Modulation of Neural Differentiation through Submicron-Grooved

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Modulation of Neural Differentiation through Submicrongrooved Topography Surface with modified Polydopamine Cheng-Hung Chen, Ching-Cheng Tsai, Po-Ting Wu, Ing-Kae Wang, Jiashing Yu, and Wei-Bor Tsai ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00556 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Applied Bio Materials

Modulation

of

Neural

Submicron-grooved

Differentiation

Topography

Surface

through with

modified Polydopamine Cheng-Hung Chena, Ching-Cheng Tsaia, Po-Ting Wua, Ing-Kae Wangb, Jiashing Yua*and Wei-Bor Tsaia* a.

Department of Chemical Engineering, National Taiwan University, No. 1, Roosevelt Rd., Sec. 4, Taipei, 106, Taiwan. Email: [email protected] (Jiashing Yu), [email protected] (Wei-Bor Tsai)

b.

Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, No. 195, Chung Hsing Rd., Sec. 4, Hsinchu, 310, Taiwan.

Keywords: Neuronal differentiation, Submicron-grooved topography, Stem cells, PC12 cells, Surface modification, Poly-dopamine

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Abstract:

Surface topography and bioactive molecules can generate physicochemical cues that control proliferation and differentiation of neural cells. In this study, polystyrene (PS) submicron-patterns with different widths (400 and 800 nm) and depths (100 and 400 nm) were prepared and subsequently modified with poly-dopamine (PDA) by a coating method. We examined neurites of PC12 cells and human adipose-derived stem cells (hADSCs) incubated in neuronal induction medium containing nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) respectively. Then, the differentiated cells on different grooved topographies were immunologically stained by Tuj-1 (a neuron marker) to compare the extent of neuronal differentiation. Our results showed that PC12 cells on grooved topography have predominantly bipolar neurite extension which align along the direction of the patterns while flat surface has multipolar neurites. We demonstrated that the depths of topography has a strong impact on neurite outgrowth and alignment. In terms of the number of neurites, neurite length and percentage of Tuj-1 positive cells, the 400/400 and 800/400 nm (widths/depths) PS grooves are appropriate for the cultivations of PC12 cells and hADSCs relative to those of other groups. In conclusion, the submicron-grooved topography and neurotrophic growth factors supported neurites outgrown and differentiated into neuron-like cells.


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1. Introduction Neuronal

differentiation

has

become

one

of

the

popular

researches

related

to

neurodegenerative disease, Parkinson’s disease, nerve injury and tissue regeneration

1-2.

Transplantation of Schwann cells has been considered as the golden standard in nerve regeneration 3. The proliferating Schwann cells form the bands of Büngner to direct regenerating axons across the nerve lesion 4. However, the source of Schwann cells restricts the application on clinical treatment and alternative cells have been investigated. As the research on stem cells becomes broader and deeper, stem cells have been a promising resource for nerve regeneration. Since stem cells can be differentiated into Schwann cell phenotype under specific stimulation, the transplantation of stem cell-differentiated Schwann cells has been a prospective treatment. Among different treatments being developed for nerve repair, the combination of stem cells and tissue engineering represents a vital treatment for generating artificial nerve tissue that could be used in the clinical setting

5-6.

It has been reported that adipose tissue is the most abundant and

accessible source of stem cells which have the ability to differentiate into neuronal cell lineages with appropriate stimulation by growth factors

3, 6-8.

In addition, PC12 cells, which are derived



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from a rat pheochromocytoma, have been widely used to investigate neuronal differentiation mechanisms and neurite outgrowth in many studies since the discovery of their neuronal differentiation ability upon exposure to nerve growth factor (NGF) 9-12. PC12 cells provide a good experimental model for studying the neuronal differentiation13-14 and human adiposed-derived stem cells (hADSCs) have the potential to differentiate into nerve-like cells 15-17. Neuronal differentiation and neurite outgrowth are affected by various factors including biophysical, biochemical, and topographic cues. Electrical gradients, stiffness, elasticity, mechanical contact guidance and topography are all biophysical cues which have great effects on the extension of growth cones

12, 18-21.

To generate biochemical cues, bioactive molecules

including forskolin, laminin, fibronectin, peptides, isobutylmethylxanthine and neurotrophins are essential elements

7, 18, 22-24.

By immobilizing bioactive molecules onto biomaterial scaffold,

biochemical cues generate and manipulate cell behavior. In addition, some kinds of stem cells including hADSCs have better performance in proliferation with the assistance of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) 25. Moreover, surface topography provides topographic cues to regulate neuronal cell proliferation and differentiation and induce orientation, migration, outgrowth and regeneration 26. Topographic influence has been reported that can greatly affect cellular response, and we know that submicron and nanoscale topography can modulate the neuronal cell differentiation. Therefore, the selection of suitable topographic cues to induce desired responses is important 3 ACS Paragon Plus Environment

8, 27-31.

In the previous

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studies, submicron-grooved topography with different feature sizes has been studied in the various cell types (from fibroblasts to stem cells)

32-35.

A previous literature summarized that

grooves and ridges with dimensions ranging from 35 nm to 25 mm in width and 14 nm to 5 mm in depth can induce cell alignment which may further influence the differentiation capability of various types of cells. For instance, 2 and 5 μm width square grooves on polydimethylsiloxane (PDMS) induced stronger orientation for rat dermal fibroblasts (RDF) than 10 μm width square grooves group. Mesenchymal tissue cells became highly polarized on 0.98-4.01 μm width square quartz grooves 36. Kim et al. (2009) exhibited that 1 μm in width of grooves and ridges pattern on PDMS significantly improved proliferation and neural differentiation of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) compared to the wider patterns

37.

Also,

Yang et al. (2016) displayed that human neural stem cells (hNSCs) have the better performance of differentiation on 300 nm in width of grooves and ridges polyurethane acrylate (PUA) substrates compared to the larger groove width revealed the importance of topographic cues

38.

39-41.

Except for those, accumulating studies have

Therefore, it is urgent to evaluate and realize

the cellular responses on submicron-grooved topography, which could provide useful information for us to design an appropriate scaffold for enhanced neuronal tissue regeneration

18, 42.

Furthermore, the effect of groove width and depth on neuronal differentiation of various cell types has been partial investigate previously and also require to be further elucidated.



In this work, we cultured PC12 cells and hADSCs on different width/depth grooved topography and studied the relationship between neuronal differentiation on cells and surface topography, as shown in Scheme 1. In addition, the neurite length, neurite distribution and neurite alignment on different submicron-patterns were also discussed. Therefore, our data suggest that submicron-grooved topography and neurotrophic growth factors supported neurites outgrown and differentiated into neuron-like cells.

2. Results

2.1. Surface Characterization

SEM images of the submicron-grooved topographic silicon wafers with different width (W) and depth (D) are shown in Figure 1A and Figure 1B (W/D (nm): 400/100, 400/400, 800/100, 800/400). The silica wafers were the masters of grooved PS while PDMS was first replicated from the silica wafers to fabricate PS molds (Figure 1C). 2.2. Surface Modification PC12 cells were cultivated on different chemical modified flat and 800/400 nm grooved PS surfaces for 24 h (Figure S1). From the figure, we could observe that fewer cells adhering on the flat and 800/400 nm grooved PS surfaces compared to the other groups. This result indicated that the cells adhesion on PS substrate could be improved after surface modification. The definition of a process is the neurite with a length longer than that of the cell body (27.45 ± 0.22 μm for PC12 5 ACS Paragon Plus Environment

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cells, over 1000 cells counted). Figure 2A shows that neurite length of PC12 cells on the grooved surface is longer than that on flat surfaces. The longer average neurite length on PDA and AMN modified grooves confirms that they are a suitable surface for PC12 cells to prolong. The immunostaining results showed fluorescent images of PC12 cells for 3 days on flat and grooved surfaces (Figure S2 and Figure S3). From the figure, the number of cells is less on PS control and PLL modified surfaces due to the poor cell adhesion. From the percentage of Tuj-1 result (Figure 2B), there is no significant difference between PS surface and the other modified groups on both flat and grooved surfaces. In this study, therefore, we use PDA modified surfaces due to the long average neurite length and the high Tuj-1 positive cells percentage.

2.3. Neuritogenesis Analysis of PC12 Cells

Figure S4 and Figure 3 are the microscope images of PC12 cells cultivated on the grooved surface with different W/D ratio for 1 day and 3 days respectively. The neurite percentage of the flat surface was significantly different from that of grooved surfaces (Figure 4A). Besides, 800/400 nm have higher neurite percentage than 800/100 nm. It suggested that the deeper grooves could stimulate neurites growth. Also, the neurite number could be influenced by the grooved surface, we estimated the percentage of neurite number, cells which were classified into monopolar, bipolar, multipolar neurites (Figure 4B). At first, the percentage of monopolar neurite on the flat and grooved surface 6 ACS Paragon Plus Environment


were almost the same. Moreover, the neurite number on grooved surfaces are mainly monopolar and bipolar because of the topographic limitation. Furthermore, as the width was fixed, the 400 nm depths have a higher percentage of bipolar neurites, and it showed that the deeper grooves have greater influences to direct neurites outgrowth on PC12 cells. On the other hand, when we kept the same depth, the 800 nm width group have a higher percentage of multipolar neurites. It could be suggested that cells have more space and freedom on wider grooves. From the figure 4A, the neurite percentage of cells on grooved surfaces is higher than that on flat surfaces. Moreover, the cells on the grooved surface also have longer average neurite length compared to those on flat surfaces (Figure 4C). There is a significant difference in average neurite length between groups with same widths but different depths, such as between 400/100 nm (48.35 ± 0.24 μm) and 400/400 nm (52.88 ± 0.76 μm), or between 800/100 nm (46.87 ± 2.07 μm) and 800/400 (53.44 ± 1.24 μm). However, there is no apparent difference between groups with different widths which indicates that the widths of grooves have less influence on neurite lengths. The accumulative percentage of neurite length were shown in Figure 4D. The median of neurite length on grooved surfaces are longer than that on flat surfaces (34.26 ± 0.90 μm). Similar to the result of average neurite length, there are significant difference between groups with different depth, such as between 400/100 nm (43.01 ± 0.96 μm) and 400/400 nm (47.34 ± 1.55 μm), or 800/100 nm (41.92 ± 2.26 μm) and 800/400 nm (47.29 ± 1.71 μm) but no apparent difference


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between groups with different width, which suggest that depths are more influential topographic factors than widths. The distribution of neurite length was presented as box plot in Figure 4E, showing the 12th, 25th, 50th, 75th and 88th percentiles. The notch interval of 400/400 nm and 800/400 nm are almost overlapped, while the notch interval of 400/100 nm is a little higher than 800/100 nm. The confidence level is 99% that the median of 400/400 nm or 800/400 nm grooves is larger than that of 400/100 nm or 800/100 nm. The results suggested that 400 nm depths are more suitable for neurite elongation. The angle between an axis of the grooves and the direction of neurite is used to estimate the neurite alignment. On the flat surfaces, the neurite direction distribution is uniform. The percentage of neurite aligned within 10° is about 90% on 400/400 nm and 800/400 nm surfaces, while it is approximately 70% of neurites aligned within 10° on 400/100 nm and 800/100 nm surfaces (Figure 5A). The results showed that the direction of neurites is greatly influenced by grooved surfaces, and depths have more effects on neurite alignment. 2.4. Immunocytochemistry Analysis of PC12 Cells PC12 cells cultivated for 3 days on flat and grooved surfaces were immunologically stained by phalloidin (an F-actin marker) or Tuj-1. DAPI was used to counterstain nuclei (Figure 6 and Figure S5). After the treatment of NGF and topographic cues, most of the PC12 cells differentiated into neuronal-like cells with Tuj-1 positive. On flat surfaces, the percentage of 8 ACS Paragon Plus Environment


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Tuj-1 positive cells is only 26.3 ± 9.1% which is much lower than grooved surfaces (Figure 5B). The result suggested that grooved topography could enhance neuronal differentiation of PC12 cells. The percentage of Tuj-1 positive cells on 400/400 nm and 800/400 nm grooves is higher than 400/100 nm and 800/100 nm grooves respectively, so the deeper grooves are suggested to have more effect on enhancing neuronal differentiation. Also, the results are consistent with the results of the average neurite length. 2.5. Neuritogenesis Analysis of Human ADSCs The microscope images of hADSCs cultivated on flat and grooved surfaces were shown in Figure S6, S7 and 7. The morphology of hADSCs grew from flat, fibroblast-like cells to elongated, spindle-shaped cells after 6 days culture. Besides, the hADSCs aligned along the grooves while grew randomly on flat surfaces The definition of a process is the neurite with a length longer than that of the cell body (47.44 ± 0.37 μm for hADSCs, over 1000 cells counted). The neurite percentage of hADSCs was shown in Figure 8A, which could be used to describe the extent of neuronal differentiation. The highest neurite percentage was 65.69 ± 3.68% on 400/400 nm surfaces, while the neurite percentage on flat surfaces was only 44.83 ± 1.78%. The neurite percentage on 800/400 nm grooves is a little higher than that on 800/100 nm grooves. It suggested that the deeper grooves are more influential to stimulate neurites growth especially on 400/400 nm grooves.


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The average neurite length on grooved surfaces is longer than that on flat surfaces (Figure 8B). There is a significant difference between 100 nm depths (400/100 nm with 89.05 ± 4.75 μm) and 400 nm depths (400/400 nm with 103.12 ± 5.52 μm). The results suggested that deeper grooves could stimulate neurite elongation. Figure 8C showed the accumulative percentage of neurite length on different surfaces. The median of neurite length on flat surfaces (71.54 ± 2.20 μm) is shorter than grooved surfaces. The median of neurite length was not dominated by certain parameters. The 800/400 nm grooves have the largest median of neurite length (91.31 ± 4.77 μm) while the 400/100 nm have the shortest (81.43 ± 4.10 μm). There is no difference between the median of 400/400 nm (88.96 ± 5.19 μm) and 800/100 nm (84.32 ± 1.47 μm) grooves. It suggested that both deeper and wider grooves can enhance neurites outgrowth. The notch interval of box plot results of 800/400 nm (94.31 ± 2.81 μm) and 400/400 nm (90.35 ± 2.92 μm) grooves do not overlap with other groups (Figure 8D). The confidence level is 99% that the median of 400/400 nm and 800/400 nm grooves are larger than other groups. The results showed that depths have more influence to neurite outgrowth. The alignment of neurites of hADSCs on different grooved surfaces was also measured. On flat surfaces, the neurite direction distribution is uniform. Over 60% of hADSCs on 400 nm depths grooved surfaces aligned the grooves within 10°, while the percentage of neurite alignment dropped to 20-30% on 100 nm depth grooved surfaces. It is suggested that the deeper grooved



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topography significantly affected hADSCs alignment. The results are similar to PC12 cells (Figure 9A). 2.6. Immunocytochemistry Analysis of hADSCs The hADSCs cultivated for 6 days on flat and grooved surfaces were immunologically stained by phalloidin, Tuj-1 antibody and DAPI were used to counterstain nuclei. In Figure 10, we displayed the neurites and F-actin filament via phalloidin staining. In Figure S8, hADSCs were stained with Tuj-1 to analyze the results of neuronal differentiation after cultivated in medium with bFGF for 3 days. However, the results showed that almost no Tuj-1 positive signal expressed, which means no cells differentiated into neuronal-like cells. When the differentiated time was prolonged to 6 days, and the medium was supplied with forskolin, the confocal images of Tuj-1 staining suggested that most hADSCs differentiated into neuronal-like cells (Figure 11). Moreover, the alignment of cells is also able to be observed. The percentage of Tuj-1 positive cells on flat surfaces (18.8 ± 0.4%) is lower than on grooved surfaces (Figure 9B). A higher percentage of hADSCs with Tuj-1 positive expressed on 400/400 nm (47.9 ± 14.8%) and 800/400 nm (49.5 ± 11.4%) than 400/100 nm (36.3 ± 4.1%) and 800/100 nm (37.4 ± 1.3%) grooved surfaces. The results demonstrated that deeper grooved surfaces could enhance neuronal differentiation of hADSCs. 3. Discussion In this work, we show that submicron-grooved topography with different widths and depths 11 ACS Paragon Plus Environment

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combined with neurotrophic growth factors could affect cellular behaviors of PC12 cells and hADSCs. The cellular morphology, neurite formation, neurite number, neurite length, neurite length distribution, neurite alignment and neuronal differentiation on various grooved surfaces are investigated. First of all, submicron-grooved PS surfaces were modified by various chemicals to enhance cell adhesion. From phase contrast photographs of PC12 cells, cells adhesion were improved after surface modification (Figure S1). Among the groups, the PDA modification has the longest average neurite length on flat or grooved surfaces. Also, Tuj-1 immunofluorescence staining was also conducted to ensure that PDA modification could enhance neuron expression (Figure 2A, Figure 2B). It is demonstrated that PDA modified surfaces could improve neuronal differentiation as proven in the previous report 9. We further displayed that PDA surface treatment was better than the other chemical treatment methods in enhancing neuronal differentiation. After the determination of chemical modified surface, we examined the effects of the submicron topology on neural cells behaviors. PC12 cells were cultured on different widths (400 and 800 nm) and depths (100 and 400 nm) grooves to evaluate the influences of topographical cues on cells. Based on our data, we found that PC12 cells have a significantly higher percentage of bipolar neurites on grooves after stimulated by NGF compared to the flat surface. On the contrary, PC12 cells on grooves performed the significantly lower percentage of multipolar neurites, which can be explained that the cells will extend along the patterns. Similarly, some studies have verified that 12 ACS Paragon Plus Environment


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neurites of PC12 cells became multipolar shape on flat or bipolar elongations on grooved surfaces 41, 43.

Most importantly, the results manifested that groove depth is the most central parameter of

submicron-grooved surfaces in the percentage of PC12 cells with neurites, average neurite length, neurite length distribution and alignment. Moreover, the confocal images analysis illustrated that 800/400 and 400/400 nm grooves have a significantly higher percentage of Tuj-1 than 800/100 and 400/100 nm grooves (Figure 5B). In the part of hADSCs differentiation, the percentage of hADSCs with neurites increased as the groove depths increased. This observation resembled the result of PC12 cells (Figure 4A, Figure 8A). It suggested that the deeper grooves could stimulate the cell to grow neurites. Furthermore, the average neurite length also prolonged on deeper grooves. Because the critical values of neurite lengths might affect the mean value of neurite lengths, we compared the median value by analysis of box plot and found deeper grooves could improve neurite outgrowth indeed (Figure 8D). The immunostaining analysis also showed that deeper grooved surface with a higher percentage of Tuj-1 positive cells which means enhancement of neuron expression for hADSCs. A Previous literature has indicated that hADSCs could be induced into neuron-like or glial-like cells with the assistance of bFGF and forskolin 17. Our study further suggested that grooves could facilitate neural differentiation compared to the flat surfaces and the deeper grooves even had better performance.


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In conclusion, both kinds of cells showed that the groove depth has significant influences on nerites elongation and alignment compared to the groove width. On the same 400 nm depths grooves, about 90% of PC12 cells aligned along the grooves within 10° and 60% of hADSCs. These results were consistent to our previous study that groove depth was more influential on cellular morphology, proliferation, and differentiation than groove width

44.

It stated that both

biochemical factors and topographical cues could affect PC12 cells and hADSCs proliferation and differentiation, which coincides the expectation.

4. Conclusion In this study, the submicron-grooved topography and neurotrophic growth factors could affect neuronal differentiation of PC12 cells and hADSCs. These cells on various width and various depth exhibited different cell morphology, neurite formation, neurite outgrowth and percentage of neuronal marker expression which were regulated by surface guidance. The result of neuronal differentiation was quantified by Tuj-1 neuronal markers which indicated that the percentage of PC12 cells and hADSCs differentiated into neuronal-like cells was higher as the grooved depth increased. Moreover, the neuritogenesis analysis also provided evidence to verify that the topographical cues could affect neurites behaviors including the percentage, numbers, lengths and alignment of neurites for two types of cells. In sum, our



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findings provide both the chemical cue and surface topology for the design of the surface of biomaterials for nerve regeneration and repair.

5. Experimental method

5.1. Materials

Polystyrene (PS) was purchased from Nihon Shiyaku Industries (Osaka, Japan). Polydimethylsiloxane (PDMS) was received from Dow Corning (Midland, MI). Goat anti-rabbit IgG- Atto 488 secondary antibody and goat anti-mouse IgG- Alexa Fluor 568 secondary antibody were purchased from Sigma (St. Louis, MO, USA) and Gibco (Grand Island, NY, USA), respectively. 4’, 6-Diamidino-2-phenylindole (DAPI) was purchased from Invitrogen (Carlsbad, CA, USA). The other chemicals not specified were purchased from Sigma-Aldrich (St. Louis, MO, USA). The rat adrenal pheochromocytoma cell line (PC12 cell) and human adipose-derived stem cells (hADSCs) were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). RPMI 1640 medium contained 1% Penicillin-Streptomycin (P/S), 5% fetal bovine serum (FBS, JRH, Australia), 10% horse serum (Gibco, New Zealand), and 2mg/mL sodium bicarbonate. Dulbecco’s modified Eagle’s medium/ Nutrient mixture F-12 (DMEM/F12, Gibco, Grand Island, NY, USA) contained 10% FBS and 1% P/S.


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5.2. The Fabrication and Characterization of PS Molds

Silicon substrate with different topography were fabricated by electron beam lithography and dry etching 33. Before the PDMS replication, the silicon wafers were cleaned in piranha solution (98% H2SO4 and 30% H2O2 with a volume ratio of 7:3) at 90°C for 20 min to remove surface oxidized layer. The cleaned silicon substrates were immersed in 1% (v/v) OTS solution (octadecyltrichlorosilane/isooctane) for 5 min. The unreacted silane was washed by isooctane (Macron, PA, USA). The PDMS molds were cast on the silicon masters and cured at 70°C for 2 h. One drop of 3% (w/w) PS/toluene solution was dropped onto the polyethylene terephthalate (PET, Nan Ya Plastics Co., ROC) substrates and pressed by a PDMS mold with weight loaded (Figure 1). The imprinted grooved PS replicates were formed on PET substrates (PS/PET) after toluene was evaporated in the hood overnight. The surface topography of silica wafers and PS substrates was characterized by scanning electron microscopy (SEM, JSM-5310, JEOL, Tokyo, Japan).

5.3. The Surface Modification of PS Substrates

The PS surfaces were treated by different chemical methods including PDA, PLL, AMN and the low-pressure oxygen plasma. For the PDA surface modification, PS substrates were coated with PDA (0.25 mg/mL in 50 mM Tris buffer, pH 8.5) for 1 h followed by rinsing with sterile PBS. The PDA-coated substrates were immersed in a buffered solution of PLL (0.1 mg/mL in 50 16 ACS Paragon Plus Environment


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mM Tris buffer, pH 8.5) for 1 h to yield PLL-linked PDA surface modification. For the AMN surface modification, the PS substrates were immersed in 2% AMN solution (100 mg AMN in 50 g PBS and adjust pH to 8.5) for 1 h followed by rinsing with sterile phosphate buffered saline solution (PBS). For low-pressure oxygen plasma modification, the PS substrates were treated by plasma under 50 standard cubic centimeter per minute (SCCM) of oxygen flow rate under 1.3 Torr and 30 W power for 3 min. The plasma-treated PS substrates were stored in 70% ethanol (R.D.H., Germany) to maintain wettability and sterilize before cell seeding.

5.4. Cell Culture

PC12 cells were seeded on the 24-well plate with chemically modified PS substrates at the concentration of 5 × 103 cells/cm2 and with 0.6 mL RPMI 1640 medium per well. After PC12 cells adhered on PS substrates for 24 h, RPMI 1640 medium containing 100 ng/mL nerve growth factor (NGF) were added to stimulate neuronal differentiation of PC12 cells. Observing and analyzing cell morphology after 24 h and 48 h of neuronal differentiation. The hADSCs were seeded on the 24-well plate with chemical modified PS substrates at the concentration of 5 × 103 cells/cm2 and with 0.6 mL culture medium per well. After hADSCs adhered on PS substrates for 24 h, DMEM/F12 medium containing 5% FBS, 100 ng/mL bFGF and DMEM/F12 medium containing 10 μM forskolin were used to stimulate neuronal


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differentiation of hADSCs for 3 days, respectively. Observing and analyzing cell morphology after 6 days of neuronal differentiation.

5.5. Immunocytochemistry

Cells on the grooved PS surface were rinsed with PBS after incubation, fixed with 4% paraformaldehyde solution for 30 min, permeabilized with 1% Triton X-100 for 10 min at 37°C. After blocking with 2% bovine serum albumin (BSA) solution at 4°C overnight, the cells were incubated with primary antibodies for 3 h at 37°C. The following primary antibodies were used for staining: mouse anti-Tuj1 (1:500) as neuronal marker. After washing with 0.01% PBST solution, secondary antibodies including Alexa Fluor 568 goat anti-mouse (1:500) and Alexa Fluor 488 goat anti-rabbit (1:500) were incubated for 1 h at 37°C. F-actin was targeted by Phalloidin-TRITC (1:200) at 4°C overnight, and Cell nuclei were stained by DAPI (1:500) for 5 min at 37°C.

5.6. Analysis of Neuritogenesis

Neurite lengths, neurite alignment, fluorescent images were analyzed by using ImageJ software. Neurites were defined as process extensions of greater than a cell body in diameter. The cell body in diameter was defined as the average of at least 1000 cells body length. The neurite alignment was determined by measuring the angle between an axis of the cells and the grooves on 18 ACS Paragon Plus Environment


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the surface. The angles were divided 10° sectors from 0° to 90°. Percentage of Tuj-1 positive cells was calculated by the cell numbers of Tuj-1 positive cells relative to DAPI positive cells. The results were evaluated by at least three rounds of experiments and conducted with four surfaces each.

5.7. Statistic analysis

These results were reported as means ± standard error of the mean. The error bar was the standard deviation (SD). The statistical analyses between different groups were determined with Student-Newman-Keuls Multiple Comparisons Test. A value of p ≤ 0.01 was considered statistically significant difference. The statistically significant difference of box plot was determined by the notch, a confidence interval around the median, which is proportional to the interquartile range (IQR) of the sample and inversely proportional to the square root of the size of the sample (n). McGill et al. illustrated that the medians of two boxes were different with a strong evidence if their notches do not overlap. The width of notches normally based on the median  1.58 IQR/√n for 95% confidence (p ≤ 0.05) and median  1.81 IQR/√n for 99% confidence (p ≤ 0.01).

Supporting Information Phase contrast photographs of PC12 cells after culturing on different chemical modified flat and 800/400 nm topographical PS surfaces for 24 h, confocal images of PC12 cells cultured on flat 19 ACS Paragon Plus Environment

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and 800/400 nm PS plates after 48 h of differentiation (Cells were stained with DAPI and Tuj-1), Phase contrast photographs of PC12 cells and hADSCs on flat and different width/depth PS surfaces (Treated by PDA) 24 h after seeding (before neuronal induction), confocal images of PC12 cells cultured on flat and different PS plates after 48 h of differentiation and hADSCs cultured on plates after 3 days of differentiation (Cells were stained with DAPI and Tuj-1) and phase contrast photographs of hADSCs on flat and different width/depth PS/PET grooves were incubated in neuronal induction medium for 3 days (Treated by PDA) Notes The authors declare no competing financial interest.

Acknowledgments This research was supported from the Ministry of Science and Technology (MOST) of Taiwan (103-2221-E-002-207-MY3).

The authors thank National Nano Device

Laboratories, Hsinchu, Taiwan, for the fabrication of the nano-grooved silicon substrates.

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Scheme 1. The illustration of submicron-grooved topography of polydopamine modified surfaces and growth factors modulating neural differentiation



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Figure 1. The SEM images of (A) the depth of the topographical silicon wafers, (B) the topographical silicon wafer surfaces and (C) the topographical PS surfaces. The grooved widths and depths were represented as width/depth. Scale bar: 1 μm.


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Figure 2. (A) Neuronal differentiations of PC12 cells cultured on different chemical modified flat and 800/400 nm surfaces for 24 h. The neuronal differentiation of the PC12 cells was quantified by measuring the neurite lengths of the PC12 cells (n = 3, **: p < 0.01 and ***: p < 0.001, compared to the PS control, #: p < 0.05 and ###: p < 0.001, compared to the dopamine modified surfaces). (B) Immunocytochemistry of Tuj-1 of PC12 cells were induced to neuronal differentiation on different chemical modified PS surfaces (flat and 800/400 nm grooves). The amount of cells positive for Tuj-1 was quantified.



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Figure 3. Phase contrast photographs of PC12 cells on (A) flat and (B-E) different width/depth PS surfaces were incubated in neuronal induction medium (RPMI 1640 Medium containing NGF) for 48 h. All surfaces were treated by dopamine for 1 h. The grooved widths and depths were represented as width/depth. Scale bar: 100 μm.


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Figure 4. (A) Quantification of PC12 cells neurite extension after 48 h of neuronal differentiation on flat and different width/depth grooved PS surfaces. The neurites were defined as processes which have a length greater than that of the cell body (27.45 μm for PC12 cells which is determined in at least 1000 cells). The data showed the percentage of cells with neurites. (n = 3, *: p < 0.05, **: p < 0.01 and ***: p < 0.001, compared to the flat control, #: p < 0.05, compared to the same width). (B) The percentage of neurite number of PC12 cells cultured on flat and different width/depth PS surfaces for 48 h. The neurite numbers were classified into monopolar, bipolar and multipolar neurites (n = 3, **: p < 0.01 and ***: p < 0.001, compared to the flat control, #: p < 0.05 and ##: p < 0.01, compared to the same width, and +: p < 0.05, compared to the same depth). (C) Neuronal differentiations of PC12 cells cultured on flat and different 30 ACS Paragon Plus Environment


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width/depth PS surfaces for 48 h. The neuronal differentiation of the PC12 cells was quantified by measuring the neurite length of the PC12 cells (n = 3, **: p < 0.01 and ***: p < 0.001, compared to the flat control, #: p < 0.05 and ##: p < 0.01, compared to the same width). (D) The accumulative percentage of neurite lengths. Neuronal differentiation of PC12 cells cultured on flat and different width/depth PS surfaces for 48 h. (E) The box plot for neurite lengths of PC12 cells cultured on flat and different width/depth grooved PS surfaces for 48 h (n = 3, **: p < 0.01, compared to the flat control and ##: p < 0.01, compared to the same width)


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Figure 5. (A) The percentage of neuronal differentiated PC12 cells aligns along the flat and different width/depth PS grooves within 10° (n = 3, ***: p < 0.001, compared to the flat control, ##: p < 0.01 and ###: p < 0.001, compared to the same width). Values indicate the mean ± SEM for 3 independent experiments. (B) Immunocytochemistry of Tuj-1 of PC12 cells were induced to neuronal differentiation on flat and different width/depth PS surfaces for 72 h. The amount of cells positive for Tuj-1 was quantified. (n = 3, *: p < 0.05 and **: p < 0.01, compared to the flat control, #: p < 0.05 compared to the same width).



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Figure 6. Influence of submicron-grooved topography on PC12 cells behavior. Confocal images of PC12 cells cultured on (A) flat and (B-E) different PS plates after 48 h of differentiation. Cells were stained with DAPI for nuclear staining (blue) and Phalloidin for F-actin staining (red). The grooved widths and depths were represented as width/depth. Scale bar: 50 μm.


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Figure 7. Phase contrast photographs of hADSCs on (A) flat and (B-E) different width/depth PS/PET grooves were incubated in neuronal induction medium (DMEM/F12 containing bFGF and forskolin) for 6 days. All surfaces were treated by dopamine for 1 h. The grooved widths and depths were represented as width/depth. Scale bar: 100 μm.



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Figure 8. (A) Quantification of hADSCs neurite extension after 6 days of neuronal differentiation on flat and different width/depth grooved PS surfaces. The neurites were defined as processes which have a length greater than that of the cell body (47.44 μm for hADSCs which is determined in at least 1000 cells). The data showed the percentage of cells with neurites. (n = 3, *: p < 0.05, compared to the flat control, #: p < 0.05, compared to the same width) (B) Neuronal differentiation of hADSCs cultured on flat and different width/depth grooved PS surfaces for 6 days. The neuronal differentiation of the hADSCs was quantified by measuring the neurite length of the hADSCs (n = 3, *: p < 0.05 and **: p < 0.01, compared to the flat control, and #: p < 0.05, compared to the same width) (C) The accumulative percentage of neurite lengths. Neuronal differentiation of hADSCs cultured on flat and different width/depth PS surfaces for 6 days. (D) The box plot for neurite lengths of hADSCs cultured on flat and different width/depth grooved PS surfaces for 6 days (n = 3, **: p < 0.01, compared to the flat control, ##: p < 0.01, compared to the same width, and %%: p < 0.01, compared to the 400/400 and 800/100 nm or 800/400 and 400/100 nm)


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Figure 9. (A) The percentage of neuronal differentiated hADSCs aligns within 10° (n = 3, ***: p < 0.001, compared to the flat control, ##: p < 0.01 and ###: p < 0.001, compared to the same width) (B) Immunocytochemistry of Tuj-1 of hADSCs was induced to neuronal differentiation on flat and different width/depth PS surfaces for 6 days. The amount of cells positive for Tuj-1 was quantified. (n = 3, *: p < 0.05, **: p < 0.01 and ***: p < 0.001, compared to the flat control). Values indicate the mean ± SEM for 3 independent experiments.



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Figure 10. Influence of submicron-grooved topography on hADSCs behavior. Confocal images of hADSCs cultured on (A) flat and (B-E) different PS plates after 6 days of cultivation. Cells were stained with DAPI for nuclear staining (blue) and Phalloidin for F-actin staining (red). The grooved widths and depths were represented as width/depth. Scale bar: 50 μm.


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Figure 11. Influence of submicron-grooved topography on hADSCs behavior. Confocal images of hADSCs cultured on (A) flat and (B-E) different PS plates after 6 days of differentiation. Cells were stained with DAPI for nuclear staining (blue) and Tuj-1 for neuron staining (red). The grooved widths and depths were represented as width/depth. Scale bar: 50 μm.



254x133mm (150 x 150 DPI)

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Modulation of Neural Differentiation through Submicron-Grooved

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