Combined Chemical Groups and Topographical Nanopattern on the

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Combined Chemical Groups and Topographical Nanopattern on the Poly (#caprolactone) Surface for Regulating Human Foreskin Fibroblasts Behavior Yan Zhang, Xiaolin Du, Dan Hu, Jing Zhang, Yan Zhou, Guoquan Min, and Meidong Lang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01361 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 10, 2016

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ACS Applied Materials & Interfaces

Combined Chemical Groups and Topographical Nanopattern on the Poly (ε-caprolactone) Surface for Regulating Human Foreskin Fibroblasts Behavior Yan Zhang1,3*, Xiaolin Du1, Dan Hu2, Jing Zhang3, Yan Zhou2, Guoquan Min3, Meidong Lang1,* 1

Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science

and Engineering, East China University of Science and Technology, Shanghai, 200237, China 2

The State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237, China 3

Shanghai Nanotechnology Promotion Center, Shanghai, 200237, China

KEYWORDS: poly (ε-caprolactone), surface chemistry, nanopattern, UV-nanoimprint, cell behaviors

ACS Paragon Plus Environment 1


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ABSTRACT

Surface chemistry and substrate topography could contribute significantly to providing a biochemical and topographical cues for governing the fate of cells on the cell-material interface. However, the synergies between these two properties have not been exploited extensively for biomaterial design. Herein, we achieved spatial-controlled patterning of chemical groups on the poly (ε-caprolactone) (PCL) surface by elegant UV-nanoimprint lithography (UN-NIL). The introduction of chemical groups on the PCL surface was developed by our newly 6-benzyloxycarbonylmethyl-ε-caprolactone (BCL) monomer, which not only solved the lack of functional groups along the PCL chain but also retained the original favorable properties of PCL materials. The synergetic effect of the chemical groups and nanopatterns on the human foreskin fibroblasts (HFFs) behaviors was evaluated in detail. The results revealed that the patterned functional PCL surfaces could induce enhanced cell adhesion and proliferation, further trigger changes in HFFs morphology, orientation and collagen secretion. Taken together, this study provided methods for straightforward fabrication of reactive PCL surfaces with topographic patterns by one-step process, and they would facilitate PCL as potential candidate for cell cultivation and tissue engineering.


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INTRODUCTION Cell-surface interactions are known to trigger various signal transductions associated with cell fates such as adhesion, migration, proliferation and differentiation.1-2 The accumulated evidence demonstrated that the topographic and chemical cues of the matrix played vital roles in cellular behaviors.3-5 As well known, the chemical nature of extracellular matrix (ECM) is elaborated with small functional groups, such as hydroxyl (OH), methyl (CH3), carboxyl (COOH), amino (NH2) and sulfhydryl (SH) etc. Thus there are abundant studies centering on clarifying how small functional groups influence the cellular behaviors.6-8 Also, more recent work revealed that the micrometer- and nanometer- scale topography of cell substrates can influence cell behavior significantly.9-11 For instance, Cho et al. fabricated nanopatterned polyurethane acrylate (PUA) substrates by nanoimprint technique, and the nanotopographical cues could manipulate focal adhesion and promote differentiation of human neural stem cells (hNSCs).12 Yang et al. addressed that the nano-topographic poly (lactic-co-glycolic acid) (PLGA) substrates were able to induce the differentiation of primary mononucleated cells to form mature muscle patches.13 However, the synergistic effects of surface chemistry and topography have not been widely studied due to the limited availability of materials and methods that can be used to fabricate surfaces with suitable combinations of topography and chemical structure. Lynn et al. presented an approach to fabricate the combinations of well-defined topography and chemical functionality surfaces by the chemically functionalized post-fabrication. These dual functionalized substrates proved to be useful for controlling over cell proliferation, differentiation, and other important behaviors.14-15 Ding et al. created gold nanopatterns on Poly (ethylene glycol) (PEG) hydrogels via block copolymer micelle nanolithography plus transfer nanolithography, and the arginine-glycine-aspartate (RGD) were conjugated to the nanopatterns.16-17 Chen et al. has



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investigated

vertically-aligned

nanowire

arrays

and

porous

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gold.18-19

However,

the

nanolithography based on the micelle assembly is expensive and low throughput for substrate fabrication. In addition, no matter coating or grafting methods would suffer from some difficulties, such as unstability under certain conditions (temperature, solvent etc.), less polymer density and so on.20 Nanoimprint lithography (NIL) has been used to fabricate topographically patterned surfaces with feature size on the order of tens of nanometers to millimeters. Particularly, NIL could facilitate cost-effective, high throughput methods for substrate fabrication.21-22 However, the thermal nanoimprint lithography (T-NIL) was confined because heat was often exploited. The significant advances of the UV-NIL have further improved the nanoimprint process, such as reduced cycle time, low pressure and polymerization at room temperature,23 which have attracted intense attention to fabricate higher resolution nanopattern. Poly (ε-caprolactone) (PCL) is a popular biodegradable polymer in tissue engineering. However, PCL generally elicits a poor cell/substrate interaction due to the lack of cell-recognition sites. Our previous study has demonstrated that the inclusion of free amino groups along with the PCL could improve the cell-matrix interaction which in turn promoted cell adhesion, proliferation and differentiation of Human mesenchymal stem cells (hMSCs).24 In addition, some research groups created micro- and nanoscale features on the PCL surface to improve

the

cell/substrate

interaction.25-27

For

examples,

Lee’s

group

developed

spatially-controlled nano-topography via T-NIL on the PCL surface, and Mesenchymal stem cells (MSCs) differentiation was greatly promoted.25 Aoyaji et al. prepared the shape-memory activation of nanopatterns to observe time-dependent changes in cell alignment, and the memorized temporal pattern could return to the original permanent pattern by heating.27


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Nevertheless, the synergic effect of topography-chemical groups on the cell behaviors on the nanopatterned functional PCL surface has not been investigated due to the lack of functional groups along the PCL chain. Herein, we developed a synergetic large-area PCL film combined topographical patterning and chemical groups based on our novel functionalized ε-caprolactone monomer and UV-NIL method in a convenient and straightforward manner, and this cell culture scaffold combinations of well-defined topography and chemical functionality enabled systematic study of the topography-chemistry cues on the HFFs behaviors. Thus this study built a straightforward approach to provide topography-chemistry cues for investigating cell-surface interactions, which will facilitate PCL as a potential candidate for cell cultivation and tissue engineering. 2. MATERIALS AND METHODS 2.1. MATERIALS. ε-Caprolactone (ε-CL, 99 %) was bought from Aladdin and dried over calcium hydride followed by vacuum distillation. The monomer (6-benzyloxycarbonylmethyl, BOM)-ε-caprolactone (BCL) was synthesized and purified according to the previous study in our lab.28 (II)-2-ethylhexanoate (Sn(Oct)2, 95 %) and charcoal coated with palladium (10 wt.%) were supplied by Aladdin and Damas-beta respectively and used as received. Acryloyl chloride was purchased from Adamas without further purification. Dulbecco’s modified Eagle’s medium (DMEM) and trypsin were purchased from Gibco(USA). Fetal bovine serum (FBS) was purchased from BioSun. RIPA lysis buffer and BCA protein assay kit were supplied by beyotime Biotechnology (Shanghai, China). Cell Counting Kit-8 was purchased from DOJINDO (Japan). F-actin Staining Kit and Sirius Red Staining were bought from Life Technology (USA) and Servicebio(Wuhan, China) respectively. Phosphate buffer saline (PBS), Ethylene diamine



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tetraacetic acid (EDTA) and Paraformaldehyde (PFA) were purchased from Sigma-Aldrich. Other reagents were bought from Shanghai Lingfeng Chemical Reagent Co., Ltd. 2.2. Synthesis of Double Bond-Terminated PB and PC Copolymers. The double bond-terminated copolymer poly (ε-caprolactone-co-benzyloxycarbonylmethyl-ε-caprolactone), (PB) was synthesized according to the following procedure (Scheme S1). Typically, the copolymer PB18.3 was synthesized by the ring opening (ROP) polymerization of ε-BCL and ε-CL using neopentyl glycol as initiator at the molar ratio of 4/16/1 and Sn(Oct)2 (0.5 % w/w) as catalyst.28 To obtain functionalized PB18.3 with acrylate end-group, the end hydroxyl groups of the linear PB18.3 were reacted with acryloyl chloride (1:10 molar ratio) and potassium carbonate (1:12 molar ratio) as catalyst in dichloromethane for 72 h at room temperature. After removing the catalyst, the double bond-terminated PB18.3 was precipitated in cold diethyl ether and then dried in a vacuum oven. The

double

bond-terminated

copolymers

poly

(ε-caprolactone-co-carboxy-methly-ε-

caprolactone) (PC) was performed as follows. Firstly, the PC18.3 was obtained by the catalytic debenzylation of PB18.3 in the presence of hydrogen gas.29 Afterwards, maleic anhydride (MAH) was used to introduce unsaturated groups into the copolymer PC18.3 by the reaction of the hydroxyl-terminated PC and MAH (1:5 molar ratio). Finally, the reaction mixture was dissolved in dichloromethane and precipitated in cold diethyl ether. 2.3. Fabrication of the Nanopatterns. Fabrication of the nanopatterned topography on the functionalized PCL film surfaces was performed according to the following process (Scheme 1A). Briefly, the polymer solution (3 wt.%) and photoinitiator (0.5 wt.% model 651 light sensitizer) in ethyl acetate (filtered through a 0.2 µm membrane syringe filter) were spin-coated and the polymer films were obtained. The process was conducted at a constant rotation speed of


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3000 RPM for 40 s in the clean room facilities. After spin-coating, films were baked at 80 °C on a hot plate for 10 s. The UV-NIL experiments were conducted using an EVG 620 (EV Group, Austria) with automated process under PC control (Scheme 1A). The optically transparent polydimethylsiloxane (PDMS) mold was placed on the top of the film and the samples were cured with UV-light (365 nm, 10 mW·cm-2) under the pressure of 300 mbar. After cross-linking for 500 s, the mold was removed and the imprinted nanogrooves were formed on the polymer membrane surface (Scheme 1B & C).

Scheme 1. A) Schematic depiction of the UV nanoimprint process. B) Benzyl groups presented on the nanopatterned surface. C) Carboxyl groups presented on the nanopatterned surface. 2.4. Structure Characterization. 1H NMR spectra was recorded on a Bruker AV (400 MHz) using CDCl3 as solvent. The molecular weight and molecular weight distribution of PB and PC copolymers were determined by gel permeation chromatography (GPC, Waters 1515, USA). Tetrahydrofuran (THF) was used as eluent at a flow rate of 1.0 mL·min-1 and calibrated by polystyrene standards. 2.5. Surface Properties Analysis. Scanning electronic microscopy (SEM, HITACHI S-4800) was used to observe the flat and width of the grooves/ridges of the nanopatterned surfaces. The



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samples were sputtered with gold prior to observation and operated at an acceleration voltage of 5 keV. The topography of the nanopatterned and flat surfaces were determined by Atomic Force Microscopy (AFM, Veeco, Dimension 3100). The measurements were carried out in Tapping Mode with scan frequency of 0.5 Hz and data analysis was determined by Nanoscope software. A contact angle goniometer (POWEREACH, Shanghai, China) was employed to determine the wettability of the substrates including the flat and nanopatterned PCL, PB and PC. Approximately 20 µL of distilled water was carefully pipetted onto the samples, and the contact angle was measured 15 seconds after the droplet was placed on the film surfaces. For each sample, the measurement was repeated at least on four different spots. The chemical composition on the film surfaces was analyzed by X-ray Photoelectron Spectroscopy (XPS, Thermo Fisher) employing a monochromatic Al Kα source (1486.69 eV). Surveys were run with 50 eV pass energy in steps of 1.0 eV and 100 ms dwell time per data point. The data analysis was determined by using XPSPeak4.1 software. 2.6. Cell Culture. Human foreskin fibroblasts (HFFs) were generated from human foreskin and cryopreserved in liquid nitrogen. After thawing, cells were cultured on tissue culture flask at 37 oC in a humidified 5 % CO2 atmosphere. When the primary culture of HFFs reached 90 % confluence, HFFs were detached using 0.25 % trypsin-0.02 % EDTA solution and used in the following study. 2.7. Protein adsorption. Prior to cell culture, FBS were used here as model protein to evaluate protein adsorption on the flat and nanogrooved surfaces. The films sterilized with 75 % ethanol for 2 h and UV light irradiation for 30 minutes. Then the samples were incubated in FBS at 37 oC in a humidified 5 % CO2 atmosphere. After incubation of 2 h, the films were rinsed with ice-cold PBS and the adsorbed proteins were extracted with RIPA lysis buffer (beyotime Biotechnology,


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Shanghai, China) on ice for 30 min. Total protein adsorption was quantified by a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). The optical density (OD) was measured at the wavelength of 562 nm by using microplate reader (Bio Tek, Elx800, USA). 2.8. Cell viability. The samples were placed in 6-well tissue culture plates and sterilized with 75 % ethanol for 2 h before cell seeding. To study cell adhesion and proliferation, HFFs were seeded at 1×105 cells per well on the polymer films, and cultured in DMEM supplemented with 10 % FBS at 37 oC in a humidified 5 % CO2 atmosphere. HFFs viability was quantified by Cell Counting Kit (CCK)-8 assay after 24 h, 48 h and 72 h incubation. The CCK-8 solution and the medium were mixed at a volume ratio of 1:10. 1 mL mixed solution was added into each well after removing the medium and re-incubated for further 2 h. The optical density (OD) was measured at the wavelength of 450 nm by using microplate reader (Bio Tek, Elx800, USA). In addition, the adhesion and proliferation of HFFs were visualized by an inverted microscope (Nikon, Japan) on 24 h and 72 h without removal of the glass substrates from the wells. 2.9. Collagen Secretion. After 72 h of culture, the substrates with adherent cells were rinsed three times with phosphate buffered saline (PBS), fixed with 4 % paraformaldehyde (PFA) for 30 min at room temperature. After washing three times with PBS, the extracellular matrix (ECM) was stained by Sirius red for 30 min to detect collagenous fiber by an inverted microscope (Nikon, Japan). 2.10. Cell Morphology Analysis. After 24 h or 72 h of culture, samples were rinsed three times with PBS to remove the residual cultured medium and unattached cells, fixed with 4 % PFA for 30 min at room temperature, then permeabilized with 0.1 % Triton X-100 (Sigma Aldrich Chemie, Munich, Germany) for 5 min. After washing three times with PBS, the cells were stained by rhodamine phalloidin (1:500 in 1 % BSA-PBS) for 20 min to detect F-actin.



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After rinsing the cells with PBS for three times, cells were stained with 1 µg/mL 4’, 6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature to detect the nuclei. The images of the HFFs skeleton on the different substrates were taken by a confocal laser scanning microscope (CLSM, Nikon AIR, Japan). The outline of each cell on the CLSM images was analyzed manually, and the cell spreading area, elongation and orientation index were determined based on F-actin staining images by the Image J software (National Institutes of Health). 3. RESULT AND DISCUSSIONS 3.1. The Composition of the Copolymers and the Characteristics of the Nanopattern. Table 1. Molecular characteristics of the copolymers. Samplea

a

Mnc

Mnd

PDI

(%)

BD

AD

BD

AD

BD

AD

PB6.5

6.5

2917

2773

4823

3605

1.28

1.16

PB18.3

18.3

2880

2592

6345

4606

1.24

1.35

PB41.7

41.7

4320

3420

5179

3748

1.28

1.30

The samples are named according to the content of BCL in the copolymer

b c

R b BCL

The percentage of P (BCL) in the PB copolymer according to 1H NMR

The number-averaged molecular weight measured by 1H NMR

d

The number-averaged molecular weight measured by GPC

BD means the copolymer before deprotection AD means the copolymer after deprotection PC copolymers are named following the copolymers before deprotection The copolymers with different content of hydrophilic/hydrophobic groups were synthesized by adjusting the feed ratio of CL to BCL. The molecular characteristics of these copolymers were characterized by 1H NMR and GPC. As listed in Table 1, the percentage of P (BCL) in the PB


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copolymer was calculated according to 1H NMR. Besides, the results of 1H NMR demonstrated that the successful removal of all the benzyl carboxylate protection groups and no chain scission occurred during the deprotection (Figure S1). The results of GPC demonstrated the molecular weights of the copolymers were well-controlled and had a narrow molecular weight distribution. Our previous study proved that the introduction of pendant groups could decrease the crystallinity of the PB and PC copolymers due to the disturbed regularity of the macromolecules,28 which was beneficial for the following spin-coated process.

Figure 1. Typical SEM and AFM images of flat (A, C) and nanopatterned (B, D) PC18.3 surface. The XYZ scan area of the AFM images are X: 10 µm, Y: 10 µm, and Z: 200 nm. The nanopattterned polymer surfaces were fabricated using UV-NIL method and performed in a three-step typical process. Firstly, the glass surfaces were spin-coated with the UV-curable polymer solution and the films were baked on a hot plate. Secondly, an optically transparent PDMS mold was used to press into the substrate, and finally UV radiation was applied to solidify the resist and formed the imprinted nanopattern. The morphology of the flat and nanopatterned polymer surfaces were examined by SEM and AFM (Figure 1 and S2). It was seen from Figure 1A that the unpatterned substrate presented a comparatively smooth surface and the AFM image (Figure 1C) demonstrated that the arithmetic average roughness (Ra) was 16.6 nm, and they were



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further used as control for the following study. Moreover, the SEM image confirmed that the desired nanopatterns were fabricated well-controllably with a good reproduction quality and uniform nanogrooves (Figure 1B), and Figure 1D demonstrated that the pitch dimensions of the nanogrooves were 820 nm wide and 175 nm deep. 3.2. Surface Properties and Protein adsorption. The chemical composition on the film surfaces were evaluated by XPS. As illustrated in Figure 2, the spectrum of the film surfaces turned out to be the similar chemical composition (C, O and H), but with different C/O ratios. According to the analysis results of relative elemental concentration, the content of O and C in P18.3 film was 22.77 % and 77.23 %, while that of PC18.3 surface was 24.42 % and C 75.58 % respectively. Particularly, the C/O ratios of PC18.3 (3.09) films surface was the lowest in comparison with PCL (3.13) and PB18.3 (3.39) film surface, which clearly indicated that the hydrophilic groups with oxygen element were bare on the surface. In addition, ATR-FTIR also confirmed that the benzyl and carboxyl functional groups situated on the PCL surface (Figure S1), which were in agreement with the results of XPS.

Figure 2. Typical XPS elemental survey of PCL, PB18.3 and PC18.3 surfaces.


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Cell adhesion, spreading and proliferation were significantly affected by substrate surface wettability. Hence, the surface wettability on both the flat and nanopatterned substrates was measured (Figure 3A). It is widely known that the chemical composition of the surface and roughness were two main parameters influencing the wetting properties of the material.30 For flat films, the water contact angle (WCA) of the PB serial films increased with the content of benzyl groups. For instance, the WCA of PB6.5 was measured to be 66.6 ± 0.4°, while the value of PB18.3 and PB41.7 film increased to 70.7 ± 1.1° and 76.6 ± 0.8° respectively. However, as for the PC serial films, the carboxyl groups move to the interface31 and the hydrophilicity of the PC surface increased accordingly, thus the WCA on the PC surface significantly decreased with the content of the carboxyl groups increased (PC6.5 = 65.1 ± 1.3°; PC18.3 = 61.2 ± 1.6° and PC41.7 = 50.0 ± 3.1°).The results indicated that surface chemistry cues could prominently modulate the wettability of polymer surfaces. Meanwhile, the surface topology also plays an important role in modulating surfaces wettability. For examples, the WCA of the PB6.5 and PB41.7 films was measured to be 68.2 ± 0.4° and 83.1 ± 1.2° respectively, while the value of WCA rose up to 86 ± 2.0° and 93.7 ± 0.8° due to the introduction of nanopatterns to these hydrophobic surfaces. As for the PC serial polymer surfaces, the nanopattern endowed the surfaces with hydrophilicity and the WCA decreased to some extent. For example, the WCA values of PC18.3 decreased dramatically from 61.2 ± 1.6° to 42.4 ± 1.4°. The results were consistent with previous study that the introduction of surface nano-topography made a hydrophobic surface more hydrophobic as well as a hydrophilic surface more hydrophilic.32-33 The protein adsorption on the flat and nanogrooved surfaces was determined by BCA assay. It was found in Figure 3B that there were significant amounts of proteins adsorbed on all films surface while the different protein adsorption occurred according to the hydrophobic/hydrophilic



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property. The nanostructure on the PB surfaces could lead to more hydrophobic and the ability of protein adsorption was enhanced accordingly. Additionally, the amount of adsorbed protein was further reduced when the nanoscale topography was introduced on hydrophilic PC surface.23 Generally, surface wettability and protein adsorption presented a certain positive correlation.34 However, other mechanisms such as hydrogen bonding and electrostatic force might be also involved in regulating protein adsorption on the film surface. It may be accounted for that the amount of adsorbed protein was not strictly dependent on the value of water contact angle.35 Collectively, the above results indicated that chemical composition and topography represent determinative factors in protein adsorption.36-37

Figure 3. Surface wettability (A) and protein absorption (B) on the flat and nanopatterned surfaces. (* indicate p

Combined Chemical Groups and Topographical Nanopattern on the

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