Effects of Nanoscale Spatial Arrangement of Arginine–Glycine

Publication Date (Web): October 27, 2015. Copyright © 2015 American .... Cells on hierarchically-structured platforms hosting functionalized nanopart...
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Effects of Nanoscale Spatial Arrangement of Arginine−Glycine− Aspartate Peptides on Dedifferentiation of Chondrocytes Shiyu Li, Xuan Wang, Bin Cao, Kai Ye, Zhenhua Li, and Jiandong Ding* State Key Laboratory of Molecular Engineering of Polymers, Collaborative Innovation Center of Polymers and Polymer Composite Materials, Department of Macromolecular Science, Advanced Materials Laboratory, Fudan University, Shanghai 200433, China S Supporting Information *

ABSTRACT: Cell dedifferentiation is of much importance in many cases such as the classic problem of dedifferentiation of chondrocytes during in vitro culture in cartilage tissue engineering. While cell differentiation has been much investigated, studies of cell dedifferentiation are limited, and the nanocues of cell dedifferentiation have little been reported. Herein, we prepared nanopatterns and micro/nanopatterns of cell-adhesive arginine−glycine−aspartate (RGD) peptides on nonfouling poly(ethylene glycol) (PEG) hydrogels to examine the effects of RGD nanospacing on adhesion and dedifferentiation of chondrocytes. The relatively larger RGD nanospacing above 70 nm was found to enhance the maintainence of the chondrocyte phenotype in two-dimensional culture, albeit not beneficial for adhesion of chondrocytes. A unique micro/ nanopattern was employed to decouple cell spreading, cell shape, and cell−cell contact from RGD nanospacing. Under given spreading size and shape of single cells, the large RGD nanospacing was still in favor of preserving the normal phenotype of chondrocytes. Hence, the nanoscale spatial arrangement of cell-adhesive ligands affords a new independent regulator of cell dedifferentiation, which should be taken into consideration in biomaterial design for regenerative medicine. KEYWORDS: RGD nanospacing, micro/nanopattern, poly(ethylene glycol) hydrogel, cell spreading, cell dedifferentiation, chondrocyte differentiated phenotype and adopt an increased fibroblast-like morphology,44 which results in an inferior repaired tissue compared with normal cartilage.45 To address this issue, researchers have found different factors to influence the chondrocyte phenotype such as soluble factors,46,47 mechanical stimulations,48 and three-dimensional culture conditions.49,50 Nonetheless, there is no report focused on the effects of nanoscale spatial organization of active ligands on cell dedifferentiation. The present work is motivated to address and answer whether or not extracellular nanoscale features can regulate cell dedifferentiation using chondrocytes as the cell model. The corresponding deterministic experiment should be based upon a well-designed nanomaterial. As a cell-binding ligand in various extracellular proteins, arginine−glycine−aspartate (RGD) has been used to modify biomaterial functions51−54 including a tissue engineering strategy in cartilage repair.55,56 Linkage between RGD motif and its transmembrane receptor, integrin, mediates specific cell adhesion through the assembly of various structural and adapter proteins, which are known as focal adhesions (FAs).1 Since the size of integrin is approximately 8−12 nm,57 features at this scale may be able to modulate integrin-based cell structures and then influence

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ells can delicately sense and respond to external nanoscale features in intricate living systems.1−4 The nanoscale sensing capacity of cells is crucial to mimic extracellular matrix (ECM). To decode the fundamental questions on how extracellular stimuli influence cell behaviors, several approaches have been established.5−12 Among them, nanopatterning affords a unique means to precisely mimic pericellular nanoenvironments and also simplify the complexity of living systems.13−20 Comprehensive studies have shown that nanocues have an impact on cell behaviors including adhesion,21−24 proliferation,24,25 migration,26,27 and differentiation.28−31 Nevertheless, little attention has been paid to nanocues of cell dedifferentiation, a process of differentiated cells to regress to an earlier differentiation stage. Cell dedifferentiation has been found to be relevant to the origins of cancer cells,32 tissue regeneration,33,34 and some diseases,35 and an in-depth understanding of dedifferentiation may bring a new perspective to regenerative medicine.36 Some uncontrolled cell dedifferentiations such as chondrocyte dedifferentiation during in vitro culture constitute a classic problem in cartilage repair.37 Cartilage tissue engineering is a challenging topic in regenerative medicine with rich science.38−41 One of the clinical approaches of cartilage repair is autologous chondrocyte implantation (ACI), and a workable amount of chondrocytes is acquired for direct implantation or seeding with supporting matrices in ACI.42,43 However, during in vitro monolayer culture, chondrocytes are liable to lose their © 2015 American Chemical Society

Received: October 5, 2015 Revised: October 23, 2015 Published: October 27, 2015 7755

DOI: 10.1021/acs.nanolett.5b04043 Nano Lett. 2015, 15, 7755−7765

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on the PEG hydrogel are schematically presented in Figure 2, panel a. The first step is to obtain gold nanoarrays on glass via block copolymer micelle nanolithography established by Spatz and Möller.67 Amphiphilic block copolymers of poly(styreneblock-2-vinylpyridine) (PS-b-P2VP, Polymer Source) were chosen as the template. After the copolymer was dissolved in toluene, micelles with core−corona structure were formed. Then, Au precursor (HAuCl4·3H2O, Alfa Aesar) was added and self-assembled into the micelle cores. A micellar monolayer was formed by dipping a glass substrate into the micelle suspension. After treated with oxygen plasma, the polymeric template was removed, and the Au precursor was reduced to gold, and thus Au nanoarray on glass was obtained. Subsequently, the nanopatterned glass was immersed in 1 mmol/L N,N′bis(acryloyl) cystamine (Sigma) solution to graft the heterobifunctional linker via the covalent bonds between Au nanodots and thiol groups. Then, PEG-DA (molecular weight 575 Da, Sigma) was poured onto the patterned glass. Photopolymerization was initiated by 2-hydroxy-4′(2-hydroxyethoxy)-2-methylpropiophenone (D2959, Aldrich) under ultraviolet radiation (365 nm). The double bonds of the linker were chemically bonded to the PEG-DA chain during polymerization, which led to the successful transfer of Au nanoarray from glass to the PEG hydrogel. The last step was to graft RGD motifs onto Au nanodots via incubating the nanopatterned PEG hydrogel in the 25 μM aqueous solution of c(−RGDfK−)−thiol (f, D-phenylalanine; K, L-lysine; Peptides International) at 4 °C for 4 h. An RGD nanopattern on a PEG hydrogel was ultimately achieved. To well control the interligand distance of RGD peptides, the molecular weights of PS-b-P2VP were regulated, with pertinent fabrication parameters listed in Supporting Information Table S1. The micellar monolayer was observed in a transmission electron microscope (TEM, Tecnai G2 20 TWIN, FEI). As shown in the upper row of Figure 3, panel a, the distances between neighboring micelle cores varied with the composition of diblock copolymer. The hexagonal arrangement of Au nanodots was well reflected from Supporting Information Figure S1, captured in an atomic force microscope (AFM, Multimode 8, Bruker). The average height of the nanodots read approximately 4 nm (Supporting Information Figure S1). Such a small size ensures that one nanodot on the material surface corresponds to only one integrin in the cell membrane after seeding cells, and thus the RGD nanospacing determines the nanospacing of integrins in cell adhesion. In this study, we prepared Au nanopatterns on PEG hydrogels with six nanospacings. The lower row of Figure 3, panel a shows some typical images observed in a field-emission scanning electron microscope (FE-SEM, Ultra 55, Zeiss). The average nanospacings were calculated using ImageJ (freely available at http://www.nih.gov). In the cell culture medium, the PEG hydrogel was swollen, with the equilibrium swelling ratio measured as 1.1. Thus, the eventual RGD nanospacings for further cell studies were 36, 50, 71, 98, 136, and 143 nm, as summarized in Supporting Information Table S1. Chondrocytes isolated from the articular cartilage of neonatal Sprague−Dawley (SD) rats were employed as the model cell type. The standard monolayer culture for several passages could lead the normal chondrocytes to a dedifferentiated phenotype, as characterized by immunocytochemistry staining of collagen II on tissue culture plate (TCP) (Supporting Information Figure S2). Therefore, chondrocytes of the first passage were chosen for the investigations of chondrocyte adhesion and

cell behaviors. Thus, nanopatterns with RGD binding sites below 10 nm achieved by block copolymer micelle nanolithography afford a powerful platform to study the interactions between RGD nanoscale organization and the corresponding cell behaviors.58,59 RGD nanospacing has been found to act as a regulator of cell functions, and a critical RGD spacing is proposed to be around 70 nm.59,60 An RGD spacing below 70 nm favors cell adhesion with a relatively larger spreading area and stronger cytoskeleton, while cells on nanopatterns with spacing above 70 nm exhibit poor adhesion and limited spreading.59,61 Meanwhile, some researchers have developed micro/nanostructured patterns as a biophysical model to manage cell responses.62,63 Herein, distinctively from previous studies, we focused on the potential direct impact of RGD nanospacing on chondrocyte dedifferentiation, as schematically illustrated in Figure 1.

Figure 1. Schematic illustration of chondrocytes on nanopatterned PEG hydrogel surfaces with cell-adhesive RGD nanoarrays to investigate the effects of RGD nanospacing on adhesion and dedifferentiation of chondrocytes during in vitro two-dimensional culture. To further decouple the RGD nanospacing effect and the spreading effect on the maintenance of the normal chondrocyte phenotype, a unique micro/nanopattern technique is used in the present study.

Hexagonal RGD nanopatterns on poly(ethylene glycol) (PEG) hydrogels with a series of RGD nanospacings were prepared. Chondrocytes derived from articular cartilage of rats were cultured in the growth media on the nanopatterns for 7 days to examine the RGD nanospacing effects on dedifferentiation. To further elucidate whether or not RGD nanospacing is an independent regulator of dedifferentiation of chondrocytes, a unique micro/nanopattern was fabricated, which enables us to decouple the straightforward effect of RGD nanospacing with the interferential factors such as cell spreading area. An unambiguous study of cell behaviors on RGD nanopatterns relies on an ideal background, which should persistently resist nonspecific protein adsorption and cell adhesion. A PEG hydrogel can prevent protein from adsorption for at least 1 week,64 which ensures the dominance of the RGDtriggered cell adhesion throughout our cell culture. However, it is technically a bottleneck to covalently generate a stable RGD nanoarray on the chemically inert PEG hydrogels, and a transfer lithography has been put forward to resolve this problem.58,65,66 PEG hydrogels were formed by radical polymerization of the corresponding macromonomer poly(ethylene glycol)-diacrylate (PEG-DA). The fabrication procedures of an RGD nanopattern 7756

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Figure 2. Schematic presentation of fabrication processes of RGD nanopattern and micro/nanopattern on a nonfouling PEG hydrogel. (a) The route of fabricating nanopatterns of RGD peptides on the PEG background. The top and middle rows demonstrate the preparation of Au nanopattern on glass surface via block copolymer micelle nanolithography. The bottom row shows the procedure of transferring the gold nanopattern to a PEG hydrogel surface by grafting linker on Au nanodots and photopolymerization of PEG-DA on the glass surface. RGD motifs were finally grafted onto Au nanodots to generate an RGD nanopattern on a PEG hydrogel. (b) Preparation procedures of a micro/nanopattern on a nonfouling PEG substrate. The top row presents the fabrication process of photoresist micro/nanopattern on the prior nanopatterned glass to protect Au nanoarrays via lift-off photolithography. The middle row shows the key step to obtain the micro/nanodomains. The region uncovered with the photoresist was selectively etched by HF/NH4F buffer solution to move out the gold nanodots and generate the height difference between micro/nanodomains and the outside areas. Then linker was grafted onto Au nanodots. The bottom row demonstrates the transfer procedure to shift gold nanoarrays inside microislands from glass to PEG hydrogel, which results in micro/nanopans on the hydrogel surface.

(red), vinculins (green), and nuclei (blue). Representative fluorescence images of chondrocytes on nanopatterns are shown in Figure 3, panel b. With the increase of RGD nanospacing, a decrease of cell density and cell spreading area was observed. In addition, clearer filamentous actin (F-actin) bundles were seen in chondrocytes growing on the nanopatterns with an interligand distance near or below 70 nm, in comparison to the ones above 70 nm. Vinculin, a key protein of

dedifferentiation on nanopatterns. We cultured chondrocytes on the sterilized nanopatterns at a seeding density of 5200 cells per cm2. The growth medium contained 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), and 2 mM L-glutamine (Gibco) in highglucose Dulbecco’s modified Eagle medium (DMEM, Gibco). After 24 h of adhesion on nanopatterns in the growth medium, cells were immunofluorescently stained to visualize F-actins 7757

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Figure 3. Nanopatterns with varied nanospacings and dependence of chondrocyte adhesion upon RGD nanospacing. (a) The upper row shows the TEM images of micelles of the indicated six PS-b-P2VP diblock copolymers with HAuCl4 loaded in the lipophobic cores. The insets show the corresponding high-magnification images. The lower row presents FE-SEM images of Au nanoarrays on PEG hydrogels. (b) Fluorescence micrographs of chondrocytes on RGD-nanopatterned surfaces with varied nanospacings. Cells were stained to visualize F-actins (red), vinculins (green), and nuclei (blue) after culturing on nanopatterns for 24 h. The upper row shows low-magnification fluorescence micrographs of chondrocytes under a relatively large field, and the lower row presents some typical laser confocal fluorescence micrographs of counter-stained single cells. The insets show the magnified portions of vinculin channel. (c) Statistics of cell density, area, circularity, and aspect ratio (n = 4). Chondrocytes on surfaces with smaller RGD nanospacings exhibited relatively higher densities, larger spreading area, and slightly elongated morphology. The pvalues of one-way ANOVA are listed in Supporting Information Tables S2−S5.

FA complex, serves as an indicator to evaluate the formation of FAs. Chondrocytes on the nanopatterns with a relative smaller nanospacing exhibited a stronger expression and a cluster distribution of vinculins near the brim of cells, indicating that nanospacings below the critical 70 nm are beneficial for cell adhesion. In contrast, unmatured FAs with vague vinculin spots were observed on the patterns with large nanospacings. To quantify the difference of cell adhesion throughout the range of RGD nanospacings, more than 150 single cells were averaged in each sample, and four independent samples were taken into statistical analysis for each group. As presented in Figure 3, panel c, the cell density and cell spreading area

decreased monotonically with the increase of nanospacing, which is consistent with preceding studies of other cell types.59,61 Meanwhile, the aspect ratio (AR) and cell circularity, as descriptors of cell shape, were acquired from the cell profile by using ImageJ. It makes sense to investigate AR and circularity of chondrocytes on different nanopatterns since our previous studies based on a micropatterning technique suggested that a more round cell shape was favorable for maintaining the phenotype of chondrocytes.68 Circularity is defined as spreading area multiplied by 4π and divided by the square of perimeter with 1.0 denoting a perfect circle. It is not hard to understand that cell circularity increased with RGD 7758

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experiments due to the same fabrication procedure of PEG hydrogels with a series of RGD nanospacings. For investigation of the effects of RGD nanospacing on cell dedifferentiation, we cultured chondrocytes on nanopatterns with nanospacings of 50, 71, 98, and 143 nm in the growth medium for 7 days, following a replacement of medium at the ends of day 1 and day 4. To attenuate the possible interference of cell−cell contact at high cell densities, we added 0.5 μg/mL of Aphidicolin (Sigma) to the growth medium after the first day to inhibit cell proliferation. Collagen II was employed as the marker of dedifferentiation of chondrocytes. After 7 days of culture, we conducted immunofluorescent staining of collagen II with a mouse monoclonal anticollagen II IgG2b (Santa Cruz, 1:100) and a streptavidin−biotin−Cy3 complex immunofluorescence staining kit (SABC-Cy3) (Boster) by following the manufacturers’ protocols. The cells with collagen II (red) and nuclei (blue) labeled were observed in an inverted microscope (Axio Observer. Z1, Zeiss) equipped with a black-white chargecoupled device (CCD, AxioCam MRm, Zeiss). Some typical fluorescence micrographs are shown in Figure 5, panel a. While normal chondrocytes were stained in red, the dedifferentiated chondrocytes secreted a very low level of collagen II with no noticeable color reaction. Then, dedifferentiation extent was conveyed by the fraction of collagen II-positive cells (Figure 5b). According to the statistical results, a decreasing trend of dedifferentiation percentage was noticed with the increase of RGD nanospacing, which indicates that relatively large RGD nanospacings favored preserving the phenotype of chondrocytes compared to small nanospacings. To confirm the phenomenon observed above, the gene expressions of collagen II, collagen I, aggrecan, and SOX9 of chondrocytes on patterns of different nanospacings were detected using quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR), with the sequences of primers (Invitrogen) shown in Supporting Information Table S7. While collagen I is expressed in most of cell types except chondrocytes, the expressions of aggrecan, SOX9, and especially collagen II are probably high in chondrocytes. The relative gene expressions of chondrocytes on varied nanospacings are presented in Figure 5, panel c, after normalization to those on 50 nm nanopatterns. The collagen II, aggrecan, and SOX9 of chondrocytes were up-regulated with the increase of RGD nanospacing, while a down-regulation of collagen I was detected. Therefore, a large RGD nanospacing disfavored the dedifferentiation of chondrocytes. The gene expression results are consistent with those immunofluorescent staining results, namely the RGD nanoscale spatial arrangement exerted a considerable influence on the phenotypic maintaining of chondrocytes. A critical question arises: whether the RGD nanospacing influences cell dedifferentiation directly or indirectly via spreading area, etc. Using the micropatterning technique, we have revealed that cell spreading area regulates dedifferentiation of chondrocytes significantly.68 The present study indicated the nanospacing effects on both spreading area and dedifferentiation of chondrocytes, as seen from Figures 3 and 5, respectively. It is, therefore, much desired to decouple the effect of cell spreading from that of nanospacing. With this motivation in mind, we designed a micro/ nanopattern, as demonstrated in Figure 6. The preparation process of the visible micro/nanopattern is schematically represented in Figure 2, panel b. The technical key is the

nanospacing due to less pseudopodia of cells on nanopatterns with a larger interligand distance. The distributions of these statistical parameters are presented in Supporting Information Figure S3. It is crucial to exclude the interference of nonspecific cell adhesion for an unambiguous conclusion of effects of nanoscale spatial arrangement of RGD peptides on cell behaviors after specific adhesion. Therefore, it is very important for us to generate nanopatterns on PEG hydrogels. A demonstration to validate the persistent antiadhesion property of the PEG hydrogel is shown in Figure 4. In the fluorescence micrograph

Figure 4. Demonstration of cell adhesion contrast of an RGD nanopattern on the nonfouling PEG hydrogel. On the left, a fluorescence micrograph of chondrocytes labeled for F-actins (red) and nuclei (blue), showing that cells were only located in the patterned area on one side of dipline, which illustrates the nonfouling capacity of the PEG hydrogel. On the right, an FE-SEM image of a cell with pseudocolor on the nanopatterned PEG surface. This chondrocyte extended several pseudopodia to probe the surface and try to bind the RGD-functionalized Au nanodots with a diameter below 10 nm.

of chondrocytes near the dipline region (Figure 4, left side), cells were only located in the nanopatterned area with a clear dipline even after 7 days of culture. Furthermore, the nanoscale contact between the cell and nanoarrays was visualized by FESEM, as shown in the right of Figure 4. Besides nonspecific protein adsorption as a surface chemistry factor, physical properties including matrix stiffness and surface roughness influence cell behaviors. In 2013, Platzman et al. made a comparative study of cell adhesion on a “PEG-based material” (a PEG hydrogel with RGD-functionalized gold nanoarray) and a “PEG-modified material” (a glass surface with corresponding nanoparticles surrounded by a passivated PEG self-assembly monolayer).69 They found that densities and spreading areas of nonanchorage-dependent hematopoietic (KG-1a) and anchorage-dependent rat embryonic fibroblast (REF52) cells were enhanced on the PEG-based RGD nanopattern, which was interpreted from the surface roughness of the PEG hydrogel compared to the glass.69 In 2015, our group reported RGD-nanopatterned hydrogels with similar RGD nanospacings but different moduli, which gave a deterministic experiment to verify the substantial impact of matrix stiffness on adhesion and differentiation of stem cells.28 Meanwhile, surface chemistry was found to regulate cell differentiation as well, for the differentiation extents depended upon RGD nanospacing.28 The present study distinguishes itself as the first examination of the nanoscale spatial arrangement of RGD peptides on cell dedifferentiation. Therefore, both matrix stiffness and surface roughness are aimed to be fixed, which can be guaranteed in our material 7759

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Figure 5. Dedifferentiation of chondrocytes on nanopatterns of RGD on PEG hydrogels. (a) Representative fluorescence micrographs of chondrocytes on nanopatterns with the indicated RGD nanospacings cultured in the growth medium for 7 days. Cells were stained for nuclei (blue) and collagen II (red) to distinguish the normal chondrocytes and dedifferentiated ones. (b) The statistical results of fractions of normal chondrocytes and dedifferentiated cells as functions of RGD nanospacing. The p-values of one-way ANOVA are listed in Supporting Information Table S6. (c) qRT-PCR detection of target genes of chondrocytes after 7 days of in vitro culture on the four indicated RGD nanopatterns. The primers of those genes are listed in Supporting Information Table S7. The p-values of one-way ANOVA between each two groups are listed in Supporting Information Tables S8−S11.

combination of photolithography and hydrofluoric acid (HF) etching based upon prior nanopatterning. First, the nanopatterned glass was spin-coated with a layer of positive photoresist (RZJ-304, Suzhou Ruihong Electronic Chemicals Co. Ltd., China) and then exposed to ultraviolet light (365 nm) covered with a predesigned chrome mask. After treated by the corresponding developer, a photoresist micropattern was obtained on the top of nanoarrays. To etch the uncovered region, the patterned glass substrate was immersed in 0.5 mol/ L HF/NH4F buffer solution. After 90 s at 35 °C, the region of glass unprotected by the photoresist was etched out together with Au nanodots. Then the photoresist was lifted off by acetone, and a micropattern with Au nanoarrays inside the microdomains was generated on the glass substrate. At the end, via a transfer technique described earlier and the modification of cyclic RGD, the RGD micro/nanopattern was realized on the PEG hydrogel. We designed micropattern masks of round micropan arrays with diameters of 15 and 25 μm (Figure 6a, left side) and chose nanospacings of 50 and 98 nm for the further cell studies. Because of the height difference between micropans and the outside regions achieved by selective etching, the micro/ nanopatterns on the PEG hydrogel substrate were visible under the optical microscope (Figure 6a, right side), which brought us

much convenience in the later experiments to judge the success of the hybrid pattern and also the cell localization. The depth of the micropan was approximately 808 nm, as shown in Supporting Information Figure S4 captured in AFM imaging. The FE-SEM images of micro/nanopatterns demonstrate the Au nanoarrays inside the micropan and the barren areas outside on the PEG hydrogel (Figure 6b). Since the RGD nanoarrays in the micropans are available for cell adhesion and the PEG hydrogel outside the micropan resists cell adhesion potently, the RGD micro/nanopattern might exhibit a capacity of selective adhesion or a predefined cell localization. Chondrocytes were seeded on the patterned surfaces with both two different micropans and nanospacings for 24 h and then immunofluorescently stained to show Factins (red), vinculins (green), and nuclei (blue). Typical optical micrographs of the counterstained single cells are shown in Figure 6, panel c. Chondrocytes only adhered inside the micro/nanodomain with approximately round shape, in both circular micropans with diameters of 15 and 25 μm and RGD nanospacings of 50 and 95 nm. Chondrocytes on the large micropan with a nanospacing of 50 nm exhibited more obvious actin stress fibers than the other three groups. Mainly due to the confinement of cell spreading, cells on neither large nor small RGD nanospacings exhibited discernible vinculin clusters. 7760

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subtracting the mean gray value of the hydrogel background (I0) from the mean gray value of a single chondrocyte (I), as presented in Figure 7, panel c. Under a given RGD nanospacing of 50 nm, which was less than the critical adhesion nanospacing (70 nm), the large cell spreading promoted chondrocytes toward a more dedifferentiated state for an increase of spreading area decreased collagen II expression (Figure 7b). This is consistent with our preceding study of chondrocyte dedifferentiation on micropatterns.68 More importantly, when the cell spreading size was kept nearly the same on the micropans with diameter of 25 μm, the integrated collagen II expression of chondrocytes on RGD arrays of nanospacing of 98 nm was significantly higher than that of RGD nanospacing of 50 nm, which indicates an accelerated loss of the normal phenotype of chondrocytes on small RGD nanospacing. This result suggests that RGD nanospacing may have direct impact on dedifferentiation of chondrocytes, regardless of cell spreading. No significant difference was found in comparison of the collagen II expression of chondrocytes between the groups of two nanospacings on the small micropans. It might arise from that the dedifferentiation process was suppressed to a great extent since the cell morphology was very similar to the normal chondrocyte on small micropans, and therefore the RGD nanospacing effect was not dominant in that case. In the case of large RGD nanospacing (98 nm), which was over the critical adhesion nanospacing (70 nm), no significant difference was found between the two micropans of diameters of 15 and 25 μm in Figure 7, panel b, indicative of the role of the large nanospacing in avoiding dedifferentiation of chondrocytes. These findings based on our micro/nanopatterning technique shed new light on the impact of nanoscale spatial arrangement of RGD ligands on cell dedifferentiation. Besides cell spreading sizes, cell shape has also been revealed to influence differentiation of MSCs70,71 and even dedifferentiation of chondrocytes. 68 Our micro/nanopatterning technique confined the cell shape (Figure 7a) and thus ruled out the possibility that RGD nanospacing influences cell dedifferentiation merely by changing shape of spread cells. By using the RGD micropatterns on PEG hydrogels, our group has set up the first semiquantitative relations between extents of cell−cell contact and extents of MSC differentiation during osteogenic/adipogenic inductions72 and interpreted the effect of cell density on cell differentiation by combination of cell spreading size and cell−cell contact.73 Very recently, a linear relation between coordination number of cells in contact and differentiation extents was reproduced in the chondrogenic induction of stem cells.74,75 In the present study, the density of adherent cells depended on RGD nanospacing (Figure 3c). Therefore, the cell density and thus cell−cell contact effect cannot be decoupled with the inherent RGD nanospacing effect merely using the nanopatterns. The micro/nanopattern enabled the statistics only for those single cells localized in micropans, and the appropriate micropan sizes designed by us resulted in high probability of single-cell occupation on micropans, with some representative cell micrographs shown in Figure 7, panel a. There was no interference of cell−cell contact at all for single cells. Therefore, the results from the micro/nanopatterns illustrate unambiguously that RGD nanospacing plays an essential role in dedifferentiation of chondrocytes regardless of cell spreading, cell shape, and cell−cell contact.

Figure 6. Morphological observations of visible micro/nanopatterns and single cell localization on RGD-nanoarrayed micropans for 24 h. (a) Left: Bright-field (BF) micrographs of designed masks with micropan arrays, and the diameters of micropans were 15 and 25 μm. Right: Phase-contrast images of visible micro/nanopatterns after being transferred to PEG hydrogels. (b) FE-SEM images of the Au nanoarrays inside the micropans and the barren areas outside the micropans on the PEG hydrogels. (c) Representative micrographs of single chondrocytes on nanoarrayed micropans with the indicated diameters and nanospacings.

To gain insight on the latent direct effect of RGD nanospacing on cell dedifferentiation, chondrocytes were cultured on the micro/nanopattern in the growth medium for 7 days. Then collagen II staining was carried out to evaluate the extent of dedifferentiation. Representative micrographs of single chondrocytes labeled for collagen II (red), F-actin (green), and nucleus (blue) are presented in Figure 7, panel a. The intensity of the excitation light and the exposure time of the CCD camera were kept constant among the groups of different RGD nanospacings and micropan sizes. A semiquantitative estimation of the collagen II expression was adopted to characterize the dedifferentiation extent of chondrocytes. We analyzed over 60 cells from three samples for each group of micro/nanodomain, and only single cells inside the micro/nanodomain were selected as statistical targets. By processing with the software ImageJ, the color images were turned into eight-bit greyscale images, and the integrated fluorescence intensity of collagen II was calculated by multiplying the effective mean gray value of a single cell by the cell area. The effective mean gray value was obtained by 7761

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Figure 7. Phenotypic maintenance of single chondrocytes on micro/nanopatterns. (a) Representative fluorescence micrographs of single chondrocytes on micro/nanopatterns after 7 days of culture. Chondrocytes were immunofluorescently stained to show the expression of collagen II (red) and distinguish single cell localization with counterstaining of F-actins (green) and nuclei (blue). The white dashed lines indicate the outlines of the RGD-nanoarrayed micropans on the PEG hydrogels. (b) Statistical results of relative integrated fluorescence intensity of collagen II. The statistical targets were single chondrocytes located inside the micro/nanodomains after 7 days of culture in the growth medium. The p-values from one-way ANOVA between each two groups are listed in Supporting Information Table S12. ∗, p < 0.05; ∗∗, p < 0.01. (c) Schematic illustration of the method to semiquantify the fluorescence intensity of collagen II of single chondrocytes by measuring an eight-bit greyscale image using ImageJ. I and I0 denote the mean gray value of collagen II and the nearby background with the same area, respectively.

Our previous study revealed that a large RGD nanospacing was favorable for chondrogenic differentiation of MSCs in twodimensional culture on nanopatterned surfaces.64 Anseth group found, using their unique technique of remote manipulation of degradation of three-dimensional (3D) PEG-based hydrogels, that photolytic removal of RGD peptides directed chondrogenesis of MSCs.76 Levenston group reported inhibition of in vitro chondrogenic differentiation of MSCs by RGD-modified 3D alginate hydrogels77 and agarose hydrogels.78 The present finding revealed that a small RGD nanospacing enhanced chondrocyte dedifferentiation into fibroblasts. Although such a conclusion about cell dedifferentiation cannot be deduced from the previous ones about cell differentiation, they share a common sense that the cues unbeneficial for cell adhesion, such as a large RGD nanospacing, are helpful for chondrogenesis. The dedifferentiated chondrocytes expanded in a monolayer culture can sometimes be reversed upon transfer into a 3D environment, and Rotter et al. observed that RGD modification of 3D agarose hydrogels inhibited cell redifferentiation.79 As the first report of RGD nanospacing effects on chondrocyte dedifferentiation, the present work found that a small RGD nanospacing below the critical value 70 nm favored not only specific cell adhesion, but also cell dedifferentiation. Hence, our conclusion is logically consistent with the previous reports of

both chondrogenic differentiation of MSCs and redifferentiation of dedifferentiated chondrocytes. RGD is an effective sequence in some ECM proteins.1 The effects of ECM proteins on dedifferentiation of chondrocytes were examined by Brodkin et al.80 They hypothesized that chondrocytes expanded on various protein monolayers might lead to different phenotypic responses. However, after bovine articular chondrocytes were cultured for up to 2 weeks on TCPs coated with fibronectin, collagen type I, or collagen type II, they concluded and honestly reported that “contrary to our hypothesis, the extracellular matrix protein substrates used in this study did not significantly alter the changes in chondrocyte morphology, gene expression, matrix formation, or cytoskeletal organization.”80 One of the improvements of the present study after 11 years comes from that RGD, the active ligand in ECM proteins, was examined in the form of nanoarrays. Another key issue to the material technique in our study is the nonfouling background of the RGD patterns. As correctly realized and kindly noted in their pioneering work, “non-coated dishes adsorb proteins from serum, including fibronectin and vitronectin, during cell culture, and therefore cannot be described as devoid of protein.”80 Our present work employed PEG hydrogels to resist protein adsorption and thus nonspecific cell adhesion, as demonstrated in Figure 4. Last but not least, we developed a micro/nanopatterning technique to 7762

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Nano Letters

Figure 8. Schematic illustration of the effects of nanoscale spatial arrangement of RGD peptides on dedifferentiation of chondrocytes. Cell-adhesive RGD nanoarrays were generated on nonfouling PEG hydrogels. A nanospacing of below 70 nm results in well-formed FA complexes and large cell spreading area. In contrast, a nanospacing of above 70 nm results in weak FAs and small cell spreading area, yet favors the phenotypic maintaining of chondrocytes. After excluding the effects of cell spreading area, etc. by utilizing micro/nanopattern technique, large RGD nanospacing still interestingly benefits maintenance of the chondrocyte phenotype. Thus, the RGD nanoscale spatial arrangement may afford an outside-in signal to influence dedifferentiation of chondrocytes.

The underlying mechanism of the effects of RGD nanospacing on dedifferentiation of chondrocyte is an open question. The integrin-mediated cytoskeleton organization may be one of the probable events. It has been known that the distributions of actin cytoskeletons of chondrocytes differ when observed under different culture conditions,81,82 and the cytoskeleton organization is suggested to be responsible for the dedifferentiation process.83 In the present experiments, the nanoscale distribution of RGD ligands can remarkably influence actin cytoskeletons through integrin-based FA. Therefore, the implicit mechanism of RGD nanospacing effect on the phenotypic maintaining of chondrocytes may be mediated by the cytoskeleton organization. Further investigations of the underlying mechanisms are necessary. In summary, the present study reveals a critical role of the nanoscale distribution of extracellular RGD ligands on the dedifferentiation extent of chondrocytes based on the material techniques of nanopatterning and especially micro/nanopatterning of RGD peptides on the persistently nonfouling PEG hydrogels. The RGD nanospacing of beyond 70 nm is not beneficial for cell adhesion. However, it favors the phenotypic maintaining of chondrocytes directly even under a given spreading area for well-controlled single cells on nanoarrayed micropans. Although the outside-in signal transduction of this nanocue is not understood at the moment, our findings indicate that the nanoscale spatial organization of RGD ligands affords a regulator of cell dedifferentiation and may bring some new perspectives for the design of advanced biomaterials in tissue engineering and regenerative medicine.

prepare RGD nanoarrays in well-defined micropans, as seen in Figures 2b, 6, and 7. Then, chondrocytes were excellently controlled on both individual cell level and molecular level. We think that their experimental conditions might essentially correspond to the case of small RGD nanospacing in our experiments, and thus their prior report does not contradict with our present study. The effects of RGD nanospacing on both adhesion and dedifferentiation of chondrocytes are schematically summarized in Figure 8. When chondrocytes are confronted with RGD nanospacing that is below the critical value of 70 nm, the precisely controlled organization of RGD peptides enables integrin clustering and formation of multiprotein FA complexes via bioconjugation between RGD ligands and their receptors integrins. The bioconjugation triggers linkage of actin filaments to the cytoplasmic domains of integrins through various anchoring proteins. Since cells can manage their morphology through organization of actin filaments, a small RGD nanospacing mediates more spreading of chondrocytes with clearer FAs and more distinct cytoskeletons. On the other hand, when the RGD nanospacing is above the critical value, chondrocytes exhibit a more round cell shape and less spreading area with vague vinculin clusters and indistinct stress fibers. Meanwhile, we found a tendency of lower proportion of chondrocyte dedifferentiation on large RGD nanospacings. It is very important that the large RGD nanospacing remains conducive to the maintenance of chondrocyte phenotype even after the effects of cell spreading, cell shape, and cell−cell contact are fixed by our micro/nanopatterning technique. 7763

DOI: 10.1021/acs.nanolett.5b04043 Nano Lett. 2015, 15, 7755−7765

Letter

Nano Letters

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b04043. AFM images of Au nanopattern on glass; optical micrographs of chondrocytes on TCP with collagen II stained; distribution diagrams of the cell area, circularity, and aspect ratio of chondrocytes on various nanopatterns; AFM images of a micropan on the PEG hydrogel; p-values in one-way ANOVA of the figures in the main manuscript (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 86 21 65643506. Fax: 86 21 65640293. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from NSF of China (Grant Nos. 51533002 and 51273046) and Science and Technology Developing Foundation of Shanghai (Grant No. 13XD1401000).

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ABBREVIATIONS ECM, extracellular matrix; RGD, arginine−glycine−aspartate; PEG, poly(ethylene glycol); FA, focal adhesion; PS-b-P2VP, poly(styrene-block-2-vinylpyridine); PEG-DA, poly(ethylene glycol)-diacrylate; D2959, 2-hydroxy-4′-(2-hydroxyethoxy)-2methylpropiophenone; TEM, transmission electron microscope; AFM, atomic force microscopy; FE-SEM, field-emission scanning electron microscopy; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; TCP, tissue culture plate; AR, aspect ratio; CCD, charge-coupled device; qRTPCR, quantitative real-time reverse transcription-polymerase chain reaction; MSC, mesenchymal stem cell

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DOI: 10.1021/acs.nanolett.5b04043 Nano Lett. 2015, 15, 7755−7765