimulation compared with no electrical stimulation. They concluded that the electrically conductive PCLF-PPy nanocomposite scaffolds possed the material properties necessary for application in nerve tissue engineering.

Polyaniline
PANI is an oxidative polymeric product of aniline under acidic conditions and is commonly known as aniline black [52]. The exploration of PANI for tissue-engineering applications has progressed more slowly than the development of PPy for similar applications. However, recently there has been more evidence of the ability of PANI and PANI variants to support cell growth [53]. Recently, Fryczkowski et al. [54] synthesized three-dimensional nanocomposite fibres of poly(3-hydroxybutyric acid) (PHB) and dodecylbenzene sulfonic acid (DBSA) doped polyaniline in chlorophorm/trifluoroethanol mixture, using electrospinning method. The morphology, electro-active properties and supermolecular structure of nanofibres webs have been analyzed and discussed. Obtained nanofibres are potentially applicable as nanocomposite scaffolds for tissue engineering. According to their results, there were limitations in composition of blended system and the PHB:PANI:solvent ratio needed to be optimized in order to obtain reasonable spinnability of compositions, and even small amount of PANI caused changes in super molecular structure of PHB/PANI nanofibres.

Poly (3, 4-ethylenedioxythiophene)
Although PPy and PANI have been the most extensively conductive polymers for tissue engineering and regenerative medicine, recently the potential of polythiophene conductive polymer for tissue engineering have been approved. This polymer has received significant attention due to a wide range of promising electronic and electrochemical applications [55,56]. PEDOT can be considered as the most successful polythiophene due to its specific characteristics [57][58][59][60][61][62][63][64][65]. PEDOT can also be considered as the most stable conducting polymer currently available because of not only high conductivity but also unusual environmental and electrochemical stabilities in the oxidized state [57][58][59][60]. Recently, Bolin et al. [66] reported electronically conductive and electrochemically active 3D-nanocomposite scaffolds based on electrospun poly(ethylene terephthalate) (PET) nanocomposite fibers. They employed vapour phase polymerization to achieve a uniform and conformal coating of PEDOT doped with tosylate on the nano-fibers. They observed that the PEDOT coatings had a large impact on the wettability, turning the hydrophobic PET fibers super-hydrophilic. According to Fig.  4, the SH-SY5Y cells adhered well and showed healthy morphology. These electrically active nanocomposite scaffolds were used to induce Ca 2+ signalling in SH-SY5Y neuroblastoma cells. Their reported nanocomposite fibers represented a class of 3D host environments that combined excellent adhesion and proliferation for neuronal cells with the possibility to regulate their signalling.

Piezoelectric polymeric nanocomposites
Recent studies on the application of conductive materials showed that piezoelectric polymeric materials can also be considered for tissue engineering applications. Piezoelectric polymeric materials can generate surface charges by even small mechanical deformations [67]. Poly(vinylidenefluoride) (PVDF) is a synthetic, semicrystalline polymer with piezoelectric properties that can be potentially used for biomedical application due to their unique molecular structure [107]. An electrical charged porous nanocomposite could be a promising ap-proach for a number of tissue engineering applications. Reported data on piezoelectric polymeric nanocomposites showed that after electrical stimulation, cellular interaction and tissue growth might be improved [68].

Applications of conducting polymers: general view
Conductive polymers exhibit attractive properties such as ease of synthesis and processing [69]. The unique properties of this type of materials have recently given a wide range of applications in the biological field. Research on conductive polymers for biomedical applications expanded extrimly in the 1980s, and they were shown via electrical stimulation, to modulate cellular activities (e.g. cell adhesion, migration, DNA synthesis and protein secretion) [70][71][72][73]. Since then many studies have been done on nerve, bone, muscle, and cardiac cells. The unique characteristics of conducting polymers have been shown to be useful in many biomedical applications, specially tissue engineering nanocomposite scaffolds and drug delivery devices [74]. In comparison to other conductive materials for biological applications, conducting polymers are inexpensive, easy to synthesize, and versatile. In addition, conducting polymers permit control over the level and duration of electrical stimulation for tissue engineering applications. Developing novel drug-delivery systems will open up new applications that were previously unsuited to traditional delivery systems. The use of conducting polymers in drug delivery is an excellent approach due to their biocompatibility and their possibility of using them in in vivo applications for real time monitoring of drugs in biological environments [75]. Controlled drug release can also be facilitated using a change in conductive polymer redox state to increase permeation of drugs such as dexamethasone [76]. Electrical stimulation of conductive polymers has been used to release a number of therapeutic proteins and drugs like nerve growth factor [77], dexamethasone [78] and heparin [79]. Another study demonstrated the use of PEDOT nanotubes polymerized on top of electrospun poly(lactic-co-glycolic acid) (PLGA) nanocomposite fibres for the potential release of the drug dexamethasone. Here, dexamethasone was incorporated within the PLGA nanocomposite fibres and then PEDOT was polymerized around the dexamethasone-loaded PLGA nanocomposite. As the PLGA fibres degraded, dexamethasone molecules remained inside the PEDOT nanotubes. These PEDOT nanotubes favoured controlled drug release upon electrical stimulation. Fig. 5 demonstrates the incorporation and release mechanism of dexamethasone from PEDOT nanotubes due to electrical stimulation. This drug-delivery system had the potential of immense interest for the treatment of cancer and tissue engineering and regenerative medicin [80]. . Simultaneously, the right film acting as the cathode contracts and shrinks because of the expulsion of the counter ions. These volume changes and the constant length of the non-conducting film promote the movement of the triple layer towards the polypyrrole film that is being contracted. (b) By changing the direction of the current, the movement takes place in the opposite direction. The muscle works in LiClO 4 aqueous solution [83] Bioactuators are devices that are used to create mechanical force, which in turn can be used as artificial muscles. The phenomenon of change in the volume of the conducting polymers scaffold upon electrical stimulation has been employed in the construction of bioactuators. In artificial muscle applications, two layers of conducting polymers are placed in a triple layer arrangement, where the middle layer comprises a non-conductive material [81]. When current is applied across the two conducting polymers films, one of the films is oxidized and the other is reduced. The oxidized film expands owing to the inflow of dopant ions, whereas the reduced film expels the dopant ions and in the process shrinks, as depicted in Fig. 6 [81]. Conducting actuators have many features that make them ideal candidates for artificial muscles, including that they:

Use and modification of conducting polymers for bioactuators
• can be electrically controlled, • have a large strain which is favourable for linear, volumetric or bending actuators, • possess high strength, • require low voltage for actuation (1 V or less), • can be positioned continuously between minimum and maximum values, • work at room/body temperature, • can be readily microfabricated and are light weight, and • can operate in body fluids [82].

Use and modification of conducting polymers for tissue engineering applications
The essential properties of conductive polymers desired for tissue engineering and regenerative medicine are conductivity, reversible oxidation, redox stability, biocompatibility, hydrophobicity, three-dimensional geometry and surface topography. Conductive polymers are widely used in tissue engineering due to their ability to subject cells to an electrical stimulation. Studies have addressed cell compatibility when a current or voltage is applied to PPy. An advantage offered by conducting polymers is that the electrochemical synthesis allows direct deposition of a polymer on the surface while simultaneously trapping the protein molecules [84].
In a recent study the release of NGF from PPy nanocomposites by using biotin as a co-dopant during the electrical polymerization was investigated [85]. In this research, NGF was biotinylated and immobilized to streptavidin entrapped within PPy nanocomposites doped with both biotin and dodecylbenzenesulfonate. The release of heparin from hydrogels immobilized onto PPy nanocomposites could also be triggered by electrical stimulation [86]. PVA hydrogels were covalently immobilized onto PPy via grafting of aldehyde groups to PPy and chemical reaction of these with hydroxyl groups from the hydrogel as shown in Fig. 7. Electrically conducting polymers have attracted much interest for the construction of nerve guidance channels. The use of conducting polymers can help locally deliver electrical stimulus. It can also provide a physical template for cell growth and tissue repair and allow precise external control over the level and duration of stimulation [87,88]. The importance of conducting polymeric nanocomposites 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 [89].
Recently, Li et al. [90] blended PANI with a natural protein, gelatin, and prepared nanocomposite fibrous scaffolds to investigate the potential application of such a blend as conductive scaffold for tissue engineering applications. As can be seen in Fig. 8, SEM analysis of the scaffolds containing less than 3% PANI in total weight, revealed uniform fibers with no evidence for phase egregation, as also confirmed by DSC. To test the usefulness of PANI/gelatin blends as a fibrous matrix for supporting cell growth, H9c2 rat cardiac myoblast cells were cultured on fiber-coated glass cover slips. Cell cultures were evaluated in terms of cell proliferation and morphology. According to Fig. 9, the results indicated that all PANI/gelatin blend fibers supported H9c2 cell attachment and proliferation to a similar degree as the control tissue culture-treated plastic (TCP) and smooth glass substrates. Depending on the concentrations of PANI, the cells initially displayed different morphologies on the fibrous substrates, but after 1week all cultures reached confluence of similar densities and morphology. Taken together they suggested that PANI/gelatin blend nanocomposite fibers could provide a novel conductive material well suited as biocompatible scaffolds for tissue engineering.

Conclusion
Tissue engineering is a new concept which is a growing area of research, in which cells are seeded on nanocomposite scaffolds and then implanted in defected part of body. Appropriate stimuli (chemical, biological, mechanical and electrical) can be applied and over a relatively short time new tissue can be formed to help restore function in the patient. The ideal scaffolds should have an appropriate surface chemistry and microstructures to facilitate cellular attachment, proliferation and differentiation. In addition, the scaffolds should possess adequate mechanical strength and biodegradation rate without any undesirable by-products. Among different materials, conducting polymers are one of the materials that can be employed to facilitate communication with neural system for regenerative purposes. In this chapter the recent methods of the synthesis of nanocomposite scaffolds using different conducting polymers was reviewed. The ability of conductive scaffolds to accept and modulate the growth of a few different cell types including endothelial, nerve, and chromaffin cells have shown a bright future in the field of tissue engineering and regenerative medicine.