Acknowledgements
5.5 Applications of Conductive Materials for Tissue Engineering
5.5.1 Applications for Nerve Tissue Engineering
the nervous system is a network of nerves and specialized nerve cells called neurons that transmit signals between parts of the body. primarily, it is the body’s way of communication among the parts of the body. Clearly, the nervous system is of great significance to the body by controlling the inter- actions in the physiological processes. harm to any of the nerves may inflict tremendous effects on the body and it is usually very difficult to recover the damaged nerve. it is extremely difficult for a nerve cell or neural tissue to regenerate on its own once neural damage has occurred. therefore, the need for neural tissue engineering arises as a promising technology to combat the negative effects of disease, aging or injury in the nervous system. the extracel- lular matrix in the body provides optimal conditions for topographical as well as chemical and electrical signals for the adhesion and growth of neural cells.
therefore, there is a significant need for a synthetic scaffold to play the role of the extracellular matrix, which must be a biocompatible, immunologically inert, infection-resistant and biodegradable biomaterial.156 Many efforts have been made to design a reasonable scaffold for nerve tissue engineering.
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00110
Table 5.1 summary of conductive materials utilized for tissue engineering application.a
applications Conductive
materials scaffolds Cell type references
nerve tissue
engineering ppy ppy/pdLCL pC12 52
ppy/siBs pC12 176
pCL–ppy drg 184
ppy–pLga pC12 185 and 186
ppy–chitosan schwann cell 174
ppy–pLLa pC12 187
ppy/pdLLa pC12 188
ppy–graphene rgCs 189
pani pani/pCL/gelatin nsCs 175
pani–pLCL–sF pC12 190
pani sh-sY5Y 191
pedot pedot–agarose schwann cells 136
piezoelectric
materials pVdF rat spinal cord
neurons 192
pVdF–trFe drg 193
pVdF–trFe hnsCs 194
Cnts Cnt rope nsCs 195
graphene graphene films Mouse hippo-
campal neurons 123 and 154
graphene foams nsCs 127
Bone tissue
engineering ppy ppy/heparin/pLLa osteoblast-like
saos-2 cells 196 pedot Bag/gelatin/
pedot:pss hMsCs 197
piezoelectric
materials pVdF MC3t3-e1
osteoblast 77 and 198
pVdF hMsCs 199
pVdF hasCs 200 and 201
pVdF–trFe/Bt human alveolar bone-derived cell 202
pVdF–trFe/Bt hpdLF 203
pVdF–trFe nih3t3 84
Cnts pdLa/MWCnts neonatal rat
osteoblasts 204 Muscle tissue
engineering pani Cpsa–pani/pLCL Mouse C2C12
myoblasts 205
pdLa/pani primary rat muscle
cells 206
pCL/pani Mouse C2C12
myoblasts 207 and 208 piezoelectric
materials pVdF Mouse C2C12
myoblasts 209
Cardiac tissue
engineering ppy ppy/pCL/gelatin primary
cardiomyocytes 210 pani pani/gelatin h9c2 rat cardiac
myoblasts 211
pani–pgs Mouse C2C12
myoblasts 212
(continued)
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00110
a ppy-coated poly(d,l-lactide-co-ε-caprolactone) (pdLCL) membrane can support the growth and differentiation of pC12 cells into neuronal pheno- types. additionally, the implantation of rats with nerve guidance channels constructed of conductive composite materials revealed myelinated axons and schwann cells comparable to those in the native nerve, indicating sciatic nerve regeneration in rats.52 Conductive core–sheath nanofibrous scaffolds, ppy–pCL, provide a good model for investigating the synergistic effect of topography and electrical stimulation on neurite outgrowth in vitro.
a dorsal root ganglion (drg) displayed good adhesion on the nanofibers and generated neurites across the material surface with nerve growth factor in the medium. Moreover, electrical stimulation was able to further enhance the neurite extension in comparison to non-stimulated nanofibers.184 Lee et al. found that ppy-coated poly(lactic-co-glycolic acid) (ppy–pLga) conductive meshes possessed excellent support for the proliferation and differentiation of embryonic hippocampal neurons and pC12 cells. Furthermore, the electri- cal stimulation showed positive effects on the neurite formation compared to non-stimulated scaffolds. stimulated cells on aligned ppy–pLga nanofibers led to an increase in neurite elongation and proportion of neurite-bearing cells related to cells on random nanofibers. these results indicated that elec- trical stimulation and topographical guidance show a combined effect on the utilization of these conductive scaffolds for nerve tissue engineering.185 also, they chemically immobilized nerve growth factor (ngF) to ppy-coated pLga fibers. the ngF modified fibers can provide support for pC12 cell growth and neuritogenesis without exogenous ngF in the medium. Moreover, elec- trical stimulation of pC12 cells through an ngF-modified ppy–pLga fiber Table 5.1 (continued)
applications Conductive
materials scaffolds Cell type references
Carbon nanomate- rials
pLga–CnF human
cardiomyocytes 135 Cnt–gelMa neonatal rat
cardiomyocytes 137 Cnt–hydrogel neonatal rat
cardiomyocytes 138 Chitosan/CnF neonatal rat
cardiomyocytes 213 Cnt–pgs/gelatin rat cardiomyocytes 99
a pdLCL, poly(d,l-lactide-co-ε-caprolactone); siBs, poly(styrene-β-isobutylene-styrene); pCL, poly(ε-caprolactone); pLga, poly(lactic-co-glycolic acid); pLLa, poly(l-lactic acid) or poly(l- lactide); pdLLa, poly(d,l-lactic acid); pLCL, poly(l-lactide-co-ε-caprolactone); sF, silk fibroin;
trFe, trifluoroethylene; Bag, bioactive glass; pss, poly(4-styrene sulfonate); Bt, barium titanate;
pdLa, poly(d,l-lactide); MWCnts, multiwalled carbon nanotubes; Cpsa, camphorsulfonic acid; pgs, poly(glycerol-sebacate); CnF, carbon nanofiber; gelMa, gelatin methacrylate. pC12, rat pheochromocytoma 12; drg, dorsal root ganglion; rgCs, retinal ganglion cells; nsCs, neural stem cells; sh-sY5Y, human neuroblastoma cell; hnsCs, human neural stem cells;
hMsCs, human mesenchymal stem cells; hasCs, human adipose stem cells; hpdLF, fibroblasts from human periodontal ligament; nih3t3, mouse fibroblasts.
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00110
increased neurite formation and neurite length by 18% and 17%, respec- tively, in comparison with non-stimulated cells on the fibers. indications of these results display that the combination of immobilized ngF with elec- trical stimulation can be used for neural tissue engineering applications.186
a ppy/chitosan membrane was found to promote schwann cell adhesion, spreading and proliferation. More importantly, electrically stimulated cells on the membrane significantly promoted the expression and secretion of ngF and brain-derived neurotrophic factor (BdnF) when measured against cells excluding electrical stimulation. this report was the first to investigate the potentiality of increasing nerve regeneration in conductive scaffolds by means of electrical stimulation-increased neurotrophin secretion.174 novel 3d fluffy ppy-coated poly(l-lactic acid) (pLLa) conductive fibrous scaffolds allowed easy cell entrance for a 3d cell culture. the number of rat pC12 cells cultured in the 3d scaffold was much higher than that on conductive fibrous meshes, indicating that the 3d scaffolds supported cell growth and prolifer- ation for a 3d culture.187
Xu et al. fabricated ppy/poly(d,l-lactic acid) (pdLLa) composite nerve conduits containing various ppy amounts. electrically stimulated pC12 cells seeded on the conduits showed increased and longer neurites than on pdLLa conduits as the content of ppy increased (Figure 5.5). interest- ingly, when using a 5% ppy/pdLLa conduit to reconstruct a rat sciatic nerve defect, the rats displayed functional recovery comparable to that of the gold standard autologous nerve graft, which was enhanced significantly more than that of the pdLLa conduits, indicating great potential for nerve tissue engineering.188
Yan et al. fabricated ppy-functionalized graphene (ppy-g)-based aligned nanofibers for electrical stimulation and controlled growth of retinal ganglion cells (rgCs). the results showed that the cell viability, neurite outgrowth and antiaging ability of rgCs were significantly increased with electrical stimula- tion (Figure 5.6). these findings provide the possibility for optic nerve regen- eration via electrical stimulation on the conductive nanofibers.189
electrospun conductive nanofibrous scaffolds prepared by mixing pani with pCL/gelatin (pani/pCL/gelatin) showed significant nsC proliferation and neurite outgrowth in comparison to non-stimulated scaffolds; indicat- ing potential applications for the attachment and proliferation of nerve stem cells.175 Zhang et al. synthesized conductive meshes of pani and poly(l-lac- tic acid-co-ε-caprolactone)/silk fibroin (pLCL-sF) coated with ngF for the investigation of electrical stimulation and ngF on neuron growth. pC12 cells on the pani–pLCL–sF scaffolds under electrical stimulation showed more and longer neurites. Furthermore, electrical stimulation was shown to help the ngF release from the conductive core–shell structure nanofiber.190 the growth and differentiation of several cells, such as human neuroblastoma sh-sY5Y cells, on pani memristive surfaces were investigated by Juarez- hernandez et al., and the results showed enhanced growth, proliferation and differentiation of cells seeding on pani films, demonstrating the suitability of pani memristors for nerve tissue engineering.191
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00110
Figure 5.5 Fluorescent images of pC12 cells labeled for actin (red) and nuclei (blue).
(a) and (B) pdLLa. (C) and (d) 5% ppy/pdLLa. (e) and (F) 10% ppy/
pdLLa. (g) and (h) 15% ppy/pdLLa. (a), (C), (e), and (g) Control cells.
(B), (d), (F), and (h) Cells stimulated with 100 mV for 2 h. scale bar: 200 µm. reprinted from Advanced Materials, 22, Y. Zhu, s. Murali, W. Cai, X.
Li, J. W. suk, J. r. potts and r. s. ruoff, graphene and graphene oxide:
synthesis, properties, and applications, 3906–3924, copyright (2010) with permission from elsevier.
Published on 03 May 2017 on http://pubs.rsc.org | doi:10.1039/9781788010542-00110
novel conductive polymer–hydrogel conduits (pedot–agarose) for axo- nal regeneration were synthesized by abidian et al. for the first time. the partially coated pedot–agarose conduit showed slightly better performance on supporting axonal growth than the fully coated pedot–agarose conduit.
this study provided a reasonable design for 3d conductive hydrogel scaf- folds for the acceleration, direction and controlling of axonal growth in the peripheral nervous system.136
electrically stimulated rat spinal cord neurons on a pVdF film substrate exhibited an increase in neuronal density and neurite number with over two times more branch points in comparison to those grown on non-stimulated film, which indicates the positive effect of electrical stimulation through the Figure 5.6 Confocal microscopy images of rgC cells seeded on (a) the random ppy-g/pLga nanofibers without es and (a′) after es; (b) the aligned ppy-g/pLga nanofibers with 1% (w/w) ppy-g without es and (b′) after es; (c) the aligned ppy/g-pLga nanofibers containing 6% (w/w) ppy-g without es and (c′) after es. (d) average cell length of rgCs without and after es. (e) Cell viability of rgCs cultured on the different substrates.
es conditions: step potential was pulsed between −700 and +700 mV cm−1. es was performed 1 h every day and lasted for 3 days. adapted from ref. 189 with permission from the american Chemical society.
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conductive film on neurite growth and branching.192 Conductive electrospun random and aligned scaffolds made of copolymer pVdF–trifluoroethylene (pVdF–trFe) showed good cell adhesion for drg neurons. however, only aligned scaffolds supported directed neurite outgrowth other than radial extension.193 human neural stem cells (hnsCs) cultured on these pVdF–
trFe substrates differentiated towards β-iii tubulin-positive cells, indi- cating neuronal cell differentiation for potential nerve tissue engineering applications.194
Cnts possess a unique array property allowing them to interact with neu- rons at a nanoscale level, and have been utilized in nerve-related research.
huang et al. developed a Cnt rope substrate to investigate the response of nsCs on the conductive substrate after electrical stimulation. the results show that the orientation of the spiral topography on the Cnt rope contrib- utes to neurite extension, and nsCs seeded on Cnt rope are differentiated towards neurons when compared with tissue culture plates (tCp). Moreover, electrically stimulated nsCs on the Cnt rope showed enhanced neuronal maturity and increased neurite outgrowth speed. their findings suggest a synergistic effect of the conductive Cnt rope substrate and electrical stimulation on promoting neurite extension as well as oriented differenti- ation of nsCs to mature neuronal cells in the application for nerve tissue engineering.195
park et al. reported a graphene substrate that showed a promotional effect on the differentiation of hnsCs into neurons. the graphene substrate was found to be a great cell-adhesion layer for persisting differentiation of hnsCs;
also, the hnsCs were more likely to differentiate toward neurons than glial cells (Figure 5.7). Moreover, the differentiated cells showed neural activity with electrical stimulation on the graphene substrate.123
Li and group’s work demonstrated that graphene films possess excellent biocompatibility for mouse hippocampal neurons. the number and average length of neurites on graphene were significantly enhanced compared to that on tCp; suggesting the potential of graphene as an implanted material for nerve tissue engineering.154 they further utilized graphene foams to fab- ricate a novel 3d porous scaffold for nsCs in vitro. the 3d graphene foam scaffold not only provides a better support for nsC growth and proliferation than two-dimensional (2d) graphene films, but also promotes the differen- tiation of nsCs to astrocytes as well as neurons. Moreover, the 3d graphene foams show good electrical coupling with differentiated nsCs for electrical stimulation. these findings indicate that the 3d graphene foams have great potential for nerve tissue engineering.127