**1. Introduction**

Advances in tissue engineering (TE) promise novel techniques to accelerate the recovery of damaged tissues by overcoming autologous, allogeneic, and xenogeneic tissue repair [1]. The three essential elements of TE are cells, scaffolds, and growth factors [2]. The success rate of TE is dependent on the ability of porous 3D scaffolds to mimic the function of the extracellular matrix (ECM) of a specific tissue. Scaffolds should also provide a compatible environment for the regeneration of tissues and the transplantation of organs [1,3]. Implantable scaffolds can meet the required criteria of biocompatibility, porosity,

**Citation:** Ahmad Ruzaidi, D.A.; Mahat, M.M.; Shafiee, S.A.; Mohamed Sofian, Z.; Mohmad Sabere, A.S.; Ramli, R.; Osman, H.; Hamzah, H.H.; Zainal Ariffin, Z.; Sadasivuni, K.K. Advocating Electrically Conductive Scaffolds with Low Immunogenicity for Biomedical Applications: A Review. *Polymers* **2021**, *13*, 3395. https://doi.org/10.3390/ polym13193395

Academic Editor: Evgenia G. Korzhikova-Vlakh

Received: 1 September 2021 Accepted: 27 September 2021 Published: 2 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cell viability, and mechanical properties [4] by ensuring: (1) a greater control over the scaffold's surface topography, surface wettability and surface charge [5]; (2) an optimised scaffold's mass transportability to facilitate nutrient exchange and waste removal; (3) optimised biocompatibility to minimise the risk of toxicity [6–8]; (4) the presence of biological cues for cell fate to induce tissue regeneration [9–11]; (5) similarity in the rate of scaffold biodegradation and tissue growth [12,13]. The continuous development of biocompatible and biomimetic scaffolds is paramount to realise its clinical applications in improving the patient's care and quality of life [8,14].

Initially, scaffolds only act as a support system for cells to attach and proliferate. Due to this limited functionality, scaffold-based tissue engineering is primarily focused on improving tissue recovery by electrical stimuli through the application of conductive polymers (CPs). CPs exhibit electrical conductivity due to a reduction in their neutral state [15,16], and because of the presence of conjugated double bonds along the backbone. Dopant ions were commonly added into the CP's chemical structure to neutralise the unstable backbone of the polymer in its oxidised state by donating or accepting electrons [17]. Poly (3,4-ethylene dioxythiophene): poly (4-styrene sulfonate) (PEDOT: PSS) is an example of a biocompatible CP that is commonly used to produce conductive scaffolds. In another study focusing on CPs, the electrical deterioration of scaffolds could be prevented by immobilising the dopant in a polyaniline (PANI) conductive scaffold patch. This was achieved as a result of the strong chelation bonding between a phytic acid dopant and chitosan [18]. In addition, several studies have demonstrated that conductive scaffolds with polypyrrole (PPy) can promote the regeneration of nerves, bones, muscles, and cardiac cells through electrical stimulation [19]. Figure 1 shows that the electrical conduction mechanism from conductive scaffolds enables cellular signalling and function in tissues to replicate normal electrophysiology; therefore, it causes electroactive cells to align in a specific direction, as well as to migrate and proliferate. Despite the advantages of using electroactive polymers in TE, the poor biodegradability of CPs is a barrier to realising their true potential [20–23].

**Figure 1.** Illustration of the electrical conduction stimulation from conductive scaffolds, which enables cellular signalling and function in tissues.

> The subject of biodegradability demands great attention in the pursuit of fabricating a conductive scaffold. The biodegradability of scaffolds is desirable, as it allows the materials to naturally dissipate, either via absorption or elimination in physiological

conditions [24]. According to previous studies, conductive scaffolds were incorporated with natural compounds to make them biodegradable. Collagen and chitosan are examples of natural compounds that can physiologically degrade, without leaving behind any toxic residues. The application of non-toxic materials in scaffolds can minimise the adverse responses of the immune systems. However, studies on the immunogenicity performances of conductive and biodegradable scaffolds are scarce [5,12,14,25,26].

It is vital to assess the efficiency of conductive scaffolds, in terms of their biodegradability and electrical conductivity. Introducing biomaterials into the human body, theoretically, triggers an immune response. However, a mild impact can be expected from a conductiveimplantable scaffold with a stable biodegradation rate, compared to the scaffolds with an unstable biodegradation rate. Regardless, the by-products of the scaffold biodegradation process should be non-toxic to the host. Toxic by-products cause excessive dead cells and damaging tissues, thus compromising the functions of organs. Therefore, the use of safer biodegradable materials with non-toxic by-products is desirable for the growth and proliferation of the cell. Figure 2 illustrates that, with the use of suitable materials, we can ideally alter the immunogenicity of scaffolds.

**Figure 2.** Illustration of the use of biodegradable and electrically conductive materials to promote cell growth with low immunogenicity.

Although organ transplantation is a proven method to address organ complications, this method is severely limited by the difficulties in securing replacement organs from donors [11]. Scaffold-based TE could be the key to this conundrum. This review article will discuss the recent fabrication techniques that can be employed to fabricate scaffolds. The contribution of the crosslinking process in regulating the degradation rate of scaffolds will be presented. A review of scaffolds' physical properties, mainly regarding their mechanical and electrical conductivity performance, will also be discussed, according to their type of CP substituents. In addition, the biodegradation trends of conductive scaffolds in phosphate-buffered saline (PBS) will be reviewed, together with a juxtaposition with human immunogenic responses. Another feature of this field of research is that its outcomes can spur innovations in drug delivery processes [27]. In the latter part of this review, we outline several of the current developments of biodegradable natural materials and synthetic polymeric materials for various biomedical applications, including tissue engineering, wound healing, and drug delivery.

#### **2. Techniques in Fabricating Electrically Conductive Scaffolds**

The fabrication and the design of conductive scaffolds influence their performance in physiological conditions. Therefore, the required properties for a specific tissue should be identified before a scaffold is produced. The selection of materials for the scaffolds is essential to promote a safe environment for cell proliferation. Scaffolds can be fabricated from natural polymers, such as silk, collagen, keratin, cellulose, and chitin. Synthetic polymers, such as polylactic acid (PLA), polyglycolide acid (PGA), and polyhydroxyalkanoates (PHA) can also act as the base material of scaffolds. Figure 3 and Table 1 list the common techniques that are used to fabricate conductive scaffolds [17,24,28–32]. Additionally, Figure 4 shows that distinct scaffold fabrication techniques yield distinct forms and structures of scaffolds, including interconnected porous, hydrogel, and nanofibrous mat [18,25,29,30,33].

At present, electrospinning and lyophilisation are two standard techniques that are used to fabricate porous scaffolds [34,35]. The electrospinning method is used to obtain the nanofiber. Nanofibrous structured scaffolds can resemble ECM tissues on the nanoscopic scale [14]. In order to produce an electrically conductive nanofibrous scaffold, CPs are usually mixed into the scaffold matrix using physical blending or through coating techniques. The combination of CPs with spinnable polymers was found to facilitate the electrospinning process, while encouraging a micro- or nano-fibre structure. A composite-conductive scaffold can be realised by adding a layer of CPs onto electrospun fibres, during the combined process of electrospinning and spin coating [25,36–38]. However, a porous scaffold that is structurally similar to foams and sponges is more stable than a nanofibrous-structured scaffold created from electrospinning. The distribution of pores in a scaffold can be rearranged randomly or by following an organised pattern. The choice of pore distribution can be accomplished by manipulating the solvent and phase separating conditions during the scaffold fabrication process. The mechanical mismatch between the host tissue and the scaffolds can be minimised by applying the best scaffold fabrication technique for the target tissue. Minimising the discrepancies in mechanical compatibility is essential to encourage the host tissue's acceptance of foreign scaffolds.

**Figure 3.** Techniques in fabricating scaffolds and their respective scanning electron microscope images [18,29–31,39,40].

A three-dimensional (3D) composite with a high concentration of PEDOT: PSS-gelatinebioactive glass scaffold was fabricated with the freeze-drying method to promote cell response, attachment, and viability. These advantages increase the number of functional human mesenchymal stem cells (hMSC) [41]. Shen Wang et al. (2020) fabricated interconnected porous hydrogel scaffolds with an ionic grafting technique. In this technique, the conductive PPy polymer was grafted onto methacrylic anhydride gelatine in the presence of ferric ions. The reversible ionic interactions between ferric ions, the gelatine, and PPy mixtures afforded the hydrogels with self-healing abilities, injectable capabilities, and high electrical conductivity [29]. In addition, the incorporation of gelatine in scaffold composites introduced injectable capabilities, which is a valuable feature for nerve tissue regeneration. Although the biodegradation processes did reduce the electrical conductivity of the scaffolds, the scaffolds still met the minimum requirement of the electrical stimulation characteristic for application in neural TE [42,43]. A strong understanding of the interaction between cells and conductive materials is indispensable in researching smart biomaterials for their application in TE. Recently, electrically conductive hydrogels have been fabricated and designed using a 3D printing method, which includes fused deposition modelling (FDM), direct ink writing (DIW), inkjet printing, and stereolithography (SLA) methods [44]. For example, conductive polymer carbon nanocomposites as the main thermoplastic filament materials for the FDM method have been employed as emerging electrochemical sensing devices [45]. However, the main drawback of 3D printing methods is the low printing resolution that is produced at the end of the fabrication process.

Mawad et al. (2016) reported that the electrical stability of the PANI scaffold patch could be improved by immobilising the dopant, phytic acid, together with a PANI on

the surface of a biocompatible chitosan film [18]. The PANI patch scaffold was prepared by drop-casting chitosan solution onto a glass slide, followed by drop-casting a mixture of aniline, phytic acid, and ammonium persulfate onto the chitosan film. The ability of the scaffold patch to sustain its conductive state under physiological conditions can instigate an electronic interface between the biomaterials and the implantation site. A dopant is typically introduced during the synthesis of CPs, to produce favourable electrical characteristics. Dimethyl sulfoxide (DMSO), ethylene glycol (EG), sulfuric acid (H2SO4), phytic acid, and *p*-Toluenesulfonic acid (*p*TSA) are commonly employed as dopants [46–54]. In their research of crosslinked porous 3D hydrogel–PEDOT CP scaffolds, Mawad et al. (2016) mixed all of the required chemicals, followed by a filtration process [55]. It was found that the electroactive region of the PEDOT hydrogel network was covalently bonded to a hydrophilic polymer, and could be further altered to fabricate a covalently linked polymeric network.

**Figure 4.** Images of scaffold structures: porous hydrogel, nanofibrous, porous hydrogel scaffold, porous sponge scaffold, and hydrogel patch scaffold [18,29,30,34,39].

Recently, the antibacterial, mechanical, and physical properties of a collagen-chitosan sponge scaffold, constructed from aquatic sources, were studied [56]. The researcher applied the lyophilisation technique (−50 ◦C, 0.5 Mbar vacuum pressure) to acquire a highly porous-structured scaffold. Furthermore, they added an alginate compound to the scaffold to improve its mechanical properties. The sponge scaffold's porosity was in the range of 88–95%, with the addition of biopolymers, such as alginate and chitosan, onto the collagen [56]. Moreover, injectable-electroconductive hydrogels have the potential to improve cell survival, which could be translated into a novel treatment protocol [29,42,57], while minimising the need for invasive surgery. Additionally, the incorporation of electroconductive nanomaterials in hydrogels may influence their bulk electrical properties and topography, which can also affect the retention and biology of living cells. For instance, the application of CPs, such as PEDOT: PSS, in hydrogel scaffolds promotes the rhythmic beating in a neonatal rat cellular matrix [34,58]. The use of PEDOT in chitosan-based gels also resulted in an excellent regenerative capacity [43].


**Table 1.** Summary of scaffold fabrication techniques.

Iandolo et al. (2020) studied biomimetic scaffolds that supported neural crest-derived stem cell osteogenic differentiation [30]. The highly porous scaffolds were prepared by combining PEDOT: PSS with collagen type I, using the ice-templated technique. Collagen type I and PEDOT: PSS were blended in an ultrasonic bath to ensure the mixture's homogeneity. Next, the mixture was poured into a specific mould with the desired shape, followed by freeze-drying and thermal treatment. (3-glycidyloxypropyl) trimethoxysilane (GOPS) was used as the crosslinker to enhance the mechanical properties of the scaffolds. Heat treatment was performed to reticulate the crosslink [30]. The outcome of this study was a porous scaffold with the desired geometry. The ice templating process was necessary to create highly porous scaffolds that were compatible with cell infiltration and proliferation.

Another technique that is used to fabricate conductive scaffolds is electrospinning. Abedi et al. (2019) studied the fabrication and characterisation of conductive nanofibrous chitosan/PEDOT: PSS for cardiac TE. PEDOT: PSS was paired with chitosan due to its chemical and thermal stability. The combination promoted the scaffold's electrical properties, mirroring the myocardium ECM. A double nozzle electrospinning apparatus was used to form a fibrous mat structure of a conductive scaffold. The scaffold was then crosslinked using the glutaraldehyde solution vapor technique [25]. They discovered that the addition of PEDOT: PSS into chitosan/polyvinyl alcohol (PVA) increased the scaffold's conductivity. The electrospinning and crosslinking of the fibrous mat scaffold increased its mechanical strength. The presence of hydrogenic bonds, between OH groups in PVA and chitosan, and SO−<sup>3</sup> groups in PSS in the CPs' dispersion, also contributed to its higher mechanical strength. Interestingly, the fibrous mat of the chitosan/PVA/PEDOT: PSS scaffold promoted the attachment and proliferation of cells.

When fabricating and designing a suitable and safe TE product, scaffolds should be able to biodegrade and should support cellular growth in vitro and in vivo. However, other aspects, such as attachment, migration, and cell proliferation, must also be taken into consideration. These aspects are highly dependent on the scaffold's surface properties and

its interaction with transmembrane proteins. Low-risk tissue regeneration processes can be achieved with advanced scaffold technology.
