PEG Nanopattern Arrays: Well-Defined Gradient

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Surfaces, Interfaces, and Applications

Graded Protein/PEG Nanopattern Arrays: Well-defined Gradient Biomaterials to Induce Basic Cellular Behaviors Peihong Xue, Wendong Liu, Zhongyi Gu, Xingchi Chen, Jingjie Nan, Junhu Zhang, Hongchen Sun, Zhanchen Cui, and Bai Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16547 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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ACS Applied Materials & Interfaces

Graded Protein/PEG Nanopattern Arrays: Well−defined Gradient Biomaterials to Induce Basic Cellular Behaviors Peihong Xue, † ‡ Wendong Liu,† Zhongyi Gu,⊥ Xingchi Chen, † Jingjie Nan,† Junhu Zhang,*,† Hongchen Sun, § Zhanchen Cui,† and Bai Yang † † State

Key Lab of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, 130012, P.R. China ‡ College of Transportation Engineering, Dalian Maritime University, Dalian, 116026, P.R. China § Liaoning Province Key Laboratory of Oral Disease, School of Stomatology, China Medical University, Shenyang, 110002, P. R. China ⊥ Department of Pathology, Stomatology Hospital, Jilin University, Changchun, 130012, P.R. China KEYWORDS gradient biomaterials, colloidal lithography, well−defined ECM, inducing cellular behavior, multifunctional substrate

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ABSTRACT Gradient biomaterials have shown enormous potential in high−throughput screening of biomaterials and material−induced cell migration. To make the screening process more rapid and precise, improving the regularity of morphological structure and chemical modification on gradient biomaterials have attract much attention. In this paper, we present a novel fabrication strategy to introduce ordered nanopattern arrays into gradient biomaterials, through combining surface−initiated atom transfer radical polymerization (SI−ATRP) and inclined reactive ion etching (RIE) based on colloidal lithography. Graded protein/PEG nanopattern arrays on quartz substrate were fabricated and applied to affect the behaviors of cells. Owing to the continuously changed ratio of two different components, the corresponding cell adhesion density along the substrate show obvious graded distribution after culturing for 24 hours. Meanwhile, the cytoskeleton show obvious polarization after culturing for 7 days, which is parallel with the direction of gradient. Additionally, oriented migration generated when mouse MC3T3−E1 cells were cultured on the graded protein/PEG nanopattern arrays. Based on the ordered and well−defined nanopatterns, the correlation between the ECM and corresponding expressions generated by different stimulus can be investigated.


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Introduction The microenvironment around cells always provides necessary stimulus for various behaviors, such as adhesion, migration, and differentiation. Extracellular matrix (ECM), as one of significant environmental parameters, has attracted broad interest for the great effect on cellular behaviors. During the past decades, works associated with various factors of ECM (composition, elasticity, and topography, etc.) have been reported to study the correlation between ECM and corresponding cellular behaviors.1−4 Through constructing predesigned microenvironment, both the topography of integrin ECM receptors and cellular processes can be controlled in a certain degree, as cells are cultured in the special microenvironment.5−8 Nevertheless, to thoroughly understand and control cellular behaviors, fundamental research on cellular behavior regulated by microenvironmental parameters is useful. To be time− and effort−saving, high−throughput materials with continuously changing microenvironments are found to be an efficient route to study the correlation between different ECM and corresponding cellular behaviors. Biomaterials with gradients of properties are a class of materials of particular interest for high−throughput studies of cell–interface interactions.9−13 Gradient biomaterials have been recognized as an effective tool in applications such as high−throughput screening of optimal biomaterial parameters and simulating the inner environment of human body.14−18 Many fabrication methods involve creating miniaturized libraries that contain massive specimens in single sample in the form of gradients, followed by data collection and analysis. The consecutive and changed



microenvironments constructed by these methods resulted in totally different expression of cultured cells.19−21 Nevertheless, numerous strategies tend to suffer from different drawbacks particularly poor−defined microenvironments. During analyzing the correlation between ECM and corresponding cellular behaviors, it is found that well−defined and ordered microenvironment are much beneficial for gradient biomaterial to be an integrated model on high−throughput investigation. In the meanwhile, improving the order and definiteness of microenvironments is an effective approach to reduce the indeterminacy and randomness of cellular behaviors in some extent. All the aforementioned factors indicate that it is of great interest to construct gradient biomaterials with well−defined structure and component for systematically analyzing the correlation between different ECM and corresponding cellular behaviors. Herein, we demonstrate a novel fabrication strategy, combining surface−initiated atom transfer radical polymerization (SI−ATRP) and inclined reactive ion etching (RIE) based on colloidal lithography,22−24 which enable the facile fabrication of gradient biomaterials with ordered and graded protein/polyethylene glycol (PEG) nanopattern arrays. The gradient biomaterials fabricated by this method were used to affect three basic behaviors of cell, adhesive density, polarization and migration. As the graded protein/PEG nanopattern arrays were composed by two different components with continuously changed ratio along the substrate, the gradually changed ECM should generate corresponding different cell adhesion density. Meanwhile, the asymmetric ECM could generate stimulation to achieve the polarization of cytoskeleton. Additionally, cells could get driving force to migrate upon the substrate when cultured


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on the graded protein/PEG nanopattern arrays. Based on the ordered and well−defined nanopattern, the correlation between the ECM and corresponding expressions generated by different stimulation were analyzed and investigated.

RESULTS AND DISCUSSION The fabrication of graded protein/PEG nanopattern arrays was based on SI−ATRP23 and inclined RIE24. Firstly, a monolayer of α−bromoisobutyryl bromide (initiator) was grafted on the quartz substrate, after the surface of quartz being processed with oxygen plasma. Subsequently, a polymer film used as protective layer was spin−coated upon the prepared initiator film, which could protect the initiator from etching by oxygen plasma in the following inclined RIE. Hexagonally close−packing polystyrene (PS) microsphere arrays were fabricated by interface method25 and transferred onto the surface of the protective layer. As shown in Scheme 1, geometric gradient was then introduced into PS microsphere arrays by inclined RIE, which was based on the graded etching rate in vertical direction.24 After removing residual PS microspheres and protective layer by their corresponding excellent solvent, nanodot arrays of the initiator with diameter gradient were fabricated. Meanwhile, the voids among the initiator nanodots were filled with silicon hydroxyl, since the thin layer of polymer without cover of etched PS microspheres was etched away during inclined RIE. Consequently, the poly(ethyleneglycol)−silane (PEG−silane) could be immobilized on the region with silicon hydroxyl. Finally, the graded PHEMA/PEG nanopattern arrays were fabricated by SI−ATRP from the graded initiator/PEG nanopattern on the quartz substrate. In addition, graded protein/PEG nanopattern arrays can also be prepared via covalently



immobilizing proteins on the PHEMA nanodots. During fabricating graded initiator/PEG nanopattern arrays, the initial diameter of PS microspheres used as etching mask is the key factor to determine the lattice constant of initiator nanodot arrays. Meanwhile, the diameter of initiator nanopattern on different positions along the substrate was affected by the diameter of etched PS microspheres from the top−view after inclined RIE. Taking an example of protein/PEG nanopattern arrays made by PS microspheres with initial diameter of 507 nm, the detailed morphological characterization of fabricating process was tracked and shown. The top−view SEM images of etched PS microsphere arrays on five different positions were characterized and shown in Figure 1. The lattice constant of etched PS microsphere arrays on all positions was 507 nm, while the diameter showed graded and continuous change from 362 nm to 492 nm. After inclined RIE by oxygen plasma, the exposed region including initiator and protective layer was all etched off. The residual protective layer upon the initiator nanodots was dissolved by ethyl alcohol, and then the graded initiator nanodot arrays were constructed on the quartz substrate. The morphology of graded initiator nanodot arrays was characterized by atomic force microscope (AFM) and shown in Figure 2. The same as the distribution of PS microsphere diameter, the diameter of initiator nanodots changed from 360 nm to 489 nm along the graded substrate. The height of initiator nanodots was all around 2 nm, which coincides with the length of initiator molecule. It is noteworthy that there are some protrusions at the edge of most nanodots. During using colloidal microspheres as etching mask in RIE, the etched material below the microspheres cannot completely


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copy the shape of mask for the existence of side etching. Similarly, the side etching can inactivate large area of initiator below the PS microspheres with a thickness of just 2 nm in our experiment. Based on aforementioned reason, a thin layer of polymer was spin−coated on the surface of initiator to protect it from the side etching. Nevertheless, instead of inactivating the initiator, the side etching results in the bonding of initiator and protective layer at the edge of initiator nanodots when they were processed by oxygen plasma. As a result, the bonding component in the edge of initiator was higher than the other area of nanodots. Finally, the sites without initiator upon the quartz substrate could be filled with PEG−silane, which were grafted with hydroxyl during oxygen RIE. Subsequently, PHEMA brushes with a thickness of 70 nm was polymerized upon the sites with initiator by SI−ATRP and shown in Figure 3. The diameter of PHEMA nanodots changed from 350 nm to 470 nm along the graded substrate while the height was all around 70 nm. Compared to the morphology of initiator nanodots, the diameter of PHEMA nanodots got shrink in some extent. It is demonstrated that the protuberant bonding component in the edge of initiator nanodots cannot initiate the polymerization of PHEMA. The PHEMA nanodots showed an obvious mastoid shape, the tops of which are relatively smooth. It is noteworthy that the height of nanodots in Figure 3e is smaller than that on other positions of graded substrate. It is generated by the existence of excess polymer brush in voids among the PHEMA nanodots when nanodots are large enough. The gradually diminishing area of voids around nanodots results in the lack of platform in the cross−section profile of PHEMA nanodots. Meanwhile, the lateral



spreading and linking of the chains at the edge of polymer nanodots is also a factor resulting in the reduction of height. Finally, graded protein/PEG nanopatten arrays were fabricated by covalently immobilizing proteins upon the PHEMA nanodots after the sites around PHEMA nanodots were grafted with PEG−silane. To evaluate the biological activity of grafted protein, human IgG and FITC−labeled goat anti−human IgG were immobilized onto the PHEMA nanodots successively. Fluorescence microscopy was used to investigate the morphology of protein under blue light excitation (shown in Figure 4). Based on the fluorescence photos in Figure 4a−e, the FITC−labeled protein nanodots kept the same trend of morphological variation with PHEMA nanodots and showed an obvious diameter gradient. Meanwhile, the clear and well−defined nanodots grafted by fluorescence protein demonstrate that protein can be specifically grafted to the PHEMA nanodots modified with succinimidyl group. Additionally, molecule with amidogen, such as RGD, fibronectin (FN) and laminin (LN), could also be grafted on the as−prepared graded PHEMA/PEG nanopattern arrays. The proteins are ideal matrix to promote cell adhesion by combining with integrin receptor, such as FN and LN. Correspondingly, PEG is an effective component to prevent cell adhesion according to its high performance on protein resistance. Hence, the protein/PEG nanopattern arrays with graded component area rate along the as−prepared substrate should exhibit different and corresponding cell adhesion behavior. Firstly, the prepared substrate was used as a high−throughtput biomaterial to investigate the correlation between different ECM provided by substrate and


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corresponding cell adhesion density. Based on aforementioned fabrication strategy, the graded FN/PEG nanopattern arrays with lattice constant of 1.1 µm were fabricated to demonstrate the capability to induce cell density gradient of rat bone marrow stromal cells (BMSC) in cell adhesion experiments. With the purpose of generating cell density gradient, the prepared graded substrate was immersed into a suspension of rat BMSCs seeded at a high planting density of 120 cells mm−2.After culturing for 24 hours, the inadhesive rat BMSCs were rinsed away with

PBS

solution

and

the

adhered

ones

were

imaged

with

4,6−diamidina−2−phenylindole (DAPI) and tetramethylrhodamine B isothiocyanate (TRITC)−labeled phalloidin. The morphology of five measured positions along the graded substrate and two control group (100% FN and 100% PEG) were characterized by AFM as shown in Figure 5a. Firstly, substrates modified with either FN or PEG exhibited small roughness based on the calculated root mean square of roughness, which demonstrate the homogeneous grafting process. Correspondingly, the area rate of region with capability of reacting with amidogen after further grafting (PHEMA nanodots) and region grafted with PEG layer showed an obvious change along the graded substrate. The diameter of the well−defined PHEMA nanodots decreased gradually from 450 ± 6 nm to 234 ± 6 nm while the area of PEG increased. Meanwhile, the height of nanodots on different positions along the graded substrate were all around 30 nm. Then, the distribution of rat BMSCs on the corresponding positions along graded substrate were characterized by optical microscope after culturing 15 min and shown



in Figure 5b. According to optical images, the suspending rat BMSCs precipitated on the prepared substrate and showed shape of spheres which had not spread. The initial density of rat BMSCs on different positions along the graded substrate and control group were all around 118 cells mm−2, which were all in reasonable agreements with the others. After culturing 24 hours, the rat BMSCs should completely spread and adhere on the substrates by the combination of integrin and FN on the substrate. Based on the fluorescent images of cell nucleus labeled by DAPI (shown in Figure 5c), the number of adhered cells on the graded substrates and control group were the mean cell number of 10 points along the direction perpendicular to the gradient and 10 random points on the homogeneously modified substrate, respectively. It is noteworthy that the cell density of cells on substrate with full FN was as high as 164 ± 8 cells mm−2 while the substrate with full PEG was only 7 ± 2 cells mm−2. The cell density on the substrate with full FN after culturing 24 hours were higher than that of 15 min for the reason of proliferation during the culturing process. The substrate was fully filled with rat BMSCs based on the enough adhesion sites on the substrate with full FN. In comparison, only several cells adhered on the substrate with full PEG due to the effective function of anti−protein adhesion, which result in preventing cells from spreading along the surface of substrate. Meanwhile, the actin of cells did not spread on the substrate with full PEG, which were all found to show a shape of sphere in the micrographs. Therein, the residual cells adhered on the substrate with full PEG was because the sites with silicon hydroxyl were not completely reacting with PEG−silane. Compared to the control groups, the cell density of rat BMSCs along the graded substrate were all between that of two


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control groups and increased gradually along the surface with modified component gradients. The cell density increased from 34 ± 1 cells mm−2 to 78 ± 3 cells mm−2 with the area of FN along the graded substrate, representing the capability of the graded materials to establish a gradient in cell density. It is supposed that the graded distribution of rat BMSCs were generated as the number of adhesion sites provided by FN nanodots on different positions along the substrate gradually increased. To further understand the mechanism of adhered cell morphology on the FN/PEG nanopattern arrays, the spreading morphology of single BMSC was characterized by SEM, as shown in Figure 6. Taking a random sample from rat BMSCs on the graded substrate as example, the cytoskeleton of rat BMSC was intactly retained after dehydrating and spreading on the protuberant FN nanodot arrays. After culturing for 24 hours, the morphology of BMSCs changed from the initial sphere to a totally spreading shape with a length about 30 µm, which demonstrated that the FN nanodot arrays could provide adhesion sites for adherent cells to spread along the substrate. Additionally, enlarged views of most filopodia were characterized and shown in Figure 6b−e. Due to the strong interaction of actin, integrin and FN, the filopodia distinctly adhered on the surface of substrate by a mass of anchored sites on the FN nanodot arrays. Meanwhile, the filopodia extended with the order of hexagonal array from the stem to the ECM around BMSC, which was extremely conspicuous in Figure 6b that the filopodia spread along a strip of continuous nanodots. Based on the spreading morphology of cell, the mechanism of the FN/PEG nanopattern arrays generating cell density gradient could be also supposed. After



arriving at the surface of substrate from the suspension, the initial shape of rat BMSCs was sphere and the contacting area was only between the bottom of sphere and the protuberant FN nanodots. Subsequently, the rat BMSCs would explore the surrounding microenvironment by spreading the filopodia to search the ECM adapted to be attached. During the above spreading process, the filopodia had to overcome the antiadhesion of PEG until detecting and combining with the adjacent FN nanodots. The distance of adjacent FN nanodots is key factor to affect the possibility of further spreading behavior. Hence, the gradually increasing distance of adjacent FN nanodots along the fabricated substrate generated the graded cell density of incubated BMSCs. It is worth mentioning that the introduction of FN/PEG ordered nanopattern with the scale of micro/nanofeature provided not only ordered and structured biomaterials with definite parameters but also visualization of adhesion and anti−adhesion area for the cells to behave with more predictability and less randomization. It is well known that the morphological structure in microfeature mostly affect cell behaviors by space confinement effect, which have an effect on large portion of the whole cell. With the decrease of morphological feature, the affected target was gradually changed from large portion of the whole cell to much small cellular structure. Compared to the structure in microfeature, the FN nanodots with diameter of hundreds nanometer tended to affect the cell morphology through controlling the number of adhesion sites to restrain the spreading of pseudopodium. As a result, the micro/nanostructure arrays could determine the cell adhesion density on the strength of their characteristic feature. Moreover, the gradient nanopattern arrays in micro/nanofeature could not only affect


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the cell density by restricting the number of adhesion sites in short range but induce the polarization of cytoskeleton by the asymmetric micro/nanostructures in long range. Benefiting from the anisotropic micro/nanostructures along the substrate, the graded FN/PEG nanopattern arrays can stimulate the cells to generate the polarized cytoskeleton after culturing for 7 days. The fabricated nanopattern arrays parallel to the direction of gradient were gradually changed and different from each other while the perpendicular was all the same. As shown in Figure 7, both SEM and fluorescence microscope were used to characterize the spreading morphology of TRITC−labeled rat BMSCs after being immobilized on the substrate. The initial culturing cell density was set as 52 ± 3 cells mm−2 to reduce the interaction of adjacent cells, which was half of cell density in the cell adhesion experiment. The BMSC showed fusiform morphology in both characterized images, which got a high aspect ratio (shown in Figure 7a and 7b). Additionally, the TRITC−labeled actin represented obvious polarization while the actin of rat BMSCs in control group was random distribution (shown in Figure 7f). It is noteworthy that the direction of polarization was parallel to the graded direction. The included angle between polarized direction and graded direction were mostly within 15° in a large area of substrate (Figure 7c and 7d). Nevertheless, most rat BMSCs upon the control substrate did not generate polarization while the others polarized with random orientation in the same area. Hence, the graded substrate successfully provided rat BMSCs with anisotropic ECM to generate necessary stimulation and activate the pathways to perform the behavior of polarization. During the process of polarization and orientation, the spreading rat BMSC could cover dozens of hexagonal FN nanodot



arrays. Therein, the parameters of covered FN nanodot arrays gradually increased along the gradient direction while remained unchanged along the direction perpendicular to the gradient. Despite the parameter difference of FN nanodots between the two sides of covered rat BMSC is small, the difference could still promote rat BMSC to generate polarization and orientation. Compared to traditional biomaterials in microfeature which could induce cell orientation (such as microstrip26) by confinement effect, the prepared graded FN/PEG nanopattern arrays were inclined to activate the pathway to induce polarization and orientation by identifying the signal difference transmitted from the ECM in nanofeature. Instead of physical pression, the origin of stimulating signal provided by the fabricated substrate was inclined to be chemical stimulation when the characteristic size decreased to micro/nanofeature. It is worth mentioning that both adhesion formation and component have showed great influence on the BMSC differentiation.27,28 Hence, constructing well−defined ECM to control the basic cellar behaviors is much beneficial for the further application in controlling the differentiation of stem cells. Besides the behavior of polarization, it was found that the number of pseudopodium on the side with large FN nanodots was much higher than that on the other side. It is well known that gradient biomaterials can provide driving force for the cells upon it. Based on the asymmetric distribution of pseudopodium, it was supposed that the cells migrated on the substrate. The dynamic behavior of MC3T3−E1 cells incubated on graded procollagen I/PEG nanopattern arrays was tracked by live cell station in 24 hours. As shown in Figure 8a, three MC3T3−E1 cells were taken as example to show


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the obvious migration during being intubated on the graded substrate. It is noteworthy that the migrating direction of the three cells were all toward the direction with larger procollagen I nanodot arrays. The initial and 24 hours later distribution of MC3T3−E1 cells on the same area were labeled and shown in Figure 8b1 and 8b2 respectively. Based on the initial (red dot) and final (blue dot) position of MC3T3−E1 cells, the migrating direction could be tracked, as shown in Figure 8b3. During the process of culturing 24 hours, the MC3T3−E1 cells generated the behavior of migration and propagation. The initial culturing cell density was also set to be sparse (52 ± 3 cells mm−2) to reduce the resistance among cells so that the migrating induction of graded substrate could play the dominant role. The number of MC3T3−E1 cells migrating toward the side with larger procollagen I nanodots (around 80%) was much more than migrating toward the opposite side (14%) or along the direction perpendicular to the gradient (6%) (shown in Figure 8b4). The directed driving force was derived from the difference of ECM under the single cell. The number of adhesion sites grew with the gradually increasing diameter of procollagen I nanodots along the gradient, which resulted in asymmetric distribution of pseudopodium. Additionally, the morphology of cytoskeleton gradually changed from symmetric sphere to asymmetric distribution under the pull of asymmetric stress. The asymmetric cytoskeleton determined the final haptotaxis of MC3T3−E1 cells on the graded procollagen I/PEG nanopattern arrays. It is worth mentioning that the migration process is nonspecific inducing which is generated by the different ECM under both ends of single cell. Hence, the inducing ability of prepared substrate should also be effective to other kinds of mature cells, such



as neuronal cells and skeletal muscle cells, which has great potential in the field of tissue repairing and regeneration.29


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CONCLUSION In conclusion, well−defined protein/PEG nanopattern arrays were successfully introduced in the gradient materials by combining SI−ATRP and inclined RIE based on colloidal lithography. Owing to the continuously changed ratio of two different components, the corresponding cell adhesion density along the substrate showed obvious graded distribution after culturing 24 hours. Meanwhile, the cytoskeleton showed obvious polarization after culturing 7 days, which paralleled with the direction of gradient. Additionally, oriented migration was generated when rat MC3T3−E1 cells were cultured on the graded protein/PEG nanopattern arrays. The gradually changed and asymmetric ECM provided the cultured cells with different number of adhesive sites and driving force to achieve the thigmotactic behavior. The well−defined and graded biomaterials could make cells generate much definite responsive behavior. Based on the aforementioned properties, this fabrication strategy has great potential in constructing well-defined biomaterials for precisely high-throughput screening of special ECM and inducing the directional migration for the further application on tissue repairing and regeneration.



EXPERIMENTAL SECTION The detailed source of materials used in this work, preparation process and process of cell seeding and staining were all shown in Supporting Information in sequence. Characterization SEM micrographs were taken with a JEOL FESEM 6700F electron microscope (JEOL, Japan) with primary electron energy of 3 kV. Before imaging, the cells were dehydrated and sputter−coated with 2 nm of Pt. Atomic force microscopy (AFM) images were recorded in tapping mode with a Nanoscope IIIa scanning probe microscope (BRUKER, Germany) from Digital Instruments under ambient conditions. The microscope images of light field were taken by using OLYMPUS BX51 instrument (OLYMPUS, Japan). The confocal fluorescent microscopy images of the cells cultured on the substrates were taken by using the laser scanning confocal microscope OLYMPUS BX81 (FluoView FV1000) (OLYMPUS, Japan). The cell migration was characterized by the combination of live cell station and inverted microscope (OLYMPUS, Japan).


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Scheme 1 A schematic illustration of the fabricating process of graded protein/PEG nanopattern arrays.



Figure 1 The SEM images of the PS microspheres mask on different positions a) 2.0 cm b) 1.5 cm cm c) 1.0 cm d) 0.5 cm e) 0.0 cm along the substrate. The scale bar in the graph is 1 μm. f) The scheme and g) the plot data of the average diameter of PS microspheres mask on different positions along the substrate. Therein Y is the direction perpendicular to gradient, while XS and XL are the direction toward the side with small and large nanodots, respectively. The error of every value is within 10 nm.


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Figure 2 The AFM images of initiator (α−bromoisobutyryl bromide) nanopattern arrays on different positions a) 2.0 cm b) 1.5 cm cm c) 1.0 cm d) 0.5 cm e) 0.0 cm along the substrate. The scale bar in the right of Figure e is for all the AFM images. f) The scheme and g) the plot data of the average diameter of initiator nanopattern arrays on different positions along the substrate. Therein Y is the direction perpendicular to gradient, while XS and XL are the direction toward the side with small and large nanodots, respectively. The error of every value is within 10 nm.



Figure 3 The AFM images of PHEMA/PEG nanopattern arrays on different positions a) 2.0 cm b) 1.5 cm cm c) 1.0 cm d) 0.5 cm e) 0.0 cm along the substrate. The scale bar in the right of Figure e is for all the AFM images. f) The scheme and g) the plot data of the average diameter of PHEMA/PEG nanopattern arrays on different positions along the substrate. Therein Y is the direction perpendicular to gradient, while XS and XL are the direction toward the side with small and large nanodots, respectively. The error of every value is within 10 nm.


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Figure 4 The fluorescent images of FITC−labeled goat anti−human IgG/PEG nanopattern arrays on different positions a) 2.0 cm b) 1.5 cm cm c) 1.0 cm d) 0.5 cm e) 0.0 cm along the substrate. The scale bar in the graph is 5 μm. f) The scheme of FITC−labeled goat anti−human IgG/PEG nanopattern arrays. Therein Y is the direction perpendicular to gradient, while XS and XL are the direction toward the side with small and large nanodots, respectively.



Figure 5 a) The AFM images for the surface morphology, corresponding initial (microscope images) and final (fluorescent images) distribution of rat BMSCs cultured on the substrate modified with full FN, full PEG and graded FN/PEG nanopattern arrays. The scale bar in the right of Figure a is for all the AFM images. b) The plot for the diameter of PHEMA nanodots on different positions along the 2 cm substrate. c) The bar graph for the initial (culturing 0.25 h) and final (culturing 24 h) cell density on different positions along the 2 cm substrate and controls.


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Figure 6 a) The SEM image for spreading morphology of single rat BMSC on the graded FN/PEG nanopattern arrays after culturing 24 h. Figure b), c), d) and e) are the enlarged views for different filopodia of the rat BMSC in Figure a.



Figure 7 The a) SEM and b) fluorescent images for spreading morphology of single rat BMSC on the graded FN/PEG nanopattern arrays after culturing 7 days. c) The fluorescent images and d) orientation distribution plot for large area of TRITC−labeled rat BMSC cells cultured on the graded FN/PEG nanopattern arrays after culturing 7 days. The fluorescent images for e) large area of TRITC−labeled rat BMSCs and f) single rat BMSC cultured on the quartz substrate without any chemical modification. g) The scheme of FN/PEG nanopattern arrays for Figure7a−f. Therein X is the direction along the gradient while Y is the direction perpendicular to gradient. XS and XL are the direction toward the side with small and large nanodots, respectively.


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Figure 8 a1−a3) The migration behavior of three rat MC3T3−E1 cells during culturing on the graded procollagen I /PEG nanopattern arrays for 24 h. b1) The initial and b2) final position of rat MC3T3−E1 cells during culturing on the graded procollagen I/PEG nanopattern arrays for 24 h. b3) The moving track and b4) statistical plot of rat MC3T3−E1 cells cultured on the graded procollagen I /PEG nanopattern arrays. XS and XL are the direction toward the side with small and large nanodots, respectively. Y is the direction perpendicular to gradient.



AUTHOR INFORMATION Supporting Information

The materials used in this work; Preparation of single−layered initiator with protective layer on quartz substrate, single−layered PS microsphere arrays, graded protein/PEG nanopattern arrays; Cell seeding and staining. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author J. Z.: E−mail: [email protected]; Fax: +86−431−85193423 Funding Sources The National Natural Science Foundation of China (Grant no. 21774043); The National Natural Science Foundation of China (Grant no. 21474037); Fundamental Research Funds for the Central Universities (3132018111)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant no. 21774043, 21474037) and the Fundamental Research Funds for the Central Universities (3132018111). REFERENCES 1.

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PEG Nanopattern Arrays: Well-Defined Gradient

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