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Fabrication of RGD micro-nanopattern and corresponding study of stem cell differentiation Jiandong Ding, Xuan Wang, Shiyu Li, Ce Yan, and Peng Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/nl5049862 • Publication Date (Web): 20 Feb 2015 Downloaded from http://pubs.acs.org on February 23, 2015

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Fabrication of RGD micro-nanopattern and corresponding study of stem cell differentiation Xuan Wang, Shiyu Li, Ce Yan, Peng Liu, JiandongDing* State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Advanced Materials Laboratory, Fudan University, Shanghai 200433, China

∗

Corresponding author. Tel.: 86 21 65643506. Fax: 86 21 65640293. E-mail address:[email protected] (JD Ding).

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Abstract:

Micropatterns of gold (Au) nanoarrays on inorganic and polymeric substrates

were fabricated by combining block copolymer micelle nanolithography to obtain gold nanoarrays on glass, photolithography plus hydrofluoric acid (HF) etching to generate macroscopic microislands, and transfer lithography to shift the gold micro-nanopatterns from glass to a bioinert poly(ethylene glycol) (PEG) hydrogel surface. Further the modification of the gold nanodots via cell-adhesive arginine-glycine-aspartate (RGD) ligands was carried out to achieve peptide micro-nanopatterns. Whereas the micro-nanopatterns of noble metals could be useful in various applications, the peptide micro-nanopatterns especially enable persistent cell localization on adhesive micropatterns of RGD nanoarrays on the background of potently non-fouling PEG hydrogels, and thus offer a powerful tool to investigate cell-material interactions on both molecular and cellular levels. As a demonstration, we cultured human mesenchymal stem cells (hMSCs) on micro-nanopatterns with RGD nanoarrays of nanospacing 46 nm and 95 nm, and with micropans of side lengths 35 µm and 65 µm (four groups in total). The osteogenic and adipogenic differentiation of hMSCs was conducted, and the potential effect of RGD nanospacing and the effect of cell spreading size on cell differentiation were decoupled for the first time. The results reveal that RGD nanospacing, independent of cell spreading size, acts as a strong regulator of cell tension and stem cell differentiation, which cannot be concluded unambiguously based on either merely micropatterns or nanopatterns.

Keywords:

Micro-nanopattern, Poly(ethylene glycol) hydrogel, RGD nanospacing, Cell

spreading, Stem cell differentiation


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Nanopatterns of noble metals and oligopeptides have numerous uses in catalysis1, 2, optics3, 4, electronics5, 6, magnetics7, 8, biological and biomaterial applications9-12. The corresponding micro-nanopatterns, namely micropatterns of the nanoarrays can indeed strengthen the applications extensively by controlling the spatial arrangements at mutiple scales. Moreover， the ability of micropatterns to make domains of different types on the same nanopatterned substrate, not only eliminates paracrined factors but also enhances research efficiency. However, the fact that the microdomain of nanoarrays can only be observed with high-resolution microscopy limits the applications of micro-nanopatterns in some scientific research. Hence, a technique to fabricate micro-nanopatterns with nanoarrays located inside microdomains arrays visible by naked eyes or under common microscopes is in great need to promote applications of micro-nanopatterns in different research applications. Whereas many techniques have been reported to fabricate micropatterns13-18, nanopatterns19-22, and even micro-nanopatterns23-27, techniques to fabricate visible micro-nanopatterns has little been reported. The present letter reports a micro-nanopattern of gold (Au) and arginine-glycine-aspartate (RGD) on glass and poly(ethylene glycol) (PEG) hydrogel with visible microdomains, and with a high resolution of nanodots located inside the microdomains observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The technique combines block copolymer micelle nanolithography developed by Spatz and Möller19 to obtain gold nanoarray on glass, photolithography28 plus hydrofluoric acid etching29,

30

to generate visualized microislands, transfer lithography

employing a heterobifunctional linker to shift the Au micro-nanopatterns from glass to PEG


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hydrogel14, 21, 31, and the modification of the Au dots by RGD ligands with a thiol end group by using the classic technique of self-assembly monolayers32, 33. Our initial motivation for preparing a visible micro-nanopattern on a non-fouling background was to reveal stem cell regulation by a well-defined RGD distribution at nanoscale. The RGD motif plays a crucial role in focal cell adhesion through its bioconjugation to integrin molecules34-37. Some researchers have prepared nanopatterns or micropatterns of RGD on non-fouling background to control cell adhesion and to study the corresponding effect of RGD organization on cell behavior38-44. The size of a single RGD receptor, integrin, is approximately 12 nm45. So, an RGD nanoarray with the size of the RGD binding site approximately 10 nm can well control the eventual cell adhesion, and thus can modulate cell behavior on a molecular level. Especially, RGD nanospacing has been found to tune specific cell adhesion significantly46, 47. The critical spacing reads approximately 70 nm, and cells adhere well only on substrates with the ligand spacing below the critical value48, 49. Cells on RGD nanopatterns with smaller nanospacing exhibited larger spreading sizes49, 50. Recently, our group has found that mesenchymal stem cells showed a significant difference in their osteogenic and adipogenic differentiation extents on RGD nanopatterns with different nanospacings51, 52. On the other hand, RGD micropatterns with RGD peptides grafted onto Au microislands on PEG hydrogels have been well applied to control cell localization and differentiation53,

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. Cell studies on different micropatterns indicate that osteogenic and

adipogenic differentiation depend upon the cell spreading size55,

56

. The different

differentiation extents of stem cells on RGD nanopatterns in our previous study could thus be influenced by cell spreading sizes. In order to demonstrate the possible direct RGD


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nanospacing effect on cell differentiation conclusively, it is strongly desired to fabricate an ideal pattern of RGD ligands on a persistently non-fouling background which can independently control cell spreading size on microscale and RGD spacing on nanoscale. Herein, we report a visible micro-nanopattern of gold dots and RGD peptides on inorganic

and

polymeric

substrates,

and

demonstrate

the

application

of

RGD

micro-nanopattern on PEG hydrogels in revealing the basic science of interactions between biomaterials and stem cells. With this unique patterning technique, we examined the effect of RGD nanospacing and cell spreading size on stem cell differentiation, and these two effects could not be otherwise decoupled from each other. Human mesenchymal stem cells (hMSCs) were employed as a model cell type, and seeded on the designed micro-nanostructured surface. We investigated cell adhesion after one day of cultivation, and cell differentiation after 14 days of induction. To disentangle the effect of cell-cell contact and cell spreading with that of RGD nanospacing, we targeted behaviors of single cells inside micropans of the RGD nanoarrays. The unique micro-nanopatterning technique enabled us to examine both the RGD nanospacing effect at controlled cell sizes, and the cell size effect at controlled RGD nanospacings (Figure 1).

Figure 1. Schematic representation of micro-nanopatterns with different RGD nanospacings and microdomain sizes, and the cell-material interactions investigated by using our unique nanomaterial technique.


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The main procedures in the fabrication of the visible micro-nanopatterns are schematically represented in Figure 2. First, the Au nanopattern on glass was prepared through block copolymer

micelle

nanolithography.

Then,

micelles

of

polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP, Polymer Source) loaded with Au precursor HAuCl4·3H2O (Alfa Aesar) were dip-coated onto a glass surface. After the coated glass was treated with oxygen plasma, the polymer was decomposed and the residues were pumped away. Meanwhile, the Au precursor was oxidized into Au2O3, which was further reduced to Au when exposed to air49, 51. The nanospacing was adjusted by the molecular weight of PS-b-P2VP, with all the fabrication parameters listed in Supplementary Table S1.


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Figure 2. Fabrication of micro-nanopattern on polymer substrate. (a) Preparation of Au nanoarrays on glass by block copolymer micelle nanolithography. Briefly, PS-b-P2VP was dissolved in toluene to form reverse micelles, then Au precursor (HAuCl4·3H2O) was loaded into micelle cores. After dip-coating, micelles loaded with gold acids self-assembled on the glass surface hexagonally. At last, PS-b-P2VP micelles were removed by oxygen plasma, and Au nanodot arrays on glass surface were obtained. (b) Preparation of RGD nanopatterned microislands. In brief, glass slides with Au nanopatterns were spin-coated with photoresist, covered by designed mask, and exposed to ultraviolet light. The photoresist after exposure was developed off. To generate visible microdomains, the glass was selectively etched by an HF/NH4F buffer solution. To further obtain a non-fouling background, the prepared Au


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micro-nanopattern on the glass was transferred onto the surface of a PEG hydrogel through a linker reagent, and then grafted with RGD motifs. The microislands on the glass, after the transfer operation, turned to micropans on the PEG hydrogel.

To prepare the microscale pattern based on the fabricated nanopatterned glass, the glass was spin-coated with photoresist (RZJ-304, Suzhou Ruihong Electronic Chemicals Co. Ltd., China). Then, the photoresist-coated surface was covered by a designed micropattern mask, and illuminated by a 365-nm ultraviolet light to generate a photoresist micropattern. The patterned substrate was subsequently immersed in 0.5 mol/L HF/NH4F buffer solution at 35

°C for 45 seconds. The regions not covered by the photoresist were etched off, and the Au nanodots within these regions were consequently wiped away. With the carving-in of the glass, the microislands of the remaining Au nanodots protruded out. After the photoresist was lifted off by acetone, the visible Au micro-nanopattern on glass was generated. To exclude the impact of other biomacromolecules and to study the individual effect of the RGD peptide distribution, the micro-nanopatterns need be transferred to a non-fouling background. The PEG hydrogel is famous for its persistent resistance of cell adhesion42, 57. Therefore, transferring Au nanoarray on glass to a PEG hydrogel is the key to the preparation of a RGD nanopattern on a PEG hydrogel. Herein, we employed a heterobifunctional reagent as a linker14, 21, 26, 31. The micro-nanopatterned glass was incubated in 1 mmol/L N, N’-bis (acryloyl) cystamine (Sigma) solution in ethanol for 1 hour. Then, poly(ethylene glycol) diacrylate (PEG-DA, molecular weight 575 Da, Sigma) was added onto the glass surface, and initiated

by

2-hydroxy-4’(2-hydroxyethoxy)-2-methylpropiophenone

(D2959,

Sigma).

During the process of PEG-DA polymerization, N,N’-bis (acryloyl) cystamine acted as a


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linker with its –SH group binding to Au dots, and its carbon-carbon double bond (–C=C–) chemically bonding to the end of the PEG-DA chain. Both Au nanoarrays and the height difference of the microdomain were transferred to the PEG hydrogel surface. The RGD micro-nanopatterns on PEG hydrogels were eventually realized after grafting RGD motifs onto the Au dots. This approach can easily achieve micro-nanopatterns of various microdomain types by designing alternative masks as demonstrated in Supplementary Figure S1. For the present cell studies, we designed a mask with a matrix of different-sized square microdomains (left image in Figure 3a). The fabricated RGD micro-nanopattern on the PEG hydrogel was observed under an inverted optical microscope (Axiovert 200, Zeiss). The images were captured with a charge coupled device (CCD, AxioCam HRC, Zeiss). The pattern outline was visible by eyes (Figure S2), and the micropan pattern was observable under a normal optical microscope (right image of Figure 3a). The height was measured by using both a 3D microscope (Bruker, ContourGT InMotion) (Figure 3b) and an atomic force microscope (AFM, Bruker Dimension Icon) (Figure S3). The characterization results were consistent with each other, both resulting in a depth of approximately 319 nm.


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Figure 3. Characterization of as-prepared micro-nanopatterns. (a) Left: designed mask for micropattern photolithography; Right: phase contrast image of a Au micro-nanopattern on the PEG hydrogel. The side lengths of the square micropans were 35 µm and 65 µm, respectively. (b) 3D optical microscopic image of a micro-nanopattern on the PEG hydrogel. The left image shows that the height of the patterned regions is lower than the non-patterned region, which was caused by the HF etching of glass. The top right is the magnification of the left image, and the bottom right shows the section of the magnified surface by the labeled lines. The depth of the micropan was measured by the vertical distance between the green triangle and the red triangle, which read 319 nm, (c) FE-SEM images of micro-nanopatterns of Au nanodots of two nanospacings on the PEG hydrogel.


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We further imaged the patterned PEG surface with a field-emission scanning electron microscope (FE-SEM, Ultra 55 Plus, Zeiss) (Figure 3c) and confirmed the existence of the hexagonally arranged Au nanodots inside the micropans. The micro-nanodomain boundary is shown in Figure S4. The average nanospacing was calculated by a self-developed plugin of ImageJ (freely available at http://www.nih.gov). The nanospacings read 42 ± 4.6 nm or 86 ± 5.0 nm, and the size of the Au dots was approximately 10 nm (Figure S5). The PEG hydrogel swelled in the cell culture medium, and the swelling ratio was measured as 1.1. The eventual nanospacings of the RGD binding site in cell studies were thus 46 nm and 95 nm. To verify the selective adhesive property of the RGD micro-nanopattern on the non-fouling PEG hydrogel within the micropans, we seeded hMSCs from Cyagen Company on another patterned surface with a side length of the micropan at 80 µm for 24 hours. Stem cells were then immunofluorescently stained to show F-actins (red) and nuclei (blue) using standard staining methods51. The images of cells were taken by a color CCD mounted on an inverted microscope, and the software we used to take photos is AvioVision Rel. 4.8. The value of color saturation was set as 1 by the software. Figure 4 demonstrates the excellent localization of stem cells inside the micropans. The images on the top row show that the middle micro-nanodomains were divided into two parts by a dipline. The region below the dipline was modified by the RGD nanoarray, whereas the region above was the non-modified bioinert background. Cells rarely passed across the dipline, and primarily occupied the modified region. In addition to confirming the resistance of the PEG background to cell adhesion, the upper row in Figure 4 also reflected that the localization of cells was not due to topological trapping but was mainly attributed to selective binding to RGD peptides through


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integrin molecules. Consequently, the micropan designed on the PEG hydrogel brings with us the convenience of macroscopic microscale distribution of cells on the micro-nanopattern under an optical microscope.

Figure 4. Cell localization on micro-nanopatterns illustrated via optical micrographs of the stem cells on the micropans of RGD nanoarrays on the PEG hydrogel. The top row shows cells near the dipline on a 46 nm nanospaced micro-nanopattern. The left image is a bright field micrograph; micelles with Au precursor loaded in the dip-coated area showed light purple. The right is a fluorescence micrograph of the hMSCs. Here we examined very large micropans in order to demonstrate diplines within micropans, which strongly illustrates the selectively adhesive property of RGD micro-nanopattern on the background of PEG hydrogels. Very large micropans were occupied by multiple cells. The bottom row shows fluorescence micrographs of the hMSCs on a 46 nm spaced micro-nanopattern. Cells were stained in orange red to show F-actins and in blue to show the nuclei. Due to the aggregation of multiple cells on very large microislands, cells on some of micropans were overexposed, leading to some yellow dots in the F-actin-strained image. Such a color-changing phenomenon can only happen in the case of a color CCD, with the optical principle interpreted in Figure S6. (Figure S6).


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All of the following data shown in this paper are for single cells on adhesive microdomains. It is important to find appropriate size of microdomain to have a high fraction of cell occupation in the long cell culture for the examination of stem cell differentiation, and a high fraction of single cell adhesion on the microdomains. This is very hard for the nanopatterned microdomains, and the visualization of the microdomains is very helpful for such studies. In our pre-tests, we recorded the spreading size of over one hundred hMSCs on RGD nanopatterns with different nanospacings (without the microarray constraint). The free spreading sizes of single cells ranged from 400 µm2 to 15000 µm2 (Figure S7), and rapidly changed with time. In our study of cell differentiation, we designed two square microislands with side lengths of 35 µm and 65 µm for our micro-nanopattern. In our mask, as shown in Figure 3a, the interval between two squares was established above 70 µm in order to avoid significant overlap of stem cells from nearby micropans, whereas maintaining high efficiency of cell culture. After swelling of PEG hydrogels in the culture medium, the area of the 35 µm micropan became approximately 1590 µm2 and that of the 65 µm micropan became approximately 4690 µm2. The two micropans were named as relatively “small” and “large” ones in this study. Although the spreading areas of any living cells dynamically fluctuated, we expected that the small micropans could show the statistically insignificant difference of the spreading areas of single cells on micropans decorated by RGD nanodots with 46 nm and 95 nm spacing, thereby enabling the examination of the nanospacing effect on stem cell differentiation under a given cell spreading area for the first time. The large micropans on the non-fouling PEG hydrogel could serve as a semi-constraint for the otherwise free cells, and many single cells in most cases could not fill a large micropan. Nevertheless, we expected


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that the comparison between the two micropans of 1590 µm2 and 4690 µm2 areas might reveal the effect of cell spreading areas on stem cell differentiation under a given RGD nanospacing, which was also enabled by our micro-nanopatterning technique for the first time. The statistics of the cell spreading areas confirmed our expectations well. We seeded hMSCs onto RGD-patterned PEG hydrogels inside 12-well culture plates at a density of 25, 000 cells per well to investigate the ability of our novel micro-nanopattern to control cell areas. After 24 hours, we randomly selected about 120 cells from 4 sample surfaces for each kind of micro-nanoisland. Only single cells inside micropans were taken into consideration. The average areas and standard deviations of different micropans were calculated and un-paired t-test was used to analyze significance of difference between any two groups. The statistical results of the spreading areas are shown in Figure S8. The t-test indicated a significant difference between spreading areas of single cells on micropans of two different areas (1443 ± 118 µm2 in the group of nanopatterned micropans (46 nm, 1590 µm2) versus 3160 ± 420 µm2 in the group of (46 nm, 4690 µm2), and 1363 ± 322 µm2 in the group of nanopatterned micropans (95 nm, 1590 µm2) versus 2834 ± 352 µm2 in the group of (95 nm, 4690 µm2)). No significant difference was found between spreading areas of two RGD nanospacings for a given micropan size with corresponding p-values shown in Table S3. So, although the cell shape could not be identically confined, the cell spreading size could be controlled by the micropans sufficiently to decouple the effects of spreading area and RGD nanospacing on the differentiation of stem cells.


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The above experiments illustrated that the novel micro-nanopattern could be used to control cell size, decouple cell-cell contact and to study the possible effects of RGD nanospacing on stem cell differentiation at both molecular (integrin) and cellular level. Before the examination of cell differentiation, the effect of RGD nanospacing on cytoskeleton and cell tension was checked because it was reported that cytoskeleton and intracellular tension could influence stem cell differentiation58. We cultured hMSCs for 24 hours, and then immunofluorescently stained F-actins to show the cytoskeleton. Vinculins and nuclei were counter-stained. Fluorescence images were observed with a CCD mounted microscope. The intensity of the excitation light and the exposure time for the CCD camera were kept constant between different experimental groups. We analyzed single cells on the micropans using ImageJ, and obtained histograms of the pixel number distributions according to the grey values of the cell image. Because F-actin bundles exhibit higher grey values than other areas, cells with clear filaments showed double peaks in the histograms, as demonstrated in Figure S9, whereas those without clear filaments showed only one peak. We counted the numbers of single cells with or without clear F-actin filaments according to their histograms. Representative images of stem cells and fractions of cells with clear F-actin filaments are shown in Figure 5a and b, respectively. The statistics illustrated that the larger RGD nanospacing led to less F-actin assembly, and the increased spreading of cells corresponded to stronger cytoskeleton.


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Figure 5. Formation of F-actin filaments in the hMSCs cultured on micro-nanopatterns for 24 hours. (a) Typical optical micrographs of the counter-stained cells. Whereas the upper row shows bright-field images observed in a DIC mode, the other rows represent corresponding fluorescence micrographs with F-actins in orange red, vinculins in green and nuclei in blue. The DAPI staining guaranteed that only single cells were used in the later statistics. Due to the confinement of micropan and the mobility of cells on RGD nanoarray, it was hard to


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observe vinculin clusters on our micro-nanopattern. (b) Fraction of hMSCs with significant actin filaments. Three samples were picked for each group (n = 3), and for each sample at least thirty single cells were analyzed. The p-values of t-tests are listed in Supplementary Tables S2. Intracellular tension can be implicated by phosphorylated myosins16,

59

. Conventional

myosins are hexameric proteins consisting of two heavy chain subunits, a pair of non-phosphorylatable light chains and a pair of phosphorylatable light chain subunits60. Myosin light chain 9 (MYL9) is one of the numerous regulatory myosin light chains61. We cultured hMSCs for 24 hours, stained phosphorylated myosin light chain 9 (p-MYL9) to show intracellular tension following the published protocol62. Cells were fixed, permeabilized, blocked, treated with a rabbit polyclonal p-MYL antibody (Santa Cruz) at room temperature for 2 hours, and then stained with anti-rabbit IgG-TRITC (Sigma) for 1 hour. Nuclei were counter-stained. Fluorescence images were captured with a CCD mounted on a microscope. The intensity of the excitation light and the duration of exposure for photographing were kept constant between the experimental groups of different RGD nanospacings and cell spreading sizes. Representative images are shown in Figure 6. The fluorescence micrographs were processed by the software ImageJ. The color images were converted to 8 bit, and the integrated fluorescence intensity of p-MYL9 was obtained by calculating the total grey value of single cells by multiplying the effective mean grey value of single cells with the cell area. The effective mean grey value was obtained by the mean grey value of single cells minus the background grey value selected from a region around the target cell with a similar area63. Only single cells on the micropans were outlined to join in the statistics. The integrated fluorescence intensity reflects the expression level of phosphorylated myosins, and thus


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resembles cell tension. The relative integrated fluorescence intensity is shown in Figure S10. The expression of phosphorylated myosin was similar to the staining tendency of the F-actin filaments. It could be promoted by smaller RGD nanospacing and larger cell size.

Figure 6. Optical micrographs of single hMSCs on micropans with RGD nanoarrays. The upper row shows the bright field images, and the lower row indicates the corresponding fluorescence images. Phosphorylated myosin cells were immunofluorescently stained with a p-MYL9 antibody to demonstrate the expression of phosphorylated myosin (red) with nuclei (blue) counter-stained. Relative fluorescence intensities of the p-MYL9 images are shown in Supplementary Figure S10. These results shed new light on the effect of RGD nanospacing on specific cell adhesion. In all preceding studies focusing on the effects of RGD nanospacing on various cells on free surfaces, the cells on patterns with small RGD nanospacings always had larger spreading sizes than the cells grown on patterns with large RGD nanospacings47, 49, 51, 52. It is therefore not conclusive whether the stronger cytoskeleton was caused by the larger cell spreading size or the smaller RGD nanospacing. Thanks to the micro-nanopatterns and the experimental design reported in this letter, we now conclude that smaller RGD nanospacing leads to stronger cytoskeleton and intracellular tension even with similar cell spreading sizes.


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For investigation of the effects of RGD nanospacings and cell spreading sizes on stem cell differentiation, hMSCs were cultured on RGD micro-nanopatterns for 1 day and then induced in osteogenic or adipogenic induction medium. The osteogenic induction medium was composed of high-glucose Dulbecco’s modified Eagle medium (DMEM, Gibco), 10% fetal bovine serum (FBS, Gibco), 50 µM ascorbic acid-2-phosphate, 10 mM β-glycerophosphate, and 100 nM dexamethasone (Sigma); the adipogenic induction medium was composed of high-glucose DMEM, 10% FBS, 1 µM dexamethasone, 200 µM indomethacin, 10 µg/ml insulin, and 0.5 mM methylisobutylxanthine (Sigma). The media were replaced every 2 to 3 days. After 2 weeks, the osteogenically induced cells were stained with Fast Blue RR (Sigma) to check their alkaline phosphatase (ALP) activity, and the adipogenic induced cells were stained with Oil Red O (Sigma) to check their oil expression51. Representative staining images are shown in the top row in Figure 7. A positive staining by Fast Blue RR indicates osteogenesis, and a positive staining by Oil Red O indicates adipogenesis. We chose only single cells inside the micropans as statistical targets, with the resultant osteogenic and adipogenic fractions shown in the bottom graphs of Figure 7. For osteogenesis of hMSCs on micropans of the same nanospacing, large cell spreading size led to higher osteogenesis fraction with a significant difference between the groups of different spreading sizes. This result is consistent with our previous study about the effect of cell size on stem cell differentiation56. When cell size was controlled, the large RGD nanospacing triggered more significant osteogenesis. This is consistent with our study on nanopatterns51. For adipogenesis of hMSCs, single cells inside smaller micropans with larger RGD nanospacing (95 nm/1590 µm2) resulted in the highest differentiation fraction at approximately 27%. In


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the present study, the differentiations of single cells on smaller RGD nanospaced or larger sized micro-nanopatterns were in such low fractions (only approximately 10%) that we cannot test out a significant difference between these groups. We also confirmed that it was not easy for single cells to conduct adipogenic differentiation as reported by Chen et al. on fibronectin micropatterns55. Meanwhile, aggregated hMSCs were well differentiated into adipocytes, as demonstrated in Figure S11. This is consistent with our previous publications about the effects of cell-cell contact and cell density on stem cell differentiation based on RGD micropatterns56.

Figure 7. Cell differentiation on micro-nanopatterns. The top row shows typical differentiated stem cells on the microdomains of RGD nanoarrays of small and large microdomain areas, with the left image demonstrating osteogenically differentiated cells stained positively by Fast Blue RR, and the right image demonstrating adipogenically


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differentiated cells stained positively by Oil Red O. The middle row presents the fluorescence images of the DAPI-stained cells corresponding to the upper row, indicating the single cells included in the statistics. The bottom row summarizes osteogenic and adipogenic fractions with different micropan sizes and RGD nanospacings. The p-values of t-tests are listed in Supplementary Tables S5-6.

To confirm the effect of RGD nanospacing on the differentiation of hMSCs, we repeated the differentiation experiments, and immunofluorescently stained osteopontin (OPN)64 for osteogenic induced cells65, and peroxisome proliferator-activated receptor gamma (PPARγ)66 for adipogenically induced cells67 to verify our conclusion. With rabbit polyclonal antibody of OPN (Santa Cruz, 1:50) as the primary antibody and anti-rabbit IgG-TRITC (Sigma, 1:150) as the secondary antibody, OPN was stained in red and imaged under a fluorescence microscope (Figure 8). PPARγ was stained green (Figure 8) with a PPARγ mouse monoclonal antibody (Santa Cruz, 1:50) as the primary antibody and anti-mouse IgG-Alexa Fluor 488 (Invitrogen, 1:150) as the secondary antibody. The relative integrated fluorescence intensities of both OPN and PPARγ images are presented in Supplementary Figure S12. Our immunofluorescence staining results of hMSC differentiation on micro-nanopatterns (Figures 8 and S11) were highly consistent with the bright-field observations of ALP activity and oil droplets (Figure 7). It is thus conclusive that the larger RGD nanospacing promotes both osteogenesis and adipogenesis.


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Figure 8. Dark-field micrographs of differentiated cells after immunofluorescence staining to show the expression of OPN (red) in the osteogenically induced stem cells, and the expression of PPARγ (green) in the adipogenically induced stem cells, with counter-stained nuclei (blue). Cells were outlined by dotted lines.

In Figure 9, we schematically present the conclusions of both adhesion and differentiation of hMSCs on our micro-nanopatterns. RGD nanospacing is a regulator of specific cell adhesion. On one hand, the small RGD nanospacing caused well-defined F-actin bundles and higher expression of phosphorylated myosin and thus more intracellular tension. On the other hand, the larger spreading size also caused more cytoskeletal tension. It has been reported that high tension inside cells promotes osteogenesis and inhibits adipogenesis55, 58, 68. Hence, the effect of cell spreading size on hMSCs could be well understood. However, the effect of RGD nanospacing on differentiation of hMSCs cannot be fully explained through cell tension. Therefore, we speculate that the nanoscale binding site of the RGD motif might trigger some unknown outside-in signaling to modulate cell differentiation. To a certain extent, a large RGD nanospacing favors, albeit is not beneficial for cell adhesion and cell tension, both osteogenesis and adipogenesis of those adhered stem cells.


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Figure 9. Schematic representation of the effects of RGD nanospacing and cell spreading size on behaviors of stem cells on micro-nanopatterned surfaces. Although it is well understood that a large adhesion area and increased intracellular tension favor osteogenesis, the nanospacing effect on differentiation of hMSCs cannot be fully interpreted by cell spreading areas and intracellular tension. So, the present work reveals that the nanospacing of adhesive sites is a new independent regulator of stem cell differentiation.

To summarize, we developed a visible micro-nanopattern by combining block copolymer micelle nanolithography, photolithography, HF etching, and transfer lithography. The


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resultant visible micro-nanopattern is very helpful for revealing cell-material interactions on both cellular level and molecular level. To demonstrate its application in biomaterials, we designed a series of micro-nanopatterns to decouple the effects of RGD nanospacing and cell spreading size. Both cell spreading size and RGD nanospacing influenced cytoskeleton and stem cell differentiation. Using our unique micro-nanopatterning technique, we revealed that small RGD nanospacing leads to stronger cell tension even under similar cell sizes. Thus, we conclude that the RGD nanospacing affords a regulator of stem cell differentiation independent of cell spreading areas. The underlying mechanism of the effect of nanospacing of adhesive sites on stem cell differentiation constitutes an interesting and important topic. In prospection of the material technique, a variety of micropatterns could be achieved by tuning the size and/or shape, and also by regulating the way of assembly of the microdomains; nanospacing of Au nanoparticles could be adjusted anywhere from a few nanometers to more than hundreds of nanometers. Patterned metals24, 69, 70 and even peptide ligands71-74 could also be altered. Hence, the applications of the visible micro-nanopatterns reported in this letter might be extended from fundamental studies of cell-material interactions to other fields such as molecular electronics, gas sensors, and cell and organ chips75-77. AUTHOR INFORMATION Corresponding author *Jiandong Ding

e-mail: [email protected]: 86 21 65643506. Fax: 86 21 65640293.

Note: The authors declare no competing financial interests.


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ASSOCIATED CONTENT Supporting Information: FE-SEM images of the micro-nanopatterns of varied shapes of microdomains, global photograph of an as-prepared hydrogel with micro-nanopattern, AFM images of micropattern, AFM image of Au nanopattern on glass, micro-nanopattern at the boundary region of micropans on PEG hydrogels, schematic presentation of the reason that an overexposure would cause images of F-actins change color after recorded by a color CCD, distribution of spreading areas of free cells on nanopatterned surfaces, spreading areas of single hMSCs on micro-nanopatterned surfaces with different nanospacings and microisland sizes, representative histograms of pixel level distributions of F-actin-stained cell images, micrographs of osteogenesis of aggregate cells, relative integrated fluorescence intensity of fluorescence images of cells stained by p-MYL9, OPN and PPARγ, tables with fabrication parameters and corresponding characteristics of nanopatterns prepared in this study, and p-values in t-tests of the figures in the main manuscript and supplementary information. These materials are available free of charge via Internet at http://pubs.acs.org.

ACKNOWLEDGMENT The authors are grateful for the financial supports from NSF of China (grant No. 51273046), Chinese Ministry of Science and Technology (973 program No. 2011CB606203), Science and Technology Developing Foundation of Shanghai (grant No.13XD1401000). ABBREVIATIONS PEG, poly(ethylene glycol); RGD, arginine-glycine-aspartate; hMSC, human mesenchymal stem cell; SEM, scanning electron microscopy; AFM, atomic force microscopy; PS-b-P2VP,


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polystyrene-block-poly(2-vinylpyridine); PEG-DA, poly(ethylene glycol) diacrylate; CCD, charge coupled device; FE-SEM, field-emission scanning electron microscopy; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; ALP, alkaline phosphatase; p-MYL9, phosphorylated myosin light chain 9; OPN, osteopontin; PPARγ, peroxisome proliferator-activated receptor gamma.


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