Biomolding Technique to Fabricate the Hierarchical Topographical

Mar 30, 2017 - Statistically significant differences from the PDL-coated substrates were determined using ANOVA variances (p < 0.05) and are marked by...
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Biomolding Technique to Fabricate the Hierarchical Topographical Scaffold of POMA To Enhance the Differentiation of Neural Stem Cells Chien-Hua Hsu,† Ting-Yu Huang,† Rui-Da Chen,‡ Yuan-Xian Liu,† Ting-Yu Chin,*,§ Yui Whei Chen-Yang,† and Jui-Ming Yeh*,† †

Department of Chemistry, Center for Nanotechnology and Institute of Biomedical Technology, ‡Master Program in Nanotechnology and Center for Nanotechnology, and §Department of Bioscience Technology, Centre for Nanotechnology and Institute of Biomedical Technology, Chung Yuan Christian University, Chung Li, Taiwan 32023, Republic of China ABSTRACT: In this paper, a biomolding technique was first used to fabricate a scaffold of hierarchical topography with biomimetic morphology for tissue engineering. First, poly(ortho-methoxyaniline) (POMA) was synthesized by conventional oxidative polymerization, followed by characterizations with Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). Moreover, the POMA scaffold with 3D biomimetic morphology was fabricated using poly(dimethylsiloxane) (PDMS) as negative soft template from natural leaf surfaces of Xanthosoma sagittifolium, followed by transferring the pattern of PDMS template to POMA. The as-fabricated POMA scaffold with biomimetic morphology was investigated by scanning electron microscopy (SEM). Subsequently, cell−scaffold interactions were carried out by culturing rat neural stem cells (rNSCs) on biomimetic and nonbiomimetic, or flat, POMA scaffolds, as well as on poly(D-lysine) (PDL)coated substrate, and evaluating the corresponding adhesion, cell viability, and differentiation of rNSCs. Results showed that there was no significant difference in the attachment of rNSCs on the three surface types, however, both the biomimetic and flat POMA scaffolds induced growth arrest relative to the PDL-coated substrate. In addition, the percentage of cells with elongated neurites after 19 days of culture was higher on the biomimetic POMA scaffold relative to flat POMA and PDL. In summary, the POMA scaffold with biomimetic morphology shows promise in promoting rNSCs differentiation and neurite outgrowth for long-term studies on nerve regenerative medicine. KEYWORDS: biomimetic, hierarchical topography, neurite, differentiation, long-term phenotype and multipotency of mesenchymal stem cells.17 Apart from this, Yang et al. developed a hierarchically patterned substrate (HPS) platform that can synergistically enhance the differentiation of human NSCs (hNSCs). It simultaneously provides microscale and nanoscale spatial controls to facilitate the alignment of cytoskeleton and the formation of focal adhesions.18 The current scaffolds found in the majority of publications in terms of studies of cell−scaffold interactions are materials with artificial patterns through microscaled, nanoscaled or micro/nano-scaled hybrid structures to give a scaffold with hierarchical topography. In the past decades, biomimetic and bioinspired materials have been explored extensively.19−22 However, materials with hierarchical topographies mimicking natural structures have seldom been mentioned as scaffolds in cell culture. We believe that the biomimetic structures inspired

1. INTRODUCTION Currently, published literature on developing versatile materials with hierarchical topographies has evoked extensive and intensive academic and industrial research interests. There were some methods based on the combination of bottom-up and top-down approaches to prepare nanoscaled and microscaled hybrid structures. For example, casting of polymer solution, phase separation,1 and electrospinning have been used to fabricate the hybrid structures.2−5 Materials with hierarchical topographies can be found to apply in different fields, e.g. paints for boats,6,7 self-cleaning windshields for automobiles,8 metal refining, stain resistant textiles, antisoiling architectural coatings,9 catalytic materials,10,11 etc. Recently, the use of materials with hierarchical topographies applied in tissue engineering (such as cell culture) have also attracted lots of attention. For instance, Bettinger et al.12 and Hoffman-Kim13 mentioned that micropatterned or aligned fibers of polycaprolactone promoted the differentiation of retinal progenitor cells into neurons and glia14 and Schwann cell maturation,15 respectively.16 On the other hand, McMurray et al. reported that nanoscale-patterned polymer surfaces support long-term maintenance of the undifferentiated © XXXX American Chemical Society

Special Issue: Biomanufacturing for Tissue Engineering and Regenerative Medicine Received: February 7, 2017 Accepted: March 30, 2017 Published: March 30, 2017 A

DOI: 10.1021/acsbiomaterials.7b00091 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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2.3. Preparation of PDMS Template. The PDMS prepolymer was achieved by mixing the elastomer base and curing agent (10:1, w/ w) (Dow Corning 184 silicone elastomer). The prepolymer was poured into molds of fixing to a piece of natural leaf of Xanthosoma sagittifolium (the veins were removed) and then cured in an oven programmed at operational temperature of 60 °C for 4 h. After curing, the PDMS blocks were detached from the molds and used as soft negative templates for the following procedure of biomolding. 2.4. Preparation of Flat Scaffold of POMA and Biomimetic Scaffold of POMA. During the biomolding procedure, the asprepared fine powder of POMA was dissolved in NMP to get typically 5 wt % solutions. The as-obtained POMA solutions were spread onto glass plate (6 × 6 cm2) and PDMS template, separately, in fume hood. The solvent was then evaporated gradually on heating plate under the hood for 8 h. After the formation of dry flat scaffold of POMA (denoted as fPOMA) and dry biomimetic scaffold of POMA (denoted as tPOMA), the as-prepared neat scaffolds could be peeled off from the inorganic glass plate as well as the PDMS negative soft template, respectively. 2.5. Isolation and Culture of rNSCs. Pregnant Sprague−Dawley rats used in this study were purchased from the National Laboratory Animal Center (Taiwan, ROC). All animal operations received humane care according to the Guidelines for Care and Use of Experimental Animals29 (National Institutes of Health publication no. 85−23, revised 1996). This study was also approved by the Animal Research Ethics Board of Chung Yuan Christian University (Taiwan, ROC). rNSCs were isolated from the brains of Sprague−Dawley rat embryos at E14.5 as described in a previous study.30 Briefly, these forebrain tissues were cut into small pieces and stably reacted in sterile papain solution containing 30 mg mL−1 papain, 50 mM ethylene diamine tetraacetic acid (EDTA), 2 mg mL−1 cysteine, and 150 mM CaCl2 (5 babies per 1 mL) at operational temperature of 37 °C for 15 min. They were then reacted with DNase I for 5 min and then added into 10% horse serum. After centrifugation and washing with Hank’s Buffered Salt Solution (HBSS) twice, the dissociated cells were collected in a serum-free medium containing Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12), 10 ng mL−1 basic fibroblast growth factor (bFGF), 20 ng mL−1 epidermal growth factor (EGF), N2 supplement, 0.5% penicillin and 1% streptomycin. The number of live cells was counted by Trypan blue exclusion assay in a hemocytometer. rNSCs were cultured in noncoated flasks in culture medium and maintained at 37 °C in a humidified atmosphere of 5% CO2. After 1−3 days in vitro, growing cells formed neurospheres, which were suspended in the medium. Several weeks later, adherent cells were discarded and suspended neurospheres were collected by centrifugation, mechanically dissociated, and subcultured as global cells in a new culture flask with fresh medium containing the same concentration of bFGF. These cells grew into new spheres after 1−3 days, that is, rNSCs proliferated and formed new neurospheres. The process of subculture was repeated again to achieve the purified rNSCs and proliferating neurospheres. The rNSCs spheres are a nonadherent culture. The serum-free medium with N2 supplement and growth factors facilitated the rNSCs selection. By 3−4 weeks, the subcultures were homogeneously nestin-positive (Figure 1). 2.6. Cell Adhesion. To assess the cell adhesion on different substrates, rNSCs were cultured at a density of 5 × 103 cells/mL on different substrates in 24-well plate. After 24 h, the wells were washed to remove unbound cells. The number of adherent cells was monitored by Trypan blue assay. Attached cells were harvested by trypsinization followed by addition of PBS to create a new cell suspension. The cell suspension was added with 0.1 mL of 0.4% Trypan blue stain and mixed thoroughly. The mixture was allowed to stand for 5 min inside a 37 °C incubator. Finally, a hemocytometer was used to count the unstained (viable) cells separately under a microscope (Nikon TS100). 2.7. Determination of Cell Proliferation. The proliferation of rNSCs cultured on PDL, fPOMA and tPOMA for 1, 3, 5, and 7 days was evaluated by mitochondrial activity through a MTS [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-

from nature leaf can facilitate cell−scaffold interactions like the artificial structures. In the last decades, the recovery prospect for patients with neurodegenerative diseases in the central nervous system is suboptimal. Nowadays, the controlled differentiation of NSCs to functional neurons is a possible treatment strategy. However, the major obstacle for clinical NSC therapy is the lack of efficient methodologies for controlled differentiation to functional cell types for transplantation and survival rate, thus enhanced control of NSC differentiation to neuronal cells becomes one of the critical issues for the success of NSC-based therapies.23,24 Maintenance of cell survival and functionality is normally highly dependent on the interactions with the extracellular matrix. In particular, matrix topographical cues appear to regulate NSCfate.25,26 Several studies demonstrate that biomimetic topography can control diverse cellular functions including attachment, proliferation, differentiation, etc.27,28 Recent attempts at new strategies to promote neural regeneration have focused on the use of engineered materials that mimic the NSCs niche, in order to establish an adequate environment for neurogenesis. In this work, we attempt to fabricate the biomimetic poly(ortho-methoxyaniline) (POMA) scaffold with PDMS negative template by biomolding technique. Our studies reveal that the 3D biomimetic morphology of POMA can facilitate the rNSCs attachment by the adhesion and proliferation assay. Furthermore, the research indicates that the biomimetic scaffold may help induce rNSC differentiation by maintaining neurite outgrowth.

2. EXPERIMENTAL SECTIONS 2.1. Chemicals and Instrumentations. Ortho-methoxyaniline (Aldrich, 97%) was distilled prior to use. Calcium chloride (Aldrich, 97%), ammonium persulfate (Aldrich, 98%) and N-methyl-2pyrrolidone (Aldrich, 99%) were used as received without further treatment. Hydrochloric acid (Riedel-delHaën, 37% HCl in H2O) and ammonia solution (Riedel-delHaën, 28% NH3 in H2O) were used as received. Polydimethylsiloxane (PDMS, Dow CorningSylgard 184) was used as received without further purification. All the reagents were reagent grade unless otherwise stated. Attenuated total reflectance FT-IR spectra were obtained at a resolution of 4.0 cm−1 with a FT-IR spectrometer (JASCO FT/IR4100) at room temperature in the range of 4000 to 650 cm−1. Biomimetic surface morphology of scaffold was investigated by scanning electron microscopy (SEM), (Hitachi S-3200). The number-average and weight-average molecular weights of the polymers were determined on a Waters GPC-150CV equipped with a differential refractometer detector and a Styragel HT column using N-methyl-2-pyrrolidone (NMP) as eluent and monodisperse polystyrenes as calibration standards. Thermal stability of 3-D biomaterial was analyzed by Thermogravimetric analysis (TGA) with DuPont TA Q50 thermal analysis system in an air atmosphere. The scan rate was 20 °C/min and the temperature range was from 30 to 800 °C. 2.2. Synthesis of Poly(ortho-methoxyaniline) (POMA). POMA was synthesized by oxidative polymerization of orthomethoxyaniline (OMA) in 2.0 M of CaCl2/HCl solution with ammonium persulfate as oxidant, followed by cooling in an ice bath (0 °C) under stirring for 12 h. The precipitate was then collected and washed for several times until gray salts were removed. Afterward, it was treated with excess amount of 1.2 M NH4OH aqueous solution, followed by drying at operational temperature of 60 °C under a vacuum oven for 24 h. The blue powder of as-synthesized POMA at yield of ca. 45% had a weight-average molecular weight of 61,000 and a number-average molecular weight of 14 400, based on the gel permeation chromatography (GPC) studies with polystyrene as calibration standard. B

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3. RESULTS AND DISCUSSION 3.1. Characterization of POMA. The FTIR spectra of POMA are shown in Figure 2. As shown in Figure 2, the

Figure 1. Cell morphology and molecular marker expression of rNScs. (a) Suspended growth of neurospheres was observed. (b) Immunostainings with neural progenitor markers nestin (green) of the rNSCs after seeded on PDL-coated glass. Cell nuclei were stained blue with Hoechst 33342. Figure 2. FTIR spectra of POMA by a KBr compressed pellet. 2H-tetrazolium] colorimetric assay (cell titer 96 Aqueous one solution Promega, Madison, WI). The reduction of yellow tetrazolium salt [3(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H tetrazolium] in MTS to generate purple formazan crystals by dehydrogenase enzymes secreted by mitochondria of metabolically active cells. The formazan crystal dye showed an absorbance at wavelength of 490 nm and the amount of formazan crystals produced is directly proportional to the number of live cells. rNSCs were plated at a density of 5 × 103 cells/mL on each substrates in 24-well plate. After seeding the cells for a period of 1, 3, and 5 days, the media were removed from well plates and the scaffolds were washed with PBS to remove unattached cells. Then the cells incubated with 20% of MTS reagent in serum free medium for 1 h at 37 °C in a 5% CO2 incubator. Subsequently, the aliquots were diluted ten times with PBS and pipetted into a 96-well plate. The absorbance of the content of each well was measured at wavelength of 490 nm by using a spectrophotometric plate reader (ELISA Reader, BIO-TEK). 2.8. Live/Dead Cell Viability Assay. To assess survival of cells, the live/dead assay (Viability/Cytotoxicity kit for mammalian cells, Invitrogen, USA) was performed. Live cells were stained by Calcein AM, a green fluorescent dye (excitation/emission 495 nm/515 nm), and dead cells were stained by Ethidium homodimer-1 (EthD-1), a red fluorescent dye (excitation/emission 495 nm/635 nm). The mixture of Calcein AM (diluted 1:500) and EthD-1 (diluted 1:1000) in PBS were added to the wells and incubated in the dark for 15 min at room temperature. After discarding the dye, the wells were washed with PBS. Subsequently, cells were observed by a fluorescence microscope (Nikon ELIPSE TE2000-U, Tokyo, Japan). Images were processed using Metafluor image software (Universal Imaging Corporation, West Chester, PA). 2.9. Immunofluorescence Staining. Cells seeded on PDL, fPOMA and tPOMA scaffolds in 24-well plate were fixed in 4% of paraformaldehyde for 15 min and subsequently permeabilized with 0.3% of Triton X-100 solution in PBS. Nonspecific binding sites were blocked using 10% of goat serum in PBS for 2 h. Thereafter, primary antibody of mature neuron specific marker protein MAP2 was added at a dilution of 1:500 overnight at a temperature of 4 °C. After washing, Alexa Fluor488-conjugated second antibody (diluted 1:500) was added for 1.5 h at room temperature. Cell nuclei were counterstained with Hoechst (diluted 1:1000). The image of the cells was taken with a fluorescence microscope (Nikon ELIPSE TE2000-U, Tokyo, Japan). 2.10. Statistical Analysis. Experiments were run in triplicates and the data presented were expressed as mean ± standard deviation (SD). Statistical differences were determined using ANOVA variance. Differences were considered statistically significant at p ≤ 0.05.

alkoxyl-substituted POMA bands were found at wavelengths of 1257 and 1026 cm−1; they were attributed to C−O−C stretching of the Ar−O−C band. In addition, the characteristic peaks found at the wavelengths of 1589 and 1512 cm−1 were attributed to the stretching modes of N-Q-N and N−B−N. Moreover, the broad absorption peak between 2792 and 3000 cm−1 was assigned to the stretching of aliphatic C−H bonds. According to these results, the positions of peaks are similar to those reported by Harada et al.31,32 3.2. Morphology Investigations of Biomimetic POMA Materials. The three-dimensional (3D) surface morphological images of natural leaf of Xanthosoma sagittifolium and tPOMA were investigated in SEM at different magnifications, as shown in Figure 3. Figure 3a shows an image of the surface of natural leaf at a magnification of X 1,000 by SEM. It should be noted that the surface has lots of microscaled mastoids, each decorated with many nanoscaled wrinkles. Figure 3b shows the surface morphology of negative soft PDMS template obtained by detaching PDMS mold from the natural leaf. Moreover, the artificial POMA scaffolds without/with 3D biomimetic surface morphological image (i.e., fPOMA and tPOMA) were prepared separately for further comparative studies in tissue engineering of neural stem cell, as shown in Figure 3c, d. For a close-look of an individual 3D biomimetic pattern morphology image, the photograph at 5000 magnification of single microscaled mastoid, decorated with nanoscaled wrinkles, was found to exhibit ∼12 μm in height and ∼10 μm in diameter, as shown in Figure 3e. The overlook photograph at magnification of X 500 for the surface of as-prepared biomimetic materials was found to reveal considerable microscaled mastoids randomly distributed in POMA coating, as shown in Figure 3f. 3.3. Thermal Properties. Thermal stability of POMA scaffold was conducted in air by TGA programmed by heating from 30 to 800 °C, as shown in Figure 4. It should be noted that the thermal stability of scaffolds of fPOMA and tPOMA was found to be higher than that of neat POMA powder. This may be attributed to the high-pressure sterilization treatment (i.e., 1.5 atm, 121 °C) of POMA scaffold before cell culture studies. This treatment of POMA scaffolds could lead to crosslinking reactions between POMA polymers. This inference can C

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Figure 5. Attachment of neural stem cells on PDL, flat POMA (fPOMA) substrate, and biomimetic template POMA (tPOMA) substrate. After 24 h of seeding, the wells were washed to remove unbound cells. The number of adherent cells was monitored by Trypan blue assay.

Effectiveness of rNSCs attachment to the tested samples was in the range 83.6−86%. 3.5. Cell Viability and Cell Proliferation. The proliferation of rNSCs cultured on different substrates was measured using MTS method at days 1, 3, 5, and 7 (Figure 6a). The rNSCs showed a time dependent growth pattern on all the samples. Among all substrate tested, PDL-coated surface was the most effective to promote rNSCs proliferation. After culturing for 7 days, the growth of cells on fPOMA and tPOMA was obviously lower than on the PDL group. To confirm that the slower growth on fPOMA and tPOMA was not caused by cytotoxity, we used the live/dead assay (Viability/Cytotoxicity kit). Viable cells were stained by Calcein AM, a green fluorescent dye, and dead cells were stained by Ethidium homodimer-1 (EthD-1), a red fluorescent dye. Fluorescent imaging showed that rNSCs were mostly alive during culture on different substrates. In addition, the numbers of green fluorescent live cells was consistent with the results of MTS assay. Overall, the data in Figure 6a, b illustrate two types of POMA substrates that did not cause any cytotoxicity but considerably lower proliferation. 3.6. Enhanced Differentiation and Maintenance of Functional Neuronal Phenotype of rNSCs. To assess the behavior and obtain fully guided differentiation, we plated rNSCs on PDL-coated glass, fPOMA and tPOMA, and then treated with differentiation inducer dibutyryl cyclic AMP (dbcAMP) for 5−19 days.34 The differentiation inducer dbcAMP, which is a membrane permeable analog of cAMP, enhanced neuronal differentiation. cAMP can induce differentiation/maturation of NSCs into functional neurons. Our previous study showed that dbcAMP treatment also induced depolarization-dependent calcium influx, an indicator of neural function.30 Shrinking of the cell body and neurite outgrowth were considered as indexes of cell differentiation.37 Differentiated rNSCs with neurite length to cell-body diameter ratios higher than two were scored as neurite-bearing cells. Results showed that the differentiation-efficiency of rNSCs was not different for cells cultured on PDL-coated glass, fPOMA, and tPOMA (Figure 7b). But the matured neuron processes preferred to grow on tPOMA rather than fPOMA or PDLcoated glass. We observed that cells seeded on tPOMA tended to form smaller cell bodies with increased elongation of neurites relative to cells on fPOMA or PDL-coated glass (Figure 7b). The tPOMA containing the biomimetic structures of micro-

Figure 3. SEM image of (a) the Xanthosoma sagittifolium leaf surface, (b) PDMS negative template, (c) flat POMA surface, (d) biomimetic template POMA substrate surface, (e) cross-sectional view of the surface of the biomimetic template POMA substrate, and (f) biomimetic template POMA substrate 3D electrode surface topography.

Figure 4. TGA curves of POMA powder, flat POMA substrate, and biomimetic template POMA substrate.

be further identified by immersing the POMA scaffolds into NMP solvent, which results in the formation of NMP swollen POMA gel. 3.4. Cell Adhesion. The quantitative evaluation of attached cells on fPOMA and tPOMA was measured by Trypan blue assay (Figure 5). PDL is the most common substrate used for seeding rNSCs.30,33 PDL molecules can be coated to a culture surface and used to enhance the electrostatic interaction between negatively charged ions of the cell membrane. The coverslips coated with PDL served as controls. In general, no significant difference can be observed after culturing for 24 h. D

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Figure 6. (a) MTS results of neural stem cell on PDL, flat POMA substrate and biomimetic template POMA substrate after 1, 3, 5, and 7 days of cell seeding. Statistically significant differences from the PDL-coated substrates were determined using ANOVA variances (p < 0.05) and are marked by stars. The magnitude and error bars represent the average and standard deviation, respectively, from 3 different experiments. (b) Cell viability was assessed with live/dead staining and examination with fluorescence microscopy (living cells stained green and dead cells stained red). Scale bars are 50 μm.

through electrostatic bonds.35 But PDL-coated substrates have a disadvantage for long-term fate of neuronal differentiation and neurite formation (Figure 8). Studies have indicated that various artificial substrates can effectively induce cell growth, morphology, and differentiation, though those shaped with nanotopographical features are well-known to be the most effective.12 For instance, more effective cell differentiation has been induced in nanometric scale surface topography of biomaterials than in flat surface topography.34 Similarly, our results showed that POMA scaffolds with biomimetic topographical structures are able to promote differentiation and neurite formation as well as influence cell maturation (Figure 8). We speculated that the specific shapes and dimensions of leaf-templated surface nanostructures induce cytoskeleton reorganization of rNSCs, resulting in cell differentiation by activation of intracellular signal transduction and relative gene expression.34,36 Previous research has predominately focused on comparing a single type of pattern shape and dimension.

scale and nanoscale patterns may guide for more efficient spatial control of stem cell differentiation via the simultaneous modulation of the alignment of cytoskeleton and enhancing focal adhesion formation. A significantly higher percentage of neurite-bearing cells were observed on tPOMA compared to other control substrate groups (Figure 8a). Moreover, longer neurites are correlated with the maturation of the differentiated cells, and thus neurite lengths were quantified on the 19th day. Neurons appeared slightly more elongated when differentiated on tPOMA (Figure 8b). We speculated that the development of successful axon guidance toward successful synaptic connections might result from more neurite-bearing cells. Therefore, these data indicate that neurite formation and outgrowth of rNSCs during differentiation were enhanced by hierarchically patterned topography of tPOMA. PDL modulates cell adhesion through a nonreceptormediated cell-binding mechanism to promote neural adhesion E

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Figure 7. Neuronal differentiation of rNSCs culture on POMA substrate. Cell differentiation of rNSCs was studied over time points of 5, 12, and 19 days. To identify the effect of different substrate on neuronal differentiation, we performed immunocytochemistry in cells treated with or without 0.5 mM dbcAMP during these time points. Immunocytochemistry of MAP2 of rNSCs were induced to differentiation on PDL, fPOMA, and tPOMA substrates. MAP2 expression is stained green and Hoechst 33342 is shown blue (200 μm of the scale bar).

differentiated rNSCs on tPOMA would be of interest for further studies.

Herein, we examined the effect of a hierarchical natural structure on rNSCs differentiation. Together our results demonstrate that hierarchical topography can enhance rNSCs differentiation but does not support self-renewal of rNSCs. How these mechanisms contribute to changes in cell behaviors have to be studied in future experiments. Functional tests containing neurotransmitter release and synaptic plasticity of

4. CONCLUSIONS In conclusion, a biomolding technique was first used to fabricate the hierarchical topographical scaffold of POMA with biomimetic morphology to enhance the differentiation of neural stem cells. The POMA was synthesized by chemical oxidative F

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Figure 8. Maintenance of functional neuronal phenotype of rNSCs seeded on POMA substrates. The level of neurites formation and the length of neurite outgrowth quantified from MAP2-stained images which results from the cells were treated dbcAMP for 19 days. (* p < 0.05, compared to the PDL group; # p < 0.05, compared to the fPOMA). (2) Ma, M. L.; Mao, Y.; Gupta, M.; Gleason, K. K.; Rutledge, G. C. Superhydrophobic Fabrics Produced by Electrospinning and Chemical Vapor Deposition. Macromolecules 2005, 38, 9742−9748. (3) Gu, Z. Z.; Wei, H. M.; Zhang, R. Q.; Han, G. Z.; Pan, C.; Zhang, H.; Tian, X. J.; Chen, Z. M. Artificial Silver Ragwort Surface. Appl. Phys. Lett. 2005, 86, 201915. (4) Singh, A.; Steely, L.; Allcock, H. R. Poly[bis(2,2,2trifluoroethoxy)phosphazene] Superhydrophobic Nanofibers. Langmuir 2005, 21, 11604−11607. (5) Acatay, K.; Simsek, E.; Ow-Yang, C.; Menceloglu, Y. Z. Tunable, Superhydrophobically Stable Polymeric Surfaces by Electrospinning. Angew. Chem., Int. Ed. 2004, 43, 5210−5213. (6) Scardino, A.; De Nys, R.; Ison, O.; O’Connor, W.; Steinberg, P. Microtopography and antifouling properties of the shell surface of the bivalve molluscs Mytilus galloprovincialis and Pinctada imbricate. Biofouling 2003, 19 (Suppl), 221−230. (7) Schultz, M. P.; Kavanagh, C. J.; Swain, G. W. Hydrodynamic forces on barnacles: Implications on detachment from fouling-release surfaces. Biofouling 1999, 13, 323−335. (8) Quere, D. Non-sticking drops. Rep. Prog. Phys. 2005, 68, 2495− 2532. (9) Zielecka, M.; Bujnowska, E. Silicone-containing polymer matrices as protective coatings: Properties and applications. Prog. Org. Coat. 2006, 55, 160−167. (10) Le Goff, A.; Artero, V.; Jousselme, B.; Tran, P. D.; Guillet, N.; Métayé, R.; Fihri, A.; Palacin, S.; Fontecave, M. From Hydrogenases to Noble Metal−Free Catalytic Nanomaterials for H2 Production and Uptake. Science 2009, 326, 1384−1387. (11) Jeong, E. Y.; Ansari, M. B.; Park, S. E. Aerobic Baeyer-Villiger Oxidation of Cyclic Ketones over Metalloporphyrins Bridged Periodic Mesoporous Organosilica. ACS Catal. 2011, 1, 855−863. (12) Bettinger, C. J.; Langer, R.; Borenstein, J. T. Engineering substrate topography at the micro- and nanoscale to control cell function. Angew. Chem., Int. Ed. 2009, 48, 5406−5415. (13) Hoffman-Kim, D.; Mitchel, J. A.; Bellamkonda, R. V. Topography, cell response, and nerve regeneration. Annu. Rev. Biomed. Eng. 2010, 12, 203−231. (14) Steedman, M.; Tao, S.; Klassen, H.; Desai, T. Enhanced differentiation of retinal progenitor cells using microfabricated topographical cues. Biomed. Microdevices 2010, 12, 363−369. (15) Chew, S. Y.; Mi, R.; Hoke, A.; Leong, K. W. The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation. Biomaterials 2008, 29, 653−661. (16) Christopherson, G. T.; Song, H.; Mao, H.-Q. The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 2009, 30, 556−564. (17) McMurray, R. J.; Gadegaard, N.; Tsimbouri, P. M.; Burgess, K. V.; McNamara, L. E.; Tare, R.; Murawski, K.; Kingham, E.; Oreffo, R.

polymerization. The morphology of surface of the assynthesized biomimetic tPOMA demonstrated lots of micromastoids, each decorated with many nanowrinkles by SEM and AFM. A series of experiments on cell-scaffold interactions was carried out by culturing rNSCs on the POMA scaffolds and evaluating the corresponding adhesion, cell viability and differentiation of rNSCs. Results suggest that bioscaffold with biomimetic topographies did not show different rNSCs attachment but induced growth arrest relative to the PDLcoated substrate based on the results of cell adhesion and proliferation assay. Moreover, a higher percentage of cells with elongated neurites were found on the biomimetic scaffold at 19 days relative to the fPOMA and PDL controls. To sum up, we found the POMA scaffold with biomimetic morphology to facilitate rNSCs differentiation efficiency and maintenance of neurite outgrowth for long-term studies on nerve regenerative medicine.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +886-3-2653530. Fax: +886-3-2653176. *E-mail: [email protected]. Tel.: +886-3-2653340. Fax: +886-3-2653399. ORCID

Jui-Ming Yeh: 0000-0003-2930-0405 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support of the Ministry of Science and Technology, Taiwan, R.O.C., NSC 102-2632-M033-001-MY3 and NSC 104-2113-M-033-001-MY3, the Center of Nanotechnology, and Institute of Biomedical Technology at CYCU is gratefully acknowledged.

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REFERENCES

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DOI: 10.1021/acsbiomaterials.7b00091 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.7b00091 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX