Multiphoton Fabrication of Fibronectin- Functionalized Protein

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Multiphoton fabrication of fibronectin-functionalized protein micropatterns – Stiffness-induced maturation of cell-matrix adhesions in human mesenchymal stem cells Jiaoni Ma, Chuen Wai Li, Nan Huang, Xinna Wang, Minghui Tong, Alfonso H.W. Ngan, and Barbara P. Chan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07064 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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ACS Applied Materials & Interfaces

Multiphoton Fabrication of FibronectinFunctionalized Protein Micropatterns – StiffnessInduced Maturation of Cell-Matrix Adhesions in Human Mesenchymal Stem Cells Jiaoni Ma, ‡,† Chuenwai Li, ‡,† Nan Huang, ‡,† Xinna Wang, ‡,† Minghui Tong, ‡ Alfonso H.W. Ngan, § and Barbara P. Chan ‡,* ‡

Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region, China.

§

Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region, China.

†These authors contributed equally to this work. *Corresponding author: [email protected] KEYWORDS ： multiphoton biofabrication, fibronectin, protein micropattern, cell matrix adhesions, maturation, mesenchymal stem cells

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ABSTRACT ： Cell-matrix adhesions are important structures governing the interactions between cells and their microenvironment at the cell-matrix interface. Focal complex (FC) and focal adhesion (FA) have been substantially investigated in conventional planar culture system using fibroblast as in vitro model. However, the formation of more mature types of cell-matrix adhesion in human mesenchymal stem cells (hMSCs), including fibrillar adhesion (FBA) and 3D matrix adhesion (3DMA), have not been fully elucidated. Here, we try to provide some hints on which niche factor(s) dominate(s) the maturation of FBA and 3DMA by using multiphoton fabrication-based micropatterning technique. Firstly, the bovine serum albumin (BSA) made protein micropatterns were functionalized by incorporating various concentrations of fibronectin (FN) in fabrication solution. The amount of crosslinked FN is positively correlated with the initial concentration of FN in the reaction liquid, as verified by immunofluorescence staining. On the other hand, the anisotropic FN-functionalized micropatterns were fabricated by varying the length (i.e., in-plane stiffness) and height (i.e., bending stiffness) of micropatterns, respectively. Finally, hMSCs were cultured on these micropatterns for 2 h and 1 day to determine the formation of FBA and 3DMA, respectively, using immunofluorescence staining. Results demonstrated that FN-functionalized micropatterns with high anisotropy in x-y dimension benefit FBA maturation. Furthermore, niche factors such as higher bending and in-plane stiffness and the presence of abundant fibronectin have a positive effect on the maturation of FN-based cell-matrix adhesion. These findings could provide some new perspectives on designing platforms for further cell niche study and rationalizing scaffold design for tissue engineering.

INTRODUCTION In native tissues, cells reside in a highly complex microenvironment or niche, in which they are subjected to various cues including chemical and mechanical signals and interact with


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neighboring cells and surrounding matrix. Therefore, traditional planar cell culture system is inadequate to mimic the physiologically relevant microenvironment for cell behavior investigation.1,2 Micropatterning, a micro-engineering method, is capable of fabricating precise confinement of cues existing in natural condition, and therefore it is superb to study the cell response to specific microenvironment features.2,3 Commonly used micropatterning techniques include photolithography, microcontact printing, pen spotting and inject printing.4-6 Multiphoton biofabrication is an alternative method to realize the micropattern fabrication in the presence of two or more photons and photosensitizer to covalently crosslink protein or monomer by reaction with singlet oxygen or via hydrogen atom abstraction.7-9 This technique permits real 3D patterning within the sample with high spatial resolution; moreover, it is suitable for processing bioactive molecules because of the mild conditions used during the fabrication.9 We have successfully fabricated complex protein microstructures utilizing femto-second laser and investigated the voxels, porosity, stiffness features and their influence on cell activity.10 Based on this concept, we have further developed the methodology to measure the single cell traction force by varying the mechanical properties of protein micropillars.11 Although BSA-based micropatterns exhibited a good cytocompatibility for fibroblast adhesion without extra extracellular matrix (ECM) protein coating.10 cells interact with multiple factors including ECM in vivo, making it highly warranted to functionalize these BSA micropatterns with ECM proteins such as fibronectin. Fibronectin (FN), an omnipresent glycoprotein in ECM, plays an essential role for tissue repair.12-14 In a complex 3D matrix, the FN assembly is crucial to physiological tissue repair not only as a structural scaffold, but also as a regulator in cellular function during wound healing.15


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Hence, it is necessary to integrate FN in micropattern fabrication to maximally mimic the native ECM niche, which would provide a better in vitro model to study matrix niche. Cell-matrix adhesion, a complex comprised of integrin and various membrane-related structures, mediates a bi-directional communication between cells and their microenvironment at the cell-matrix interface.16 It regulates lots of physiological processes such as cell proliferation, differentiation, migration, signaling transduction and matrix remodeling.17 Cell-matrix adhesion can be categorized into four types with increasing level of maturity, that is, focal complex (FC), focal adhesion (FA), fibrillar adhesion (FBA) and 3D-matrix adhesion (3DMA).18 These different types of cell adhesions are changed dynamically depending on the environment where the cell exists. Among others, the evolution towards mature FN-based adhesions upon FN fibrillogenesis, including FBA, as characterized by the molecular components of FN, integrin α5 and tensin, 17,18 and 3DMA, as characterized by the molecular components of FN, integrin α5 and paxillin. 17,18 It is suggested that several factors, including external force, substrate stiffness and topography, are also important for the maturation of FN-based adhesions.19 Mesenchymal stem cells (MSCs) have been substantially used in tissue engineering and regenerative medicine since their multipotency to differentiate, easy isolation and amplification from bone marrow as well as immunological toleration as an allogeneic transplant.20 Similarly, cell-matrix adhesion of MSCs is a vital process to determine cell fate. Some researchers have engineered artificial substrates with different stiffness and topography using micropatterning techniques to decipher the formation and maturation of cell-matrix adhesion upon seeding MSCs on substrate in vitro. For example, culturing human mesenchymal stem cells (hMSCs) on poly (dimethylsiloxane) (PDMS) and tissue-culture polystyrene (TCPS) dishes with nanopatterns showed that stiffness and topography are two important determinants of cell mechanical


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properties, but topography plays a major role in determining the cytoskeleton organization and focal adhesions.21 Another study showed that the formation and maturation of FAs of murine MSCs is highly dependent on the height of grid micropatterns but not on the rigidity of substrates.22 However, these studies mainly focus on the formation of FAs, little evidence is available on the relationship between substrate mechanical properties and maturation of FNbased matrix adhesions including FBA or 3DMA. Technologically, we hypothesize that the BSA-based protein micropatterns could be functionalized with FN in a dose-dependent manner. In the meantime, the stiffness of these structures could be controlled by varying the length and height of the micropatterns using the two-photon fabrication platform. Scientifically, we hypothesize that anisotropic FNfunctionalized micropatterns could induce the maturation of FN-based cell matrix adhesions. Defining the matrix and mechanical niche factors inducing the formation and maturation of cellmatrix adhesions is critical in designing and rationalizing biomimetic scaffold for future tissue engineering applications. MATERIALS AND METHODS Fabrication System The two-photon confocal laser scanning microscopy (CLSM) system equipped with a modelocked Ti:Sapphire femtosecond near infrared (NIR) laser (Coherent Inc., Santa Clara, CA) was used for protein micropatterning as previously described.10,11 Figure 1 shows the schematic diagram of the fabrication system. In brief, a glass-bottomed 35 mm confocal culture dish (P25G-1.5-10-C, MatTek Corp., Ashland, MA) with a silicon-insert isolator (ibidi, Martinsried, Germany) adhered to the bottom center was mounted onto the stage of the inverted Carl Zeiss 710 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). After loading the


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fabrication solution into the isolator, the liquid-solid interface between the solution and the glass was scanned with an Argon laser at 488 nm excitation wavelength in a reflection mode and then marked as the zero-position in the z-direction before fabrication. Afterwards, the laser was adjusted to the wavelength at 800 nm and a 40×/1.3 N.A. oil-immersion objective lens (PlanApochromat) was used for the fabrication. The actual laser power was measured before each fabrication using a power meter (Coherent). The default software (Zen 2010, Carl Zeiss) was used to design the region of interest (ROI) and control the fabrication process.


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Figure 1. Schematic diagram of fabrication system. A: Solution loading system (top view); B: Whole fabrication system (side view). Fabrication and Characterization of FN-Functionalized Micropatterns As FN is one of ECM components and plays an important role on various cellular functions, we functionalized BSA microstructures with different concentrations of FN by incorporating soluble FN into the fabrication solution and then crosslinked the mixture of BSA, FN and Rose bengal (RB), a xanthene compound used as photoactivator to produce singlet oxygen,7 by a twophoton laser. Specifically, a mixture of BSA (100 mg/ml; Sigma-Aldrich Corp., St Louis, MO), RB (0.1% w/v; Sigma-Aldrich) and FN (0, 50, 150 and 350 µg/ml; 356008, Corning) was loaded into the wells of isolator as described above. Square micropatterns with dimension (length × width × height) of 101 × 101 × 3 µm were fabricated according to the pre-designed ROI. The fabrication parameters used were as follows: (1) z-stack interval was kept at 0.5 µm and starting position was set to 1 µm below zero-position to make a stable adherence of structure obtained to the glass bottom; (2) scanning power, speed and scan cycle were set to 60 mW, 1.27 µs and 1, respectively; (3) scanning zoom was 2.1 to get the scanning area of 101µm × 101 µm; (4) frame size was kept at 512 × 512 to get the pixel size of 0.2 µm × 0.2 µm. Meanwhile, the structures with the same dimension but fabricated with RB and BSA only were immersed in soluble FN, which served as a control group (FN-adsorbed group) to exclude the fluorescence signal from the non-specific binding of FN to BSA structure surface. After fabrication, the structures were washed carefully with phosphate-buffered saline (PBS) three times (5 min/time) and then sterilized by soaking in PBS containing 4% (v/v) Antibiotic-Antimycotic (Gibco, Grand Island, NY) overnight at 4° C. Afterwards, these ready-to-use structures were incubated with rabbit polyclonal anti-FN primary antibody (1:300, sc-9068, Santa Cruz Biotecnology, Santa Cruz, CA)


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at 4° C overnight. And then they were washed with PBS three times (5 min/time) followed by incubation with Alexa Fluor 488 goat anti-rabbit (1:400, A11034, Invitrogen，Carlsbad, CA) at room temperature (RT) in the dark for 1 hour. After washing off the secondary antibody, crosslinked FN was characterized by immunofluorescence, which was visualized by z-stack images with interval of 0.5 µm using the Zeiss LSM 710 confocal system with a 40x/1.3 N.A. objective lens and analyzed with the 3D image reconstruction software Imaris (Bitplane, Zurich, Switzerland). Stiffness Control of FN-Functionalized Protein Micropatterns On the other hand, micropatterned arrays with multiple shapes, in-plane dimensions and heights were fabricated to obtain a series of FN-functionalized microstructures with different stiffness. According to our previous work11, the bending stiffness of the protein microstructures can be calculated by the following formula:

s = 3EI / L3 (1) where s represents the stiffness; E is the reduced elastic modulus measured by atomic force microscopy (AFM) as described before11 and it is around 30 kPa in this study; L is the height of the structures; I is the moment of inertia. If the height and the reduced modulus of the structures stay constant, then s is determined by I. For a rectangular structure:

I a = a 3b /12 I b = ab3 /12 in which a and b represent the length and width of rectangle, respectively. The ratio of stiffness (Sa/Sb) equals (a/b)2, which represents the stiffness in the x-y axis (in-plane stiffness). So firstly we fabricated anisotropic rectangular FN-functionalized micropatterned arrays with constant height (5 µm) and three different in-plane dimensions (length × width) of 1 × 1 µm, 3 × 1 µm and


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10 × 1 µm to realize stiffness control. The corresponding in-plane stiffness can be summarized in Table 1. Theoretically, stiffness control can be alternatively achieved by changing the height of microstructures and keeping I constant. Herein, we fabricated rectangular micropatterned arrays with fixed in-plane dimension (length × width = 1 × 1 µm) but with different heights of 2 µm and 5 µm, respectively (Table 2). After fabrication, the structures were washed gently with PBS for three times (5 min/time) and fluorescence images were captured directly by excitation wavelength at 543 nm due to the inherent fluorescence of RB. Afterwards, these structures were either used for cell cultures in the subsequent experiments or fixed with 2.5% glutaraldehyde (Sigma Aldrich) for 20 min for structural evaluation by scanning electron microscopy (SEM). Followed by serial dehydration with ethanol (25, 50, 70, 80 and 90%) for once and absolute ethanol for twice, these structures underwent further dehydration by critical point drying and sputter coating with gold for 100 s before observation under SEM (Hitachi S-4800 FEG SEM, Japan). Table 1. In-plane stiffness of the rectangular prism micropillars with different in-plane dimensions Micropattern in-plane dimensiona (length × width ) (µm)

Stiffness in length (Sa, nN/µm)

Stiffness in width (Sb, nN/µm)

In-plane stiffness (Sa/ Sb)

1×1

0.06

0.06

1:1

3×1

1.62

0.18

9:1

10 × 1

60

0.6

100:1

a: Constant height: 5 µm. Table 2. Bending stiffness of the rectangular prism micropillars with different heights

Micropattern heighta (µm)

Stiffness in length (Sa, nN/µm)

Stiffness in width (Sb, nN/µm)

In-plane stiffness (Sa/ Sb)


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2

0.06

0.06

1:1

5

0.06

0.06

1:1

a: Constant in-plane dimension: length × width = 1 × 1 µm. Cell Culture hMSCs derived from human bone marrow were obtained from the Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine (Scott & White Hospital, Bryan, TX, USA) and cultured in low-glucose Dulbecco’s Modified Eagle Medium (DMEM-LG, Gibco) containing 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin (Gibco), 100 µg/ml streptomycin and 2 mM GlutaMax (Gibco). The cells were maintained in humidified conditions with 5% CO2 at 37 ° C and the medium was changed every 3 days. Cells at P5 or P6 were detached from culture flask by 0.05% trypsin-EDTA (Gibco) when the cell confluence reached to around 80% and then re-suspended in fresh culture medium at a density of 1 × 105 cells/ml for cell adhesion study by seeding them on the pre-fabricated protein micropatterns. Determination of the Cell-matrix Adhesion Formation of hMSCs Cultured on Micropatterns Initially, FBA maturation was investigated in terms of immunofluorescence staining of paxillin, tensin and integrin α5 upon culturing hMSCs on FN-functionalized micropatterns with different stiffness by changing the length (1, 3 and 10 µm) in vitro for 2 h. Then 3DMA maturation was visualized by immunofluorescence staining of paxillin, FN and integrin α5 upon culturing hMSCs on micropatterns incorporated with different concentrations of FN (0 µg/ml, 150 µg/ml and 350 µg/ml), or on micropatterns with different stiffness either varied by the length or the height in vitro for 1 day. Briefly, when micropatterns with different properties (e.g., incorporation of different concentrations of FN or different stiffness) were ready to use, 10 µl of hMSCs (1000 cells) from above obtained cell suspension were seeded on micropatterned arrays.


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Cell culture was terminated by fixing the cells with 4% paraformaldehyde at RT in the dark for 10 min followed by PBS washing three times (5 min/time). Samples were then permeabilized in 0.5% (v/v) Tween 20-containing PBS (Sigma-Aldrich) at RT for another 10 min and washed again with PBS three times (5 min/time) to completely remove the Tween 20 residual. Subsequently, the fixed cells and protein structures were blocked in 3% BSA for 30 minutes at RT and then incubated with rabbit polyclonal anti-FN primary antibody (1:300, sc-9068, Santa Cruz), sheep polyclonal anti-paxillin primary antibody (1:40, AF4259, R&D Systems, Minneapolis, MN) and mouse monoclonal anti-integrin α5 primary antibody (1:300, ab23589, Abcam, Cambridge, MA) at 4° C overnight. After removal of primary antibodies, the samples were incubated with secondary antibodies including Alexa Fluor 488 goat anti-rabbit (1:400, A11034, Invitrogen), Alexa Fluor 350 goat anti-mouse (1:400, A21049, Invitrogen) and Alexa Fluor 647 donkey anti-sheep (1:400, A21448, Invitrogen) at RT in the dark for 1 hour. Finally, the samples were mounted with Fluoro-gel II mounting medium containing DAPI (EMS, Hatfield, PA). All the samples were observed under Zeiss LSM 710 confocal microscope with a 63x/1.4 N.A. objective lens (Plan-Apochromat). Alexa Fluor 488 fluorescence was detected using 488 nm excitation and 493-555 nm detection; Alexa Fluor 647 fluorescence was captured using 633 nm excitation and 638 or 755 nm detection; Alexa Fluor 350 and DAPI fluorescence was obtained via 700 nm multi-photon excitation and 437 or 479 nm detection. Image Analysis of Colocalization of Cell Adhesion Molecules Colocalization of cell adhesion molecules was analyzed using free software ImageJ (National Institutes of Health, Bethesda, Maryland; http://rsbweb.nih.gov/ij/) and detailed image analysis process is depicted in Figure 2. Briefly, Z-stack images taken in corresponding channel were firstly stacked into a single slice with the ‘maximum intensity’ function in ‘Z-project’.


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Afterwards, they were converted into binary images using ‘threshold function’ under the default setting to define the shape and size of structures to be analyzed. Next, the obtained binary images in different channels were merged together such that only fluorescence signals positive in all three detection channels were retained. Finally, colocalized immunopositive regions larger than 0.5 µm2 (exclude the noise signals) were selected for aspect ratio calculation by ‘analyze particles’ function. Statistical analysis was performed after data analysis on colocalization of different adhesion proteins.

Figure 2. Image analysis process of colocalization of cell adhesion molecules after hMSCs culturing on FN-functionalized micropillars with different stiffness by Image J. Statistical Analysis All quantitative data were presented in box plots. Linear regression analysis was conducted to reveal the association between the input concentration of FN during fabrication of the


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micropatterns and the intensity of the immunofluorescence staining of FN after the crosslinking, and the intensity of the colocalized immunofluorescence of 3DMA marker molecules. Welch’s one-way ANOVA with Games-Howell post-hoc tests were conducted to analyze the aspect ratio of FBA among different in-plane stiffness groups. SPSS 22.0 was used to execute all statistical analyses and the significant level was set at 0.05. RESULTS Functionalizing Protein Micropatterns with FN in a Controllable Manner Figure 3A shows the surface of FN-crosslinked BSA micropatterns in terms of immunofluorescence staining of FN. The immunofluorescence signal increases as the input concentration of FN in the fabrication solution increases (Figure 3A (a-d)). Fluorescence intensity of FN in crosslinked group dramatically and linearly increases in a FN dose-dependent manner, in contrast, fluorescence intensity of FN is extremely low in FN-adsorbed group though it shows a shallow trend at increasing FN concentration (Figure 3B), illustrating the FN dosedependency is from the photo-crosslinking process rather than from simple adsorption. Moreover, a strong positive linear relationship between FN immunofluorescence intensity and concentration of FN is detected (linear regression coefficient (R linear) = 0.976 (p < 0.001)) with an excellent fitting (coefficient of determination (R2 linear) = 0.953 (p < 0.001)) (Figure 3C).


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Figure 3. Immunofluorescence staining of two-photon based FN-BSA structure. A: Fluorescence images of surface of FN-BSA structures with different concentrations of FN incorporated in fabrication solution. a: 0 µg/ml; b: 50 µg/ml; c: 150 µg/ml; d: 350 µg/ml. Scale bar: 5 µm. B: Box plot of fluorescence intensity against FN concentration in FN-crosslinked group and FNadsorbed group. C: Linear regression analysis on relationship between immunofluorescence intensity and FN concentration (R linear = 0.976 (p < 0.001), R2 linear = 0.953 (p < 0.001), n=10). Characterization of the FN-Functionalized Protein Microstructures We previously reported that the stiffness of BSA micropillars could be controlled by varying their heights (L) and using a constant reduced elastic modulus (E) and the moment of inertia (I).11 Here, we kept E and L of the FN-functionalized protein microstructures constant but changed I by varying the length of the microstructures ranging from 1µm to 10 µm (Figure 4).


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Figure 4A-F display the orthogonal view (A-C) and 3D view (D-F) of fluorescence images of the fabricated protein microstructures with different length of 1 µm, 5 µm and 10 µm. SEM images show the morphology of protein micropatterned arrays at lower magnification (×4000) (Figure 4G-I) and close-up of each single microstructure as illustrated by insert in Figure 4G-I with higher magnification (×22000 for Figure 4G, ×20000 for Figure 4H and ×12000 for Figure 4I). Orthogonal view of fluorescence images, which were taken immediately after fabrication, present regular shape of micropatterns with different in-plane dimension. In contrast, micropatterns exhibit obvious shrinkage in SEM images, especially in Figure 4I, which might be caused by processing of SEM samples, such as serial dehydration or critical point drying.


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Figure 4. Characterization of FN-functionalized protein structures with different dimensions by CLSM (A-F) and SEM. (G-I). A-C: Orthogonal view of fluorescence images of fabricated structures with length of 1 µm (A), 3 µm (B) and 10 µm (C). Scale bar: 1 µm in (A), 3 µm in (B) and 10 µm in (C). D-F: 3D view of fluorescence images of fabricated structures with length of 1 µm (D), 3 µm (E) and 10 µm (F). Scale bar: 10 µm. G-I: Side view of micropatterned arrays with length of 1 µm (G), 3 µm (H) and 10 µm (I) under SEM. Scale bar: 10 µm. Inserts show the


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close-up of single pattern in each group with higher magnification. Scale bar: 2 µm in (G, H) and 4 µm in (I). Higher Aspect Ratio (AR) of FBA on FN-Functionalized Micropatterns with Higher InPlane Stiffness Fibrillar adhesion is developed by focal adhesion and marked by clusters of colocalizing integrin α5, FN and tensin.23 We are interested in studying the relationship between fibrillar adhesion formation and stiffness of micropatterns. Figure 5A shows that triple colocalization of tensin (green), FN (blue) and integrin α5 (red) were present on all micropatterns after 2 h culture, indicating that FN-functionalized micropatterns were able to induce FBA maturation regardless of the in-plane stiffness. However, as shown in Figure 5B (a), the ARs of adhesion clusters formed on micropatterns with highest in-plane stiffness (Sa/Sb=100:1) span from < 5 to > 20, in comparison, the ARs of adhesion clusters are mainly distributed below 10 when hMSCs were cultured on micropatterns with lower in-plane stiffness. Moreover, further calculated ARs of colocalized clusters demonstrate that micropatterns with highest in-plane stiffness favor the formation of more elongated and hence mature FBA (median: 9.444). In contrast, hMSCs prefer to form shorter FBA clusters on micropatterns with middle (median: 2.287) and lowest (median: 1.915) in-plane stiffness (Figure 5B (b)) (p < 0.001). Taken together, the data indicate that hMSCs prefer to form more fabrillar adhesions with higher AR on FN-functionalized micropatterns with higher in-plane stiffness.


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Figure 5. Colocalization of immunofluorescence of FBA markers FN, tensin and integrin α5 after culturing hMSCs on FN-crosslinked micropatterns with different in-plane stiffness for 2 h


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and the dimension of FBA as measured by the aspect ratio of the colocalized clusters. A: Immunofluorescence staining of tensin (green), FN (blue), integrin α5 (red) after culturing hMSCs on FN-functionalized micropatterns with different in-plane stiffness (top row: Sa/Sb = 1:1, middle row: Sa/Sb = 9:1, bottom row: Sa/Sb = 100:1). Colocalizations of FN, paxillin and integrin α5 in selected regions were magnified in close-up images. Scale bar: 10 µm. B: Bar chart (a) showing the distribution of aspect ratio of fabrillar adhesions formed on FN-functionalized micropatterns with different in-plane stiffness. Box plot (b) illustrating the differences of aspect ratios of FBA clusters among groups with different in-plane stiffness by Welch’s one-way ANOVA with Games-Howell post-hoc tests. * p < 0.001. Exploration on Niche Factors Inducing Adhesion Maturation (1) Dose-dependent crosslinking of FN on micropatterns In order to study whether ECM-functionalized micropatterns with different dosages of exogenous ECM component (e.g., FN in the present study) could possibly induce 3DMA formation, a unique type of mature cell matrix adhesion present in 3D, we further evaluated the formation of 3DMA on micropatterns with constant dimension (length × width × height: 10 × 1 × 5 µm) but with different concentrations of photocrosslinked FN (0 µg/ml, 150 µg/ml and 350 µg/ml) undergone photo-crosslinking. Immunofluorescence signal of integrin α5 (Figure 6A (c, h, m)) is actually as high as that from the photosensitizer RB. Therefore, it cannot be discerned and analyzed. But the size of colocalization of FN and paxillin is dependent on the exogenous FN present (Figure 6B), that is, more FN incorporated can result in more elongated clusters of colocalization of FN and paxillin. Box plot in Figure 6C shows the positive relationship between the correlation coefficient of the colocalized FN and paxillin and the dose of the exogenous FN input, suggesting that colocalization of FN and paxillin is more positively associated with


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micropatterns with higher concentration of exogenous FN crosslinked. Hence, we speculate that hMSCs would prefer to form FN-based cell-matrix adhesion maturation on micropatterns with more exogenous ECM present.


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Figure 6. The expression of other FN-based mature matrix adhesions, FN, paxillin and integrin α5 and their colocalization after culturing hMSCs on micropatterns with constant stiffness but different concentrations of FN (0 µg/ml, 150 µg/ml and 350 µg/ml) crosslinked for 1 day. A: Immunofluorescence staining of FN (green), paxillin (blue) and integrin α5 (red) after culturing hMSCs on protein micropatterns incorporated with FN at concentration of 0 µg/ml (a-e), 150 µg/ml (f-j) and 350 µg/ml (k-o). Arrows denote the colocalization of FN, paxillin and integrin α5 in (e), (j) and (o) and magnified side view of denoted regions are shown in (e1), (j1) and (o1). Scale bar: 10 µm. B: Distribution of aspect ratio of colocalized FN and paxillin formed on micropatterns with different dosages of exogenous FN input. C: Box plot of the correlation coefficient between the colocalization of FN and the paxillin and concentrations of exogenous FN in fabrication solution. (2) Stiffness of micropatterns Stiffness is an important mechanical property of substrate in matrix adhesion formation.20 As described in ‘Materials and Methods’, we could fabricate micropatterns with different stiffness either in plane or in Z-axis. Therefore, we fabricated series of micropatterns with different stiffness through changing the length or height of the structures and meanwhile, functionalized these structures with FN at 350 µg/ml, which could induce more colocalized FN and paxillin as investigated earlier to explore the effect of substrate stiffness on the formation of mature FNbased matrix adhesions. Figure 7A shows the expression markers of mature FN-based matrix adhesions including FN, paxillin and integrin α5 after culturing hMSCs on FN-functionalized micropatterns with different in-plane stiffness (Sa/Sb). With an increase of Sa/Sb from 1:1 to 100:1, the expression of FN (Figure 7A (a, f, k)) and paxillin (Figure 7A (b, g, l)) spreading along the length of micropatterns increases. Similarly, colocalization of these molecules is


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incremental with the increase of Sa/Sb (Figure 7A (e, e1, j, j1, o, o1)). On the other hand, we changed the height of structures to explore the influence of bending stiffness on FN-based adhesion maturation. As shown in Figure 7B, more abundant FN (Figure 7B (a, f)) and paxillin (Figure 7B (b, g)) were found on micropillars with higher stiffness at a height of 2 µm than those with lower stiffness at a height of 5 µm, illustrating that higher bending stiffness of micropatterns (i.e., lower height of micropatterns) could facilitate the maturation of FN-based cell-matrix adhesion. Furthermore, SEM images in Figure 8 show that hMSCs pull on and bend the micropatterned arrays with lowest in-plane stiffness (Sa/Sb =1:1, Figure 8 (b, e)) and moderate one (Sa/Sb = 9:1, Figure 8 (c, f)), whereas they spread along the length of micropatterned arrays with highest in-plane stiffness (Sa/Sb = 100:1, Figure 8 (d, g)).


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Figure 7. Immunofluorescence staining of FN, paxillin and integrin α5 and their colocalization after culturing hMSCs on micropatterns with FN (350 µg/ml) crosslinked under different stiffness for 1 day. A: Immunofluorescence staining of FN (green), paxillin (blue) and integrin α5 (red) after culturing hMSCs on micropatterns with different in-plane stiffness. Top row: Sa/Sb


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= 1:1, middle row: Sa/Sb = 9:1, bottom row: Sa/Sb = 100:1 (same images as Figure. 6A (k, l, m, n, o) here just to illustrate the difference induced by variation of in-plane stiffness). Arrows indicate the colocalization of FN, paxillin and integrin α5 and magnified side view of colocalization is shown in e1, j1 and o1, respectively. Scale bar: 10 µm. B: Immunofluorescence staining of FN (green), paxillin (blue) and integrin α5 (red) after culturing hMSCs on micropatterns with different bending stiffness by varying the height. Height in top row: 2 µm, height in bottom row: 5 µm (same images as Figure. 7A (a, b, c, d, e) here just to illustrate the difference induced by variation of bending stiffness). Arrows indicate the colocalization of FN, paxillin and integrin α5 and magnified side view of colocalization is shown in e1and j1, respectively. Scale bar: 10 µm.


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Figure 8: SEM images of hMSCs cultured on micropatterned arrays with different Sa/Sb. (a): Cell cultured on 2D glass. (b,e): Cell cultured on micropatterns with Sa/Sb of 1:1. (c,f): Cell cultured on micropatterns with Sa/Sb of 9:1. (d,g): Cell cultured on micropatterns with Sa/Sb of 100:1. Scale bar: 10 µm. DISCUSSION This work realizes the functionalization of inert protein micropatterns with ECM protein-FN in a controllable manner, based on multiphoton fabrication technology. The stiffness of these functionalized micropatterns is controlled by varying the dimension of structures, including length and height. In the current study, we found that the maturation of fibrillar adhesions is positively affected by increased in-plane stiffness of FN-functionalized micropatterns, which is demonstrated by the increase of AR of FBA molecular markers. Furthermore, higher concentration of FN incorporated would induce the formation of more 3DMA-like structures. On the other hand, 3DMA-like structures are found on the lower and longer micropatterns, suggesting that a higher stiffness favors the formation of mature adhesions such as 3DMA. It is well known that ECM is one of indispensible constituents of cell niche since it takes part in cell adhesion, proliferation, differentiation and migration.24,25 FN, a protein assembling into a fabrillar network and interacting with cell surface receptors directly, is a common and abundant ECM component.26 In addition, it has been reported that FN-rich environment is benefit to the fibroblasts to form FBA.27 Realization of functioning substrates is important in cell niche study because this manipulation provides a system that is better mimicking a native environment. A myriad of technologies are focusing on this, such as photolithography, microcontact printing, etc., which have been introduced previously. However, our fabrication platform outweighs other technologies in terms of convenience, non-toxicity and retaining functional molecules’


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bioactivities. We successfully immobilized different dosages of FN to BSA micropatterns based on two-photon photochemical crosslinking technique in this study, and most importantly, the epitope of crosslinked FN is uninterrupted and hMSCs can attach well on these FNfunctionalized protein structures, indicating the bioactivity of crosslinked FN can be retained. Further work on incorporation of other type of ECM proteins such as laminin, and soluble bioactive molecules such as growth factors is warranted. Cell-matrix adhesion formation starts from the initial encounter of a cell with substrates around.28 Hence, the properties of substrates, such as the type of ECM proteins present, stiffness, topography and roughness affect the formation and maturation of cell-matrix adhesions. Bulk of studies reported the nano-/micro-topography plays a crucial role in FA formation and/or maturation.20,21,29-34 Fibrillar adhesions, a more matured type of cell-matrix adhesion than FA, allow cells to form stable anchor to the substrate and might be a pivotal mechanism in regulating cell migration.35 In addition, FBA can be considered as an early event of cell-adhesion maturation since it can facilitate fibronectin fibrillogenesis by applying tension to fibronectin and the ongoing fibrillogenesis induces the accumulation of a thick matrix, which provides cell a new 3D environment and leads to 3DMA maturation.17 Here, we engineered FN-functionalized BSA micropatterns with different in-plane stiffness, which is actually realized by changing the length of micropattens, to investigate its effect on FBA maturation in hMSCs after a short culture period (2 h), and therefore to provide a useful platform to elucidate some physiological and pathological processes related to matured cell-matrix adhesion formation. The results demonstrate that FBA size is positively correlated with the length and hence the in-plane stiffness of micropatterns, a similar finding to previous studies on relationship between the lateral dimensions of micropatterned substrate and the FA formation.20 This might be explained by inferring that FBA


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is developed from FA, and therefore, the factors affecting FA formation could also regulate FBA maturation. Chang Ho Seo, et al.21 reported that the underlying mechanism of micropatterntopography induced FA formation is associated with RhoA-ROCK pathway, however, it is unclear whether this signaling pathway is involved in FBA maturation as well. In addition, micropatterns tend to show an anisotropic property when the in-plane stiffness is increased, which could induce cell alignment along the direction of anisotropy.36-38 Interestingly, our results show that FBA maturation increases along the direction of anisotropy of micropatterns, which triggers the speculation of the relationship between FBA maturation and cell alignment, and therefore provides a new perspective to investigate the mechanism of cell alignment. The term ‘3D matrix adhesion’ (3DMA) was firstly proposed by Cukierman, et al. in 2001, which describes a distinct type of cell-matrix adhesion existing in vivo and on cell-derived matrix on culture dish18 3DMA is able to enhance many cellular functional activities, such as increasing cell attachment, enhancing migration rate as well as accelerating to get cells in in vivo-like morphology, compared to 2D adhesions (FA and FBA). Cukierman, et al. also pointed out that factors to induce 3DMA maturation include three-dimensionality, FN, other matrix component(s) and pliability.18 Therefore, these requirements should be taken into consideration when designing scaffolds capable of providing an environment that mimics the 3D cell niche. Our previous study has shown that cells exhibit more 3D-like morphology when they were cultured on BSA micropillars than those cultured on 2D system.10 Moreover, these micropatterns can be further functionalized with ECM proteins and their mechanical properties, such as stiffness, can be readily controlled by varying the dimension of micropatterns. We attempted to find out the niche factors in the protein micropatterns affecting the formation of the mature FN-based matrix adhesions, specifically 3DMA, by detecting the co-expression of FN, paxillin and integrin α5


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after culturing hMSCs on micropatterns for 1 day (Our separate in-house study proved that 3DMA could be observed within 1 day if the culture environment is appropriate). However, the autofluorescence of the photosensitizer used in the 3DMA experiment affected the successful detection of triple colocalization of the three 3DMA markers. Nevertheless, colocalization of FN with paxillin and the elongated morphology of these adhesions can still lead to a conservative conclusion on the formation of mature FN-based matrix adhesions on micropatterns with increasing stiffness or increasing concentrations of FN input. It is obvious that more FN-based cell-matrix adhesion in hMSCs will form on longer but lower micropatterns with more FN incorporated. This observation might provide a useful perspective on designing micropatterned substrate for cell-matrix adhesion studies and rationalizing biomimetic scaffolds for tissue engineering applications. CONCLUSIONS ECM-functionalized protein micropatterned arrays with different stiffness can be successfully engineered using two-photon-induced photochemical crosslinking technology. This versatile micropatterns could be used to elucidate the role of stiffness on FBA maturation in hMSCs. Our findings show that micropatterns with high in-plane stiffness can induce formation of mature FN-based matrix adhesion, specifically FBA. Furthermore, micropatterns with niche factors including increasing bending and in-plane stiffness and increasing FN concentration are favorable to the maturation of FN-based cell-matrix adhesions. This work provides some new perspectives on designing platform for biomimetic cell niche study and rationalizing the scaffold design for tissue engineering.

AUTHOR INFORMATION


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Corresponding Author * E-mail: [email protected] Author Contributions J.N.M. collected data, participated in the study design and wrote the manuscript, C.W.L. collected and analyzed data and participated in the study design, N.H. analyzed the data and wrote the manuscript, X.N.W. collected and analyzed data, and wrote the manuscript, M.H.T. analyzed data and participated in the study design, A.H.W.N. participated in the study design, analyzed the data and edited the manuscript, B.P.C. conceived the study, participated in the study design, edited and approved the manuscript and provided funding. ACKNOWLEDGMENT This work was supported by grants from NSFC/RGC Joint Research Scheme (N_HKU713/14 and 51461165302). REFERENCES (1) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Cells on Chips. Nature 2006, 442, 403-411. (2) Théry, M. Micropatterning as a Tool to Decipher Cell Morphogenesis and Functions. J. Cell Sci. 2010, 123, 4201-4213. (3) Ito, Y. Surface Micropatterning to Regulate Cell Functions. Biomaterials 1999, 20, 23332342.


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(35) Esteban-Barragán, M. A.; Ávila, P.; Álvarez-Tejado, M.; Gutiérrez, M. D.; García-Pardo, Á.; Sánchez-Madrid, F.; Landázuri, M. O. Role of the Von Hippel-Lindau Tumor Suppressor Gene in the Formation of β1-Integrin Fibrillar Adhesions. Cancer Res. 2002, 62, 2929-2936. (36) Caliari, S. R.; Harley, B. A. C. The Effect of Anisotropic Collagen-Gag Scaffolds and Growth Factor Supplementation on Tendon Cell Recruitment, Alignment, and Metabolic Activity. Biomaterials 2011, 32, 5330-5340. (37) Teixeira, A. I.; Abrams, G. A.; Bertics, P. J.; Murphy, C. J.; Nealey, P. F. Epithelial Contact Guidance on Well-Defined Micro-and Nanostructured Substrates. J. Cell Sci. 2003, 116, 1881-1892. (38) Tzvetkova-Chevolleau, T.; Stéphanou, A.; Fuard, D.; Ohayon, J.; Schiavone, P.; Tracqui, P. The Motility of Normal and Cancer Cells in Response to the Combined Influence of the Substrate Rigidity and Anisotropic Microstructure. Biomaterials 2008, 29, 1541-1551.


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Multiphoton Fabrication of Fibronectin- Functionalized Protein

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