Plant Flavonoid-Mediated Multifunctional Surface Modification

Article pubs.acs.org/cm

Plant Flavonoid-Mediated Multifunctional Surface Modification Chemistry: Catechin Coating for Enhanced Osteogenesis of Human Stem Cells Jung Seung Lee,† Jong Seung Lee,† Min Suk Lee,‡ Soohwan An,† Kisuk Yang,† Kyueui Lee,§ Hee Seok Yang,‡ Haeshin Lee,§ and Seung-Woo Cho*,†,∥ †

Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Republic of Korea § Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea ∥ Center for Nanomedicine, Institute for Basic Science (IBS), Seoul 03722, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Application of surface chemistry using bioactive compounds enables simple functionalization of tissue-engineering scaffolds for improved biocompatibility and regenerative efficacy. Recently, surface modifications using natural polyphenols have been reported to serve as efficient multifunctional coating; however, there has yet to be any comprehensive application in tissue engineering. Here, we report a simple, multifunctional surface modification using catechin, a phenolic compound with many biological functions, found primarily in plants, to potentiate the functionality of polymeric scaffolds for bone regeneration by stem cells. We found that catechin hydrate can be efficiently deposited on the surface of various substrates and can greatly increase hydrophilicity of the substrates. While identifying the chemical mechanisms regulating catechin surface coating, we found that catechin molecules can self-assemble into dimers via cation−π interactions. Interestingly, the intrinsic biochemical functions of catechin coating provided the polymer scaffolds with antioxidative and calcium-binding abilities, resulting in enhanced adhesion, proliferation, mineralization, and osteogenic differentiation of human adipose-derived stem cells (hADSCs). Ultimately, catechin-functionalized polymer nanofiber scaffolds significantly promoted in vivo bone formation by hADSC transplantation in a critical-sized calvarial bone defect. Our study demonstrates that catechin can provide a biocompatible, multifunctional, and cost-effective surface modification chemistry to produce functional scaffolds with improved tissue regenerative efficacy.

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proliferation, differentiation, and maturation.12−15 However, covalent modifications usually require complicated chemical reactions, treatment with γ-rays or plasma, and the use of strong acids/bases, which alter intrinsic surface properties and denature the bioactive molecules.13−16 The efficiency of this strategy is also entirely dependent upon the type of surface material used.13,17 Therefore, the development of a novel, simple surface modification chemistry in a material-independent manner with biocompatible and cost-effective compounds is ideal for the generation of functional scaffolds with tissueengineering applications. Organic phenolic compounds can be used for highly efficient surface functionalization due to their biocompatibility, intrinsic biochemical properties, and accessibility. In particular, phenols and polyphenols containing catechol groups (e.g., dopamine,

INTRODUCTION

Surface chemistry is essential for improving the functionality and efficacy of tissue-engineering scaffolds and biomedical devices. Numerous chemical and physical approaches for surface modification have been reported to immobilize chemicals, peptides, proteins, and genes on the surfaces of various materials.1−4 Physical adsorption of chemicals or biomolecules can allow for simple functionalization of material surfaces by changing surface properties (e.g., surface charge and hydrophilicity) to promote protein and cell adhesion;5−8 however, this method usually mediates only weak interaction of immobilized molecules with the substrate, leading to limited long-term stability and significant loss of surface modification.9 Electrostatic interaction with polycations (e.g., poly-L-lysine, poly-L-ornithine) have been used for surface functionalization of substrates and scaffolds; however, this method may cause cytotoxicity.10,11 Covalent grafting of bioactive molecules on the substrate allows for more stable immobilization and longlasting functionality of adhered molecules for cellular adhesion, © 2017 American Chemical Society

Received: February 24, 2017 Revised: April 22, 2017 Published: April 24, 2017 4375

DOI: 10.1021/acs.chemmater.7b00802 Chem. Mater. 2017, 29, 4375−4384

Article

Chemistry of Materials

Figure 1. Characterization of catechin hydrate surface coating. (A) Schematic illustration of catechin hydrate surface coating and its multifunctional application in biomedical engineering. (B) Water contact angle measurement on a PS dish before and after catechin coating (n = 3). (C) SEM images of uncoated and catechin-coated surfaces. Scale bar = 500 nm. (D) Gross views (upper) and SEM images (lower) of each group before and after AgNO3 solution treatment. Scale bar = 500 nm. (E) Atomic surface composition measured by XPS analysis for C 1s (upper) and O 1s (lower) on uncoated and catechin-coated PS dishes. (F, G) Confirmation of catechin coating on various substrates. Water contact angle on each substrate was measured before and after catechin surface modification (n = 3, *p < 0.05, and **p < 0.01 versus no coating group for each substrate).

norepinephrine, taxifolin, and quercitrin) have become popular multifunctional bioinspired coating materials for modifying surface properties and adding specific functionalities to substrates.18−23 Oxidation of the catechol moiety in phenolic compounds is known to induce self-polymerization and π−π stacking of the molecules in basic conditions, providing material-independent surface coating.24 In addition, bioactive molecules containing various nucleophiles (e.g., amine, thiol, and imidazole) can be efficiently immobilized on the catecholmodified substrates, which further improves functionality of the surface.25,26 Recent studies have reported highly efficient, biocompatible surface modification methods using polyphenols containing galloyl groups from plant flavonoids.27−34 The galloyl group-based surface modification is especially advantageous as it can be readily achieved by a short and simple dipping process and it does not induce color or morphological changes in the substrates.27−29

In the present study, we established a surface modification mediated by catechin, a representative plant flavonoid, to functionalize tissue-engineering scaffolds. Catechins are mainly present in plants (e.g., tea leaves, coffee beans, and cocoa) and have many interesting biological functions, such as antioxidation, anti-inflammation, and anticarcinogenesis. 35−37 Although biological and pharmacological activities of soluble catechin have been widely studied, there have been no studies using catechin as a surface coating material to prepare scaffolds with improved tissue regenerative capacity. Among various plant flavonoids, catechin hydrate is more economically advantageous than the most widely studied catechin derivatives such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin. Here, we propose that catechin hydrate is a simple, multifunctional, material-independent coating compound, and we describe its 4376


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High-Performance Liquid Chromatography Mass Spectrometry Analysis. Catechin solution (2 mg/mL in saline) was incubated at room temperature for 1 and 14 h and filtered using 0.45 μm filter (Minisart, Sartorius Stedim Biotech, Goettingen, Germany) before being injected into the high-performance liquid chromatography instrument (HPLC; 1260 Infinity, Agilent Technologies, Palo Alto, CA, USA). Mass spectrometry (MS; 120, Agilent Technologies) was then performed with the positive ion mode using the Discovery BIO wide pore C18 column (3 μm, Supelco Inc., Bellefonte, PA, USA). A mobile phase (DW/acetonitrile [ACN] mixture in 0.1% (v/v) trifluoroacetic acid [TFA]) was flowed (0.5 mL/min−1) at the following intervals: (1) 95 to 5% water for 45 min, (2) 5 to 95% water for 50 min, and (3) 95 to 5% water for 60 min. The separated samples were measured using ultraviolet−visible spectrophotometer (UV−vis, HP8453, Hewlett-Packard, Palo Alto, CA, USA) at a 280 nm wavelength. Protein Adsorption Test. To determine the amount of protein adsorption, uncoated and catechin-coated (5 mg/mL) substrates were incubated with 10-fold diluted serum solution for 6 and 12 h at 37 °C. Mouse serum was acquired by the retro-orbital bleeding method.38 At each time point, the remaining protein in the retrieved serum solution was quantified using BCA protein assay (Thermo Scientific, Waltham, MA, USA), and the amount of protein adhered to the substrates was calculated by subtracting the values from the total amount of initial protein. Cell Culture and Adhesion Tests. HUVECs were expanded and cultured in endothelial growth medium-2 (EGM-2, Lonza, Walkersville, MD, USA). hADSCs were purchased from ATCC (Rockville, MD, USA) and cultured in mesenchymal stem cell growth medium (ATCC) during expansion and in vitro experiments. For cell adhesion tests, HUVECs (1.0 × 104 cells/cm2) and hADSCs (5.0 × 103 cells/ cm) were seeded on either uncoated or catechin-coated (2 and 5 mg/ mL) substrates and then fixed with 4% paraformaldehyde (Sigma) at 4 and 24 h after seeding. Subsequently, the fixed cells were immunofluorescently stained with the focal adhesion staining kit (Millipore, Temecula, CA, USA) following the manufacturer’s protocol. Cell nuclei were counterstained with 4′,6-diamidino-2phenylindole (DAPI, TCI, Nihonbashi-honcho, Chuo-ku, Tokyo), and the staining was visualized using confocal microscopy (LSM 700, Carl Zeiss, Oberkochen, Germany). The cell coverage area was calculated by measuring the area covered by the cells using ImageJ (National Institute of Health, Bethesda, MD, USA). The total area of the acquired images was set as 100%. Cell Viability and Proliferation. Cells were seeded (HUVECs, 5.0 × 103 cells/cm2; hADSCs, 4.0 × 103 cells/cm2) on either uncoated or catechin-coated (2 and 5 mg/mL) substrates, and viability was examined using the Live/Dead viability/cytotoxicity kit (Invitrogen, Carlsbad, CA, USA) after 1 and 4 days of culturing. Cell proliferation was evaluated by measuring mitochondrial metabolic activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma) after 1 and 4 days of culturing. To test the reactive oxygen species (ROS)-scavenging ability of the catechin coat, cells were seeded on uncoated and catechin-coated (2 and 5 mg/mL) PCL nanofiber scaffolds and allowed to adhere to the scaffolds for 12 h. Subsequently, cells were treated with 0.2 mM hydrogen peroxide (H2O2, Sigma) for 12 h, and the relative viability of cells on each substrate was examined with the MTT assay. Osteogenic Differentiation of hADSCs. Osteogenic differentiation of hADSCs and characterization of the differentiated stem cells were performed as previously described with minor modifications.17 For in vitro osteogenic differentiation of hADSCs, the cells were seeded (1.5 × 104 cells/cm2) and induced to differentiate to the osteogenic lineage by culturing them in osteogenic induction medium (Dulbecco’s modified Eagle’s medium [DMEM], high-glucose [Gibco, Gaithersburg, MD, USA], supplemented with 100 nM dexamethasone [Sigma], 50 μg/mL L-ascorbic acid [Sigma], 10 mM β-glycerophosphate [Sigma], 10% [v/v] fetal bovine serum [FBS, Gibco], 3.7 g/L sodium bicarbonate [Sigma], and 1% [v/v] penicillin/streptomycin [Gibco]) when cells reached 70% confluency. After 3 weeks of differentiation induction, cells were characterized by immunofluor-

potential use in cell and tissue engineering, especially in stem cell-mediated bone regeneration (Figure 1A). For surface modification, catechin was deposited onto the substrates made of various materials by using the simple dipcoating method.29 Catechin surface coating increased hydrophilicity of material surfaces without inducing color or morphological changes. We found that physical assembly of catechin molecules mediated by cation−π interactions might contribute to material-independent surface modification with thin catechin layer formation. The catechin coating remarkably improved adhesion and proliferation of human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs), even under oxidative stress conditions, thanks to its antioxidative activity. Interestingly, mineralization and osteogenic differentiation of hADSCs was enhanced due to the calcium-binding affinity of the catechin modification. Catechinfunctionalized polycaprolactone (PCL) nanofibrous scaffolds transplanted with hADSCs facilitated new bone formation in a mouse model with a critical-sized calvarial bone defect. To the best of our knowledge, this is the first comprehensive study reporting catechin-mediated surface modification chemistry and its potential application for stem cell-mediated tissue engineering.

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EXPERIMENTAL SECTION

Catechin Coating. (+)-Catechin hydrate (Sigma, St. Louis, MO, USA) was dissolved in saline (0.9% sodium chloride, CJ Healthcare, Seoul, Korea), 0.1 M calcium chloride (CaCl2, Sigma), or 0.1 M iron chloride (FeCl3, Sigma) solution. Subsequently, various substrates (polystyrene [PS], silicon oxide [SiO2], titanium oxide [TiO2], polycaprolactone [PCL] nanofiber, glass coverslip, gold [Au], polytetrafluoroethylene [PTFE], and polydimethylsiloxane [PDMS]) were dipped in the catechin solution and incubated for 12 h at room temperature. The coated substrates were then gently washed with distilled water (DW) three times before use. To test the role of Na+ in catechin coating chemistry, solvents without Na+ supplementation 10 mM Tris-hydrochloride buffer (Tris-HCL, pH 8.5, USB Corp., Cleveland, OH, USA) and DWwere used for the catechin coating tests. Surface Characterization of Catechin-Coated Substrates. Surface morphology of substrates was characterized using field emission scanning electron microscopy (FE-SEM; JSM-7001F, JEOL Ltd., Tokyo, Japan). The surface roughness of substrates was evaluated by atomic force microscopy (AFM; JPK Instrument, Berlin, Germany). Hydrophilicity of substrates before and after catechin coating was evaluated by measuring the water contact angle of 10 μL drops of DW using an automatic contact angle analyzer (n = 3, Phoenix 300, SEO Co., Suwon, Korea). Surface atomic composition of each substrate was analyzed using X-ray photoelectron spectroscopy (XPS; Escalab 220iXL, VG Scientific Instruments, East Grinstead, U.K.). For macroscopic visualization of even distribution of the catechin coat, uncoated and catechin-coated PS substrates were treated with 100 mM silver nitrate (AgNO3, Sigma) solution and incubated for 6 h at room temperature. After washing with DW, the substrates were photographed and silver particle formation was further confirmed by FE-SEM images. The thickness of the catechin coating was measured using a spectroscopic ellipsometer (n = 6, J. A. Woollam Co., Lincoln, NE, USA) at a 70° angle of incidence. To confirm calcium deposition mediated by catechin, uncoated and catechin-coated (2 and 5 mg/mL) PS substrates were incubated with 2.5 mM calcium chloride (CaCl2, Sigma) solution at 37 °C for 7 days, and then surface atomic composition was examined using XPS analysis. The stability of the catechin coating on the substrates was indirectly evaluated by measuring the water contact angle before and after sonication using a probe-type sonicator with 30% amplitude for 5 min. In surface characterization experiments, 2 mg/mL catechin hydrate solution was used to coat the substrates. 4377


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Figure 2. Surface chemistry of catechin hydrate coating. (A) HPLC analysis after incubation of catechin solution (2 mg/mL in saline) for 1 and 14 h. (B) Mass spectrometric analysis of the eluate after 14 h of incubation. (C) Proposed chemical mechanism of physical dimeric self-assembly of catechin molecules during incubation for surface coating. (D, E) Evaluation of the stability of catechin surface coating. Water contact angle on uncoated and catechin-coated PS dishes before and after sonication for 5 min with 30% amplitude (n = 4). escence staining, real-time quantitative polymerase chain reaction (qPCR), and alizarin red staining. For immunostaining, the differentiated cells were fixed with 4% paraformaldehyde (Sigma) and stained using antiosteopontin (OPN; 1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anticollagen type I (COL I; 1:50, Calbiochem, San Diego, CA, USA) antibodies. The cells were further stained with Alexa-Fluor 488- and 594-conjugated secondary antibodies (Invitrogen) and DAPI, and then examined using confocal microscopy (Carl Zeiss). qPCR was performed with osteogenic marker genes (human COL I [COL1A2; Hs01028969_m1], human OPN [OPN; Hs00959010_m1], and human osteocalcin [OCN; Hs01587814_g1]) and endogenous control (human glyceraldehyde3-phosphate dehydrogenase [GAPDH; Hs02758991_g1]) as previously reported.17 Calcium deposition by the differentiated hADSCs was evaluated and calculated using alizarin red staining and image processing as previously described.39 Bone Regeneration Assay with Mouse Calvarial Defect Model. Randomly aligned 4 mm wide PCL nanofiber scaffolds were prepared and coated with catechin solution (5 mg/mL in saline) overnight at room temperature. To evaluate enhanced bone regeneration by cells transplanted with catechin-coated scaffolds, hADSCs were seeded (1.0 × 106 cells/scaffold) onto uncoated and catechin-coated PCL nanofiber scaffolds and cultured in osteogenic

induction medium to commit the cells to the osteogenic lineage for 5 days before transplantation. The critical-sized calvarial defect mouse model (defect size, 4 mm) preparation and cell transplantation were performed as previously described.40 The groups were divided as follows: (i) no treat, (ii) only PCL scaffold (PCL), (iii) only catechincoated PCL scaffold (PCL-Cat), (vi) PCL scaffold with hADSCs (PCL-hADSC), and (v) catechin-coated PCL scaffold with hADSCs (PCL-Cat-hADSC). All animal experiments were performed with the protocol approved by Institutional Animal Care and Use Committee (IACUC) of the Yonsei Laboratory Animal Research Center (YLARC) (IACUC-201606-449-01). Eight weeks after transplantation, the calvarial bones were collected, fixed with 4% paraformaldehyde (Sigma), and analyzed using microcomputed tomography (micro-CT, SkyScan-1172, SkyScan, Kontich, Belgium). Bone volume was quantified using an image analysis software (CTAn, Skyscan). Soft X-ray images were acquired through three-dimensional reconstitution of the micro-CT results using the DataViewer program (Skyscan) to evaluate mineral density of the regenerated bone. For histological analysis, the fixed samples were embedded in paraffin and sectioned into 6 μm tissue slides. The sectioned slides were stained with Goldner’s Trichrome method.40 4378


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Chemistry of Materials Statistical Analysis. Quantitative data are presented as mean ± standard deviation. Statistical significance was evaluated using unpaired Student’s t test and one-way analysis of variance (ANOVA) followed by Tukey to compare all pairs of columns with GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). A p value under 0.01 and 0.05 was considered statistically significant.

coating of catechin hydrate would be more beneficial on tissueengineering scaffolds, substrates, and devices with nano- or microscale structures on their surface because self-polymerized catecholic surface modifications usually induce formation of thick coating layers with large aggregates that may interfere with the effect of inherent surface geometry and structures on cellular behavior control.26 Surface Chemistry of Catechin Coating. The surface chemistry of the catechin coating was elucidated by examining chemical reaction modes of self-assembling catechin molecules. Physical self-assembly of material-independent surface modifying polyphenols is known to be as crucial as covalent polymerization in oxidative conditions.24 In order to investigate physical assembly of catechin hydrate during surface coating processes, HPLC-MS analysis was performed after incubating catechin solution (2 mg/mL in saline, pH 7) at room temperature for 1 and 14 h. In both conditions, only a single peak appeared after a 10 min elution, demonstrating that there was no polymerization of catechin molecules (Figure 2A). Subsequently, the eluate from the solution incubated for 14 h was analyzed using mass spectrometry and three mass-to-charge ratios (m/z) were detected at 291, 313, and 603 m/z (Figure 2B). The mass peak at 291 m/z represents a single catechin hydrate molecule, and the peak at 313 m/z indicates a catechin−sodium ion (Na+) integrated form. There was no indication of the formation of catechin−catechin adducts with or without Na+ up to 14 h postincubation, which is represented by the lack of peaks at 579 or 601 m/z, respectively (Figure 2B). Interestingly, a peak was detected at 603 m/z, which indicates physical self-assembly of two catechin hydrate molecules with Na+; however, there was no peak at 581 m/z, indicating catechin molecules did not self-assemble without Na+ (Figure 2B). These results suggest that cation−π interaction (Na+− catechin) is essential for physical dimeric self-assembly of catechin molecules during the catechin coating process. When catechin was dissolved in solvents (DW or Tris-HCl buffer) without Na+ and used for PS surface coating, the water contact angle was only slightly reduced (Figure S2; DW, 64.8 ± 6.0°; Tris-HCl, 64.5 ± 4.1°) compared to substrates coated in catechin dissolved in a saline (Na+-rich) solution (Figure 1b, 27.8 ± 3.6°). This suggests that coating efficiency is significantly decreased in the absence of Na+. From mass spectrometry analyses and water contact angle measurements, we conclude that physical assembly of catechin dimers mediated by cation−π interactions and subsequent interactions between the physically stacked dimers and the substrates (e.g., π−π interaction, hydrogen bonding, and so on) are primary chemical mechanisms of catechin surface coating (Figure 2c), rather than chemical polymerization, which was previously regarded as the main mechanism for polyphenolic compoundbased surface modification.24,41 Because catechins are known to form polymers only under specific conditions in the presence of enzymes or chemicals,42,43 it can be inferred that physical selfassembly of two catechin molecules with Na+ is a primary mode of catechin surface coating chemistry in our study. We also tested the feasibility of catechin surface coating by using other cations (e.g., Ca2+ and Fe3+). To this end, catechin (2 mg/mL) was dissolved in 0.1 M calcium chloride (CaCl2) or 0.1 M iron(III) chloride (FeCl3) solution, and then coated onto PS substrates. From the analysis of water contact angle measurement, it was confirmed that the hydrophilicity of catechin-coated substrates with Ca2+ and Fe3+ was increased

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RESULTS AND DISCUSSION Characterization of Catechin Surface Coating. The catechin surface coat was characterized on the commonly used PS cell culture substrate. For surface modification, catechin hydrate was dissolved (2 mg/mL) in saline and coated on the PS plate, which was then incubated overnight at room temperature. Similar to other surface modification approaches with phenolic compounds,27,28 the catechin coating significantly increased surface hydrophilicity (no coating of 77.6 ± 2.3° versus catechin coating of 27.8 ± 3.6°) (Figure 1b). However, there was no color change or morphological difference observed in the substrate before and after catechin coating, which was confirmed by FE-SEM (Figure 1C). When the surface roughness of the substrates before and after catechin coating was compared by using AFM analysis, a thin layer of catechin coating reduced the roughness of the substrates from 6.863 to 2.443 nm without significant morphological changes of the surface (Supporting Information Figure S1). In order to macroscopically visualize the uniform distribution of catechin coating, the catechin-coated PS surface was treated with AgNO3 solution. It has been well-documented that catechol groups can bind to silver ion (Ag+) through reduction and oxidation, forming a thin silver layer on the catechol-modified surface.27 Upon addition of AgNO3, brown-colored silver nanoparticles were observed only on the catechin-coated substrate, while there were few aggregate formations on the uncoated substrate (Figure 1D). The change in surface atomic composition by catechin coating was confirmed by XPS. The C 1s shoulder peak near 286 eV and O 1s peak at 531.8 eV appeared on the catechin-coated surface, indicating successful catechin deposition (Figure 1E). In order to confirm the versatility of catechin surface modification, the catechin coat was tested on other material substrates (SiO2, TiO2, PCL nanofiber, glass coverslip, Au, PTFE, and PDMS) that are used as tissue-engineering scaffolds and medical devices (Figure 1F). In all tested substrates, water contact angle was significantly reduced after catechin surface coating (Figure 1F,G). Interestingly, the hydrophilicity of surfaces coated with catechin varied among the substrates and was closely dependent on intrinsic surface properties of each material (Figure 1G). We speculate that catechin coating may be too thin to completely decouple the original surface properties of the bare substrates, yet is still thick enough to increase the hydrophilicity and specific functionality of the substrates. Indeed, when the thickness of the catechin coating on different substrates including Si, SiO2, and TiO2 was measured by using ellipsometer, catechin formed a thin nanolayer on both Si (2.3 ± 0.8 nm) and SiO2 (3.4 ± 0.4 nm) substrates. Although there was a slight increase in the thickness on TiO2 substrate (15.4 ± 1.3 nm), thin catechin coating was confirmed on all tested metal or metal-oxide substrates. The thickness of the catechin coating might be varied dependent upon the degree of interactions between catechin molecules and surface materials and could also be controlled by adjusting coating conditions such as pH, coating time, and cation concentration.27,29 In particular, a thin surface 4379


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Figure 3. Adhesion, viability, and proliferation of hADSCs on catechin-coated substrates. (A) Immunofluorescent staining of vinculin (green), Factin (red), and DAPI (blue). Scale bar = 100 μm. (B) Surface area covered by attached cells measured 4 and 24 h after seeding (n = 6, *p < 0.05, and **p < 0.01 versus no coating group, ##p < 0.01 versus catechin (2) group). (C) Live/Dead staining and (D) MTT assay of hADSCs after 1 and 4 days of growth on uncoated and catechin-coated (2 and 5 mg/mL) substrates (n = 3, *p < 0.05, and **p < 0.01 versus no coating group; #p < 0.05 and ##p < 0.01 versus catechin (2) group). Scale bar = 200 μm. (E) Relative viability of hADSCs cultured on uncoated and catechin-coated (2 and 5 mg/mL) PCL nanofiber scaffolds after 12 h of 0.2 mM H2O2 treatment (n = 3, *p < 0.05, and **p < 0.01 versus no treat group for each substrate). The viability of hADSCs was normalized to the value of the no treat group for each substrate.

(CaCl2, 38.1 ± 0.2°; FeCl3, 29.7 ± 1.9°), compared to that of bare substrate (DW, 64.8 ± 6.0°) (Figure S2), indicating that those cations could also contribute to surface modification by catechin. It has been known that various metal ions can form complexes with catecholates, though the degree of interactions with catecholates may differ among various cations.44 In addition, natural polyphenolic compounds were previously confirmed to form thin film on various surfaces via metal-oxide coordination (e.g., Fe3+).30 Although more detailed chemical mechanisms of other cation-mediated catechin coating should be further elucidated in future studies, we could conclude that other cations (Ca2+ and Fe3+) in addition to Na+ may also be able to induce catechin coating. The stability of surface modification chemistry is important for long-term functionality of the coated substrates. The stability of the catechin surface coating was tested by inducing physical dissociation using a probe-type sonicator. Uncoated and catechin-coated PS substrates were sonicated for 5 min (30% power), and the change in hydrophilicity of each substrate was compared (Figure 2D,E). We found that the water contact angle on catechin-coated substrates slightly increased from 27.6 ± 2.3° to 38.6 ± 5.2°, demonstrating that although there was partial detachment of catechin molecules, the coating was largely maintained on the surface even after harsh physical dissociation. Although the catechin coating seems to be thin and induced mainly by physical self-assembly of catechin molecules, the interaction of the catechin polymers with the substrates was found to be stable and durable.

Enhanced Cellular Adhesion and Proliferation by Catechin Coating. Enhancement of cell adhesion and proliferation on scaffolds and substrates is crucial for successful tissue regeneration. Previous studies have reported that catecholic compound-based surface modification greatly improved cellular adhesion and proliferation by increasing surface hydrophilicity and interactions of catechol moieties with serum proteins, cell surface molecules, and immobilized bioactive molecules.22,26,45 We confirmed that catechin coating also improved adsorption of serum proteins on substrates (Figure S3). The applicability of catechin surface modification for tissue engineering was first tested by examining adhesion and proliferation of hADSCs (Figure 3). To evaluate initial cell attachment, hADSCs were cultured on uncoated or catechincoated (2 and 5 mg/mL) PS substrates, and underwent immunofluorescent staining for focal adhesion proteins (vinculin) and cytoskeletal proteins (filamentous actin, Factin) (Figure 3A). Quantification of the cell adhesion area showed that the catechin coat accelerated adhesion and spreading of cells at 4 and 24 h after seeding (Figure 3A,B). Live/Dead staining indicated that the catechin coat did not cause cytotoxicity (Figure 3C). Increased spreading morphology of the attached cultured cells was observed in catechincoated groups compared to the uncoated group at days 1 and 4 (Figure 3C). In addition, hADSCs cultured on the catechincoated substrates showed greater proliferative ability in a dosedependent manner (2 and 5 mg/mL) compared to those on uncoated substrates at day 1 and 4, as confirmed by MTT assay (Figure 3D). Similarly, catechin coating (2 and 5 mg/mL) 4380


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Figure 4. In vitro osteogenic differentiation of hADSCs on catechin-coated substrates. (A) Immunofluorescent staining of OPN (green), COL I (red), and DAPI (blue). Scale bar = 100 μm. (B) Gene expression analysis of COL1A2, OPN, and OCN by qPCR (n = 3, *p < 0.05, and **p < 0.01 versus no coating group; ##p < 0.01 versus catechin (2) group). The gene expression in each group was normalized to that of the no coating group. (C) Alizarin red staining for evaluating calcium deposition of osteogenically differentiated hADSCs (scale bar = 200 μm) and (D) its quantification (n = 8, **p < 0.01 versus no coating group; ##p < 0.01 versus catechin (2) group). (E) XPS analysis of Ca 2p peak after incubating uncoated and catechin-coated (2 and 5 mg/mL) substrates with CaCl2 solution for 7 days.

significantly improved initial cellular adhesion, spreading, and proliferation of HUVECs, compared to the uncoated group (Figure S4A−D). Overall, catechin coating enhanced cell adhesion, spreading, and proliferation, most likely due to increased surface hydrophilicity and surface energy, and subsequent serum protein adsorption. However, the exact mechanisms of the interaction between cells and catechinmodified substrates need to be elucidated in future studies. In addition to the ability to alter surface properties, catechin coating may contribute to cell maintenance, and increase cell viability and proliferation on the tissue-engineering scaffolds due to catechin’s intrinsic biochemical properties. The catechol

moiety in catechin hydrate is crucial for its antioxidative properties and its ability to scavenge ROS.46 The ability of catechin-coated surfaces to protect cells from oxidative stress was evaluated by treating hADSCs and HUVECs cultured on PCL nanofiber scaffolds with 0.2 mM H2O2. Electrospun PCL nanofiber scaffolds with a larger surface area than conventional microporous scaffolds were used to maximize the ROSscavenging ability of the catechin coating. FE-SEM images showed that there was no structural or morphological change after catechin was coated on the PCL nanofiber scaffolds (Figure S5). H2O2 treatment reduced the viability of hADSCs and HUVECs grown on the uncoated PCL nanofiber scaffolds; 4381


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Figure 5. In vivo bone regeneration by hADSC transplantation with catechin-coated PCL nanofiber scaffolds in critical-sized calvarial bone defect mouse model. (A) Micro-CT images of the defect site (scale bar = 1 mm) 8 weeks after transplantation. Scale bar = 1 mm. Quantification of (B) the bone coverage area and (C) bone volume of the defect site after hADSC transplantation, evaluated from the micro-CT images (n = 10, **p < 0.01 versus no treat group; #p < 0.05 and ##p < 0.01 versus PCL group; +p < 0.05 versus PCL-Cat group). (D) Colorized mineral maps reconstituted from the cross-sectioned micro-CT images. White arrowheads indicate the defect region. Scale bar = 1 mm. (E) Goldner’s Trichrome staining of each group (left, scale bar = 1 mm) and their enlarged images (right, scale bar = 100 μm). Black arrowheads indicate the defect region.

and HUVECs. It has been shown that reduction in oxidative stress promotes osteogenic differentiation of stem cells by controlling ROS-related signaling pathways such as Wnt and Hedgehog signaling.49 Increased calcium deposition induced by the catechin coat points to a different explanation for the improved osteogenesis of hADSCs. Ryu et al. found an increase in hydroxyapatite crystallization on polydopamine coats due to high binding affinity of the catechol group of polydopamine to calcium.50 Therefore, we next examined catechin-mediated calcium deposition on substrates using XPS analysis (Figure 4E). After incubating uncoated and catechin-coated (2 and 5 mg/ mL) PS substrates with 2.5 mM CaCl2 solution for 7 days, distinct peaks of Ca 2p representative of enhanced calcium deposition were observed in catechin-coated groups, whereas a negligible increase of Ca 2p peaks was observed in the uncoated group (Figure 4E). Therefore, we conclude that catechin coating facilitates deposition of calcium from both osteogenically differentiated hADSCs and supplemented medium on substrates, thereby promoting mineralization and ultimately enhancing osteogenesis of hADSCs. Improved in Vivo Bone Regeneration of hADSCs by Catechin Surface Modification. Finally, in vivo bone regeneration of hADSC transplantation using catechinmodified scaffolds was evaluated in a mouse model with critical-size calvarial bone defects. Undifferentiated hADSCs were seeded on uncoated and catechin-coated (5 mg/mL) PCL nanofiber scaffolds and cultured in osteogenic induction medium to induce hADSCs to assume the osteogenic lineage for 5 days before transplantation. Eight weeks after hADSCs were transplanted with PCL scaffolds into calvarial bone defects, bone regeneration was evaluated using micro-CT and histological analyses. Micro-CT data revealed that the control

however, relative cell viability increased on catechin-coated nanofiber scaffolds after H2O2 treatment, especially on the group with 5 mg/mL−1 catechin (Figure 3E and Figure S4E). These results suggest that catechin coating reduces oxidative damage by scavenging ROS, highlighting the potential of catechin coats to promote cell viability in tissues with high oxidative stress due to ischemia or inflammation.47,48 Enhanced Osteogenic Differentiation of hADSCs by Catechin Coating. In addition to the ability to improve cell attachment and proliferation, the effect of catechin surface coating on differentiation was confirmed by investigating osteogenesis and bone regeneration with human stem cells. For in vitro osteogenic differentiation, hADSCs were cultured on uncoated and catechin-coated (2 and 5 mg/mL) PS substrates in osteogenic differentiation induction medium. After 3 weeks of differentiation, the cells were immunofluorescently stained for OPN and COL I, which are representative markers for osteogenic differentiation (Figure 4A). We found that higher levels of OPN and COL I proteins were expressed in differentiated hADSCs on the catechin-coated surface compared to the cells on the uncoated surface (Figure 4A). To evaluate osteogenic marker (COL1A2, OPN, and OCN) gene expression, real-time qPCR was performed after 3 weeks of differentiation (Figure 4B). In the catechin-coated groups, all osteogenic markers were significantly upregulated, especially in the group treated with 5 mg/mL catechin, indicating enhanced osteogenic differentiation of hADSCs (Figure 4B). A larger amount of calcium deposition was observed with alizarin red staining in the catechin-coated groups, indicating enhanced osteogenic differentiation and mineralization of hADSCs (Figure 4C,D). Catechin’s antioxidative properties may explain the enhanced osteogenesis of hADSCs, as it was responsible for the increased viability in oxidative stress conditions in hADSCs 4382



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group (no treat) and uncoated PCL scaffold group without hADSCs (PCL) had minimal new bone formation (Figure 5A,B). Catechin-coated PCL nanofiber scaffolds alone induced greater bone formation than no treatment and uncoated PCL scaffold groups; however, the level of bone regeneration was still low (Figure 5A,B). Bone regeneration was significantly enhanced when hADSCs were transplanted with PCL scaffolds. Importantly, mineralized bone formation was further increased by transplantation of hADSCs using the catechin-coated scaffolds (Figure 5A,B). Three-dimensional bone remodeling analysis using micro-CT revealed that the greatest volume of new bone formation with high mineral density was observed in hADSC transplanted with catechin-coated scaffolds (Figure 5C,D). Lastly, Goldner’s Trichrome staining, used to evaluate bone-specific collagen deposition, showed that the greatest amount of collagen deposition was found in the damaged regions of the calvarial bone tissue treated with transplanted hADSCs and catechin-coated scaffolds, and little collagen deposition was found in the control groups (Figure 5E). Enhanced bone regeneration of hADSC transplantation by catechin-coated scaffolds can be explained by biochemical effects of the catechin coat confirmed through in vitro experiments. Damaged tissue is typically exposed to high levels of oxidative stress due to local hypoxia and ROS generated by inflammation.49,51 Catechin modification of the scaffolds may relieve the oxidative damage of cotransplanted hADSCs, thereby enhancing osteogenic differentiation of hADSCs in the damaged tissue. Furthermore, increased calcium deposition from osteogenically differentiated hADSCs and body fluids by high calcium binding affinity may also promote osteogenesis of human stem cells on the catechin-coated scaffolds. Our results demonstrate that catechin coating can potentiate osteogenic functionalities of tissue-engineering scaffolds for bone regeneration.

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CONCLUSION

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ASSOCIATED CONTENT

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Seung-Woo Cho: 0000-0001-8058-332X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant (2016R1A5A1004694) from the Translational Research Center for Protein Function Control funded by the Ministry of Science, ICT and Future Planning, Republic of Korea and a grant (2015R1A2A1A15053771) from the National Research Foundation (NRF), Republic of Korea. This work was supported by the Institute for Basic Science (IBS-R026-D1).

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REFERENCES

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Here, we report a novel, simple surface modification chemistry using a plant flavonoid, catechin hydrate, to improve surface functionalities of substrates and scaffolds for tissue-engineering applications. Catechin was stably coated on the substrates via Na+-mediated physical self-assembly of catechin molecules with no harmful side effects to the materials, and without inducing color or structural changes. The catechin-functionalized substrates not only improved cellular adhesion and proliferation but also significantly enhanced osteogenesis of hADSCs in vitro and in vivo due to the intrinsic biochemical properties of catechin, including antioxidation and high calcium binding affinity. Our study suggests that naturally derived flavonoid compound-based surface modification chemistry can produce functional substrates and scaffolds to improve tissue engineering and stem cell therapy.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b00802. AFM analysis data, water contact angle measurement, quantification of serum protein adsorption, supplemental data of in vitro assays using HUVECs, and SEM images (PDF) 4383


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Plant Flavonoid-Mediated Multifunctional Surface Modification

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