## 2 Tissue-Engineered Scaffolds with GBM and Neural Tissues
### Conduits for Nerve Guide
\begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Biomaterial(s)** & **GBMs Concentration** & **Target and Cell Type** & **Outcomes** & **Year** \\ \hline \multicolumn{6}{p{113.8 pt}}{* Grafted GO/PEI Core–shell microfiber arrayed hydrogel (GO-PEI) 1:10, (SOF-1\(\alpha\)) and GO-PEI/pDNAs-bFGF microparticles} & \multirow{6}{*}{Mass ratio 1:40, and 1:70 PEI/pDNAs-bFGF microparticles} & \multirow{6}{*}{Recruit and stimulate the neural-like differentiation BM-MSCs} & \multirow{6}{*}{Optimum ratio: GO-PEI 1:10, \(\uparrow\) neuronal endogenous stem cell therapy} & \multirow{6}{*}{2021} \\ \cline{3-4} \cline{6-6} \cline{5-6} \  
considerable progress to encourage nerve repair and regeneration in a targeted manner with their physical and chemical features; however, there is still a gap to achieve structural and functional restoration that mimics the natural properties. Current research supports the use of NGCs to repair large nerve defects, particularly in the PNS [90].  
NGCs are used to bridge the nerve stumps and eliminate scar formation, help the axon stretch, and form a suitable microenvironment to regenerate the injured nerve [91]. NGCs require great mechanical strength, as well as adequate elasticity to allow regular muscle motions around the conduit without tube collapsing [72]. The recovery is extremely effective as nerve extension is hampered through the generation of scar tissue during the repair process [92; 93]. Injuries related to the PNS are common (13\(-\)23 out of 100,000 individuals are effect annually in developed countries [94]); so, research has been focused on developing appropriate NGCs through several techniques to improve treatment. To meet the various needs of a suitable NGC for nerve regeneration, \"multifunctional NGCs\" are needed; hence, multiple biomaterials, biomolecules, and fabrication techniques have been used in most case studies [25; 93; 95; 96; 93].  
The properties of GBMs mean that they are promising biomaterials for the successful fabrication of NGC. One successful example includes an NGC synthesized by a 3D graphene mesh tube (GMT) and subsequently filled with alginate-gelatinmethacryloyl (GelMA) hydrogel, which was also evaluated by animal studies (Sprague-Dawley rats). The addition of alginate enhances the mechanical properties through a double network structure and supports tube formation. To enable axonal guidance and neurons migration, Netrin-1 (100 ng/mL) has been loaded (Figure 8A) [95]. The presence of G not only resulted in the enhanced proliferation of Schwann cells and guided their alignments but also led to a satisfactory Young's modulus (725.8 \(\pm\) 46.52 kPa) and electrical conductivity (6.8 \(\pm\) 0.85 S/m). The release of netrin-1 was significant in directing axon pathfinding and neuronal migration, which optimized tube formation ability at 100 ng/mL. In vivo studies showed that the NGC successfully supported peripheral nerve regeneration, restored denervated muscle, and was superior to the positive control (autologous graft). Additionally, the revascularization of denervated muscle was achieved, which is a crucial factor for regeneration and recovery after PNI. Another achievement of this study was enhancing the survival and function of Schwann cells both in vitro and in vivo. The extraction and cultivation of Schwann cells is complex and inconvenient, so many studies concentrate on stem cells such as adipose-derived stem cells (ADSCs) that can differentiate into Schwann-like, neuron-like, and endothelial cells [95].  
A further study utilized conductive hydrogel-based NGCs, made of GelMA/GO and followed by chemical reduction, to improve the electrical properties of the hydrogel, r(GO/GelMA) [97]. This is because restoring sp\({}^{2}\)-carbon bonds while minimizing rGO aggregation can enhance the electrical characteristics of GO-loaded hydrogels [15]. The fabricated NGCs had adequate electrical conductivity (r(GO/GelMA)\(>\)GO/GelMA), flexibility, mechanical strength, and permeability, and r(GO/GelMA) showed higher neuritogenesis enhancement of PC12 neuronal cells compared to previous studies. Muscle regeneration after NGC implantation was investigated by Sprague-Dawley rats by calculating the weight of the gastrocnemius muscles (both left and right). The left gastrocnemius muscle showed serious atrophy at 4 weeks, and the GelMA group underwent considerable shrinkage by 8 weeks. However, the other groups did not display atrophy and shrinkage; instead, they exhibited muscle recovery, particularly the r(GO/GelMA) and autograft (Figure 8B) [97].  
In order to optimize and evaluate the G concentration, a collagen-based NGC based was prepared using the cryogel technique to regenerate the spinal cord. The cryogels were stabilized by using amine-functionalized graphene as a nano-crosslinker and resulted in super-macroporous cryogel (Figure 9A). With the addition of 0.1%, 0.5%, and 1% \(w/v\) of G, an upward trend in electrical conductivity was recorded; however, there was no further increment despite enhancing the concentration above 1% \(w/v\), so this was found to be the plateau for the greatest amount of conductivity. The differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into neuronal-like cells has been demonstrated in the presence of graphene and ES. Based on the cell studies and organotypic spinal explants on samples, optimum neuronal differentiation was seen with 0.5% \(w/v\) G/collagen cryogels. Research has shown that BM-MSCs-seeded cryogels were able to secrete ATP energy upon ES. 0.5% \(w/v\) crosslinked collagen cryogels supplied adequate mechanical and electrical cues that encouraged the significant extracellular secretion of ATP. The cryogels have also shown that having a mixed population of M1 and M2 macrophages is necessary for nerve tissue repair [98].  
The introduction of micropatterns and bioactive substances into the inner wall of NGCs play a significant role in regulating Schwann cells behavior, axons elongation, and macrophages phenotype. Linear micropatterns with various ridges and grooves (3/3, 5/5, 10/10 and 30/30 \(\upmu\)m) were made on poly(D,L-lactide-co-caprolactone) (PLCL) films; following this, surface aminolysis and GO electrostatic adsorption were conducted (Figure 9B). GO has been used to provide enhanced cell attachment and proliferation, as well as electrical conductivity, in turn improving guiding. The GO-modified micropatterns accelerated the collective migration of Schwann cells and directed cells along with stripes by the fastest rate on the 3/3-GO film that contains the largest force of cell adhesion. It also resulted in tending macrophages to differentiate into the M2 type on the 3/3-GO film (optimum NGC). The NGCs were implanted to bridge the 10 mm rat sciatic  
Figure 8: (**A**) Schematic of netrin-1-loaded GMT/hydrogel conduit preparation. (**a**,**b**) Growing G onto a nickel mesh (CVD method), (**c**) covering G/nickel mesh with a precursor solution, (**d**) formation of strong ionic bonds between alginate and Ca\({}^{2+}\) ions due to immersing GMT into CaCl\({}_{2}\) solution, (**e**,**f**) polymerization of GelMA under UV light, (**g**) etching nickel template, and (**h**) Immersing the conduit in high concentrated netrin-1 solution, (**i**) peripheral nerves regeneration. Reused with permission from [95]. (**B**) Images of regeneration of muscle after NGC implantation in different groups. Reused with permission from [97].  
nerve defects for 4 and 8 weeks. To investigate function recovery of regenerated sciatic nerves, at 4- and 8-weeks post-operation, nerve conduction velocity and compound motor action potential were measured. The 3/3-GO group represented higher values than other groups. Additionally, in the 3/3-GO group, myelinated nerve fibers and blood vessels were generated more significantly than others after 8 weeks [99].  
Figure 9: (**A**) Schematic of projected collagen graphene cryogel mechanism: due to spinal cord injury, inflammatory cytokines and infiltration of inflammatory cells have been produced. By implanting the cryogels, it will promote proliferation and stemness maintenance of BM-MSCs and secrete anti-inflammatory biomolecules. Further, the presence of cryogels and macrophage infiltration will stimulate high polarization of the M2/M1 phenotype. Reused with permission from [98]. (**B**) The illustration depicts the PLCL film fabrication with stripe micropatterns and GO nanosheets and its use in four steps: (1) creating micropatterns by thermal pressing of a polydimethylsiloxane template onto a PLCL film, (2) aminylog by 1,6-hexanediamine then GO adsorption electrostatically, (3) manufacturing micropatterned PLCL/GO conduit, and (4) implanting into a rat with sciatic nerve defects. The middle schematic shows that the micropatterned PLCL/GO film can improve the directional migration of Schwann cells from their cell spheroids, induce the macrophages differentiation into M2 type, and guide the neurites of N2a cells along with the patterns. Reused with permission from [99].  
**Electrospun** & PCL/collagen/G & 0.5\%, 1\%, 1.5\%, & & & & & \(\uparrow\) concentration \(\Rightarrow\)\(\uparrow\) \\ **fiber** & & & & & & conductivity; (1\%): 5.27 \(\times\) \\  & & & & & 10\({}^{-6}\) S/m \\  & & & & & \(\uparrow\) concentration \(\Rightarrow\)\(\downarrow\) tensile \\  & & & & & & strength and elastic modulus \\  & & & & & & Optimum: 2\(\%\) PAG \(\Rightarrow\)\(\uparrow\) CPM \\  & & & & & & Conductivity: \\  & & & & & 10.8 \(\times\) 10\({}^{-5}\) S/cm \\  & & & & & & Elastic modulus: 2.67 MPa \\  & & & & & & Conductivity: 25 S/m \\ Foam & G/PCL & 2\% wt & PC12 & NI & \(\uparrow\) porosity & 2021 \\  & & & & & \(\uparrow\) cell proliferation and \\  & & & & & & extension \\  & & & & & No cytotoxicity \\  & & & & & & \(\uparrow\) piezoelectricity and \\  & & & & & electrical conductivity \\  & & & & & & High flexibility \(\Rightarrow\) easy and \\  & & 5\% wt & & & & appropriate NGC formation \\  & & & & & & \(\uparrow\)CPM (particularly 0.5\% and \\  & & & & & 1\%) \\  & & & & & & Elastic modulus: 720 kPa \\  & & & & & & Conductivity: 6.8 S/m \\
**Hydrogel** & & & & & & & \\  & & & & & & GMT \(\Rightarrow\) orientation of PNR, \\  & & & & & O2 and nutrition transport, \\  & & & & & \(\uparrow\) levels of S100 and Sox10 \\  & & & & & (1 PNR). \\  & & & & & Conductivity: 4.4 \(\times\) 10\({}^{-3}\) S/m \\  & & & & & (GO/GelMA) and 8.7 \(\times\) 10\({}^{-3}\) S/m \\  & & & & & S/m (rGO/GelMA) \\  & & & & & 2020 \\  & & & & & 1\(\uparrow\) neutrogenesis: \\  & & & & & \(\uparrow\) rGO/GelMA \(\Rightarrow\) GO/GelMA \\  & & & & & & \\  & & & & & \\  & & & & & & \\  & & & & & \\  & & & & & Optimum: 8\% \\  & & & & & Conductivity (5.27 \(\times\) \\  & & & & 10\({}^{-4}\) S/cm) + ES \(\Rightarrow\) \\  & & & & & 1\({}^{-}\) differentiation and \(\uparrow\) CPM. \\  & & & & & Asaiticoside released \(\Rightarrow\) no \\  & & & & & growth and collagen secretion \\  & & & & & \\  & & & & & of fibroblasts \(\Rightarrow\) No scars for \\  & & & & & NR. \\ \hline \hline \end{tabular}
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