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1. Introduction
The interfaces between material surfaces and cells play important roles in cell func-tions, including adhesion, proliferation, and differentiation. Many studies have reported that topographical features can be used to stimulate cells to guide and enhance cell performance. [ 1 ]
Topograph-ical surfaces with microscale roughness were reported to have particular use in neural cell growth because entire neu-rites can be trapped within the micro-structures, restricting the direction of neurite growth or generating tension at the cellular level. [ 2,3 ] This strategy has been
employed by designing and coating novel materials [ 4 ] to increase surface roughness,
immobilize cells, and enhance cell differ-entiation. Additionally, electrical stimula-tion is crucial for increasing the number of cells with neurites or shortening the dif-ferentiation time. [ 5 ] This stimulation can
be achieved by coating with conducting materials, such as carbon nanotubes, [ 6 ] a
conducting polymer, [ 7 ] or potentially previously unused
mate-rials. Moreover, the incorporation of neuronal guidance and drugs in materials can increase cell performance. [ 8 ] Certain
studies grafted nerve growth factor (NGF) onto the nerve guide structure or encapsulated NGF in polymeric microspheres [ 9 ] for
delivery. [ 2,10 ] However, to the best of our knowledge, most
plat-forms cannot seal great amounts of growth factor into spheres and control its release precisely in real time, which may lead to undesirable release, causing a low effi ciency of cellular growth and differentiation. The integration of precisely controlled neuron/material interfaces with multifunctionality and guid-ance modalities (physical cues, chemical cues, and electrical stimulation) into a single platform to enhance cell growth and differentiation is one possible approach to rapidly reconnecting severed nerve ends.
The need to address this challenge stimulated us to design optimal scaffold architecture, including the use of new mate-rials to design specialized and effective neural interfaces that
Arrayed rGO
SH
/PMA
SH
Microcapsule Platform Integrating
Surface Topography, Chemical Cues, and Electrical
Stimulation for Three-Dimensional Neuron-Like Cell
Growth and Neurite Sprouting
Heng-Wen Liu , Wei-Chen Huang , Chih-Sheng Chiang , Shang-Hsiu Hu , Chia-Hsin Liao ,
You-Yin Chen , * and San-Yuan Chen *
The biocompatible thiol-functionalized rGO
SH/PMA
SHmicrocapsules
encapsulating nerve growth factor (NGF) are arrayed onto a transparent
and conductive substrate, i.e., indium tin oxide (ITO), to integrate
electri-cally stimulated cellular differentiation, electrielectri-cally controlled NGF release,
and topographically rough nano-surfaces into a 3-D platform for nerve
regeneration. The rGO
SH/PMA
SHmicrocapsules with microscale topography
function not only as an adhesive coating to promote the adhesion of PC12
cells but also as electroactive NGF-releasing electrodes that stimulate NGF
release and accelerate the differentiation of PC12 cells during electrical
stimulation. Once electrical treatment is applied, NGF release and
electri-cally enhanced cellular differentiation lead to an obvious increase both in
the percentage of cells with neurites and in the neurite length. This length
can reach nearly 90 µm within 2 days of cell culture. The average neurite
length is signifi cantly increased (four-fold) after culture on the rGO
SH/
PMA
SHmicrocapsule substrate for 2 days compared with culture on a
substrate without an rGO
SH/PMA
SHcoating. These multifunctional rGO
SH/
PMA
SHmicrocapsules may be used as potential 3-D patterned substrates
for neural regeneration and neural prosthetics in tissue engineering
applications.
DOI: 10.1002/adfm.201303853
H.-W. Liu, W.-C. Huang, C.-S. Chiang, Prof. S.-Y. Chen Department of Materials Science and Engineering National Chiao Tung University, No. 1001 Ta-Hsueh Rd. , Hsinchu , Taiwan 300, R.O.C E-mail: sanyuanchen@mail.nctu.edu.tw Prof. S.-H. Hu
Department of Biomedical Engineering and Environmental Sciences
National Tsing Hua University Hsinchu , Taiwan
Prof. C.-H. Liao
Department of Medical Research Buddhist Tzu Chi General Hospital
No. 707, Sec. 3, Chung-Yang Rd. , Hualien , Taiwan 970, R.O.C. Prof. Y.-Y. Chen
Department of Biomedical Engineering National Yang Ming University
No. 155, Sec. 2, Linong St. , Taipei , Taiwan 112, R.O.C. E-mail: irradiance@so-net.net.tw
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control neuronal development and neurite outgrowth. Recently, graphene and its derivatives, such as graphene oxide (GO) and reduced graphene oxide (rGO) sheets, have received great atten-tion in biomaterial applicaatten-tions because of their outstanding electrical, mechanical, thermal, and biocompatible proper-ties. [ 11 ] Using a simple layer-by-layer (LbL) assembly method,
a two-dimensional graphene nanosheet can be developed into three-dimensional (3-D) functional materials. For example, Hong et al. [ 12 ] formed multilayer hollow graphene capsules
using electrostatic interactions to produce (rGO-NH 3 +
/rGO-COO − ) n . Ju et al. [ 13 ] proposed a novel approach for the
fabrica-tion of positively charged polystyrene beads, or polymer nano-spheres, coated with negatively charged graphene with electrical conductivity. The use of the LbL approach and graphene mate-rial to form hollow spheres has shown advantages over tradi-tional capsule formulations. To address the present limitations in neuronal cell culture, our group combined a thiol polymer with rGO to provide a unique and suitable platform for neuron-like cells. A mesoporous microcapsule core with an rGO and polymer shell was designed and developed in this study to increase NGF encapsulation in the core, without decreasing electrical stimulation of the cells, and to enhance the surface roughness of the shell and its surface’s affi nity for cells. Fur-thermore, the substrate containing rGO-coated microcapsules has several advantages, particularly with regard to its fl exibility in pattern coating, electrical characteristics, surface roughness, and cell activity.
Herein, we describe a graphene/polymer LbL microcapsule substrate for the integration of multiple cues into one structure that enhanced the differentiation of rat pheochromocytoma
(PC12) cells. We fi rst functionalized the thiol groups on poly(methacrylic acid) (PMA) as well as rGO; we then oxidized the thiol groups into disulfi de linkages between the rGO SH and
PMA SH chains within the multilayer to provide structural
sta-bility. These microcapsules made from the two-dimensional rGO nanosheet can promote NGF release under electrical stim-ulation due to electroosmosis coupled with electrophoresis. [ 14 ]
Moreover, due to the deposition of rGO on the shell surface of the microcapsule to retain its intrinsic conductive nature, we can manipulate the proliferation and differentiation of PC12 cells by integrating electrical stimulation, surface roughness, and the NGF cue into one responsive, multifunctional rGO SH /
PMA SH LbL microcapsule substrate. Based on our
under-standing of the interaction between PC12 cells and the rGO SH /
PMA SH microcapsule substrate, we will apply this responsive
substrate to neural regeneration and neural prosthetics through neural tissue engineering.
2. Results and Discussion
2.1. Synthesis of rGO SH /PMA SH Mesoporous Silica
Figure 1 a schematically illustrates the preparation of rGO SH /
PMA SH LbL microcapsules in which the preloading technique
was conducted to encapsulate 90% of NGF (135 ng) in rGO SH /
PMA SH microcapsules. The LbL shell acquired versatile
func-tions by integrating inorganic and organic molecules. We used the small size of the rGO sheet to modify the thiol groups on its surface. The size of the rGO sheet was measured by DLS,
Figure 1. a) Preparation of rGO SH /PMA SH multilayers on the surface of MS particles by hydrogen bonding and formation of the disulfi de linkages.
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as shown in Figure S1 (see Supporting Information). Inorganic rGO SH was used in this study based on its electrical
proper-ties, biocompatibility, and functionalized thiol groups, whereas organic PMA SH , which has been widely used in LbL capsules in
recent years, [ 15 ] was used due to its biocompatibility, hydrogen
bond donors, and stability via disulfi de linkages. The thiol-func-tionalized PMA (PMA SH ) is deposited alternately with rGO SH
on the the surface of amine-functionalized, positively charged mesoporous silica (MS) particles through the hydrogen bonding between PMA SH and rGO SH . The coating step was repeated to
control the shell thickness and 6 bilayers of PMA SH /rGO SH
were used in this study. After the conversion of the thiol groups into disulfi de cross-links by chloramine T, an oxidative reagent, the LbL microcapsules became stable in the extracellular envi-ronment due to the disulfi de bonds.
Figure 1 b schematically shows the arrayed rGO SH /PMA SH
MS as a growth factor reservoir that can be used to study release behavior from extracellular matrices and as a template that can be applied to improve PC12 cell proliferation and differentia-tion in response to an electrical stimulus. The microcapsule template combines the effects of surface topography, the NGF chemical cue, and electrical stimulation to enhance PC12 cell performance on this substrate.
2.2. Structural Morphology of rGO SH /PMA SH MS
The MS core particles displayed a structurally uniform mor-phology and had a mean particle size of 3 µm. Figure 2 a displays
an optical micrograph of the orderly confi guration of the NGF-loaded rGO SH /PMA SH microcapsules on a transparent ITO
substrate. In Figure 2 b, SEM imaging of the rGO SH /PMA SH
microcapsules further demonstrates that the dried particles were uniformly and compactly arranged on the ITO substrate. The self-assembly of these microcapsules on the ITO susbtrate was regulated and fi xed by particle interactions through the solvent evaporation and 2-hydroxyethyl methacrylate (HEMA) polymerization processes. As the solvent evaporated slowly, the microcapsules were gathered together and arranged on the substrates to form 3-D structure as illustrated in Figure S2, Supporting Information. The enlarged SEM image in Figure 2 c shows that the surface of the MS after assembly with rGO SH /
PMA SH molecules displayed a rough texture, implying that the
rGO SH layer was adsorbed on polymer spheres. The coating
quality was directly dependent on the concentration of PMA SH
and rGO SH, the lateral length of the rGO SH sheet, and the
coating time. We repeatedly used ultracentrifugation with high-power ultrasonication to obtain a narrow size distribu-tion of rGO SH sheets, which were generally smaller than the
diameter of the MS core. Although we precisely controlled the concentration and size to obtain individual particles that were homogeneously coated with PMA SH and rGO SH , a few rGO SH
sheets adhered to more than one sphere and formed a bridge linking nearby particles simultaneously, as shown in Figure 2 c. The SEM image in Figure 2 d shows the collapsed morphology of a sample after removing the MS core using HF/NH 4 F,
indi-cating that the LbL microcapsules exist in a hollow state. The rGO SH /PMA SH microcapsules’ core shell structure is clearly
Figure 2. a) Optical micrograph of the arrayed microcapsules. b) SEM image of the rGO SH /PMA SH microcapsules. c) High-magnifi cation SEM image
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shown in the PL microscopy images in Figure S3, Supporting Information.
2.3. Characterization of rGO SH /PMA SH Microcapsules
The size of the rGO sheet used for coating was measured by DLS, as shown in Figure S1, Supporting Information. To verify the coating of rGO onto the microcapsules, we used a Raman spectrometer coupled with an OM to evaluate the microcap-sules before and after coating with rGO. As shown in Figure 3 a, a laser beam was directly focused on the microcapsules. In Figure 3 b, the Raman spectrum of the MS microcapsules dis-plays only two peaks, one at 520 cm −1 and the other at 943 cm −1 , which are the fi rst-order and the second-order phonon scat-tering of silica, respectively. The G-band and the D-band were attributed to the fi rst-order scattering of the E 2g vibration mode
in the graphite sheets and structural defects (disorder-induced modes), respectively. For the rGO SH /PMA SH microcapsules,
the following were clearly observed: a D-band at 1322 cm −1 ; a G-band, which had sp 2 -hybridized carbon-carbon bonds, [ 16 ] at
1602 cm −1 ; and a 2D-band, the overtone (second harmonic) of the D-band, at 2918 cm −1 . The relative ratio of D- to G-band intensity ( I D / I G ) was 1.24, which corresponds to the Raman
spectrum of rGO. Moreover, the thiol groups on the rGO and PMA appeared at 2605 cm −1 . The results demonstrated that rGO and thiol molecules were successfully deposited and adsorbed on MS.
To assess whether the rGO SH /PMA SH MS substrate can retain
the intrinsic electrical conductivity of rGO, we assessed the elec-trical conductivity of the rGO, rGO SH and rGO SH /PMA SH MS
substrates using the four-probe method. The rGO sheets, rGO SH
sheet and rGO SH /PMA SH MS showed electrical conductivities of
1.74, 1.62, and 1.01 S m –1 , respectively. Although a lower
elec-trical conductivity was obtained with rGO SH /PMA SH MS, our
study revealed that the electron transfer behavior still can occur in the substrate, even after the microcapsules are covered with PMA. After the rGO SH /PMA SH MS was subjected to cell culture
and electrical stimulation for 1 h at 1 V at 2 days, the rGO SH
sheets became somewhat exposed in some places which were demonstrated on the SEM images in Figure S4, Supporting Information. The morphology indicated that the electrical stimulation still can be applied on the cells adhered on rGO SH /
PMA SH due to the exposure of the rGO SH sheet and bridge
linking neighboring microcapsules. The possible mechanism can be illustrated in Figure S5, Supporting Information. When the NGF@rGO SH /PMA SH MS substrate was exposed to the
electric fi eld, the mobile counter ions tended to migrate toward the cathode and drag water and NGF molecules with them. At the same time, the negatively charged rGO SH sheets in the
PMA SH matrix may be subjected to an action toward the anode
and exposed on the surface. The NGF release from the rGO SH /
PMA SH MS substrate without and with electrical stimulation
was further studied and is shown in Figure S6, Supporting Information. A greater NGF release was observed in the pres-ence of electrical stimulation than with no electrical stimulation.
2.4. Evaluation of Cell Viability, Proliferation, and Differentiation in the Absence and Presence of Electrical Stimulation
To determine whether the microcapsule materials could act as suitable substrates, PC12 cells were grown for 1 or 4 days on the ITO, MS, and rGO SH /PMA SH MS substrates, after which the
cells were fi xed. The cell viability was measured using a live/ dead viability/cytotoxicity kit (Molecular Probes, Eugene, OR). Figure 4 a shows that the cell viability on the MS and rGO SH /
PMA SH MS substrates after 1 day was 47 ± 8% and 53 ± 9%,
respectively, which was slightly higher than the viability on the ITO substrate (43 ± 5%) at 1 day. There was no signifi cant dif-ference between the ITO and rGO SH /PMA SH MS substrates at
1 day. Moreover, an increased rate of effective cell viability was observed after 4 days when PC12 cells were cultured on the rGO SH /PMA SH MS substrate. We observed a signifi cant
differ-ence (** P < 0.01) in the cell viability between the ITO (66 ± 7%) and the rGO SH /PMA SH MS (99 ± 10%) substrates at 4 days.
The rGO SH /PMA SH MS substrate greatly enhanced cell viability
compared with the ITO and MS substrates. Representative
(a)
0 500 1000 1500 2000 2500 3000 3500 500 1000 1500 2000 2500 3000 Intensity (a.u.) Raman shift (cm-1) MS S-H D G 2D(b)
5 um
rGOSHPMASHMSFigure 3. a) Optical microscopy images of laser-irradiated rGO SH /PMA SH microcapsules. The red spot in this fi gure indicates that the laser beam was
focused on the rGO SH /PMA SH microcapsules. b) Raman spectrum of the MS and rGO SH /PMA SH microcapsules. The G-band and the D-band were
attributed to the fi rst-order scattering of the E 2g vibration mode in the graphite sheets and structural defects (disorder-induced modes), respectively.
For the rGO SH /PMA SH microcapsules, the D-band was at 1322 cm −1 , the G-band was at 1602 cm −1 , the 2D-band was at 2918 cm −1 , and thiol (S-H)
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live/dead images of cells cultured on the ITO, MS, and rGO SH /
PMA SH MS substrate for 4 days are shown in Figure 4 b–d.
These observations provided evidence for the importance of using the rGO SH /PMA SH MS substrate for the PC12 cell
cul-ture. An MTT assay [ 17,18 ] was also used to evaluate the cell
viability in this study, as shown in Figure S7 (Supporting Infor-mation), which presents the consistent change of PC12 cell viability on the ITO, MS, and rGO SH /PMA SH MS substrates
compared with those measured by a fl uorescent live/dead cell assay.
This trend is also apparent in Figure S8 (Supporting Informa-tion), which shows PC12 cell adhesion to and proliferation on the ITO and rGO SH /PMA SH microcapsule substrates without
electrical stimulation in the culture medium. These results imply that this rGO SH /PMA SH MS substrate possesses high
biocompatibility. The results can be attributed to the surface nano-roughness and the biocompatibility of rGO SH /PMA SH , which
has been reported to enhance cellular adhesion and activity, [ 19 ]
thereby leading to cell proliferation. [ 7,8 ] Furthermore, compared
with other substrates, rGO nanosheets have an intrinsic advan-tage in building interfaces with cells and tissue and are thus a good raw material to support neuronal cell proliferation. [ 20 ] For
example, Park et al. [ 21 ] suggested that graphene can be used as an
excellent nanostructured scaffold for enhancing human neural stem cell adhesion and differentiation in the long term. There-fore, as expected, PC12 cells grew very well on these regions, indicating that rGO SH /PMA SH MS does not have a harmful effect
on PC12 cells compared with other cell-growth substrates, such as ITO and MS. Furthermore, the rGO SH /PMA SH MS substrate
provided an optimal rough surface structure for the enhance-ment of PC12 cell adhesion as well as proliferation.
We expanded this study to observe the effects of electrical stimulation on cell behavior on the ITO, MS, and rGO SH /
PMA SH MS substrates (22 mm × 22 mm) by applying a
con-stant voltage of 1 V for 1 h. In this experiment, no NGF was added to the system; rather, only electrical stimulation was applied to each substrate. Figure 5 quantifi es the cell viability, the average neurite length and the number of cells with neur-ites after 2 days of electrical stimulation in Figure 5 a–c, respectively. A P value < 0.05 was considered to be statistically signifi -cant. Clearly, a substrate with electrical stimulation exhibited a statistically signifi cant improvement in cell performance over the substrate without electrical stimulation.
Although the electrical effect on the cell viability was not signifi cant, as shown in Figure 5 a, PC12 cells showed slightly enhanced viability on the rGO SH /PMA SH MS substrates.
How-ever, the enhancing effect of an electric fi eld on differentiation has been reported by several publications. [ 22 ] The proportion of
the neurite length and cells with neuriteswere greatly increased under an electric fi eld, as shown in Figures 5 b,c, compared with those on ITO because of the dominant surface topography. Moreover, the rGO SH /PMA SH MS substrate still induced
statis-tically signifi cantly greater PC12 cell differentiation than did the MS substrate. This result indicates that PC12 cell activity can be largely enhanced on the rGO SH /PMA SH MS substrate because
the coating of rGO SH and PMA SH onto MS particles can
increase surface roughness, conductivity, and biocompatibility. Consequently, MS with an rGO SH /PMA SH coating offers major
Figure 4. a) PC12 cell viability (%) using a live/dead assay at one day and four days. ( N = 5) Data are presented as the mean ± SEM. * P < 0.05 was
considered statistically signifi cant with respect to the MS substrate and ITO substrate after 4 days. ** P < 0.01 compared with the rGO SH /PMA SH MS
substrate and the ITO substrate after 4 days. b–d) Fluorescent (100×) images of live (calcein AM, green) and dead (ethidium homodimer-1, red) cells cultured for 4 days on b) ITO, c) MS, and d) rGO SH /PMA SH MS substrates.
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advantages over conventional fl at ITO and non-coated MS as a neural interface.
2.5. Neuronal Changes of PC12 Cells Cultured on NGF-Loaded rGO SH /PMA SH Microcapsules Following Electrical Stimulation
The effects of the combined NGF cue and electrical stimula-tion on neuron markers of β-III-tubulin and neuron-specifi c cytoskeletal protein in PC12 cells cultured on the rGO SH /
PMA SH microcapsules were observed by fl uorescence and
con-focal imaging. Figure 6 a shows representative fl uorescence images of PC12 cells cultured on rGO SH /PMA SH microcapsules
for 2 days in the presence of electrical stimulation. The cell nucleus was stained in blue, the actin cytoskeleton was stained in green, and the rGO SH /PMA SH microcapsules were stained
in red to observe the neurite outgrowth and cell-matrix inter-action. Based on the overlaid fl uorescence images, we found much longer neurite extensions among PC12 cells cultured on rGO SH /PMA SH microcapsules. To further study the PC12 cell
interaction with the arrayed microcapsules during electrical stimulation, cross-sectional confocal images were collected to understand the neurite outgrowth behavior. As shown in Figure 6 (b), the neurite did not grow straight and seemed to interact with surface structures, such as trenches, microcapsule surfaces, and gaps between the microcapsules. PC12 cells were found to be well interconnected with the rGO SH /PMA SH
micro-capsules via the neurites of the cells. The longitudinal section also indicates that the neurites grew deep into the rGO SH /
PMA SH microcapsules, whereas the nuclei remained above the
microcapsules. The cross-sectional image shows that the neu-rites were widely splayed above the rGO SH /PMA SH
microcap-sules, directly indicating the high differentiation of the PC12 cells. The longest neurites achieved nearly 90 µm in length within 2 days of cell culture, which is much longer than the length reported by Cho et al. illustrating that PC12 cells grown on a Ppy/MSN-NGF composite substrate can possess neurites of approximately 20 µm in length after 2 days in culture. [ 23 ]
In addition, Kang et al. [ 24 ] reported that NGF-loaded, 200-nm
porous polypyrrole conducting polymers used as a substrate for PC12 cells can enhance neurite outgrowth to approximately 23 µm, which was much shorter than the neurites of PC12 cells cultured on our microcapsule substrate.
Figure 7 shows fl uorescence images of PC12 cells cultured on rGO SH /PMA SH microcapsules imaged in a z -stack at depth
intervals of 10 µm along the cross-section at each depth. In the fi rst and second cross-sectional images, we observed that the
Figure 5. Cell behavior of PC12 cells attached on ITO, MS, and rGO SH /PMA SH MS substrates by electrical stimulation or not. a) Cell viability, b) the
neurite length, and c) percentage of cells with neurites with and without electrical stimulation. PC12 cells were exposed to a constant voltage of 1 V for 1 h. For the rGO SH /PMA SH MS substrate under different conditions (NGF +: with NGF; NGF -: without NGF; E +: with electrical stimulation; E –:
without electrical stimulation) ( N = 5), * P < 0.05 was considered statistically signifi cant.
Figure 6. a) Low-magnifi cation fl uorescence images of PC12 cells adhered on rGO SH /PMA SH microcapsules after electrical stimulation for
2 days. b) Confocal microscopy images demonstrating that the electrical stimulation passed through the rGO SH /PMA SH microcapsules and then
stimulated the PC12 cells to extend the neurite outgrowth into the rGO SH /
PMA SH microcapsules. (red = microcapsules; green = β-III-tubulin/Alexa
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cell nuclei were above the arrayed rGO SH /PMA SH
microcap-sules. Neurites adhered to and extended into the microcapsules in the following intervals (the third and fourth cross-sectional images). Certain healthy neurites even extended into the deeper part and sprouted fi lopodial (microspikes) extensions toward the arrayed rGO SH /PMA SH microcapsules. It was noted that the
neurites could grow in a particular downward direction when cultured on the arrayed rGO SH /PMA SH microcapsules, which
served as a 3-D platform for the PC12 cells and guided the sub-sequent directional neurite growth of PC12 cells. Such surface guidance of cells can be employed toward the repair and regen-eration of the nervous system. This result also suggested that a combined effect of surface topography, a chemical cue, and electrical stimulation can enhance neurite extension. There-fore, the NGF-loaded rGO SH /PMA SH microcapsules showed a
positive effect on cell viability, proliferation, and differentiation
because of the combined effects of the sur-face topography of the arrayed microcapsules, NGF release, and electrical stimulation. More importantly, when electrical stimuli were applied, greatly enhanced neurite outgrowths were detected in response to the increased differentiation. The success in inducing neu-rite growth with a longer length in the NGF-loaded rGO SH /PMA SH microcapsule system
is promising for the further investigation of neural regenerative medicine for long axonal connections.
2.6. Effect of NGF Release on Cell Viability, Proliferation, and Differentiation in the Absence and Presence of Electrical Stimulation
We continued to evaluate neuron differen-tiation and neurite outgrowth by combining electrical, chemical, and surface topograph-ical cues. Thus, we used the rGO SH /PMA SH MS substrate to
study the effects of NGF encapsulation and electrical stimula-tion on cell behavior by co-culture with PC12 cells under dif-ferent stimulated conditions, including with/without electrical stimulation and with/without NGF encapsulation in rGO SH /
PMA SH MS. The cell viability, the neurite length, and the
per-centage of cells with neurites at 2, 4, and 7 days are shown in Figures 8 a–c, respectively. There was no signifi cant difference ( P > 0.05) in cell viability between NGF-encapsulating and non-NGF-encapsulating rGO SH /PMA SH MS substrates by electrical
stimulation or not after 2 days as shown in Figure 8 a. At 4 days, the cell viability on the NGF@rGO SH /PMA SH MS substrate
without/with electrical stimulation showed no signifi cant differ-ence, as shown in Figure 8 (a), even though NGF was released at 25 ng and 111 ng, respectively, as shown in Figure S6. In addition, during 7 days of cell culturing, the cell viability
Figure 7. Confocal microscopy images of the neurite outgrowth of the PC12 cells that adhered onto surfaces of rGO SH /PMA SH microcapsules by electrical stimulation for 2 days. Positively
expressed β-III-tubulin was observed in PC12 cells in the areas of the neurite that were extended in response to the combined NGF cue and electrical stimulation. The entire culture system was imaged in a z -stack at depth intervals of 10 µm along the cross-section at each depth (red = microcapsules; green = β-III-tubulin/Alexa Fluor 488, cytoskeleton; blue = DAPI, nuclei).
Figure 8. Comparison of PC12 cell behavior on NGF-encapsulating and non-encapsulating rGO SH /PMA SH MS substrates with and without electrical
stimulation at 2, 4, and 7 days. a) Cell viability, b) the neurite length, and c) the percentage of cells with neurites of PC12 cells There was no signifi cant difference ( P > 0.05) in cell viability for NGF-encapsulating rGO SH /PMA SH MS substrates by electrical stimulation or not after 2, 4, and 7 days. There
were apparent differences ( P > 0.05) in cell viabilities for non-NGF-encapsulating rGO SH /PMA SH MS substrates when submitted to electrical
stimula-tion after 4 and 7 days, respectively. Importantly, a signifi cant enhancement of cell viabilities ( P < 0.05) was found for the respective time points when PC12 cells received the simultaneous application of NGF and electrical stimulation. There were signifi cant increases in PC12 cell differentiation on the NGF-encapsulating rGO SH /PMA SH MS substrate compared with those on the non-NGF-encapsulating rGO SH /PMA SH MS substrate after 2, 4, or 7 days
of culture. PC12 cells were exposed to a constant voltage of 1 V for 1 h each day ( N = 5; * P < 0.05, ** P < 0.01 was considered statistically signifi cant)
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with electrical stimulation was 8.9, whereas the cell viability on the rGO SH /PMA SH MS substrate without electrical stimulation
was 8.5. These results suggest that the amount of NGF does not play an important role in the viability of PC12 cells. In con-trast, for the non-NGF-encapsulating rGO SH /PMA SH MS
sub-strate, the cell viability was signifi cantly different in response to 4d E– and 4d E+ versus 7d E– and 7d E+. This result suggests that electrical stimulation can increase PC12 cell viability on the non-NGF rGO SH /PMA SH MS substrate ( P < 0.05).
PC12 cells with their neurite outgrowth length were sig-nifi cantly increased ( P < 0.05) on the NGF@rGO SH /PMA SH
MS substrate compared with those on the non-NGF@rGO SH /
PMA SH MS substrate in response to 2d E+ or 2d E– as shown
in Figure 8 b,c. Additionally, following 2 days of electrical stim-ulation, the neurite outgrowth of PC12 cells was greater than that of cells that were not exposed to an electrical stimulus.
When we observed the neurite length at 4 days, as shown in Figure 8 b, this parameter increased dramatically, from 80 µm to 145 µm, with electrical stimulation. At the same time, the percentage of cells with neurites, as shown in Figure 8 c, was enhanced from 33% to 85% because of an 86-ng increase in NGF release in response to electrical stimulation. During 1 week of co-culture, the percentage of cells with neurites and the neurite length were also increased. Moreover, the non-NGF-encapsulating rGO SH /PMA SH MS substrate under the same
condition was used as the control group. Smaller increases in neurite length and the percentage of cells with neurites were found on the non-NGF-encapsulating rGO SH /PMA SH
MS substrate compared with those on the NGF-encapsulating rGO SH /PMA SH MS substrate. For the
non-NGF-encapsu-lating rGO SH /PMA SH MS substrate, the neurite length was
1 ± 0.3 µm, 66 ± 5 µm, 2 ± 0.35 µm, 111 ± 11 µm, 5 ± 2 µm, and 150 ± 16 µm for samples 2d E–, 2d E+, 4d E–, 4d E+, 7d E-, and 7d E+, respectively, as shown in Figure 8 b. In Figure 8 c, we also observed that the percentage of cells with neurites was 2 ± 0.4%, 18 ± 2.3%, 3 ± 1%, 66 ± 4%, 5 ± 3%, and 85 ± 3% for samples 2d E–, 2d E+, 4d E–, 4d E+, 7d E–, and 7d E+, respectively, for the non-NGF-encapsulating rGO SH /PMA SH MS substrate. The
neurite length and the percentage of cells with neurites were statistically signifi cantly different ( P < 0.05) between NGF and non-NGF encapsulation rGO/PMA MS substrates. The above reported data demonstrate that both NGF and electrical stimu-lation show obvious effects on PC12 cell differentiation; never-theless, the contribution from NGF release has a greater effect on neurite length and the percentage of PC12 cells with neur-ites during a longer duration (7 days) of cell culture compared to that from electrical stimulation.
3. Conclusion
In summary, we have developed multifunctional microcapsules to encapsulate NGF by functionalizing the thiol groups on the surface of rGO nanosheets and depositing self-assembled rGO SH /PMA SH LbL onto the MS. The stimulus-responsive,
well-ordered rGO SH /PMA SH microcapsules with microscale
topography were arrayed into a 3-D extracellular matrix on a fl exible ITO substrate. The goal was to accelerate the prolif-eration and differentiation of PC12 cells by controlling NGF
release behavior and manipulating rGO SH /PMA SH
microcap-sule interfaces during electrical stimulation. Our results show that a combination of surface topography, a chemical cue, and electrical stimulation not only has a positive effect on cell via-bility but also strongly enhances the neurite outgrowth of PC12 cells. This biocompatible and conductive rGO SH /PMA SH
micro-capsule can also be integrated into a patterned substrate to pro-vide a new platform for tissue engineering applications.
4. Experimental Section
Materials : Graphite with an average mesh size of 325 and a purity of 99.8 mol% was supplied by Alfa Aesar. Concentrated sulfuric acid (95-97 mol% H 2 SO 4), nitric acid (69–70 mol% HNO 3 ), potassium
chlorate and phosphate-buffered saline (PBS) (pH 7), used for the preparation of buffers, were purchased from Sigma-Aldrich Co. Mesoporous silica (MS) (3-µm diameter, pore size = 2 nm) was used as a core. Poly(methacrylic acid, sodium salt) (PMA) (MW = 15000 g/mol) was purchased from Polysciences (USA), and dithiothreitol (DTT), N -chloro- p -toluenesulfonamide sodium salt (chloramine T), 2-hydroxy-2-methyl-propiophenone (Darocur 1173), cysteamine hydrochloride, 5,5′-dithiobis-2-nitrobenzoic acid (Ellman’s reagent), 2-hydroxyethyl methacrylate (HEMA), and 3-aminopropyltrimethoxysilane (APS) were purchased from Sigma-Aldrich. Fluorescein isothiocyanate (FITC) (MW = 389.382 g mol –1; Sigma) and rhodamine B isothiocyanate
(RITC) (W = 536.08 g/mol; Fluka) were used as an MS core-shell dye. NGF 2.5S (26 kDa, male mice; Invitrogen) was used as a model drug to characterize the release behavior. An NGF enzyme-linked immunosorbent assay (ELISA) kit was obtained from ChemiKine TM to
measure the amount of NGF.
Preparation of Mesoporous SiO 2 + Particles : A suspension of
3-µm-diameter SiO 2 particles dispersed in 1 mL of ethanol was reacted
with 250 µL of APS and 50 µL of 30% ammonia solution for 2 h. Next, the particles were washed several times in ethanol and then three times in distilled water. The resulting particles had a ξ-potential of 45 ± 3 mV, as measured in 10 m M sodium acetate buffer (pH 4).
NGF Loading into the Mesoporous SiO 2 + Particles : The mesoporous
SiO 2 + particles were washed with PBS (pH 5) and then washed with
deionized water. NGF (150 ng mL –1 ) was dissolved in PBS (pH 7.4) in
advance. The NGF was loaded into the mesoporous SiO 2 + particles by
exposing the particles to the NGF solution for 12 h followed by washing with water. The NGF diffused into the microcapsules in the pH 7.4 buffer solution.
Preparation of PMA SH : PMA solution (250 mg of 30 wt% solution)
was diluted with 5 mL of potassium phosphate buffer (0.1 M , pH 7.2).
The resulting solution was charged with EDC (70 mg) and NHS (45 mg), and the mixture was stirred for 15 min. Next, cysteamine dihydrochloride (7.5 mg, target modifi cation 20 mol%), which had been preoxidized in air for several days, was added to the mixture, and the reaction was allowed to proceed overnight. The resulting mixture was dialyzed extensively against distilled water, and the polymer was isolated via freeze drying. The resulting solid was purifi ed further by reprecipitation from water into dioxane. [ 25 ] A PMA sample with thiol groups at 6.125 mol% was
synthesized from PMA and cysteamine dihydrochloride via carbodiimide coupling. [ 26 ] Fluorescence labeling of PMA
SH was performed using a
1 g L –1 dimethylsulfoxide (DMSO) solution of FITC (typically 10 µg)
mixed with 1–10 mg of PMA SH at a concentration of 10 g L –1 in pH 7.2
phosphate buffer. The reaction between the maleimide in the fl uorescent dye and the thiol groups along the polymer was allowed to proceed overnight; then, the polymer was purifi ed via gel fi ltration and isolated via freeze drying. The PMA SH was incubated in a solution of 100 g L –1
DTT at pH 8 for at least 12 h and diluted with 10 m M sodium acetate
buffer (pH 4) to the required concentration.
Preparation of rGO SH : Graphite oxide (GO) was prepared from
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to a modifi cation of Hummers’ method. [ 27 ] Sonication promotes the
exfoliation of stacked GO sheets in oxidized graphite. Centrifugation allows the separation of large and small size graphene sheets. rGO sheets of an average size of 200 nm were obtained after sonication for 30 min, and centrifugation at 12 000 rpm was repeated three times. The size of the rGO sheets was determined by dynamic light scattering (DLS), and the results are presented in Figure S1, Supporting Information. The small-sized rGO sheets were thiol functionalized by reacting with cysteamine in ethanol suspension with the aid of the condensation agent dicyclohexylcarbodiimide (DCC). In a typical procedure, functionalized rGO was added to ethanol at 0.3 mg mL –1 and sonicated for 10 min. An
excess (relative to the calculated amounts of carboxylic groups present on the rGO) of DCC ethanolic solution (0.5 mg mL –1 ) was added, and
the solution was stirred for several minutes. Finally, 5 m M cysteamine solution was added, and the mixture was stirred again for 24 h at room temperature. The amount of cysteamine was double the amount that was estimated for the carboxylic groups. The thiol-functionalized rGO obtained was relatively unstable and tended to precipitate slowly after the reaction. The precipitate obtained after the reaction was separated from the solution by fi ltration and washed to remove unreacted molecules; the byproducts were then dispersed in ethanol by prolonged sonication (30 min).
Assembly of rGO SH /PMA SH LbL MS : A suspension of the SiO 2 +
particles (0.25 wt%) was washed with pH 4 buffer via several water centrifugation/redispersion cycles. The resulting suspension was combined with an equal volume (1 mL) of a 1 mg mL –1 solution of
rGO SH (thioled graphene) in 10 m M sodium acetate buffer, pH 4, and
adsorption of the rGO SH was allowed to proceed for 15 min with constant
shaking. Next, the particles were washed with fresh pH 4 buffer (three times), redispersed, and used in 1 µL of solution that was combined with 2 µL of reduced PMA SH in 10 m M sodium acetate buffer to a fi nal
concentration of PMA SH of 0.186 g mL –1 . PMA SH adsorption was allowed
to proceed for 15 min, after which the particles were washed with fresh 10 m M acetate buffer, pH 4. This procedure describes the assembly of a single bilayer, and the process was repeated until the desired number of bilayers was assembled. In this study, the PMA SH /rGO SH LbL structure
was designed to have 6 bilayers of PMA SH /rGO SH , and its outermost layer
was a PMA SH . The particles were treated with a solution of chloramine T
(2.5 m M ) in 2-( N -morpholine) ethane sulfonic acid (MES) buffer solution
(50 m M , pH 6) for 1 min; this was followed by two washing cycles with
MES buffer solution and sodium acetate buffer solution (20 m M , pH 4)
to achieve the oxidation of thiol groups into disulfi de linkages between the PMA SH and thioled graphene layers. After forming the disulfi de
bonds, the coating layers outside the core were stabilized. The rGO SH /
PMA SH LbL MS, as synthesized above, was deposited onto an indium tin
oxide (ITO, or tin-doped indium oxide)-coated conducting fl exible plate (PET substrate, JoinWill Tech. Co., Ltd., Taiwan; electrical resistance of sq/50 Ω) with dimensions of 20 mm × 20 mm × 0.02 mm. We used the HEMA to fi x the microcapsules on the ITO substrate to form 3D arrays by free-radical photopolymerization.
Determination of the Thiol Group Content : The thiol content of the resulting polymer was characterized using Ellman’s reagent. [ 26 ] The
degree of modifi cation, that is, the number of thiol groups immobilized on the PMA, was determined spectrophotometrically with Ellman’s reagent. First, 0.50 mg of conjugate was hydrated in 250 µL of demineralized water. Then, 250 µL of phosphate buffer (0.5 M , pH 8.0)
and 500 µL of Ellman’s reagent (3 mg) in 10 mL of 0.5 M phosphate
buffer, pH 8.0) were added. The samples were incubated for 3 h at room temperature. The supernatant was separated from the precipitated polymer by centrifugation (10 000 g , 10 min). Thereafter, 300 µL of the supernatant was transferred to a microtitration plate and subjected to an ELISA, and the absorbance was measured at a wavelength of 412 nm (DV990BV4, GDV). A cysteamine standard curve was used to calculate the number of thiol groups immobilized on the polymer.
Morphology of the rGO SH /PMA SH LbL MS : The microcapsules were
visualized using an optical microscope (OM), whereas the surface morphology and diameter of the rGO SH /PMA SH LbL MS were examined
using scanning electron microscopy (SEM) (JEOL JSM S6700, JEOL Ltd.,
Akishima-shi, Japan). For SEM analysis, the microcapsules were fi xed on a 0.5 cm × 0.5 cm silicon wafer and then critical-point dried. Finally, the microcapsules were coated with an ultrathin layer of platinum and examined by SEM under operation at an accelerating voltage of 15 kV. Furthermore, to investigate the thickness of the six-layer rGO SH /PMA SH
LbL coating on the MS, photoluminescence (PL) microscopy was used. RITC (MW = 536.08 g mol –1 , Fluka) and FITC-dextran (MW =
389.382 g mol –1 , Sigma) were used to tag the MS core and the PMA SH
layer, respectively, which allowed for microcapsule visualization under a PL microscope by sharply defi ning the inner and outer perimeters of the LbL microcapsule.
Study of NGF Release from the rGO SH /PMA SH MS Substrate : NGF@
rGO SH /PMA SH MS substrates ( N = 5) were placed into a six-well culture
dish with a well size of 20 mm and incubated in PBS for 7 days at 37 °C. Similarly, NGF@rGO SH /PMA SH MS substrates ( N = 5) in a six-well
culture dish with an electrical stimulation setup, as shown in Figure S9 (Supporting Information), were incubated under the same conditions. In total, 10 µL of supernatant was collected from each well at each time point and analyzed with a commercially available sandwich ELISA kit. An NGF ELISA (Catalog No.: CYT304, ChemiKine TM, USA and
Canada) was performed strictly in accordance with the manufacturer’s manual to examine the concentration of NGF released from the rGO SH /
PMA SH MS substrate into the PBS. The ELISA for NGF expression
involved the measurement of the change in optical density (OD), which was proportional to the NGF concentration, with a linear standard curve from 7.8–500 pg mL –1 ( R 2 = 0.997, P ≤ 0.001). [ 28 ] In this study,
supernatants were directly added to a 96-well ELISA microtiter plate in a buffer provided with the kit, after which the NGF ELISA was performed as previously described. Finally, the absorbance at 450 nm (Sunrise Absorbance Reader, Tecan Group Ltd., Switzerland) was used to calculate the NGF concentration present in each sample. Each NGF release experiment was performed in triplicate.
Cell Culture on the Test Substrates : The PC12 cell line (ATCC), derived from a rat pheochromocytoma of adrenal medullary origin, [ 29 ] has been
extensively used as a model system for neuronal differentiation. PC12 cells stop dividing and terminally differentiate when treated with NGF (Sigma). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to compare the cell viability of an ITO substrate, an MS substrate, and the rGO SH /PMA SH microcapsule
substrate. Cells were cultured in 75-cm 2 fl asks at a density of 10 000 cells
cm –2 and maintained in culture until the plates reached > 95% confl uence.
The PC12 cells were maintained in Dulbecco’s modifi ed Eagle’s medium (DMEM) (Invitrogen) supplemented with 85% RPMI 1640, 2 m M
L-glutamine (Gibco), 10% heat-inactivated horse serum (Gibco), and 5% fetal bovine serum (Gibco) at incubator settings of 5% CO 2 and 37 °C.
In all experiments, cells were harvested from subconfl uent cultures using trypsin and were then resuspended in fresh complete medium before plating. A comparison of the in vitro cell viability and proliferation on the ITO, MS, and rGO SH /PMA SH MS microcapsule substrates over
time was performed for PC12 cells. Briefl y, 2 × 10 5 cells were plated in
six-well plates in 1 mL of culture medium for 12 h or 48 h. At the end of the incubation, 100 µg of MTT solution was added and incubated for an additional 4 h. The medium was then replaced with 1 mL of DMSO. In total, 200 µL of the mixed solution was added to a 96-well plate to monitor the absorbance at a wavelength of 570 nm using a Sunrise absorbance microplate reader (CTECAN). The OD of the media was proportional to the number of viable cells. The media were changed every 2 days. All experiments were performed in triplicate.
Neurite Formation Observation and Assay : The PC12 cells cultured on the ITO, MS, or rGO SH /PMA SH MS microcapsule substrates were fi xed
in 3.7% formaldehyde in PBS for 30 min and permeabilized in 0.1% Triton X-100 for 30 min in 37 °C. The cell nuclei were visualized by incubation for 30 min with 200 ng mL –1 4′,6-diamidino-2-phenylindole
(DAPI) (Sigma). The actin cytoskeleton was stained with a secondary goat anti-mouse IgG1 Alexa Fluor 488 antibody (Invitrogen, Darmstadt, Germany) overnight, and each step was washed with PBS three times. Morphological changes in the PC12 cells were observed and photographed using confocal laser scanning microscopy (CLSM). Ten
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fl uorescence images were taken of randomly selected areas in each substrate. To quantify the number of neurite-bearing cells and their neurite length on each substrate, ImageJ software was used (NIH ImageJ ver.1.42, National Institutes of Health, Bethesda, MD, USA). The neurite length of PC12 cells was defi ned as the distance from the tip of a neurite to the junction between the neurite base and the cell body; the value was expressed relative to the respective cell soma. Each experiment was independently repeated fi ve times for each substrate. For statistical analyses, the Wilcoxon rank sum test or the two-tailed unpaired Student t -test was performed using the SPSS software package (version 13.0). A P -value < 0.05 was considered to be statistically signifi cant. All data are presented as the mean value ± SEM ( N = 5).
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgments
This work was fi nancially supported by the National Science Council of the Republic of China, Taiwan under Contract of NSC 102-2221-E-009 -023-MY3 and NSC100-2320-B-009-006-MY2 and by the “Aim for the Top University” program of the National Chiao Tung University and the Ministry of Education, Taiwan, R.O.C.
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Received: November 13, 2013 Revised: January 29, 2014 Published online: March 4, 2014
