**3. The Crosslinking Process in Fabricating Conductive-Polymeric Scaffolds**

During the fabrication of polymeric scaffolds, the crosslinking process is adopted to chemically bind the molecules within the scaffold's structure. This, in turn, significantly influences their mechanical properties. For example, the crosslinking process upon a nanofibrous-structured scaffold causes the fibres to attach and stack between each other, resulting in better resistance performance to the application of shearing forces. The crosslinker can be divided into three types: chemical crosslinker, biophysical crosslinker, and enzymatic crosslinker. Adding crosslinkers can enhance their mechanical properties; however, this can affect their physical condition, causing the scaffolds to become contracted or shrunk. However, in the presence of dehydrothermal (DHT), crosslinking can prevent scaffold contraction, thus making scaffolds more stable for a long period of time. Meanwhile, crosslinking with ultraviolet (UV) light can enhance the scaffold's stability in an aqueous environment, and does not cause any physical changes to the scaffolds [58–60]. This may be due to the fact that the UV light crosslinking process does not take place chemically, and interacts with the compound that is present in the scaffolds. UV crosslinking hardened the liquid polymeric material to be more stable and to possess a rigid shape, with no heat exposure exerted on the material [61]. In addition, crosslinking can also avoid the premature dissolution of the scaffold at the normal body temperature [62]. Figure 5 shows an illustration of the mechanism of the scaffold's crosslinking processes. Extensive research has been performed to develop biomedical scaffolds that meet the criteria of cost-effective fabrication, ease of customisation, and safe application in clinical settings, particularly in the areas of infection treatment and drug delivery [38,63].

**Figure 5.** Illustration of the enhancement of a scaffold's mechanical properties by employing the crosslinking processes.

The intermolecular crosslinking of collagen-based scaffolds, either through chemical or physical methods, can modify their mechanical properties [12,56]. However, some wellknown crosslinking agents, such as glutaraldehyde (GTA), glyoxal, and glycol diglycidyl ether, are toxic [64,65]. According to Hua et al. (2020), sodium tripolyphosphate (TPP)

is a safe crosslinker for collagen and chitosan, with excellent biocompatibility [66]. A comparative study of crosslinkers reported that genipin (GP) and TPP had better biocompatibility, compared to GTA, towards the collagen/chitosan scaffold. Nonetheless, TPP was recommended as the most suitable crosslinking agent for myocardial TE [67]. In their study, collagen and chitosan were dissolved in a 1% (*v*/*v*) acetic acid solution at room temperature. After fabrication was carried out using the lyophilisation method (−20 ◦C for 48 h), a 1:1 ratio of a lyophilised collagen/chitosan scaffold was crosslinked. In the case of collagen, crosslinking could be achieved through the formation of an amide, from the activation of the carboxylic group with the amine. In comparison, 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide were employed as crosslinkers to strengthen the covalent attachment between the carbonyl group of gelatine and the amino group of chitosan in the porous PEDOT/Chitosan/Gel scaffolds [41].

Interestingly, a study focusing on poly(vinyl) alcohol-gellan gum-based nanofiber proved that a scaffold's degradation rate could be controlled by various crosslinking agents [38]. In addition, for electroconductive hydrogels (EH), there are several types of suitable crosslinking processes, such as physical crosslinking, covalent crosslinking, and supramolecular crosslinking. However, each of these crosslinking techniques may affect the biodegradability of the EH scaffold. The density and concentration of the crosslinker can alter the properties of the scaffolds, including their water content, mesh size, porosity, diffusivity, and mechanical characteristics [60,68–70]. Overall, the use of crosslinkers could physically change the properties of scaffolds, which can affect the biodegradability of the scaffolds. This idea warrants further investigation, focusing on the execution of scaffolds' optimum degradation rates by varying the concentration of any crosslinker.

#### **4. Physical Properties of Conductive-Polymeric Scaffolds**

No single polymeric system can be considered the ideal biomaterial for all medical applications, due to the complexity of the human body and the scope of applications that polymeric biomaterials are currently utilized for. Thus, a mixture of natural and synthetic polymers can overcome the limitations of a monocomponent system [12]. Theoretically, a composite scaffold that is composed of more than two sub-materials exhibits a greater characteristic value, due to the combination of materials that hold some desired properties. Due to the various needs of scaffolds, composite materials with excellent properties are commonplace in TE [71]. For example, Lari et al. (2016) fabricated nanohydroxyapatite/chitosan (nHAp/CS) composite scaffolds that were embedded with PEDOT: PSS through a lyophilization technique. They found that nHAp and PEDOT: PSS were homogeneously dispersed in the chitosan matrix. The CS/nHAp/PEDOT: PSS scaffolds exhibited a high cell attachment rate, due to their surface roughness. The electrical conductivity that was recorded for the CS/nHAp/PEDOT: PSS sample was 9.72 ± 0.78 µS/m. They also found that both the compressive modulus and the yield strength of the chitosan increased to 0.5–1.0 MPa and 5–5.5 MPa, respectively, with the addition of nHAp and PEDOT: PSS. Unfortunately, the CS/nHAp/PEDOT: PSS had poor biodegradability, due to the strong bonding between PEDOT: PSS and the rest of the substrate chains [26]. Meanwhile, Iandolo et al. (2020) prepared highly porous biomimetic scaffolds by combining the PEDOT: PSS with collagen type I (the most abundant protein in bone) for inactive support. However, aggregation took place when collagen was a part of the mixture, due to the interaction between the positively charged protein chains and the negatively charged polystyrene sulfonic acid groups, which caused the restructuring of the CPs. Unfortunately, this process contributed to the de-doping of the conductive PEDOT segments.

Abedi et al. (2019) demonstrated that the addition of PEDOT: PSS in chitosan scaffolds could improve their biocompatibility, cell viability, and mechanical and electrical properties. In their study, the electrical conductivity, elongation at break (%), ultimate strength, and toughness of CS/PVA/PEDOT: PSS scaffolds were recorded at 7.63 <sup>×</sup> <sup>10</sup>−<sup>3</sup> S/m, 5.6 <sup>±</sup> 0.3%, 18.78 <sup>±</sup> 0.95 MPa, and 48.87 <sup>×</sup> <sup>10</sup><sup>6</sup> J m−<sup>3</sup> , respectively. They also discovered that the attachment length of the cells increased as the nanofiber diameter was decreased to 40 nm.

Further shrinkage of the fibre diameter to less than 40 nm affected the attachment of cells. However, the addition of PEDOT: PSS can be toxic, depending on the weight composition. The addition of more than 0.6 wt.% PEDOT: PSS in gelatine scaffolds causes toxicity in stem cells; this means that the gelatine amount must be lower than 0.6 wt.% in a single scaffold composition [25]. Overall, previous studies established that PEDOT: PSS, which is favourable for cell proliferation, can be the predecessor for a highly biomimetic, electroactive scaffold for stem cell expansion and differentiation [30].

It is essential to establish a link between materials degradation in vitro and mechanomorphological characteristics, since biodegradation can negatively affect the mechanical and structural integrity of the scaffolds. Biodegradation is manifested by material loss in grafted scaffold structures because of ageing in the PBS medium. The biodegradation rate of a scaffold can be determined by the calculation of weight loss in PBS conditions. A higher percentage of mass loss in the scaffolds can be attributed to the high surface area and porosity of scaffolds [72]. For example, Zhang et al. (2008) investigated the in vitro biodegradation of an electrospun tubular protein scaffold, by immersing the scaffold in a PBS condition (pH = 7.3) for various timeframes. This was then followed by determining the scaffolds' weight loss percentage. Ideally, the degradation rate of the scaffold should be similar to the rate of new tissue formation. The mechanical and electrical properties of various scaffolds are summarised in Table 2. In addition, the conductivity of the fabricated scaffold should refer to the sheet conductivity of the material, which is different from the commonly evaluated electrochemical impedance responses of the coated electrodes.


**Table 2.** Mechanical and electrical properties of conductive-based scaffold materials.
