Topologically Controlled Cell Differentiation Based on Vapor

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Topologically Controlled Cell Differentiation Based on Vapor-Deposited Polymer Coatings Ya-Ting Tsai, Chih-Yu Wu, Zhen-Yu Guan, Ho-Yi Sun, Nai-Chen Cheng, Shu-Yun Yeh, and Hsien-Yeh Chen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01984 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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Topologically Controlled Cell Differentiation Based on Vapor-Deposited Polymer Coatings

Ya-Ting Tsai1,£, Chih-Yu Wu1,£, Zhen-Yu Guan1, Ho-Yi Sun1, Nai-Chen Cheng2, Shu-Yun Yeh1, Hsien-Yeh Chen1,*

1

Department of Chemical Engineering, National Taiwan University, Taipei, 10617

(Taiwan). 2

Department of Surgery, National Taiwan University Hospital, Taipei, 10018

(Taiwan)

Keywords: surface pattern; poly-p-xylylene; proliferation; osteogenesis; multifunctional

1

ACS Paragon Plus Environment

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ABSTRACT In addition to the widely adopted method of controlling cell attachment for cell patterning, pattern formation via cell proliferation and differentiation is demonstrated using precisely defined interface chemistry and spatial topology. The interface platform

is

created

using

a

maleimide-functionalized

parylene

coating

(maleimide-PPX) that provides two routes for controlled conjugation accessibility, including the maleimide-thiol coupling reaction and thiol-ene click reaction, with a high reaction specificity under mild conditions. The coating technology is a prime tool for the immobilization of sensitive molecules, such as growth factor proteins. Conjugation of the fibroblast growth factor 2 (FGF-2) and bone morphogenetic protein (BMP-2) was performed on the coating surface by elegantly manipulating the reaction routes, and confining the conjugation reaction to selected areas was accomplished

using

microcontact

printing

(µCP)

and/or

UV

irradiation

photopatterning. The modified interface provides chemically and topologically defined signals that are recognized by cultured murine preosteoblast cells for proliferation (by FGF-2) and osteogenesis (by BMP-2) activities in specific locations. The reported technique additionally enabled synergistic pattern formation for both osteogenesis and proliferation activities on the same interface, which is difficult to perform using conventional cell attachment patterns. Because of the versatility of the coating, which can be applied to a wide range of materials and on curved and complex devices, the proposed technology is extendable to other prospective biomaterial designs and material interface modifications.

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INTRODUCTION Cell patterning is of interest in the fields of biomaterials and tissue engineering,1-3 which are important fields for the study and prediction of cell behaviors and for the organization and assembly of cells into engineered tissues and organ structures.4-6 The approaches exploited are based on controlling the cell-surface interactions via chemical or physical modifications on material substrates in spatially specified locations, and such topologically defined functional constructs at the cell/material interface play important roles during cell development and are recognized by the attached cells.7-9 For instance, using chemical means to address cell adhering molecules, e.g., RGD or fibronectin protein,10-11 on defined locations can effectively change the attachment of the cells to a different, desired location. On the other hand, using physical means to alter the hydrophobic and hydrophilic properties at the addressed locations can also create a patternable platform on which the cell can attach (usually on hydrophilic patterns). These cell patterns can further develop into the desired

physiological

pattern

and

have

demonstrated

promise

for

future

applications.12-13 Despite the myriad of cell patterning methods available, vapor-based coating using functionalized parylene has been shown to be a robust tool to generate cell patterns. Because the vapor deposition coating can conform to a variety of surfaces, cell patterns have been created on curved substrates and complex devices.14-16 Producing multifunctional patterns has been demonstrated to be possible.17-20 In the present study, instead of using a cell attachment pattern, we show that the cell differentiation patterns can be directly manipulated and controlled through precise and synergistic immobilization of growth factor proteins at selected areas of interests, and, in addition, an elegant pathway to a multifunctional and topological cell differentiation interface was established. The proposed technology exploited a maleimide-functionalized parylene coating (hereafter referred to as maleimide-PPX) on the substrate material, which has the advantage of providing site-specific conjugation accessibility via a Michael-type nucleophilic addition to the target molecules that contain thiols, e.g., growth factor proteins.21-23 The maleimide moiety is also clickable via light-induced thiol-ene chemistry, which allows the addition of thiols across the vinyl groups on the maleimide-forming thioether adducts (Figure 1a).24 Both conjugation routes can

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occur with high specificity and rapid kinetics and proceed under mild conditions at room temperature in the presence of oxygen/water. Additionally, the routes provide the flexibility to confine the reaction to selected areas using microcontact printing (µCP, for the maleimide-thiol coupling reaction) and/or UV light photopatterning (for the thiol-ene reaction). Conjugation of the growth factor proteins via these reaction mechanisms accesses the native disulfide bonds of the protein molecules, and only the preferentially

reduced, exposed

disulfide

bonds

undergo

conversion

upon

immobilization via the use of mild reaction conditions, which alleviate the concern of denaturing the proteins or destroying the biological activities.25-26 The cell proliferation patterns were produced by selectively immobilizing fibroblast growth factor 2 (FGF-2) at defined locations of interest, and cell recognition was further validated to resolve the proliferation pathway/pattern. Using the same idea and conjugation mechanism, the patterned formation of osteogenetic activity was validated via the spatially defined tethering of bone morphogenetic protein 2 (BMP-2) to the registered regions. In addition, producing a synergistic presentation of both the cell proliferation and osteogenetic activities and patterned formation of these activities on the same substrate interface were attempted and validated. The patterned formation of multifunctional cell activities was, in contrast, difficult to perform for the conventional cell attachment patterns. State of the art control over the biointerface was established with chemically and topologically defined surface properties, and the multifunctional and timed controls for sophisticated biological activities can be manipulated. The present technique offers an opportunity to replicate the complex architectural and characteristic boundary conditions for cells and tissues to develop discrete and distinct cellular processes or tissue phenotype in vitro and in vivo.

RESULTS AND DISCUSSION The maleimide-PPX was synthesized via a chemical vapor deposition (CVD) polymerization process from 4-N-maleimidomethyl-[2.2]paracyclophane (dimer). During the CVD process, the dimers were sublimated at approximately 110 °C under a reduced pressure (75 mTorr) and were subsequently transferred to a pyrolysis zone where the temperature was maintained at approximately 560 °C. The pyrolysis temperature ensured the cleavage of the C–C bonds of the dimers to form the

4


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corresponding quinodimethanes (monomers).27 During the last step of the CVD process, the monomers polymerized upon condensation on the sample substrate at a temperature of 15 °C to result in the maleimide-PPX coating. The synthesis of the maleimidomethyl-[2.2]paracyclophane dimer and the refined CVD process have also been reported elsewhere.28 The resulting coating thickness was approximately in the range from 100 nm to150 nm, which was estimated by using spectroscopic ellipsometry on a reference silicon substrate. Theoretically, the coating thickness has a linear dependence on the CVD processing time.29-31 Combined characterizations using infrared reflection absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS) were used to verify the maleimide-PPX. The IRRAS spectra showed peaks at 1398 cm−1 (C–N–C) and 1720 cm−1 (C=O), which corresponded to the characteristic band stretches for the maleimide groups. The high-resolution C1 s spectra from the XPS also revealed the binding energy and positions for the aliphatic and aromatic carbons (C–C, C–H) and the C–N, N–C=O maleimide group, and the results were in good agreement with the theoretical values of the proposed coating structure. The IRRAS and XPS data are included in the supporting information in Figure S1. The confirmed maleimide functionality can undergo (i) a maleimide-thiol coupling reaction through a Michael-type nucleophilic addition to the target molecules that contain thiols at pH 6.5–7.5.28 In addition, the maleimide moiety is also clickable through (ii) light-induced thiol-ene chemistry in which the thioether bond is formed by the addition of thiols across the vinyl groups on the maleimide moiety.32 With respect to controlling the reaction routes, the light-induced thiol-ene reaction was initiated under reductive conditions at pH > 8 to suppress the maleimide-thiol coupling reaction and was accelerated by the addition of a photoinitiator, 2,2-dimethoxy-2-phenylacetophenone (DMPA).24 The accelerated thiol-ene click reaction additionally suppressed the cross-reaction of maleimide with possible amino groups and ensured the specificity of the reaction.24, 33 Verification of the reactivity of (i) and (ii) was conducted by immobilizing the FGF-2 and the BMP-2 under the controlled reaction conditions. The resulting immobilization surfaces were further characterized using IRRAS, and the recorded spectra in Figure 1b shows characteristic –N–H adsorption peaks in the range from at 3200 cm-1 to 3600 cm-1, which are indicative of the successful immobilization of FGF-2 and BMP-2. On the other hand, the reaction specificity was verified by performing the same immobilizations under reductive reaction conditions without initiating the 5


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photochemical process, and the –N–H adsorption peaks were absent from the IRRAS spectra because of the non-reactive conditions. The data are included in the supporting information in Figure S2. The reactivity and specificity were further enhanced by selectively immobilizing the growth factor proteins at confined locations using microcontact printing (µCP, for the maleimide-thiol coupling reaction) and/or UV light photopatterning (for the thiol-ene click reaction). As shown in Figure 2, the fluorescent molecules of Alexa Fluor® 555 and fluorescein (FITC), which both have a terminal thiol group, were selected as the reporter molecules during the experiment. The spatial confinement using µCP and/or the light-initiated thiol-ene click reaction with a photomask was cross-examined using these fluorescence molecules, and distinct patterns, including 50 µm × 50 µm square array patterns (by µCP) and 300 µm × 300 µm square array patterns (by photomask), were used to guide the eyes. The resulting fluorescence images show the Alexa Fluor® 555 and FITC fluorescent signals in either the µCP-stamped regions or the photochemically defined regions, as indicated in Figure 2a. The results unambiguously verified the reactivity and specificity of these reactions. More importantly, the experiment further demonstrated the feasibility of parallel immobilization of two fluorescence molecules using a stepwise approach to control the reaction routes of (i) and (ii). Specifically, the thiol-Alexa Fluor® 555 was first photochemically patterned on the maleimide-PPX surface (with 300 µm × 300 µm square array patterns) via a thiol-ene reaction, and, subsequently, the remaining, unreacted maleimide groups on the same surface underwent a second, thiol-maleimide coupling reaction to conjugate the thiol-FITC. The overlaid fluorescence image of the spatially controlled, co-immobilized thiol-Alexa Fluor® 555 and thiol-FITC is shown in Figure 2b. The control over the reaction routes and locations and the conjugation flexibility with other terminated molecules were further used with thiol-terminated poly(ethylene glycol) (thiol-PEG) and a thiol-terminated functional peptide (RGDYCC). The thiol-PEG was first photochemically immobilized in confined areas (photomask, 300 µm × 300 µm square arrays), and in the second step, the RGDYYC peptide was conjugated via a thiol-maleimide reaction on the remaining unreacted maleimide groups, based on the aforementioned stepwise procedure. In light of the antifouling property of the PEG moieties and the cell-adherent property of RGD,34-35 a cell culture of murine preosteoblasts (MC3T3-E1) was allowed to grow on the PEG/RGD-patterned surface. Figure 3 shows the results after 24 h for the MC3T3-E1 6


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grown on the areas where RGDYYC was immobilized, and significant inhibition of cell attachment was found in the PEG-modified regions. Cell numbers of 0.41 ± 0.06 × 104 cells/cm2 and 8.13 ± 0.45 × 104 cells/cm2 were quantified for PEG and RGDYYC areas, respectively. A sharp boundary between these two regions, RGDYYC (cell-attached) and PEG (cell-repellent), was identified, which indicated the precise modification of the surface chemistry and the effectiveness of the immobilization process. Additional images were provided for a wider field of view to verify the reliability and reproducibility of the proposed patterning method, and the data are included in the Supporting Information in Figure S3. In addition, the challenge of spatially controlling cell differentiation was demonstrated by precisely conjugating the growth factors of FGF-2 and BMP-2 in defined regions. The conjugations of FGF-2 and BMP-2 were performed in a manner analogous to the previously mentioned reaction routes via a thiol-maleimide reaction or the thiol-ene reaction under controlled conditions, as well as the stepwise patterning/conjugation procedure to enable synergistic presentation of the two growth factors on the corresponding areas and on the same sample surface. Conjugation via preferential reduction of the exposed disulfide bonds under mild reaction conditions ensured a negligible impact on denaturating the proteins or destroying the biological activities.25-26 To confirm if the immobilization of FGF-2 and BMP-2 was successful (on the defined locations), the corresponding antibodies, including anti-FGF-2 and anti-BMP-2, were incubated with the sample surfaces, and subsequently, the fluorescently labeled (FITC and rhodamine) second anti-bodies were allowed to react with the same samples for detection. The resulting fluorescence images showed strong signals and sharp contour lines from the patterns due to the self-assembled binding of the second anti-bodies, and the patterns agreed well with the proposed confinement patterns that formed during the immobilization processes (Figure 4). The topographical characteristics and nanomechanical properties of the immobilized FGF-2 and BMP-2 on the surface were investigated using atomic force microscopy (AFM)-based nanoindentation in a liquid phase (in a phosphate-buffered saline (PBS) solution). The results from the modulus mapping revealed the granular structure boundaries of the immobilized BMP-2 and FGF-2 on the surfaces, and the results indicated tenacious and irreversible immobilization of the growth factors compared to the loosely bound proteins on the surfaces, which showed stranded structures that were reported previously in the literature.36-39 The quantitative results of the 7


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root-mean-square roughness (Rms) showed approximately 5.85 ± 0.9 nm for the FGF-2 areas compared to 2.97 ± 0.3 nm for the BMP-2 areas. Additionally, the indented results from Young’s modulus indicated an average value of 0.74 GPa for the FGF-2 areas and 0.63 GPa for the BMP-2 areas. Although the boundary line between the areas of the two growth factors was vaguely distinguishable, the differences in the surface roughness and modulus was able to determine during the AFM analysis (Figure 5). An Rms value of 4.97 ± 0.8 nm and a modulus of 0.70 GPa were recorded for the area above the boundary line (FGF-2 areas), while 3.01 ± 0.4 nm and a modulus of 0.59 GPa were recorded for the BMP-2 area below the boundary line. The immobilization of FGF-2 and BMP-2 in defined spatial locations was again confirmed. The question of whether the FGF-2 and BMP-2 patterned areas on the culture surface can provide guided cell proliferation and osteogenesis cues, and, more importantly, synergistic and paralleled manipulation of these two activities on the same culture surface, was further examined. MC3T3-E1 cells were cultured on the FGF-2 and BMP-2-modified surfaces, and the proliferation and differentiation behaviors of the cells were observed over time to identify these activities. Control experiments, including non-patterned surfaces of pure FGF-2 and pure BMP-2, were also performed for comparison. The first observation was performed on day 1, and the cell growth and quantified cell number were similar for the FGF-2 and BMP-2 areas on the same sample and were comparable to the control groups (Figure 6). Specifically, the cell number was approximately 1.27 ± 0.02 × 104 cells/cm2 on the FGF-2-patterned regions and 1.11 ± 0.11 × 104 cells/cm2 on the BMP-2-patterned regions. Cell migration activity would occur only with limited space in the neighborhood under such populated number of cells.40 Continued cell growth was observed on day 4 (a time when the cell proliferation characteristics become detectable), and the recorded images indicated more cell growth of MC3T3-E1 on the FGF-2-patterned regions compared to the BMP-2 regions. Cell numbers of 1.75 ± 0.41 × 105 cells/cm2 and 1.23 ± 0.19 × 105 cells/cm2 were quantified for FGF-2 and BMP-2, respectively. Statistical analysis was also performed by comparing the normalized cell number ratio from day 4 to day 1 to evaluate the cell proliferation activity. An approximately 40% higher ratio was found for the FGF-2 regions while no significant difference was discovered for the BMP-2 regions compared to the control surface of the pure maleimide-PPX coating. The results were comparable to 8


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the control experiments and suggested an enhancement of the MC3T3-E1 proliferation activity was induced by FGF-2 on the corresponding regions. The data for the control experiments are included in the Supporting Information in Figure S4. An additional observation was performed on day 10 when the early-stage osteogenesis of alkaline phosphatase (ALP) expression is detectable. The enhanced expression of ALP was, as anticipated, discovered on the BMP-2 regions compared to the FGF-2 regions on the same sample surface (Figure 7a and 7b). More specifically, the statistical results from normalizing the signal intensity per area for the entire sample surface suggested 3-fold more ALP was expressed on the BMP-2 regions than the FGF-2 regions. The observation on day 21 was performed to detect the mature osteogenesis stage, and staining with Alizarin red was performed to examine the degree of calcium mineralization.[31] As shown in Figure 7c and 7d, a positive result and enhancement in the calcium formation was observed in the BMP-2 regions compared to the suppressed signals detected on the FGF-2 regions. By comparing the normalized signal with respect to the patterned areas, the results indicated 4-fold more calcium deposition on the BMP-2 regions at the mature osteogenesis stage and positively supported the ALP expression data from the early stage. Additional images of the regiospecific ALP expression and calcium mineralization activities are included Supporting Information in Figure S5 to provide the views in different locations of the same modified sample surface and to demonstrate the pattern reliability or reproducibility. The consistent results were comparable to the control experiments, and the data are included in the Supporting Information in Figure S6. The results demonstrated that precisely patterned FGF-2 and BMP-2 on one interface can be used to control cell differentiation activities (resolved in time latency) and manipulate synergistic multifunction in spatially defined regions.

CONCLUSIONS Based on the established feasibility and versatility of depositing functionalized parylene on substrate materials without restrictions on the substrate material or geometry, the present coating technique and immobilization and patterning technique can be used and transferred from one material/device to another. In addition, the proposed coating technology provides site-specific accessibility for maleimide-thiol

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coupling without compromising the functional properties of the immobilized molecule, and the technique is particularly amenable for the immobilization of growth factor proteins, which requires precise topological and chemical control. The coating and immobilization technology is extendable beyond applications related to the control of cell proliferation and the osteogenesis differentiation pathways demonstrated in the current study. We foresee the use of this technology for developing prospective biomaterials devices, tissue engineering scaffolds, and regeneration medicine applications.

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Figure 1. (a) Schematic illustration of controlling the reaction routes using maleimide-PPX to immobilize the growth factor proteins. (b) IRRAS spectra show the immobilized FGF-2 and BMP-2 proteins on the maleimide-PPX surface from the two route, controlled conjugation reactions. The peaks in the range from 3200 cm-1 to 3600 cm-1 indicate the characteristic –N–H band adsorptions of BMP-2 and FGF-2.

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Figure 2. Verification using fluorescence probes to examine the reactivity and specificity of the two conjugation reactions. Additional topological control to confine the reactions was enabled using µCP or a photochemical process with the assistance of a photomask. (a) Fluorescence images indicated successful immobilization of thiol-terminated FITC (green channel) and Alexa Fluor® 555 (red channel) molecules on selected areas via controlled conjugation reactions. (b) Parallel immobilization of the thiol-terminated FITC and Alexa Fluor® 555 on defined locations via a stepwise conjugation procedure by controlling the reaction routes, and the overlaid image of the red and green channels indicates the precise immobilization of the two molecules at specific locations. The images were captured at the same location on the same sample.

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Figure 3. A controlled cell attachment pattern was demonstrated by culturing MC3T3-E1 on the maleimide-PPX surface composed of selectively modified PEG and RGDYCC moieties. A stepwise modification procedure was used to perform PEG modification within the squares, and the remaining tunnel areas were modified with RGDYCC. (a) Optical micrograph showing the controlled MC3T3-E1 attachment on the areas where RGDYCC was immobilized, and suppressed cell growth was found on the PEG areas. (b) Fluorescence micrograph of cell nuclei stained by DAPI to unambiguously verify control of the cell attachment. The images (a) and (b) were captured at the same location on the same sample.

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Figure 4. Immobilization of FGF-2 and BMP-2 growth factor proteins was selectively performed on the maleimide-PPX at confined areas via controlled reaction routes and conditions. A stepwise immobilization procedure was devised by patterning the first growth factor (FGF-2 or BMP-2) through µCP or the photomask-assisted photochemical process, and, subsequently, the remaining, unreacted maleimide groups on the same surface underwent a second, thiol-maleimide coupling reaction to conjugate the second growth factor (BMP-2 or FGF-2, respectively). Antibodies, including anti-FGF-2 and anti-BMP-2, were allowed to incubate on these modified surfaces to induce self-assembled binding. Fluorescently labeled second antibodies were then used for detection via a secondary self-assembled binding to verify the activity and specificity of the formerly established FGF-2 and BMP-2 pattern surfaces.

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Figure 5. Surface analysis using AFM to characterize the immobilized FGF-2 and BMP-2 on the maleimide-PPX surface. Mappings of (a) surface roughness and (b) modulus were recorded at the areas where FGF-2 and BMP-2 were immobilized. Acquisition of the surface roughness and modulus was recorded in the vicinity of the border between the FGF-2 and BMP-2 regions in (c). Cross section profiles along the red or blue line were shown below correspondingly. The dotted line was drawn to indicate the border. The averaged values of the surface roughness and Young’s modulus are summarized in (d). The AFM images were collected with a field of view of 1 µm × 1 µm and were analyzed using a NanoScope Analysis software.

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Figure 6. MC3T3-E1 proliferation activity was examined on the surfaces composed of parallel FGF-2 and BMP-2 immobilization with topological confinement. A stepwise modification procedure was used to perform BMP-2 modification within the squares, and the remaining tunnel areas were modified with FGF-2. Pure maleimide-PPX coating surfaces were served as a control for the comparison. Optical and fluorescence micrographs recorded at (a) day 1 and (b) day 4 to observe the cell growth behaviors. (c) Cell number was calculated for day 1 and day 4 with respect to the FGF-2 and BMP-2 regions. (d) The normalized ratio of day 4 to day 1 for the cell number was compared with respect to the FGF-2 and BMP-2 regions. Each bar represents the mean value (±SD) of three independent experiments.

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Figure 7. Osteogenesis activities of MC3T3-E1 were examine on the surfaces with confined immobilization of FGF-2 and BMP-2. A stepwise modification procedure was used to perform BMP-2 modification within the squares, and the remaining tunnel areas were modified with FGF-2. Pure maleimide-PPX coating surfaces were served as a control for the comparison. (a) Early stage marker of ALP expression was analyzed at day 10, and (b) the statistical analysis of ALP expression was compared for the two patterned regions of FGF-2 and BMP-2. (c) Mature stage marker of calcium deposition was observed at day 21 using Alizarin red staining, and the statistical analysis of the Alizarin red signals for both regions is also compared in (d). Each bar represents the mean value (±SD) of three independent experiments.

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ASSOCIATED CONTENT Supporting Information. Experimental details, spectroscopic data, control experiments of cell proliferation and osteogenesis. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Fax: (+)886-2-33669476 E-mail: [email protected]

Author Contributions £

Y.-T. Tasi and C.-Y. Wu contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT H.-Y. Chen gratefully acknowledges financial support from the Ministry of Science and Technology of Taiwan (104-2628-E-002-010-MY3), National Taiwan University (103R7745 and 104R7745).

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Table of Contents Graphic and Synopsis Topological and synergistic control over cell differentiation pathways via confined immobilization of growth factor proteins using vapor-deposited polymer coatings.

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Topologically Controlled Cell Differentiation Based on Vapor

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