## 2 Tissue-Engineered Scaffolds with GBM and Neural Tissues
### Hydrogels  
Hydrogels are 3D networks containing crosslinked hydrophilic polymer chains that can absorb a large amount of water; it is this characteristic that allows them to mimic the natural ECM [68]. Hydrogels are remarkably promising for application of the sustained delivery of biomolecules and can be directly implanted defect site, as well as for TE applications [12]. Hydrogels are the most promising choice for the delivery of genes or cytokines for nerve proliferation and differentiation. The addition of GBMs to hydrogels has gained recent attention due to its desired resultant properties: (1) higher stability and controlled delivery of molecules, GO in particular; (2) overcoming the notable weak point of hydrogels, and low mechanical properties, by acting as a capable reinforcement; and (3) creating conductive hydrogels that are highly useful for mimicking the natural environment in the nervous tissues or organs [69; 70; 71; 72; 73]. There are different aspects to investigate the effects of GBMs on hydrogels for NTE, such as the effect of concentration, pore size, morphology, chemistry (related to the functional groups), mechanical strength (elastic modulus in particular), and electrical conductivity [69; 70; 71; 72; 73]. GBMs are a candidate as a carrier for gene delivery and cell transfection, which can be better controlled and sustained within hydrogels [58; 63]. In a study focusing on promoting BMSCs' recruitment and stimulating sensory nerve regeneration, the stromal cell-derived factor-1\(\alpha\) (SDF-1\(\alpha\)), a member of the chemokine family of pro-inflammatory mediators, and pDNAs were used. To this aim, 25 kDa polyethylenimine (PEI) was conjugated to nanoscale GO sheets to deliver pDNAs encoding bFGF (GO-PEI-bFGF) and crosslinked by matrix metalloproteinase (MMP)-2. The crosslinking of GO-PEI sheets keeps the DNA inside to prevent degradation and induce the responsive BMSCs transfection. This gene delivery system was encapsulated by a GO-based hydrogel that contains SDF-1\(\alpha\). SDF-1\(\alpha\) can recruit distant endogenous BMSCs, and MMP-2 activated the hydrolysis of crosslinked GO-PEI and then started the BMSCs transfection towards the neural-like cells (Figure 6A). Using GO resulted in several improvements, such as stable biomolecule delivery and release in a controlled and sustained way. Although there are still some concerns regarding GO biocompatibility, this study confirmed the dose-dependent cytotoxicity of nano (significantly >10 ug/mL) and microscale (even noticeable at 1 ug/mL) GO. Conjugating the cationic PEI to the GO surface improved GO biocompatibility remarkably (GO-PEI 1:10 was the optimum) [69]. Gene delivery with the help of GBMs promises hope by maximizing therapeutic efficacy, the substantial factor to consider for delivery efficiency with gene transfection, and according to the reports; this type of engineered delivery system can introduce treatments for CNS diseases [69; 74]. To evaluate the impact of GBMs on the electrical conductivity of hydrogels, GBMs can be applied solely, with a combination of other conductive biomaterials or even with the addition of electric charges, to result in an appropriate range of conductivity. Introducing electric charges is a strategic method to stimulate the proliferation and differentiation of nerve cells [72; 75; 76]. This is because excitable cells rely significantly on electrical conductivity, and ion accumulation and flow, to coordinate cellular functions and signal transduction [75; 77]. In order to form a positively charged hydrogel, oligo(poly(ethylene glycol) fumarate) (OPF) was crosslinked with [2-(methacryloyloxy)ethyl]trimethylammonium chloride (MTAC). The results have shown enhanced neural cell adhesion, proliferation, and differentiation. Furthermore, from the dissociated embryonic chick dorsal root ganglion (DRG) explant, it can be said that OPF hydrogels resulted in a combination of neurons, neuronal support cells, and Schwann cells [75].