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MSC-03529; No of Pages 10

Materials Science and Engineering C xxx (2012) xxx-xxx

Contents lists available at SciVerse ScienceDirect

Materials

journal

Science

homepage: www

and Engineering C

.elsevier.com/locate/msec

13 4 5 6 8 9 10

Immobilization of epidermal via dopamine treatment

growth factor on titanium and stainless steel surfaces

Jeonghwa Kang a.b, Makoto Sakuragi a, Aya Shibata a, Hiroshi Abe a, Takashi Kitajimaa, Masay0shi Mizutani `, Hitoshi Ohmori `, Hiroto Ayame d, Tae II Son e, Toshiro Aigaki,b, a Nano Medical Engineering Laboratory , RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351 0198, Japan

Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-Osawa, Tokyo, 192-0397 Japan Material Fabrication Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

Diagnostic Biochip Laboratory, RIKEN Center for Intellectual Property Strategies, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan Bioscience Biotechnology, Chung-Ang University, 40-1 San, Nae-Ri, Daeduck-myun, Ansung-si, Kyungki-do , 456-756, Republic of Korea

Seiichi Tada a, Yoshihiro Ito a,b,d,*

11 12 13 14 13 16

20 21 22 23 24

ARTICLEINFO ABSTRACT

.44 -13

Article history:

Received 15 November 2011 Received in revised form 22 June 2012 Accepted 26 July 2012

Available online xxxx Keywords:

Titanium Stainless steel

Dopamine

Epidermal growth factor

Titanium and stainless steel were modified with dopamine for the immobilization of biomolecules, epidermal 25 growth factor (EGF). First, the treatment of metal surfaces with a dopamine solution under different pH con- 26 ditions was investigated. At higher pH, the dopamine solution turned brown and formed precipitates. Treat- 27 ment of the metals with dopamine at pH 8.5 also resulted in the development of brown color at the surface of 28 the metals. The hydrophobicity of the surfaces increased after treatment with dopamine, independently of 29 pH. X-ray photoelectron spectroscopy revealed the formation of a significant amount of an organic layer on 30 both surfaces at pH 8.5. According to ellipsometry measurements, the organic layer formed at pH 8.5 was 31 about 1000 times as thick as that formed at pH 4.5. The amount of amino groups in the layer formed at pH 32 8.5 was also higher than that observed in the layer formed at pH 4.5. EGF molecules were immobilized 33 onto the dopamine-treated surfaces via a coupling reaction using carbodiimide. A greater amount of EGF 34 was immobilized on surfaces treated at pH 8.5 compared with pH 4.5. Significantly higher growth of rat fibro- 35 blast cells was observed on the two EGF-immobilized surfaces compared with non-immobilized surfaces in 36 the presence of EGF. The present study demonstrated that metals can become bioactive via the surface immo- 37 bilization of a growth factor and that the effect of the immobilized growth factor on metals was greater than 38 that of soluble growth factor.39

© 2012 Published by Elsevier B.V. 40 -1R

15

-16 47 48 -19 30 51 32 53 54 55 56 57 58

1. Introduction

Metals, such as titanium, titanium alloy, and stainless steel implants, are used widely in medicine based on their biocompatibility, nontoxicity, good mechanical properties, and excellent corrosion resistance. Over 70%

of implant devices are made of metal [1]. Recently, various surface mod- ifications have been developed to endow biological functionality on metals [1-4]. Surface properties are of prime importance in establishing the response of tissues to biomaterials and provide a set of very power- ful signals for cells.

To modify the surface of metals, various organic molecules, from synthetic polymers to proteins, have been developed by many re- searchers. In addition to surface modifications aimed at preventing biofouling [1,5,6], extracellular matrix, active peptides, or derivatives have been immobilized on metals to promote cell adhesion [7-13].

* Corresponding author at: Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. Tel.: +81 48 467 5809; fax: +81 48 467 9300.

E-mail address: y-ito@riken jp (Y. Ito).

Moreover, immobilization of proteins on metals was also developed to regulate higher levels of cell function, such as the control of growth or differentiation [14-17]. Various methods, including physical, ionic, and covalent immobilization, have been used for the immobilization of organic molecules on metal surfaces. Among the immobilization methods available, covalent immobilization is considered very useful, because it provides stable immobilization and leads to a biosignaling effect that is maintained for a long period [4,18,19].

Treatment of metal surfaces with silane derivatives has been used widely to achieve covalent immobilization [12,13]. In addition to si- lane derivatives, Weber et al. synthesized 1-aziglycoses, which react with the hydroxyl groups on the surface of oxidized titanium [20].

The chemicals generated singlet carbenes that were inserted readily into H-0 bonds, leading to the glycosidation of titanium. Mikulec and Puleo used p-nitrophenyl chloroformate to immobilize trypsin on the titanium alloy Ti-6A1-4V [21]. Puleo et al. developed a plasma surface modification via the polymerization of allylamine, to enable the immobilization of bioactive molecules on a "bioinert" metal, Ti-6A1-4V [22]. A photoimmobilization method using perfluorophenyl azide groups has also been reported [23]. Recently, Li and coworkers

59 60 61 62 63 (i-I 65 66 67 68 69 70 71 72 73 74 75 76 77 78

0928-4931/$ - see front matter © 2012 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.msec.2012.07.039

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012) , http://dx.doi.org/1O.1016/j.msec.2012.07.039

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79 80 81 52 53 8-1 5.3 86 57 88 89 90 91 9.2 93 94 95 96 97 9,5 99 100 101 102 103 104

105

1(16

107 108 109 110 111 112 113 114 115 116 117 115 119 120 121 122 123 124 125 126 127 125 129 130 131 132 133 134 135

139

137 138 139

J. Kang et a!. / Materials Science and Engineering C xxx (2012) xxx-xxx

develop a new method to treat the surface of titanium via photochem- ical grafting using alkene-containing compounds attached covalently to the TiO2 group, probably via Ti- 0- C bondage [24]. Li et al. reported the development of a "clickable" organic layer via extension of this method [25].

In addition to these methods, biomimetic approaches inspired by mussel adhesive activity have also been devised [26-29]. Because mus- sel proteins contain 3,4-dihydroxy-L-phenylalanine (DOPA), DOPA has been used by many researchers as a surface treatment to prepare organ- ic layers [30-38]. The similar compound dopamine has also been used [39,40]. Dopamine was attached to polymers and the polymer deriva- tives were used to achieve surface modification of metals [41-44]. Sub- sequently, Lee et al. found that the coexistence of a catechol (DOPA) and an amine (Lys), which are rich in mussel's adhesive, was crucial to achieve adhesion to a wide spectrum of materials [45]. This reaction is considered as the formation of a dopamine-melanin layer on the sur- face [46,47]. These treatments provide not only organic layers but also further immobilization of biological molecules on the surface of various materials [48-62].

In this study, we immobilized covalently a biosignaling molecule (a growth factor) on dopamine-treated metal surfaces. We aimed to optimize the conditions of dopamine treatment that are suitable for surface modification by providing an immobilization site, such as an amino group, and investigated quantitatively the effect of immobiliza- tion of a growth factor, the epidermal growth factor (EGF), on metal surfaces.

2. Materials and methods

2.1. Materials

The rat kidney adherent fibroblast cell line NRK49F was provided by the RIKEN Cell Bank (Tsukuba, Japan) and was maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS; Moregate Inc., Hamilton, Waikato, New Zealand) and 1% penicillin-streptomycin, purchased from Wako Pure Chemical Industries (Osaka, Japan). Trypsin containing 1 mM ethylenediaminetetraacetic acid (EDTA) was purchased from Wako Pure Chemical Industries. Recombinant human EGF was obtained from R&D Systems Inc. (Minneapolis, USA).

3,4-Dihydroxyphenethylamine hydrochloride (dopamine) and N-hydroxysuccinimide (NHS) were purchased from Wako Pure Chemical Industries. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochlo- ride (water-soluble carbodiimide, WSC) was obtained from Dojindo

(Kumamoto, Japan).

The monoclonal anti-human EGF antibody was purchased from R&D Systems Inc. The fluorescein isothiocyanate (FITC)-conjugated secondary antibody was obtained from Cappel Research Reagents (Costa Mesa, USA). Block Ace Powder was obtained from DS Pharma Biomedical (Sapporo, Japan).

A glass plate (diameter, 15 mm; thickness, 1 mm) was coated with titanium by Osaka Vacuum Ind. Co. (Osaka, Japan). The glass plate was cleaned via nine rounds of ultrasonication in ultrapure water and was dried using heated gas. Pure titanium was vacuum deposited on the plate using an electron beam with a thickness of 400 nm (±25%). Tissue-culture polystyrene plates with 24 wells (well diameter, 16 mm) were purchased from BD Falcon (New Jersey, USA) and 48-well plates were obtained from Corning (New York, USA).

Stainless steel (SUS316; diameter, 10 mm; thickness, 1 mm) was pur- chased from Iwasaki Co. (Osaka, Japan).

2.2. Surface treatment

The surface of the plates was washed in hexane solution, cleaned with 6 M hydrogen chloride for 10 min, rinsed twice with triple distilled water, dried in a vacuum oven for 24 h, and cleaned photochemically

E 0 0 10 i~

a L

0.2

0.16

0.12

0.08

0.04

pH

—-10min --U-24 hour

Fig. 1. Turbidity of dopamine solutions at different pH values.

using an excimer UV lamp (USHIO Inc., Tokyo, Japan) for 10 min before incubation in the solution of dopamine. This method was applied to achieve complete removal of C— C bonds and avoid subsequent decompo- sition of the organic molecules [5,7]. The complete removal of the organic material was confirmed by the decrease of the water contact angle, a value of 6 = 0° being indicative of a surface that is absolutely hydrophilic.

Dopamine treatment was performed thereafter. The cleaned plates were placed in a flask containing 1 or 2 mg/mL of dopamine solution in water (the resulting pH was 4.5) or 10 mM Tris-buffer (adjusted to pH 8.5). The reaction was performed at room temperature for 24 h.

The dopamine-treated TiO2 and SUS316 plates were rinsed in fresh water, followed by drying in a clean vacuum oven at room temperature for 24 h.

For immobilization of EGF on the surface of the plates, an EGF so- lution was mixed with an aqueous solution of 50 mM of WSC and 20 mM of NHS. The dopamine-treated plates were immersed in the mixed solution for 48 h at 4 °C [40]. After the incubation, the plates were washed three times with phosphate-buffered saline (PBS).

2.3. Surface analysis

The static water contact angles of the sample surfaces were mea- sured at 25 T in air using a contact-angle meter (Kyowa Interface Science Co., Tokyo, Japan) based on the sessile drop method. All contact angles were determined by averaging 10 different point values mea-

sured on each dopamine-treated surface.

Non 1 mg mL-1

(pH 4.5)

2mgmL-1 (pH 4.5)

1 mg mL-1 (pH 8.5)

2 mg mL-1 (pH 8.5)

Non 1 mg mL-1

(pH 4.5)

2 mg mL-1 (pH 4.5)

1 mg mL-1 (pH 8.5)

2 mg mL-1 (pH 8.5) Fig. 2. Images of dopamine-treated titanium (a) and stainless steel (b) materials.

140 141 142 14:3 144 145 146 147 144 149 151) 151

152 1:i3 154

155 156 157

158

159 166 161 162 16:3

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012) , http://dx.doi.org/10.1016/j.msec.2012.07.039

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J. Kang et al. / Materials Science and Engineering C xxx (2012) xxx-xxx 3 t1.1

t1.2 t1.3 tl.-1 t1.5 11.6 0.7 0.8

Table 1

Water contact angle and thickness of the dopamine layer formed on the metal surface .

pH Dopamine

concentration

Water contact angle Thickness

On titanium On stainless steel On titanium 4.5

8.5

1 mg/ml 2 mg/ml 1 mg/ml 2 mg/ml

46.82 ± 1.07°

50.71 ±0.64°

46.92 ± 0.84°

48.82 ± 0.79°

52.67±3.51°

58.76± 1.66°

41.23±3.40°

46.78+ 1.79°

3.40±0.49 nm 3.42±0.64 nm 3.36±0.55 pm 4.97±0.63 pm

164 165 166

The surfaces were also analyzed using X-ray photoelectron spectros- copy (XPS; AXIS-HS, Kratos, Manchester , UK) in an in vacuo condition of less than 10-7 Pa. Al-Mot monochromatic X-ray with a source power of

150 W was used. Wide and narrow scans were acquired at a pass ener- gy of 80 and 40 eV, respectively. Overview spectra were obtained in the range of 0-1100 eV using an analyzer pass energy of 80 and 40 eV.

The thickness of the polymer was measured using an ellipsometer M-2000DI (JA Woollam Company, Nebraska, USA) in the spectral range of 195-1500 nm using three angles (65°, 70°, and 75°). The roughness of the surface was analyzed using a New View 5032 appa- ratus (Zygo Co., Middlefield, USA). The surface was observed by a scanning electron microscope (SEM, JSM6330F, JOEL Ltd., Akishima, Japan) without any treatments.

• FITC was used to determine the amount of amino groups on sur - faces. One hundred microliters of a 10 mg/mL FITC solution in dimethylsulfoxide was mixed with 1 mL of 0.1 M sodium bicarbonate

167 168 169 170 171 172 17:3 174 175 176 177 178 179

(a)

C, spectra Non

pH 4.5 pH 8.5

N,s spectra Non

pH 4.5 pH 8.5

295 290 285 280 410 405 400 395

O, spectra Non

pH 4.5 pH 8.5

540

Ti2p spectra

535 530 525 470 465 460 455

Non pH 4.5 pH 8.5

450

(b)

C1 spectra

295 290 285

Non pH 4.5 pH 8.5

280

N,s spectra

410 405 400

Non pH 4.5 pH 8.5

395

O,S spectra

540 535 530

Non pH 4.5 pH 8.5

525

Fe2p spectra

740 730 720 710

Non pH 4.5 pH 8.5

700

Fig. 3. XPS spectra of titanium (a) and stainless steel (b).

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012), http://dx.doi.org/10.1016/j.msec.2012.07.039

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180 181 182 183 184 185 186 187 188 189 190 191 192

J. Kang et al. / Materials Science and Engineering C xxx (2012) xxx-xxx

solution (pH 9.0). The sample plate was incubated in the solution for 1 h at room temperature and rinsed with PBS 10 times. The amount of FITC was measured using an AxioVision instrument (Zeiss, Oberkochen, Germany) with a Cool SNAP HQ camera (Photometrics , Tokyo, Japan).

The amount of immobilized EGF was determined using an anti- EGF antibody. The plate was rinsed with PBS-Tween (0.2%) and blocked by incubation in an aqueous solution of 1% nonfat milk for 30 min . Subsequently, the plate was incubated with an and-EGF antibody (diluted 1000 times) overnight at 4 °C and washed three times with PBS-Tween (0.2%) before incubation with an FITC-conjugated second- ary antibody (diluted 1000 times) for 1 h at room temperature . After washing three times with PBS-Tween (0.2%), the amount of FITC was measured using an AxioVision instrument with a Cool SNAP HQ camera.

(a)

Titanium

• .'.t.i~y 'i rkn

0Pm 144

To make calibration curve, various concentrations of FITC-conjugated secondary antibody were spotted on the surface.

2.4. Cell culture

NRK49F cells were cultured in DMEM supplemented with 5% FBS and 1% penicillin-streptomycin at 37 °C in 95% humidified air/5% CO2. The cells were then washed using 5 mL of PBS and harvested using a 0.25%

trypsin solution containing 1 mM EDTA for 3 min at 37 C. Finally, the re- covered cells were suspended in medium for the following in vitro exam- ination. The cell suspension was added to 24- or 48-well tissue culture polystyrene plates (1.0 mL/well, 5 103 cells/mL) containing the samples, which had been washed previously with sterilized PBS. The cells were

+1.36 nm -1 .37 106 Pm

+1.00

E +0.38

r -0.25 .0) -0.88 -1.50

rI~ 1Fy~rt---'~

1 j

193 194

195

190 197 198 199 200 201 202 2(13

Titanium

Treated at pH 4.5

Titanium

Treated at pH 8.5

(b)

Stainless steel

0

•s•: •

0 um

+1.46 nm

E -2.51

108 Pm

+1.00

g +0.50

.c +0.00 a)

-0 .50 -1 .00 0

'fr

50100 Distance (pm)

144

r I +I I{

~ k i

+4.01 nm

-5 .01 108 Pm

E L a,

+1.50 +0.88 +0.25 - 0 .38

-1 .00 0

^i`'

am100 Distance (pm)

x| /

» Pm144

0

+1264.6 nm -1937.9 108 Um

+1000 E +250 s -500

CD

_ -1250

-2000

0 50100

Distance (pm)

_y Tr- i~

Stainless steel Treated at pH 4.5

Stainless steel Treated at pH 8.5

0

0 pm 144

0 144

+1256.9 nm

-1824 .7 108 Pm

+500

g +125

s -250 a)

_ -625

-1000 0

0 Pm

- -

50100

Distance (pm)

711

0 m

' • 0 144

+1219.5 nm -2003 .31

108 Pm

+1000

f+375

-250

2 -875

-1500

0 50100

Distance (pm)

0 50100

Distance (pm)

Fig. 4. Surface roughness of dopamine-treated and nontreated titanium (a) and stainless steel (b). (c) Illustration of the roughness parameters, Ro and R ,.

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012), http://dx.doi.org/10.1016/j.msec.2012.07.039

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J. Kang et al. / Materials Science and Engineering C xxx (2012) xxx-xxx 5

(c) R= —IZ(x)dx 1 fir

trp

R.

R = *I-6- 1 fir Z2(x)dx

0

R.'

Fig. 4 (continued).

204 205 206 207

208

209

210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230

231 232

233 234

59.1

t2.2 t2.3

cultured under a 5% CO2 atmosphere at 37 °C for 5 days and were studied using a phase-contrast microscope. The number of cells was counted using the microscope. Statistical analyses were performed using analysis of variance.

3. Results

3.1. Appearance and measurement of the water contact angle First, the properties of the dopamine solution were investigated based on turbidity, as shown in Fig. 1. The solution was transparent at low pH, even after 24 h, and turned to turbid and brown at high pH.

After 24 h, some precipitate was found at high pH. In this study, dopa- mine treatment was performed at pH 4.5 or 8.5, which were used as typical low and high pH values, as described by He et al. [40] and Lee et al. [45], respectively. Because the concentration of the solutions used by He et al. [40] and Lee et al. [45] were 1 and 2 mg/mL, respectively , we also used these two concentrations.

The surface of the metals turned brown when the titanium and stainless steel were treated at pH 8.5 (Fig. 2). In contrast, no signifi- cant color change was observed when they were treated at pH 4.5

(Fig. 2). However, the assessment of surface hydrophilicity based on contact angle measurements revealed that the water contact angle of surfaces increased with dopamine treatment, regardless of the type of metal or pH (Table 1). This indicates that the surfaces of titanium and stainless steel were fully covered by dopamine, regardless of pH. Li et al. [57] and Wei et al. [58] reported contact angles of a polydopamine- treated surface of 51 ± 1.5° and -55°, respectively. The static water con- tact angles of the treated surfaces analyzed in the present study were sim- ilar to these values.

3.2. XPS analysis, ellipsometry analysis, roughness measurement, SEM image, and measurement of amino groups

XPS analysis was performed on titanium and stainless steel surfaces treated with dopamine at different pH values (Fig. 3). Cis and Nis spectra

Table 2

Roughness of the surface of the titanium and stainless steel materials.

Surface treatment with dopamine

Ra (nm)'' RQ (nm)'

demonstrated that significant peaks corresponding to an organic layer 2:i5 appeared on titanium and stainless steel surfaces after treatment at pH 236 8.5. Ols spectra indicated that a chemical shift of oxygen was observed 237 on titanium and stainless steel surfaces only after treatment at pH 8.5. 238 Ti20 and Fe20 spectra showed that the metal surfaces were not detectable 239 by XPS after treatment at pH 8.5. These results indicate that a thick layer of 240 dopamine was formed on the metal surfaces treated with dopamine at pH 241 8.5, as the probing depth of the XPS technique is about 8 nm in an organic 242 matrix [44,62].2.13

This result was confirmed by thickness measurement using 244 ellipsometry, as shown in Table 1. Surface roughness was also measured, 215 as shown in Fig. 4 and summarized in Table 2. The surface of the stainless 216 steel material used in this study exhibited a degree of roughness that pre- 247 cluded the measurement of the thickness of the layer formed (Table 2). In 248 contrast, the thickness of the layer covering the titanium surface could be 249 measured: the thickness of the dopamine layer formed at pH 8.5 was 250 about 1000 times as thick as that formed at pH 4.5. The roughness of 251 the metal increased slightly after treatment at pH 8.5. For the stainless 252 steel material, this variation was not significant because of its high degree 253 of surface roughness, although the surface roughness of the stainless steel 254 material decreased slightly after dopamine treatment.255

The surface was also observed by SEM as shown in Fig. 5. The sur- 256 face of stainless steel was rougher than that of titanium. The dopamine 257 treatment reduced the roughness and the treatment at pH 8.5 reduced 258 more than that at pH 4.5. Microphotos with high magnification show 259 nano-particle deposition on the surfaces at pH 8.5 as reported by Jiang 260 et al. [63].261

The amount of amino groups present in the dopamine layer was 262 measured using FITC (Fig. 6). The amount of amino groups in the organ- 263 is layer formed at pH 8.5 was higher than in that formed at pH 4.5, for 264 both titanium and stainless steel materials. However, the variation in 265 amino group content was smaller than that observed for thickness 266 (Table 1). It is known that amino groups in dopamine are consumed 267 by polymerization at pH 8.5. Therefore, this result suggests that some 268 amino groups remained available for reactivity, even after the polymer- 269 ization of dopamine. The differences observed for the two metals were 270 considered as being caused by differences in roughness. The surface of 271 the stainless steel material was so rough that the apparent surface 272 area was larger and the amount of amino acids produced was higher 273 than that observed on the smoother titanium surface.274

52.4 52.5 52.6 t2.7 t2.8 t2.9

Titanium

Stainless Steel

No pH 4.5 pH 8.5 No pH 4.5 pH 8.5

0.27+0.01 0.27+0.01 0.34+ 0.03

175+4.99 155+3.35 157+0.48

0.34+0.01 0.34 f 0.01 0.45+0.06 236+6.93 216 f 3.14 220+0.02

3.3. Immobilization of EGF

t2.10 a Ro=rSid lZ(x)Idx and Rq=1r fdZ2(x)dx

275

On both metals, the amount of immobilized EGF increased with the 276 increase of EGF in solution, as shown in Fig. 7. In addition, the amount of 977

EGF immobilized on surfaces treated with dopamine at pH 8.5 was 278

higher than on those treated at pH 4.5, for both metals. There were no 279 significant differences in the degree of EGF immobilization between280

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012) , http://dx.doi.org/10.1016/j.msec.2012.07.039

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(a)

Titanium

Titanium

Treated at pH 4.5

J. Kang et al. / Materials Science and Engineering C xxx (2012) xxx—xxx

Titanium

Treated at pH 8.5

Fig. 5. SEM images of dopamine-treated and nontreated titanium (a) and stainless steel (b).

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012), http://dx.doi.org/10.1016/j.msec.2012.07.039

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(b)

Stainless steel 4

J. Kang et al. / Materials Science and Engineering C xxx (2012) xxx—xxx

~~ .`^

rr ~-(.

~"` ~. ..:':' . 4

7

Stainless steel Treated at pH 4.5

Stainless steel Treated at pH 8.5

t r ' r....` 41° gta., •

enwiT1

N

,k,',74,41t5.'4,2. Nt *Zs',

Fig. 5 (continued).

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012), http://dx.doi.org/10.1016/j.msec.2012.07.039

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3

1

3

2 3

5 :i

N C

N 0 7 0 0) 0 c

E 05 70 60 50 40 30 20 10 0

J. Kang et al. / Materials Science and Engineering C xxx (2012) xxx—xxx

pH 4.5 pH 8.5 pH 4.5 pH 8.5

4. Discussion

4.1. Surface treatment with dopamine

Mussels are promiscuous fouling organisms that attach to virtually all types of inorganic and organic surfaces. The identification of dopamine as a small-molecule compound that contains both functionalities, by Lee et al. [45], led to the assumption that the coexistence of catechol (DOPA) and amine (lysine) groups is crucial to achieve adhesion to a wide spec- trum of materials. The formation of polydopamine or dopamine-mela- nin has been described and is used for surface modification under mild alkali conditions. Simple immersion of substrates in a diluted aqueous solution of dopamine buffered to a pH typical of marine environments (2 mg of dopamine per mL of 10 mM Tris, pH 8.5) resulted in spontane- ous deposition of a thin layer of adherent polymer. Protein immobiliza- tion was performed by some researchers on dopamine-treated surfaces.

Lee et al. immobilized trypsin on dopamine-treated surfaces via a reac- tion between nucleophiles and the polydopamine surface [56]. Simi- larly, Poh et al. immobilized the vascular endothelial growth factor on dopamine-treated titanium [17], Yang et al. immobilized avidin on a polydopamine surface [51], and Wei et al. immobilized bovine serum albumin on dopamine-treated polymeric membranes [58].

Conversely, at low pH, the formation of polydopamine does not occur as shown in Fig. 1. However, Xu et al. [39] and He et al. [40] treated iron oxide or titanium surfaces with dopamine dissolved in pure water (the resulting pH was about 4.5), respectively, using the binding affinity of the catechol structure. He et al. immobilized RGD peptide or collagen on dopamine-treated titanium [40]. To date, no comparison of the effect of pH on the dopamine treatment has been performed. Therefore, in this study, we investigated the effect of pH on the treatment of metal surfaces using dopamine. The results of this analysis are illustrated in Fig. 9. At low pH, a dopamine monolayer was formed on the titanium and stainless steel materials; however, the amount of immobilized EGF was higher at high pH. As a thicker layer was formed at high pH, the apparent increases of surface roughness and content of amino groups were considered.

298

299

(a) (b)

Fig. 6. Amount of amino groups on dopamine-treated titanium (a) and stainless steel (b) surfaces (n=5; p"<0.005; p""<0.05).

titanium and stainless steel. This result indicates that, after dopamine treatment, the surfaces of these metals were similar to each other.

3.4. Cell proliferation on EGF-immobilized surfaces

Fig. 8 shows the growth of NRK49F cells in the presence of soluble and immobilized EGF. Cell growth was enhanced with the increase of the amount of EGF in a well. Immobilized EGF enhanced cell growth ef- ficiently compared with soluble EGF, on both metals. Lower amounts of

immobilized EGF were sufficient to enhance cell growth compared with soluble EGF. In addition, the maximum enhancement effect of

immobilized EGF was higher than that of soluble EGF. These phenome- na were observed on several polymer surfaces, as reviewed previously [4,18,19]. The present investigation confirmed the effectiveness of the immobilization of a growth factor on surface-treated metals.

In addition, there were no significant differences between titanium and stainless steel regarding the effect of immobilized vs soluble EGF.

This result also indicates that, after dopamine treatment, the surfaces of the two metals were similar to each other.

E U C U.J

a) N O E E

1r aI L W a) N .0 0 E E

0

(a)

100---

so(b)

40

—1 -- pH 4 .5

—6-- pH 8 .5

20 40 60 80 100

EGF concentration (pg/mL)

120

0

—0-- pH 4 .5 --*-- pH 8 .5

20 40 60 80100

EGF concentration (pg/mL)

120

Fig. 7. Amount of EGF immobilized on titanium surfaces treated with dopamine at pH 4.5 (-4—) and at pH 8.5 (~) (a) and stainless steel surfaces treated with dopamine

T

at pH 4.5 () and at pH 8.5 (-10—) (b) (n=3; p'<0.05; p"<0.05).

2.5

_c 2.2 0 +a 1.9 a)

0 1.6 a) •1.3

1.0

2.5

2.2 0 a

) 1.9 C) o 1.6 c0

•1.3 102

1.0111.

10'

(a) —•— pH 4 .5

--1— pH 8 .5

—~ Soluble EGF

10' 100 101102 103

amount of EGF (ng/well)

104

(b)

10

—+— pH 4.5

—0— pH 8 .5

—1—Soluble EGF

10° 10' 102103 104 amount of EGF (ng/well)

1 0°

Fig. 8. Growth of NRK49F cells on an EGF-immobilized titanium surface treated with dopamine at pH 4.5 (--4,—) and at pH 8.5 (-0—) (a) and on a stainless steel surface treated with dopamine at pH 4.5 (-4—) and at pH 8.5 (-0—) (b) (n=3).

300 301 302 303 304 305 306 307 308 309 310 311 312 :113 :114 :315 :116 317 318 :319 32(1 :121 :322 323 :124 325 326 :127 :128 329 :330

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012) , http://dx.doi.org/10.1016/j.msec.2012.07.039

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J. Kang et al. / Materials Science and Engineering C xxx (2012) xxx-xxx

pH 4.5

OH OH OH

I I

metal

pH 8.5

9

Dopamine coating

NH2

O O

I I metal

OH

HOL

NH2

O 0 I metal

immobilization of EGF

EGF

; HNO

0 0

EGF

HN

0 0

metal

EGF

HN

0 0

Dopamine layer

HO

HO

HO OH

EGF:) HN

EGF O~C/

NH

EGF

o IEGF : 0

NH

EGF • HO OH NH

s'" 40 OH

OH

O HN~

O O o 9

metal

9 9

Fig. 9. Schematic illustration of the treatment of a metal surface using dopamine.

331 332 333 334 335 336 337 338 339 3-10 341 312 343 344 345 346

347

348 319 350 351 352 353 354 355 356 357

The interaction between the dopamine layer and the metal include covalent and non-covalent interactions. The former includes the reac- tions of o-quinone yielded by oxidation of catechol with catechol by quinone-phenol dismutation, or with amine by Michael addition and Schiff base reaction [64]. The latter includes the hydrogen bonding inter- action, n—n interaction, and electrostatic interactions. However, the polymerization mechanism and deposition behavior of dopamine on various substrates are in dispute and not yet clearly known. The possible structure evolution of dopamine in aqueous solution was considered by Jiang et al. [63] recently. According to their consideration, dopamine is easily oxidized by dissolved oxygen under alkaline conditions, creating 5,6-dihydroxyindole and 5,6-indolequinone via intramolecular cycliza- tion, oxidation, and rearrangement. After the multistep reaction proceed- ing by these compounds, a mass of melanin-like dopamine aggregates was generated in the solution and a tightly adherent dopamine layer was formed on the surfaces of the substrates simultaneously.

4.2. Immobilization of EGF

Regarding the amount of immobilized EGF, different surface densities have been reported. Ito et al. [65], Chen et al. [66], Nakaji-Hirabayashi et al. [67], Liberelle et al. [68], Klenkler et al. [69], and Goncalves et al. [70]

reported EGF surface densities of 90 ng/cm2, 0.2 EGF molecules/nm2 (200 ng/cm2), 168-380 ng/cm2, 14-30 pmol/cm2 (84-180 ng/cm2), 300 ng/cm2, and 4-18 ng/cm2, respectively. Because of the difficulties in the evaluation of surface area, which depends on the roughness of the surface, it is not possible to compare the surface concentra- tions reported. However, considering that the densely packed mono- layer of immobilized EGF (2.97 x 10-13 cm2) [71 ] theoretically

provides about 31 ng/cm2 of surface concentration, the maximum surface concentration of immobilized EGF in the layer formed at pH 4.5 indicates the formation of an EGF monolayer. The organic layer formed on another surface prepared at pH 8.5 was thick and the surface was considered as rough. The roughness increased the surface area on which EGF could be immobilized.

4.3. Effect of immobilized EGF

358 359 360 361 362 363

364

After the demonstration that immobilized insulin enhanced cell :oi5 growth significantly, EGF was also immobilized on surfaces. The effect 366 of immobilized growth factors was discussed by Ito [4,18,19]. The effec- 367 tiveness of immobilized growth factors was confirmed by micropattern 36s immobilization, antibody blocking, radioisotope labeling, microarray- :169 based comparison with other proteins, etc. [4,18,19]. It is known that :370 the effect of immobilized growth factors is higher than that of soluble 371 ones. [18,19,72]. In addition, immobilized EGF induced some effects 372 different from those of soluble EGF on some cells [18,19,73]. The re- 373 sults of the present study demonstrated that a lower amount of 374 immobilized EGF was sufficient for cell growth and that its maximum 373 enhancement effect was higher than that of soluble EGF, on both tita- 376 nium and stainless steel surfaces. This observation can be explained 377 mainly by the following factors: the high local concentration of 378 growth factors and the multivalency of immobilized growth factors. 379 In addition, the main reason for the observation of a higher activity of 380 immobilized EGF vs soluble EGF may be the inhibition of downregulation, 381 considering recent reports on long-lasting activation by immobilized EGF 382 [4,18,19,65,73].383

Please cite this article as: J. Kang, et al., Materials Science and Engineering C (2012) , http://dx.doi.org/10.1016/j.msec.2012.07.039

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384

385 386 387 388 389 390 391 392

393

394 395

396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 413 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 513

5. Conclusions

Metal (titanium and stainless steel) surfaces were treated via a biomimetic method using dopamine, and a growth factor (EGF) was immobilized on the treated surfaces. At high pH, a dopamine-melanin layer was formed on the surface of these metals that provided a larger amount of amino groups for coupling with EGF. Immobilized EGF pro- moted cell growth more efficiently than soluble EGF. Surface modifica- tion methods will provide new biologically functional metals that can be used for the development of medical devices.

Acknowledgment

The authors thank Mr. D. Inoue at RIKEN Advanced Science Institute and Dr. M. Takemasa for their surface observation.

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i

VT

,.t t•

it 11;2 16

,t(;1 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 312

Materials S ience and EngineeringAl_

Fig.  1.  Turbidity  of  dopamine  solutions  at  different  pH  values.
Fig. 3. XPS spectra  of titanium  (a) and  stainless steel  (b).
Fig. 4. Surface roughness  of dopamine-treated  and  nontreated  titanium  (a) and stainless steel  (b)
Fig.  5.  SEM images  of dopamine-treated  and  nontreated  titanium  (a)  and  stainless  steel  (b).
+3

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