Stepwise and Programmable Cell Differentiation Pathways of

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Stepwise and Programmable Cell Differentiation Pathways of Controlled Functional Biointerfaces Zhen-Yu Guan, Chih-Yu Wu, and Hsien-Yeh Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00312 • Publication Date (Web): 24 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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ACS Biomaterials Science & Engineering

Stepwise and Programmable Cell Differentiation Pathways of Controlled Functional Biointerfaces Zhen-Yu Guan, Chih-Yu Wu, and Hsien-Yeh Chen* Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan

KEYWORDS: stepwise cell differentiation, programmable biointerface, poly-p-xylylene coating, growth factor protein, osteogenesis

ABSTRACT

An advanced control of biomaterial surfaces was created to enable the stepwise and switchable activities of the immobilized growth factor (GF) proteins for a programmed manipulation over cell differentiation pathways. The GF protein was immobilized on an advanced vapor-based coating of poly[(4-2-amide-2’-amine-dithiobisethyl-p-xylylene)-co-(pxylylene)], and the equipped disulfide exchange mechanism of the coating enables the detachment and/or the displacement of the previously installed GF to reinstall a second GF protein. In this study, the controlled immobilization and displacement of the fibroblast growth factor (FGF-2) and bone morphogenetic protein (BMP-2) were demonstrated on cell culture substrates, and the resulting surfaces provided a programmable induction of cellular responses in proliferation and osteogenesis toward the cultured murine preosteoblasts (MC3T3-E1). A

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depreciated or stopped activity of the previously induced biological function, i.e., proliferation or osteogenesis, was found for MC3T3-E1 on the modified surface after the cleavage of the corresponding GF. In addition, with the approach to devise the displacement and reinstallation of FGF-2/BMP-2 proteins, a combined induction of proliferation and osteogenesis can be initiated in a timed latency to resolve programmable biological activities.

INTRODUCTION The immobilization of growth factor (GF) proteins by covalent conjugation on biomaterials surface is considered superior because of the prolonged availability of the biological function, controlled delivery to the local biological environment, and confined dosage of GF concentration by immobilization.1-5 By contrast, the conventional approach of delivering GFs through direct injection can be rapidly degraded through endocytosis pathways and consequently requires refills; in addition, these nonlocalized GFs increase the chance of overuse that damages cells and tissues.5-6 Herein, for the first time, we demonstrate that the switching and displacement of growth factor (GF) proteins on biomaterial surfaces are feasible, and the resulting control parameters of the cell differentiation pathways are effectively directed and/or reversibly altered. The dynamic control of the surface chemistry using an advanced vapor-based coating of poly[(42-amide-2’-amine-dithiobisethyl-p-xylylene)-co-(p-xylylene)] was exploited to provide (i) a covalent coupling site to immobilize a GF protein and (ii) the accessibility of a disulfide bond for the disulfide re-dox reaction to detach or reinstall a second GF protein during the cell culture.


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FGF-2 (fibroblast growth factor; a first GF of choice) was immobilized on the modified surfaces with the anticipated cell physiological pathway of proliferation for the cultured murine preosteoblasts (MC3T3-E1), and the subsequent cleavage to remove FGF-2 molecules from the culture substrate further demonstrates the slowed or ceased activities for cell proliferation. In addition, the reinstallation of BMP-2 (bone morphogenetic protein, a second GF) on the same culture substrate can continue the detrimental effect on the cell fate in a temporal sequence, and the divergent cell physiological activity of osteogenesis was initiated (Figure 1).

RESULTS AND DISCUSSION An advanced poly-p-xylylene system comprised a 4-(2-amide-2’-amine-dithiobisethyl) side group featuring an integrated disulfide moiety with a reactive amino end group (hereafter referred to as PPX-SS-NH2 coating), which was exploited as an interfacial material to modify biomaterial substrates. The modification of cell culture substrates with a PPX-SS-NH2 coating was performed in one step via a chemical vapor deposition (CVD) polymerization process from a dimeric 4-(2-amide-2’-amine-dithiobisethyl)-substituted [2.2]paracyclophane. The detailed synthesis route of the dimer and the characterization are included in the Supporting Information. During

the

CVD

polymerization,

the

dimer

of

4-(2-amide-2’-amine-dithiobisethyl)

[2.2]paracyclophane was used as the starting material for the CVD process and was sublimated at approximately 125 °C before being thermally treated at a pyrolysis temperature of approximately 550 °C. Pyrolytic cleavage occurred only for the two carbon-carbon bonds in the dimers and converted the dimers into quinonoid structures (monomers). Finally, the monomers


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were spontaneously polymerized at room temperature (25 °C). The decomposition or side reactions did not occur during the CVD polymerization. A coating thickness of a 100–150 nm was obtained, as estimated by in situ quartz crystal microbalancing (QCM). The as-deposited PPX-SS-NH2 coating was also mechanically stable under a cross-cut tape adhesion test7 on different materials, including stainless steel, titanium alloy, gold, silicon, glass, and polymeric substrates as well as a thermo-stability test carried out at 150 °C. After the coating and modification was performed on the tissue culture polystyrene (TCPS) microplates, the immobilization of fibroblast growth factor (FGF-2) was subsequently realized by conjugating FGF-2 through a reductive Mannich reaction with the addition of glutaraldehyde to form an imine bond between the formaldehyde group and the amine group. Infrared reflection absorption spectroscopy (IRRAS) was used for the characterization of the resulting FGF-2-conjugated surface, and the recorded spectra showed a characteristic band adsorption of –N-H at 3036 to 3601 cm-1, which indicated there was successful immobilization of FGF-2 after the conjugation reaction (Figure 2a). The idea of detaching the already conjugated FGF-2 was then enabled by using the integrated disulfide bond of the PPX-SS-NH2 coating, and the cleavage of the disulfide bond could occur under reductive conditions of glutathione (GSH) to resolve a sulfhydryl (–S-H) surface.8-10 In addition, the reinstallation of a second growth factor, i.e., bone morphogenetic protein 2 (BMP-2), could finally be approached by a thiol-disulfide interchange reaction to conjugate a thiol-terminated molecule of sulfosuccinimidyl 6-(3'-(2 pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP) and to catch the BMP-2 protein through an amine-sulfosuccinimidyl coupling reaction.11 The thiol-specific affinity of forming a disulfide bond between the thiol-terminated BMP-2 and the sulfhydryl surface ensured the specificity required to achieve successful conjugation in the vast array of


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functionality present in biological systems.12-13 The IRRAS analysis also confirmed the cleavage of FGF-2 with decreases in the peak intensity of –N-H stretches and the reinstallation of BMP-2 with increases in the peak intensity of –N-H at 3098-3452 cm-1 on the same sample surface. The switching of GF molecules from FGF-2 to BMP-2 with a conversion rate of 84.0% was estimated by comparing the normalized integrated peak intensities of the –N-H bands (30603452 cm-1) between the reinstalled BMP-2 surface and the first immobilized BMP-2 surface. With respect to the surface density (concentration) of approximately 150 ng·cm-2 BMP-2 can be estimated from a previous discovery,14-15 and the resulting density on the surface after such cleavage and reinstallation process was calculated with a value 128 ng·cm-2 in the margin of error. The IRRAS spectra of the first immobilized BMP-2 surface are shown in Figure S3 (Supporting Information). The normalized integrated peak intensities of the –N-H bands were normalized by the integrated peak intensities of –C-H bands (2762-3060 cm-1) for each spectrum. The switching of the FGF-2 and BMP-2 growth factors was further dynamically characterized and verified using a quartz crystal microbalance (QCM) analysis. The binding affinities of two specific antibodies (human FGF-2 antibody and human BMP-2 antibody) were used to crossexamine the specific binding efficiency of two growth factor proteins after the immobilization (first GF) and reinstallation (second GF). The displacement rate (efficiency) was calculated based on the specific binding affinity ratio of each GF protein (FGF-2 and BMP-2) at the reinstallation state to the first immobilization state. As shown in Figure 2b, 336.0 ± 11.0 ng/cm2 of FGF-2 antibody (reduced frequency of 62.1 ± 2.0 Hz) and 94.7 ± 6.8 ng/cm2 of BMP-2 antibody (reduced frequency of 17.5 ± 1.2 Hz) were bound to the surfaces of the first immobilized FGF-2 and the reinstalled BMP-2, respectively, and an anticipated binding specificity was detected. Further examination by reversing the immobilization sequence of BMP-


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2 (first GF) and FGF-2 (second GF) also showed consistency for such binding specificity: 108.2 ± 8.0 ng/cm2 for the BMP-2 antibody (reduced frequency of 20.0 ± 1.5 Hz) and 301.9 ± 13.7 ng/cm2 for the FGF-2 antibody (reduced frequency of 55.8 ± 2.5 Hz), which were detected for the former BMP-2 and latter FGF-2 surfaces, respectively. The displacement rate (efficiency) of the reinstallation of the second GF was calculated from the QCM results: 90.0% for FGF-2 and 87.5% for BMP-2, which were consistent with the conversion rate (84.0%) of the IRRAS analysis. The results collectively have also verified the low fouling activity of the surface by the first immobilized GF layer, and showed consistency with reported results in the literature.15-18 The saturated surface with the first GF allows only the binding of secondary antibody with specific binding affinity, and prevents non-specific proteins from adsorbing to such a surface. As to whether a cleavage of the FGF-2 or BMP-2 growth factor protein can depreciate or stop the activity of the previously induced biological function, it is not clear whether a divergent activity of a specific biofunction is re-initiated with a response to the change in the corresponding growth factor of displacement. These questions were further examined. Murine preosteoblasts (MC3T3-E1) were used and cultured on the first immobilized FGF-2 surfaces with various time frames (day 1, day 2, and day 3) to cleave the FGF-2 growth factors from the culture surface. A control experiment for the immobilized FGF-2 surface without further cleavage of the growth factors was also performed in parallel for comparison. To better understand and characterize the proposed surface chemistry, the cells were removed by trypsin from the culture surfaces during the modifications and subsequently re-seeded to the same surfaces after modifications. All the surfaces were continued with the MC3T3-E1 culture until day 4 (a time where the cell proliferation characteristics become detectable), and the results were observed and captured as shown in Figure 3a. The images indicated that a confluent cell


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growing pattern of MC3T3-E1 was discovered on the surface with a longer responsive time toward FGF-2, i.e., cleavage of FGF-2 at a later time in day 3, compared to a lower number for MC3T3-E1 growth, which was found on the surface of the FGF-2 cleaved on day 1. Statistically, the normalized cell number ratio of day 4/day 1 was calculated to evaluate cell proliferation, and the results unambiguously supported the observed images that relatively high intensive enhancement of MC3T3-E1 proliferation was induced with a longer exposure to the FGF-2 surface (cleaved at day 3 or no cleavage) compared to less proliferation on the day 1 cleaved FGF-2 surface (Figure 3b). As anticipated, the proliferation ratio for the cleaved FGF-2 surface on day 3 was approximately 95% similar to the control experiment, for which no FGF-2 was cleaved. However, surfaces with various cleavage times (day 3, day 7, and day 10) for previously immobilized BMP-2 were also used for the culture of MC3T3-E1, and the resulting osteogenesis activities were studied by examining calcium formation and the osteocalcin protein expression. The cleavage of BMP-2 was performed by using the aforementioned procedure involved in the addition of reductive GSH under mild conditions. The cell culture continued, and the resulting osteogenesis examinations were performed at day 14, for which the characteristics of calcium formation and osteocalcin expression have become detectable. Control experiments of a BMP-2 surface without further cleavage were also examined in parallel for the comparison. The calcium deposit formation and the bone-specific protein (osteocalcin) expression at day 14 were examined by using Alizarin red staining and immunofluorescence staining of fluorescein-labeled goat anti-rabbit IgG, respectively. As shown in the images in Figure 4a, the signals of Alizarin red, which indicate calcium deposition, revealed an increased trend of intensity with increasing exposure time for BMP-2 on the culture surfaces. The detection of osteocalcin has additionally provided evidence to support the Alizarin red data that showed a similar trend for increasing the


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osteocalcin expression (specifically bound FITC-IgG signals) and demonstrated more MC3T3E1 differentiation toward the direction of mature osteoblasts, and osteocytes formation corresponded well to the surfaces with longer exposure time for BMP-2 (green channel images in Figure 4b). An additional investigation of cell number variation was conducted by staining cell nuclei with DAPI (blue channel) for the cultured surfaces on day 14, and the results showed an insignificant difference of the cell number for different surfaces with various cleavage times for BMP-2. The blue-channeled images were overlaid with FITC-IgG images and are also as shown in Figure 4b. The statistical results of these osteogenesis activities are summarized in Figure 4c and were consistent with the observations from the images that revealed an increased trend of intensity with a longer exposure time for BMP-2 on the surfaces. A separate experiment was additionally performed by directly cleaving BMP-2 on the cell culture system without removing the cells from the culture surfaces. The experiment was compared in parallel with a similar functional-PPX coating (PPX-NH2) but without the disulfide cleavage mechanism. The results unambiguously indicate the similar activity of increasing ALP expression for the sample groups with longer exposure to BMP-2 on the surfaces. The cleaved BMP-2 were expected to be removed from the culture system via medium change or degrade through endocytosis pathways.1, 3, 19

Furthermore, the biological impact from the cleavage modification was insignificant because

> 90% viable cells were discovered on the modified surface compared to the control surface of the bare TCPS. These data are included in the supporting information in Figures S4 and S5. The results from cell proliferation (induced by FGF-2) and osteogenesis (induced by BMP-2) collectively indicate that: (i) the induced biological activities such as cell proliferation or osteogenesis can slow or terminate with the controlled approach by removing (i.e., cleaving) the corresponding functional GF protein from the culture surfaces; and (ii) the redox disulfide


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mechanism of the PPX-SS-NH2 coating provides a switchable (dynamic) control and effectively induces the biological activity to enable a timed and/or programmed biofunctional interface platform. Finally, the combined activities of cell proliferation and osteogenesis were demonstrated with time latency and functional specificity via a stepwise immobilization and displacement of FGF-2 and BMP-2 on the PPX-SS-NH2 coating-modified surfaces. The cell culture surfaces were first modified with FGF-2 immobilization by using the PPX-SS-NH2 coating, culturing MC3T3-E1 on such modified surfaces and studying the resulting physiological activities. The cleavage of FGF-2 was devised on day 3, and the displacement to reinstall BMP-2 was subsequently performed via a redox thiol-disulfide interchange reaction, which was similar to the approach to the foregoing procedures. Two control experiments, including immobilized FGF-2 and BMP-2 surfaces with no further cleavage, were both examined for comparison. An expected cell proliferation of MC3T3-E1 was observed at day 4 by showing a consistent cell growing pattern and the cell proliferation ratio (day 4/day 1) (Figure 5a) compared to the previously described results in Figure 3a, which were comparable to the results on the control FGF-2 surface (with no cleavage). In contrast, the cell proliferation was suppressed on the other control surface of BMP-2, and the approximately 2.5-fold less proliferation activity was discovered on such a surface. While the FGF-2/BMP-2 displacement was performed and the cell proliferation was confirmed, the cell culture of MC3T3-E1 on the studied surface continued for another 14 days (at day 17), and the resulting cultured surfaces were then examined with the osteogenesis activities. Alkaline phosphatase (ALP) expression, which is an indicative marker of the early stage of osteogenesis,20-21 was analyzed on day 10, and as shown in Figure 5b, the images and the statistical results indicated significant ALP signals for the reinstalled BMP-2 surface, which


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was comparable to the control BMP-2 surface (no cleavage), and by comparison, low expression of ALP was found on the pure FGF-2 surface. Late-stage activities of osteogenesis, including calcium formation and the osteocalcin protein expression, were finally examined at day 17, and the staining by Alizarin red demonstrated a high potency of calcium deposition for the reinstalled BMP-2 surface, and the differentiation of MC3T3-E1 toward osteoblasts and osteocytes were also confirmed by a high concentration of expressed osteocalcin protein on such a surface (Figure 5c and Figure 5d). The statistical results in Figure 5e have also compared the experiment with control surfaces of permanent immobilizations of FGF-2 and BMP-2 and showed consistency with the observations from the images that osteogenetic differentiation was induced by the reinstalled BMP-2 surfaces. The results above confirmed that (i) the first immobilized FGF-2 effectively provided induction of cellular response with enhanced proliferation; (ii) the detachment/displacement of FGF-2 to reinstall BMP-2 has additionally provided re-initiative induction toward osteogenesis, for which a divergent cell physiological activity was induced; and (iii) a programmable interfacial engineering approach of using Parylene-S-S can enable a time-dependent and function-defined control and render synergistic biological activities.

CONCLUSIONS With the requirement of more sophisticated mechanisms and functions for biotechnological needs, advancing biomaterials designs can emerge to seek programmed biofunctional conducts to enable timed and/or a temporal series of growth factor manipulations to mimic embryonic development. A biointerface that is marked by a stepwise restriction in developmental potential and by changes in the expression of key regulatory genes is characterized by the introduced


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switchable interfacial material of PPX-SS-NH2 technology, and with the coating technology and the immobilization technique, this gene can be ideally and accurately transferred from one substrate to another regardless of composition or geometry and without compromised coating fidelity.22-23 We are a step closer to the ideal dynamic and programmable biomaterial interface.

EXPERIMENTAL SECTION CVD polymerization to synthesize PPX-SS-NH2 coating The coating of PPX-SS-NH2 was prepared via a CVD polymerization process from the starting material of dimeric 4-(2-amide-2’-amine-dithiobisethyl) [2.2]paracyclophane. The dimer was

first

synthesized

with

a

three-step

reaction

route.

Briefly,

non-substituted

[2.2]paracyclophane was obtained commercially (Sigma Aldrich, USA) and subsequently underwent a Friedel-Crafts acylation to afford 4-trifluoroacetyl-[2.2]paracyclophane (95% yield) by treatment with trifluoroacetic anhydride (TFAA, Sigma Aldrich, USA) and aluminum chloride (AlCl3, Alfa Aesar, USA). Then, the resulting 4-trifluoroacetyl-[2.2]paracyclophane was hydrolyzed with potassium hydroxide (KOH, SHOWA, Japan) to give 4-carboxyl-functionalized paracyclophane (85% yield). Finally, 4-carboxyl[2.2]paracyclophane was dissolved in a mixture of tetrahydrofuran (THF) and N,N’-dicyclohexylcarbodiimide (DCC, Sigma Aldrich, USA) for 20 min and then further mixed with cystamine dihydrochloride (Sigma Aldrich, USA) for 16 h. The resulting sediment was filtered, and the remaining supernatant was collected and further purified by column chromatography eluting ethyl acetate/hexane (1:5) to form 4-(2-amide-2’amine-dithiobisethyl) [2.2]paracyclophane in 68% yield. The NMR, IRRAS, and ESI-MS characterizations data and spectra of the resulting dimer are included in the Supporting


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Information. For the CVD polymerization process, the dimer was sublimated in the sublimation zone at approximately 125 °C. The sublimated species was then transferred to a stream of argon carrier gas at a flow rate of 30 sccm to be the pyrolysis zone in which the temperature was controlled at 550 °C. Following pyrolysis, the radicals were transferred into the deposition chamber and then polymerized onto substrate materials on a rotating holder at 25 °C to ensure a uniform deposition of the PPX-SS-NH2 coating. The chamber wall was held at 90 °C to prevent any residual deposition. A pressure of 75 mTorr was maintained throughout the CVD polymerization process, and all deposition rates were regulated at approximately 0.5 Å/s and monitored on the basis of in situ quartz crystal microbalancing analysis (STM-100/MF, Sycon Instruments, USA).

Growth factor immobilization and displacement Recombinant human BMP-2 and recombinant human FGF-2 were obtained commercially (R&D Systems, USA). BMP-2 was reconstituted as a stock solution at a concentration of 100 µg/mL in sterile 4 mM HCl and stored at -20 °C. FGF-2 was reconstituted as a stock solution at a concentration of 100 µg/mL in phosphate-buffered saline (PBS, pH 7.4, Sigma Aldrich, USA) and stored at -20 °C. The switching growth factor of the PPX-SS-NH2 coated surfaces was achieved by altering the surface chemical compositions via disulfide interchange reaction with thiol-terminated linkers. For the first immobilization of FGF-2 and BMP-2 to the PPX-SS-NH2 coated surfaces, 2.5% glutaraldehyde (Sigma Aldrich, USA) in 50 mM carbonate buffer (pH 9.6) was reacted with the PPX-SS-NH2 modified surfaces for 2 h at 37 °C. After washing with PBS containing Tween 20 (PBS-Tween 20) (pH = 7.4) (Sigma Aldrich, USA) three times, once more with pure PBS (pH=7.4) (Sigma Aldrich, USA), and finally rinsed with distilled water, the BMP-


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2 and FGF-2 stock solutions were incubated with the resulting surfaces for 1 h at 37 °C. For the cleavage and reinstallation of the second proteins of FGF-2 or BMP-2, 10 mM glutathione (GSH) (Sigma Aldrich, USA) aqueous solution was incubated with the resulting samples for 2 h at 37 °C, followed by a rinse process using PBS-Tween 20 and pure PBS to remove the cleaved substances. The reinstallation of the second growth factor (BMP-2 or FGF-2) was finally performed by incubation with BMP-2 or FGF-2 in the presence of 20 mM sulfosuccinimidyl 6(3'-(2 pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP, Thermo Fisher Scientific, USA) aqueous solution for 6 h at 25 °C. The resulting samples were cleaned thoroughly three times with PBS-Tween 20 and one time with pure PBS.

Surface characterizations Infrared reflection absorption spectroscopy (IRRAS) spectra were recorded using a 100 FT-IR spectrometer (PerkinElmer, USA) equipped with an advanced grazing angle specular reflectance accessory (AGA, PIKE Technologies, USA) and a liquid nitrogen-cooled MCT detector. The samples were mounted in a nitrogen-purged chamber, and the recorded spectra were corrected for any residual baseline drift. The dynamic binding analysis exploited a QCM instrument (ANT Technologies, Taiwan) equipped with a flow injection analysis (FIA) device and a continuous frequency variation recording device. Flow rate was controlled at 36.5 µL/min using a peristaltic pump connected to the FIA device. The sensing element of this instrument was an AT-cut piezoelectric quartz disc with a 9 MHz resonant frequency and a 0.1 cm2 total sensing area. The quartz discs were also coated with PPX-SS-NH2 via the CVD polymerization process and used for switching growth factors. Anti-FGF-2 and anti-BMP-2 solutions had a mass


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concentration of 50 µg/mL in PBS, which were injected through the FIA device to the analysis chamber, and the time-dependent change in frequency was continuously monitored.

Cell proliferation and osteogenesis Tissue culture polystyrene (TCPS) microplates (BD Falcon, USA) were coated with PPXSS-NH2, and FGF-2 or BMP-2 was immobilized on the PPX-SS-NH2-coated surfaces after the aforementioned procedures. Cell lines including the murine preosteoblast cell line (MC3T3-E1 subclone 4, CRL-2593, ATCC, USA) and bone marrow mesenchymal stem cells (BMMSCs, isolated from mouse epiphysis

24

) were used to examine the cell proliferation or osteogenesis

activities. The culturing of these cells was seeded at a density of 1×104 cells/cm2 on the studied surfaces. MC3T3-E1 was removed with trypsin and re-seeded to the same well before and after the PPX-SS-NH2-FGF-2 surfaces were treated with GSH at various times on day 1, day 2, and day 3. To examine the MC3T3-E1 proliferation, the studied surfaces included cleaved FGF-2 protein on day 1, day 2, and day 3 and a non-cleaved FGF-2 surface. A control experiment of a surface without the FGF-2 immobilization was conducted in parallel. During the cleavage of FGF-2, the cultured MC3T3-E1 was removed with trypsin for cryopreservation at -80 °C, and the cells were re-seeded back to the same sample surfaces after the completion of surface modifications. The resulting cultured sample surfaces were observed and photographed using an inverted microscope (Leica, German) for their proliferation activities. The cell number was calculated as follows: the cell suspensions were diluted in a 1:1 ratio with a 0.4% Trypan Blue solution (Sigma Aldrich, USA), and then the cell number and viability were determined by dye exclusion of live cells on a hemocytometer. To examine the osteogenesis activities of the cultured MC3T3-E1 or BMMSCs on surfaces, the characteristics of the early-stage ALP


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expression were examined 7 days later (day 10) after the first BMP-2 immobilization, and the late-stage calcium deposition and/or osteocalcin expression was examined 14 days later (day 14 or day 17). The studied surfaces included cleaved BMP-2 protein on day 3, day 7, and day 10 and a non-cleaved BMP-2 surface; a control experiment of a surface without the BMP-2 immobilization was conducted in parallel. A similar procedure of removing/re-seeding the MC3T3-E1 cells on the same sample surface was performed during the cleavage/reinstallation step of the surface modification with BMP-2, while the identical surface modification was performed on the cultured BMMSCs without removing the cells from the culture surface. Specifically, the cells were fixed with 4% paraformaldehyde for 30 min and then stained with an Alkaline Phosphatase Detection kit (Millipore, USA) in the dark for 30 min for the ALP expression analysis. The fixed cells were stained with a 2% Alizarin red staining solution (Sigma Aldrich, USA) for 30 min for the calcium deposition analysis. Three washing cycles were performed using deionized water during each staining process. The osteocalcin expression was analyzed using an immunofluorescence assay in which the fixed cells were further treated in 0.5% TritonX-100 (Sigma Aldrich, USA) at 4 °C for 10 min, rinsed three times with PBS, and then incubated in 1% bovine serum albumin (BSA; Sigma Aldrich, USA) at room temperature for 30 min. Then, the samples were examined with the corresponding rabbit polyclonal antibody (Abcam, USA) against osteocalcin for 1 h. The cells were further stained with FITC-labeled goat anti-rabbit IgG (Abcam, USA) and DAPI at room temperature for 1 h. A careful rinsing process using PBS (pH = 7.4) three times was performed during each conjugation and staining procedure. Each experiment was conducted in triplicate.


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Figure 1. Schematic illustration of using a PPX-SS-NH2 coating to enable a stepwise and programmable biointerface. The cleavage of growth factor proteins can slow down or cease the already induced biological activity, and the reinstallation of a second factor protein of another kind can re-initiate a divergent differentiation activity.


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Figure 2. (a) IRRAS spectra of the stepwise immobilization and displacement of FGF-2 and BMP-2 on the PPX-SS-NH2 coating-modified surfaces. The peaks at approximately 3036 to 3601 cm-1, attributed to the characteristic -NH band adsorption from FGF-2 or BMP-2 and showed a significant higher intensity for the immobilized FGF-2 or after the reinstallation of BMP-2 on the same sample surface, compared to low -NH intensity for the cleavage of FGF-2. (b) QCM dynamic analysis of the binding affinity for the immobilized first growth factor protein (FGF-2 or BMP-2) and the reinstalled second growth factor (BMP-2 or FGF-2). Two specific


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antibodies, including human FGF-2 antibody and human BMP-2 antibody, were used to crossexamine the specific binding efficiency toward the two growth factor proteins.


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Figure 3. Cell proliferation of MC3T3-E1 was examined on the culture surfaces with a varied time frame of cleaving the previously immobilized FGF-2 proteins from the culture surfaces. (a) Cell growing pattern of MC3T3-E1 on the surfaces with immobilized FGF-2, which was cleaved on day 1, day 2, and day 3. Control experiments with no cleavage of FGF-2 and surfaces without FGF-2 were used for comparison. The images were observed and captured on day 4. (b) The resulting cell number of the grown MC3T3-E1 was calculated, and the day 4/day 1 ratio was analyzed to evaluate the cell proliferation efficacy for the studied culture surfaces. Each bar represents the mean value (± SD) of three independent experiments.


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Figure 4. Osteogenesis activities of MC3T3-E1 were examined on the culture surfaces with varied time frames for cleaving previously immobilized BMP-2 proteins from the culture surfaces. The BMP-2 was cleaved on day 3, day 7, and day 10 for the culture surfaces. Control experiments of no BMP-2 cleavage and surfaces without BMP-2 were conducted for comparison. The characteristics of osteogenetic patterns, which include the (a) calcium deposition and (b) osteocalcin expression, were analyzed on day 14. Alizarin red was used to stain mineralized calcium, and FITC-IgG was used to detect osteocalcin in the green channel. The overlaid blue channel images indicate the DAPI-stained cell nuclei, and the cell density is


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indicated at the bottom of each image. (c) The statistical results of (a) and (b) were analyzed and compared. Each bar represents the mean value (± SD) of three independent experiments.


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Figure 5. The combined activities of the MC3T3-E1 proliferation and osteogenesis were examined via a stepwise immobilization and displacement of FGF-2 and BMP-2 on identical cell culture surfaces. (a) Induced cell proliferation of MC3T3-E1 by the previously immobilized FGF-2 (cleaved at day 3) was confirmed by showing the growing pattern and the cell number ratio of day 4/day 1. Osteogenesis activities including the early-stage marker of (b) ALP expression were observed on day 10, and the late-stage markers of (c) calcium deposition and (d) osteocalcin expression were detected on day 17. The cell density is indicated at the bottom of each image. (e) The statistical results of the osteogenesis activities in (b)-(d) were analyzed and compared. Each bar represents the mean value (± SD) of three independent experiments.


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ASSOCIATED CONTENT Supporting Information.

1

H-NMR,

13

C-NMR and ESI-MS spectrums of 4-(3-((3-

methylamido)disulfanyl)propanoic acid) [2,2]paracyclophane; XPS high-resolution C 1 s and S 2p spectra of PPX-SS-NH2 coating; IRRAS characterization of PPX-SS-NH2 coating; control experiments of non-displaceable poly-p-xylylenes. 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 £

Z-Y Guan and C-Y Wu contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT


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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). The authors would like to thank the National Taiwan University Mass Spectrometry-based Proteomics Core Facility for conducting the ESI-MS analysis.

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For Table of Contents Use Only Stepwise and Programmable Cell Differentiation Pathways of Controlled Functional Biointerfaces Zhen-Yu Guan, Chih-Yu Wu, and Hsien-Yeh Chen

An advanced biointerface is established by using vapor-based polymer coatings to enable stepwise and switchable activities of the immobilized growth factor (GF) proteins for a programmed manipulation over cell differentiation pathways.


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Stepwise and Programmable Cell Differentiation Pathways of

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