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Fabrication and biocompatibility investigation of TiO2 films on the polymer substrates obtained via a novel and versatile route
Colloids and Surfaces B: Biointerfaces 76 (2010) 123–127
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Fabrication and biocompatibility investigation of TiO2 films on the polymer substrates obtained via a novel and versatile route Junfei Ou a,b , Jinqing Wang a,∗ , Dong Zhang c , Puliang Zhang c , Sheng Liu a,b , Penghua Yan a , Bin Liu c , Shengrong Yang a a b c
State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China Graduate University of Chinese Academy of Sciences, Beijing 100080, China School of Stomatology, Lanzhou University, Lanzhou 730000,China
a r t i c l e
i n f o
Article history: Received 8 July 2009 Received in revised form 13 October 2009 Accepted 14 October 2009 Available online 23 October 2009 Keywords: Implanted polymer materials Polydopamine coating TiO2 films Biocompatibility
a b s t r a c t Titanium oxide (TiO2 ) films were successfully deposited onto the polymer substrates of polytetrafluoroethylene (PTFE), polyethylene (PE), and polyethylene terephthalate (PET), which were pre-modified with polydopamine coating (polydopamine and its coating are coded as PDA and PDAc, respectively), by a simple liquid phase deposition (LPD) process. The morphology and chemical state of the obtained TiO2 films were characterized by field emission scanning electron microscope (FE-SEM) and X-ray photoelectron spectroscopy (XPS), respectively. Subsequently, the biocompatibility of the samples was investigated by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and acridine orange staining of MC-3T3 osteoblast cells, and the results demonstrated that the fabricated TiO2 films could markedly improve the in vitro cytocompatibility. So, the presented route is anticipated to be a promising surface modification methodology to improve the practical outcome of the implanted materials for its versatility and validity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Recently, some inactive polymers with different physicochemical properties such as chemical composition, surface hydrophobicity and reactivity, typically, polytetrafluoroethylene (PTFE), polyethylene (PE), and polyethylene terephthalate (PET), are widely employed as the clinical implanted materials [1–3] for their desirable mechanical and thermodynamic characteristics [4–6]. However, the practical outcome of these materials is greatly restricted by the poor tissue-material interactions after implantation owing to their inert and hydrophobic surfaces. So, the surface modification for improving the biocompatibility of the polymer substrates is quite necessary [6,7]. One of the most promising modification procedures is to construct bioceramic coatings, especially, titanium oxide (TiO2 ) films on the implanted materials since surface TiO2 films can tailor the biological reaction and interaction between artificial and living matter [8,9]. However, it was very difficult to construct bioceramic coatings on the inactive polymers (e.g. PTFE and PE, etc.) surfaces unless special large-scale apparatus [10] or plasma pretreatment [11] was applied.
∗ Corresponding authors. Tel.: +86 931 4968076; fax: +86 931 8277088. E-mail addresses: [email protected] (J. Wang), [email protected] (S. Yang). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.10.024
In this work, a novel and versatile route of fabricating adherent and uniform TiO2 films on different polymer substrates have been realized by a simple liquid phase deposition (LPD) process for the first time. Our inspiration profits from a magic compound of dopamine, a functional molecule containing catechol and amine groups, and both of which may be crucial for the high adhesive property of Mytilus edulis foot protein 5 (Mefp-5) [12]. It has been demonstrated that this kind of simple structure is a powerful building block for spontaneous deposition of thin polymer coatings on almost all material surfaces. Moreover, the active groups of –NH2 and –OH on the formed coating surfaces can facilitate the further modification [12]. Here, three polymer substrates of PTFE, PE, and PET were respectively immersed into a dilute dopamine aqueous solution and then the spontaneously formed thin polymer film was anticipated to provide a versatile platform for fabricating TiO2 films through a relative simple route, as schematically shown in Fig. 1(a). 2. Materials and methods 2.1. Materials 3-Hydroxytyramine hydrochloride (dopamine hydrochloride) and tris(hydroxymethyl) aminomethane (Tris) were purchased from Acros Organics. Ammonium fluotitanate ((NH4 )2 TiF6 ) was purchased from Shanghai SSS Reagent Co., Ltd. Boric acid
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Fig. 1. (a) A schematic view of the TiO2 deposition on the polymer substrates mediated by PDAc. (b) The “oxidation–polymerization” mechanism for the formation of PDA proposed by Messersmith and co-workers [12].
was obtained from Beijing Xinguang Chemical Reagent Factory. Dimethyl sulfoxide (DMSO) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco. Trypsin–EDTA solution and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were supplied by Sigma. Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. MC-3T3 osteoblast cells were supplied by the Fourth Military Medical University.
The cells were routinely cultured in DMEM containing 10% FBS and incubated at 37 ◦ C in a humidified chamber with 10% CO2 . The culture medium was refreshed every 2 days. When the cells became almost confluent, they were released by treating with 0.25% trypsin–EDTA solution for 3 min at 37 ◦ C and resuspended in the DMEM with a final concentration of 104 cells/ml.
2.2. Preparation of TiO2 films on the polymer substrates
In vitro cytocompatibility was investigated by MTT colorimetric assay. Briefly, the sterilized samples were placed into a 24-well culture plate and cells were seeded for 2 h, 4 h, 2 days, and 4 days, respectively. At the prescribed time point, 100 l of MTT phosphate buffer solution (PBS) (5 mg/ml, pH 7.4) was injected into each well containing the test sample and culture medium. The cultures were then kept at 37 ◦ C for 4 h. At the end of incubation, the supernatant was removed and 1 ml of DMSO was added into each well. The 24well plate was then shaken to dissolve the purple formazan crystals and the optical density (OD) value of the resulting solution was recorded using a micro-plate reader (Model 550, BIO-RAD, Japan) at the wavelength of 490 nm. The assay was conducted in triplicate for each sample and three parallel experiments were performed. To calibrate the cellular survival rate, blank and control groups were set. In the blank group, only culture media was added into the well. While, in the control group, cells and culture media without polymer substrates were added. The blank and control groups were treated with the same procedures and incubated for the same time as those in the experimental group. The measured OD values of the blank, control, and experimental groups were coded as ODbla , ODcon , and ODexp, respectively. Finally, the cellular survival rate was calculated by the following equation:
PET, PE, and PTFE plates were used as the substrates and cleaned thoroughly in acetone and water by ultrasonication (59 kHz, 45 W) in succession, followed by drying in ambient conditions. Then, the pretreated substrates were dipped into a 9 mM dopamine Tris–HCl solution (pH 8.5) for 12 h, followed by ultrasonication in ultra-pure water and blowing dry with N2 [10]. The PDA coated substrates were immersed into a fresh prepared aqueous solution containing 0.1 M (NH4 )2 TiF6 and 0.3 M H3 BO3 with a pH value of 3.88 at room temperature for 12 h, and then ultrasonically cleaned in water and blown dry with N2 . The prepared samples were coded as PET (or PE, PTFE)–PDAc–TiO2 . 2.3. Characterizations Contact angle meter (DSA100, Krüss, Germany) was used to measure the static water contact angle of the prepared samples. An ellipsometer (L116-E, Gaertner, USA) equipped with a He–Ne laser (632.8 nm) set was adopted to measure the film thickness. X-ray photoelectron spectroscopy (XPS, PHI-5702, Physical Electronics, USA) was performed using a monochromated Al–K␣ irradiation to determine the chemical state of certain elements with the C 1s binding energy of 284.8 eV as the reference. The morphologies of the pristine and TiO2 film coated polymer plates were observed by a field emission scanning electron microscope (FE-SEM, JEOL, JSM-6701F, Japan).
2.5. In vitro cytocompatibility assay
survival rate =
ODexp − ODbla . ODcon − ODbla
2.6. Acridine orange staining 2.4. Cell culture MC-3T3 osteoblast cells were employed to evaluate the biocompatibility of the pristine and TiO2 film coated polymer plates.
MC-3T3 osteoblast cells cultured on the pristine and TiO2 film coated polymer plates were visualized by fluorescent acridine orange staining. After being seeded for 2 days, all samples were
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Fig. 2. XPS spectra of various samples: survey spectra of PET (a-i), PTFE (b-i), and PE (c-i); survey spectra of PET–PDAc (a-ii), PTFE–PDAc (b-ii), and PE–PDAc (c-ii); survey spectra of PET–PDAc–TiO2 (a-iii), PTFE–PDAc–TiO2 , (b-iii) and PE–PDAc–TiO2 (c-iii). N 1s (d) and O 1s (e) spectra of the PDAc on different substrate surfaces (e); Ti 2p (f) and O 1s (g) spectra of the as-deposited TiO2 films on different substrate surfaces.
rinsed in PBS, and then cells were fixed in the ethanol for about 10 min. Subsequently, the cells attached to the sample surfaces were stained with acridine orange fluorescent dye in PBS for 3 min and examined by a fluorescence microscope (BX51, Olympus, Japan).
performed with Student’s t-test and values were considered to be significantly different when p < 0.05. 3. Results and discussion 3.1. The formation of TiO2 films on the polymer substrates
2.7. Statistics Data were expressed as the mean ± SD (standard deviation) from three independent experiments. Statistical analysis was
The formation of TiO2 films on polymer substrates mainly involved the following steps: substrate cleaning, dopamine polymerization, and TiO2 film deposition. Firstly, the polymer
Fig. 3. FE-SEM images of different samples: PET (a), PTFE (b), PE (c), PET–PDAc–TiO2 (d, g), PTFE–PDAc–TiO2 (e, h) and PE–PDAc–TiO2 (f, i).
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Fig. 4. Cellular survival rate obtained from MTT colorimetric assays. For all polymer substrates coated with TiO2 films, the differences as compared to the pristine polymer substrates were significant (p < 0.05).
substrates were cleaned under sonication in acetone and water successively, and then were dipped into a dopamine Tris–HCl solution with a pH value of 8.5. As proposed by Messersmith and co-workers, the PDAc formed on the polymer substrates mainly underwent an oxidation–polymerization process, i.e., the dopamine monomer molecules were firstly oxidized into 5, 6-dihydroxyindole under an alkaline condition, whose further oxidation caused the intermolecular cross-linking to yield PDA which can be adhered to almost all materials surfaces via a strong physisorption to form PDAc [12], as schematically illustrated in Fig. 1. The formation of PDAc can be confirmed by the characterizations of water contact angle measurement and X-ray photoelectron spectroscopy (XPS) analysis. After being immersed in dopamine solution and subsequently ultrasonicated in water and blown dry with N2 , the water contact angles of all samples decrease from above 90◦ for pristine polymer substrates (122◦ , 107◦ and 90.5◦ for PTFE, PE and PET, respectively) to about 50◦ , which was very close to the average value of PDAc on various substrates [12]. Moreover, XPS spectrum revealed the appearance of N 1s signal at 399.6 eV after deposition of PDAc (see Fig. 2(a–d)), which could be assigned to the N element of amino groups in the dopamine molecules. In this case, the exact thickness of PDAc was impracticable to be measured due to the rather rough surfaces of polymer plates. Alternatively, the polished Si wafer was employed as a substitute and the ellipsometric thickness of PDAc was measured to be about 20 nm under the same deposition conditions with the polymer plates. In addition, the adhesion strength of the PDAc to the polymer substrate was also very hard to be obtained directly. However, it can be reflected
indirectly by the ultrasonication treatment. As the results showed, the formed PDAc on the PET surface had the highest adhesion strength, which remained intact even after 30 min ultrasonication. The PDAc on the PTFE and PE substrates could keep stable and intact under the ultrasonication within 2 min, however, there was almost nothing left on the surfaces of PTFE and PET with prolonging ultrasonication time to 5 min, which could be indicated by the increase of water contact angle and color change from brown (PDAc) to white (polymer substrates of PTFE or PE). Finally, the fabrication of TiO2 film was accomplished by two processes: the heterogeneous nucleation and the homogeneous growth. It has been reported that the hydroxyl groups in catechol derivatives can react with certain metal oxide [13,14] and PDAc is hydrophilic with plentiful active hydroxyl groups [12]. In addition, in the precursor solution, the TiO2 nano-particles were formed by the hydrolysis of (NH4 )2 TiF6 and H3 BO3 [15–19]. Therefore, the heterogeneous nucleation was actualized through the chelation between the hydroxyl groups on the PDAc surface and the TiO2 nano-particles in the solution. The homogeneous growth was induced by the nucleated TiO2 layer on the substrate, which could be served as the seed layer to boost the homogeneous condensation of the TiO2 nano-particles. The formation of TiO2 films was further approved by characterizations of FE-SEM and XPS. From the FE-SEM images in Fig. 3, it can be seen that the polymer substrates were covered by TiO2 films. Moreover, the morphology of the TiO2 film was influenced by the morphology of the substrate. As displayed in Fig. 3(a–c), the pristine PET and PTFE plates were relatively flat, while PE was
Fig. 5. Fluorescent acridine orange staining of the osteoblast cells on the pristine polymer substrates (a: PET, c: PTFE, and e: PE) and TiO2 films coated polymer substrates (b: PET–PDAc–TiO2 , d: PTFE–PDAc–TiO2 , and f: PE–PDAc–TiO2 ) for 2 days, respectively; the images were magnified 40 times.
J. Ou et al. / Colloids and Surfaces B: Biointerfaces 76 (2010) 123–127
apparently rough. Consequently, the TiO2 film on PE surface was uneven with some wrinkles (see Fig. 3(f) and (i)). Obviously, all the as-deposited TiO2 films are composed of plentiful TiO2 nanoparticles with the diameter of several nanometers, as displayed in Fig. 3(g–i). Due to the lateral shrinkage during the drying process [18], there are some cracks generated on the TiO2 films, as shown in Fig. 3(d–f). On the other hand, the spectral peaks in XPS corresponding to Ti 2p (458.9 eV) were observed from the deposited thin films on PDAc (see Fig. 2(f)). This binding energy value is very close to that of TiO2 (458.4–458.7 eV) [20–22] and higher than that of Ti metal (454.0 eV), suggesting that the titanium elements in the films are positively charged by formation of direct bonding with oxygen. While the O 1s signals of TiO2 modified polymer substrates are located at 530.2 eV and 532.2 eV, respectively (see Fig. 2(g)). The former value is similar to that of TiO2 (529.9 eV [20], 530.1 eV [21], and 530.2 eV [23]) and lower than that of neutral oxygen molecules (531.0 eV [23]), indicating that the oxygen is negatively charged, possibly through the formation of chemical bonding with Ti. The binding energy value of the latter one is almost the same as that of O 1s in PDAc (Fig. 2(e)), suggesting that the signal is most likely to be resulting from the exposed PDAc, which was facilitated by the cracks of TiO2 film, as clearly shown in Fig. 3(d–f) [22,24]. 3.2. In vitro cytocompatibility assay To evaluate the in vitro cytocompatibility of the samples, MTT colorimetric assay was performed. As shown in Fig. 4(a), the cellular survival rates on the TiO2 coated polymer substrates over 2 and 4 h were about 10% higher than that on the corresponding pristine substrates. Moreover, the difference of the cellular survival rates increased to about 20% after 2 and 4 days of cell seeding, indicating that the fabricated TiO2 film apparently enhanced the cytocompatibility (Fig. 4(b)) [25,26]. Moreover, for the samples of PTFE–PDAc–TiO2 and PE-PDAc-TiO2 , the survival rates were a little lower than that on PET-PDAc-TiO2 , which might be attributed to the fact that the TiO2 films on edges of PTFE and PE substrates were partially destroyed by ultrasonication owing to their less adhesive strength to substrates. In addition, the fluorescent staining result was shown in Fig. 5, which revealed that the greater and denser osteoblasts were proliferated well on the TiO2 coated polymer substrates as compared with the corresponding pristine ones. Based on the above mentioned experimental results, it can be concluded that the cellular survival rate of the polymer substrates has been improved greatly, which can be definitely attributed to the excellent intrinsic biocompatibility of the TiO2 films. On the other hand, the TiO2 films fabricated via this simple process were hydrophilic (water contact angle values of about 0◦ ) with abundant hydroxyl groups on the surfaces (as depicted in Fig. 1), which markedly promoted the cell-material interaction [27]. 4. Conclusion For the first time, a novel and versatile route of fabricating TiO2 films on different polymer surfaces, even the high hydrophobic
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PTFE surface, has been realized via a simple LPD process. The formed TiO2 film plays a significant role in improving the biocompatibility of the polymer materials, demonstrating this simple approach has overcome the obstacles restricting the application of polymer in medical implanted materials and will be a promising surface modification methodology for its versatility and simple process. Acknowledgements The authors are grateful to the National Natural Science Foundation of China (Grant Nos. 50801065 and 20823008) and “Top Hundred Talents Program” of Chinese Academy of Sciences for financial support. References [1] K. Vallières, É. Petitclerc, G. Laroche, Macromol. Biosci. 7 (2007) 738. [2] W. Bonfield, M.D. Grynpas, A.E. Tully, J. Bowman, J. Abram, Biomaterials 2 (1981) 185. [3] J.M. Brynaert, M. Deldime, I. Dupont, J.L. Dewez, Y.J. Schneider, J. Colloid. Interf. Sci. 173 (1995) 236. [4] J.N. Meussdoerffer, H. Niederpriim, Chem. Zeitung 104 (1980) 45. [5] S. Ramakrishna, J. Mayer, E. Wintermantel, K.W. Leong, Comp. Sci. Technol. 61 (2001) 1189. [6] L. Yang, J. Chen, Y. Guo, Z. Zhang, Appl. Surf. Sci. 255 (2009) 4446. [7] V. Moby, C. Boura, H. Kerdjoudj, J.-C. Voegel, L. Marchal, D. Dumas, P. Schaaf, J.-F. Stoltz, P. Menu, Biomacromolecules 8 (2007) 2156. [8] K.-L. Ou, Y.-H. Shih, C.-F. Huang, C.-C. Chen, C.-M. Liu, Appl. Surf. Sci. 255 (2009) 2046. [9] L. Giorgetti, G. Bongiorno, A. Podestà, G. Berlanda, P.E. Scopelliti, R. Carbone, P. Milani, Langmuir 24 (2008) 11637. [10] M. Kemell, E. Färm, M. Ritala, M. Leskelä, Eur. Polym. J. 44 (2008) 3564. [11] G. Acharya, T. Kunitake, Langmuir 19 (2003) 2260. [12] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007) 426. [13] M. Guvendiren, P.B. Messersmith, K.R. Shull, Biomacromolecule 9 (2008) 122. [14] M.J. Sever, J.T. Weisser, J. Monahan, S. Srinivasan, J. Wilker, Angew. Chem. Int. Ed. 43 (2004) 448. [15] S. Deki, Y. Aoi, O. Hiroi, A. Kajinami, Chem. Lett. (1996) 433. [16] S. Deki, Y. Aoi, H. Yanagimoto, K. Ishii, K. Akamatsu, M. Mitzuhata, A. Kajinami, J. Mater. Chem. 6 (1997) 1879. [17] S. Deki, Y. Aoi, Y. Asaoka, A. Kajinami, M. Mizuhata, J. Mater. Chem. 7 (1997) 733. [18] H. Pizem, C.N. Sukenik, U. Sampathkumaran, A.K. McIlwain, M.R. De Guire, Chem. Mater. 14 (2002) 2476. [19] A. Razgon, C.N. Sukenik, J. Mater. Res. 20 (2005) 2544. [20] R.J. Collins, H. Shin, M.R. DeGuire, A.H. Heuer, C.N. Sukenik, Appl. Phys. Lett. 69 (1996) 860. [21] F. Zhang, S. Jin, Y. Mao, Z. Zheng, Y. Chen, X. Liu, Thin Solid Films 310 (1997) 29. [22] D. Huang, Z.-D. Xiao, J. -H. Gu, N.-P. Huang, C.W. Yuan, Thin Solid Films 305 (1997) 110. [23] Y. Masuda, N. Saito, R. Hoffmann, M.R. DeGuire, K. Koumoto, Sci. Technol. Adv. Mater. 4 (2003) 461. [24] H. Shin, R.J. Collins, M.R. De Guire, A.H. Heuer, C.N. Sukenik, J. Mater. Res. 10 (1995) 699. [25] X. Yang, Y. Chen, F. Yang, F. He, S. Zhao, Dent. Mater. 25 (2009) 473. [26] K. Mustafa, A. Wennerberg, K. Arvidson, E.B. Messelt, P. Haag, S. Karlsson, Clin. Oral Impl. Res. 19 (2008) 1178. [27] G.A. Crawford, N. Chawla, K. Das, S. Bose, A. Bandyopadhyay, Acta. Biomater. 3 (2007) 359.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Fabrication and biocompatibility investigation of TiO2 films on the polymer substrates obtained via a novel and versatile route Junfei Ou a,b , Jinqing Wang a,∗ , Dong Zhang c , Puliang Zhang c , Sheng Liu a,b , Penghua Yan a , Bin Liu c , Shengrong Yang a a b c
State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China Graduate University of Chinese Academy of Sciences, Beijing 100080, China School of Stomatology, Lanzhou University, Lanzhou 730000,China
a r t i c l e
i n f o
Article history: Received 8 July 2009 Received in revised form 13 October 2009 Accepted 14 October 2009 Available online 23 October 2009 Keywords: Implanted polymer materials Polydopamine coating TiO2 films Biocompatibility
a b s t r a c t Titanium oxide (TiO2 ) films were successfully deposited onto the polymer substrates of polytetrafluoroethylene (PTFE), polyethylene (PE), and polyethylene terephthalate (PET), which were pre-modified with polydopamine coating (polydopamine and its coating are coded as PDA and PDAc, respectively), by a simple liquid phase deposition (LPD) process. The morphology and chemical state of the obtained TiO2 films were characterized by field emission scanning electron microscope (FE-SEM) and X-ray photoelectron spectroscopy (XPS), respectively. Subsequently, the biocompatibility of the samples was investigated by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and acridine orange staining of MC-3T3 osteoblast cells, and the results demonstrated that the fabricated TiO2 films could markedly improve the in vitro cytocompatibility. So, the presented route is anticipated to be a promising surface modification methodology to improve the practical outcome of the implanted materials for its versatility and validity. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Recently, some inactive polymers with different physicochemical properties such as chemical composition, surface hydrophobicity and reactivity, typically, polytetrafluoroethylene (PTFE), polyethylene (PE), and polyethylene terephthalate (PET), are widely employed as the clinical implanted materials [1–3] for their desirable mechanical and thermodynamic characteristics [4–6]. However, the practical outcome of these materials is greatly restricted by the poor tissue-material interactions after implantation owing to their inert and hydrophobic surfaces. So, the surface modification for improving the biocompatibility of the polymer substrates is quite necessary [6,7]. One of the most promising modification procedures is to construct bioceramic coatings, especially, titanium oxide (TiO2 ) films on the implanted materials since surface TiO2 films can tailor the biological reaction and interaction between artificial and living matter [8,9]. However, it was very difficult to construct bioceramic coatings on the inactive polymers (e.g. PTFE and PE, etc.) surfaces unless special large-scale apparatus [10] or plasma pretreatment [11] was applied.
∗ Corresponding authors. Tel.: +86 931 4968076; fax: +86 931 8277088. E-mail addresses: [email protected] (J. Wang), [email protected] (S. Yang). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.10.024
In this work, a novel and versatile route of fabricating adherent and uniform TiO2 films on different polymer substrates have been realized by a simple liquid phase deposition (LPD) process for the first time. Our inspiration profits from a magic compound of dopamine, a functional molecule containing catechol and amine groups, and both of which may be crucial for the high adhesive property of Mytilus edulis foot protein 5 (Mefp-5) [12]. It has been demonstrated that this kind of simple structure is a powerful building block for spontaneous deposition of thin polymer coatings on almost all material surfaces. Moreover, the active groups of –NH2 and –OH on the formed coating surfaces can facilitate the further modification [12]. Here, three polymer substrates of PTFE, PE, and PET were respectively immersed into a dilute dopamine aqueous solution and then the spontaneously formed thin polymer film was anticipated to provide a versatile platform for fabricating TiO2 films through a relative simple route, as schematically shown in Fig. 1(a). 2. Materials and methods 2.1. Materials 3-Hydroxytyramine hydrochloride (dopamine hydrochloride) and tris(hydroxymethyl) aminomethane (Tris) were purchased from Acros Organics. Ammonium fluotitanate ((NH4 )2 TiF6 ) was purchased from Shanghai SSS Reagent Co., Ltd. Boric acid
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Fig. 1. (a) A schematic view of the TiO2 deposition on the polymer substrates mediated by PDAc. (b) The “oxidation–polymerization” mechanism for the formation of PDA proposed by Messersmith and co-workers [12].
was obtained from Beijing Xinguang Chemical Reagent Factory. Dimethyl sulfoxide (DMSO) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco. Trypsin–EDTA solution and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were supplied by Sigma. Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. MC-3T3 osteoblast cells were supplied by the Fourth Military Medical University.
The cells were routinely cultured in DMEM containing 10% FBS and incubated at 37 ◦ C in a humidified chamber with 10% CO2 . The culture medium was refreshed every 2 days. When the cells became almost confluent, they were released by treating with 0.25% trypsin–EDTA solution for 3 min at 37 ◦ C and resuspended in the DMEM with a final concentration of 104 cells/ml.
2.2. Preparation of TiO2 films on the polymer substrates
In vitro cytocompatibility was investigated by MTT colorimetric assay. Briefly, the sterilized samples were placed into a 24-well culture plate and cells were seeded for 2 h, 4 h, 2 days, and 4 days, respectively. At the prescribed time point, 100 l of MTT phosphate buffer solution (PBS) (5 mg/ml, pH 7.4) was injected into each well containing the test sample and culture medium. The cultures were then kept at 37 ◦ C for 4 h. At the end of incubation, the supernatant was removed and 1 ml of DMSO was added into each well. The 24well plate was then shaken to dissolve the purple formazan crystals and the optical density (OD) value of the resulting solution was recorded using a micro-plate reader (Model 550, BIO-RAD, Japan) at the wavelength of 490 nm. The assay was conducted in triplicate for each sample and three parallel experiments were performed. To calibrate the cellular survival rate, blank and control groups were set. In the blank group, only culture media was added into the well. While, in the control group, cells and culture media without polymer substrates were added. The blank and control groups were treated with the same procedures and incubated for the same time as those in the experimental group. The measured OD values of the blank, control, and experimental groups were coded as ODbla , ODcon , and ODexp, respectively. Finally, the cellular survival rate was calculated by the following equation:
PET, PE, and PTFE plates were used as the substrates and cleaned thoroughly in acetone and water by ultrasonication (59 kHz, 45 W) in succession, followed by drying in ambient conditions. Then, the pretreated substrates were dipped into a 9 mM dopamine Tris–HCl solution (pH 8.5) for 12 h, followed by ultrasonication in ultra-pure water and blowing dry with N2 [10]. The PDA coated substrates were immersed into a fresh prepared aqueous solution containing 0.1 M (NH4 )2 TiF6 and 0.3 M H3 BO3 with a pH value of 3.88 at room temperature for 12 h, and then ultrasonically cleaned in water and blown dry with N2 . The prepared samples were coded as PET (or PE, PTFE)–PDAc–TiO2 . 2.3. Characterizations Contact angle meter (DSA100, Krüss, Germany) was used to measure the static water contact angle of the prepared samples. An ellipsometer (L116-E, Gaertner, USA) equipped with a He–Ne laser (632.8 nm) set was adopted to measure the film thickness. X-ray photoelectron spectroscopy (XPS, PHI-5702, Physical Electronics, USA) was performed using a monochromated Al–K␣ irradiation to determine the chemical state of certain elements with the C 1s binding energy of 284.8 eV as the reference. The morphologies of the pristine and TiO2 film coated polymer plates were observed by a field emission scanning electron microscope (FE-SEM, JEOL, JSM-6701F, Japan).
2.5. In vitro cytocompatibility assay
survival rate =
ODexp − ODbla . ODcon − ODbla
2.6. Acridine orange staining 2.4. Cell culture MC-3T3 osteoblast cells were employed to evaluate the biocompatibility of the pristine and TiO2 film coated polymer plates.
MC-3T3 osteoblast cells cultured on the pristine and TiO2 film coated polymer plates were visualized by fluorescent acridine orange staining. After being seeded for 2 days, all samples were
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Fig. 2. XPS spectra of various samples: survey spectra of PET (a-i), PTFE (b-i), and PE (c-i); survey spectra of PET–PDAc (a-ii), PTFE–PDAc (b-ii), and PE–PDAc (c-ii); survey spectra of PET–PDAc–TiO2 (a-iii), PTFE–PDAc–TiO2 , (b-iii) and PE–PDAc–TiO2 (c-iii). N 1s (d) and O 1s (e) spectra of the PDAc on different substrate surfaces (e); Ti 2p (f) and O 1s (g) spectra of the as-deposited TiO2 films on different substrate surfaces.
rinsed in PBS, and then cells were fixed in the ethanol for about 10 min. Subsequently, the cells attached to the sample surfaces were stained with acridine orange fluorescent dye in PBS for 3 min and examined by a fluorescence microscope (BX51, Olympus, Japan).
performed with Student’s t-test and values were considered to be significantly different when p < 0.05. 3. Results and discussion 3.1. The formation of TiO2 films on the polymer substrates
2.7. Statistics Data were expressed as the mean ± SD (standard deviation) from three independent experiments. Statistical analysis was
The formation of TiO2 films on polymer substrates mainly involved the following steps: substrate cleaning, dopamine polymerization, and TiO2 film deposition. Firstly, the polymer
Fig. 3. FE-SEM images of different samples: PET (a), PTFE (b), PE (c), PET–PDAc–TiO2 (d, g), PTFE–PDAc–TiO2 (e, h) and PE–PDAc–TiO2 (f, i).
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Fig. 4. Cellular survival rate obtained from MTT colorimetric assays. For all polymer substrates coated with TiO2 films, the differences as compared to the pristine polymer substrates were significant (p < 0.05).
substrates were cleaned under sonication in acetone and water successively, and then were dipped into a dopamine Tris–HCl solution with a pH value of 8.5. As proposed by Messersmith and co-workers, the PDAc formed on the polymer substrates mainly underwent an oxidation–polymerization process, i.e., the dopamine monomer molecules were firstly oxidized into 5, 6-dihydroxyindole under an alkaline condition, whose further oxidation caused the intermolecular cross-linking to yield PDA which can be adhered to almost all materials surfaces via a strong physisorption to form PDAc [12], as schematically illustrated in Fig. 1. The formation of PDAc can be confirmed by the characterizations of water contact angle measurement and X-ray photoelectron spectroscopy (XPS) analysis. After being immersed in dopamine solution and subsequently ultrasonicated in water and blown dry with N2 , the water contact angles of all samples decrease from above 90◦ for pristine polymer substrates (122◦ , 107◦ and 90.5◦ for PTFE, PE and PET, respectively) to about 50◦ , which was very close to the average value of PDAc on various substrates [12]. Moreover, XPS spectrum revealed the appearance of N 1s signal at 399.6 eV after deposition of PDAc (see Fig. 2(a–d)), which could be assigned to the N element of amino groups in the dopamine molecules. In this case, the exact thickness of PDAc was impracticable to be measured due to the rather rough surfaces of polymer plates. Alternatively, the polished Si wafer was employed as a substitute and the ellipsometric thickness of PDAc was measured to be about 20 nm under the same deposition conditions with the polymer plates. In addition, the adhesion strength of the PDAc to the polymer substrate was also very hard to be obtained directly. However, it can be reflected
indirectly by the ultrasonication treatment. As the results showed, the formed PDAc on the PET surface had the highest adhesion strength, which remained intact even after 30 min ultrasonication. The PDAc on the PTFE and PE substrates could keep stable and intact under the ultrasonication within 2 min, however, there was almost nothing left on the surfaces of PTFE and PET with prolonging ultrasonication time to 5 min, which could be indicated by the increase of water contact angle and color change from brown (PDAc) to white (polymer substrates of PTFE or PE). Finally, the fabrication of TiO2 film was accomplished by two processes: the heterogeneous nucleation and the homogeneous growth. It has been reported that the hydroxyl groups in catechol derivatives can react with certain metal oxide [13,14] and PDAc is hydrophilic with plentiful active hydroxyl groups [12]. In addition, in the precursor solution, the TiO2 nano-particles were formed by the hydrolysis of (NH4 )2 TiF6 and H3 BO3 [15–19]. Therefore, the heterogeneous nucleation was actualized through the chelation between the hydroxyl groups on the PDAc surface and the TiO2 nano-particles in the solution. The homogeneous growth was induced by the nucleated TiO2 layer on the substrate, which could be served as the seed layer to boost the homogeneous condensation of the TiO2 nano-particles. The formation of TiO2 films was further approved by characterizations of FE-SEM and XPS. From the FE-SEM images in Fig. 3, it can be seen that the polymer substrates were covered by TiO2 films. Moreover, the morphology of the TiO2 film was influenced by the morphology of the substrate. As displayed in Fig. 3(a–c), the pristine PET and PTFE plates were relatively flat, while PE was
Fig. 5. Fluorescent acridine orange staining of the osteoblast cells on the pristine polymer substrates (a: PET, c: PTFE, and e: PE) and TiO2 films coated polymer substrates (b: PET–PDAc–TiO2 , d: PTFE–PDAc–TiO2 , and f: PE–PDAc–TiO2 ) for 2 days, respectively; the images were magnified 40 times.
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apparently rough. Consequently, the TiO2 film on PE surface was uneven with some wrinkles (see Fig. 3(f) and (i)). Obviously, all the as-deposited TiO2 films are composed of plentiful TiO2 nanoparticles with the diameter of several nanometers, as displayed in Fig. 3(g–i). Due to the lateral shrinkage during the drying process [18], there are some cracks generated on the TiO2 films, as shown in Fig. 3(d–f). On the other hand, the spectral peaks in XPS corresponding to Ti 2p (458.9 eV) were observed from the deposited thin films on PDAc (see Fig. 2(f)). This binding energy value is very close to that of TiO2 (458.4–458.7 eV) [20–22] and higher than that of Ti metal (454.0 eV), suggesting that the titanium elements in the films are positively charged by formation of direct bonding with oxygen. While the O 1s signals of TiO2 modified polymer substrates are located at 530.2 eV and 532.2 eV, respectively (see Fig. 2(g)). The former value is similar to that of TiO2 (529.9 eV [20], 530.1 eV [21], and 530.2 eV [23]) and lower than that of neutral oxygen molecules (531.0 eV [23]), indicating that the oxygen is negatively charged, possibly through the formation of chemical bonding with Ti. The binding energy value of the latter one is almost the same as that of O 1s in PDAc (Fig. 2(e)), suggesting that the signal is most likely to be resulting from the exposed PDAc, which was facilitated by the cracks of TiO2 film, as clearly shown in Fig. 3(d–f) [22,24]. 3.2. In vitro cytocompatibility assay To evaluate the in vitro cytocompatibility of the samples, MTT colorimetric assay was performed. As shown in Fig. 4(a), the cellular survival rates on the TiO2 coated polymer substrates over 2 and 4 h were about 10% higher than that on the corresponding pristine substrates. Moreover, the difference of the cellular survival rates increased to about 20% after 2 and 4 days of cell seeding, indicating that the fabricated TiO2 film apparently enhanced the cytocompatibility (Fig. 4(b)) [25,26]. Moreover, for the samples of PTFE–PDAc–TiO2 and PE-PDAc-TiO2 , the survival rates were a little lower than that on PET-PDAc-TiO2 , which might be attributed to the fact that the TiO2 films on edges of PTFE and PE substrates were partially destroyed by ultrasonication owing to their less adhesive strength to substrates. In addition, the fluorescent staining result was shown in Fig. 5, which revealed that the greater and denser osteoblasts were proliferated well on the TiO2 coated polymer substrates as compared with the corresponding pristine ones. Based on the above mentioned experimental results, it can be concluded that the cellular survival rate of the polymer substrates has been improved greatly, which can be definitely attributed to the excellent intrinsic biocompatibility of the TiO2 films. On the other hand, the TiO2 films fabricated via this simple process were hydrophilic (water contact angle values of about 0◦ ) with abundant hydroxyl groups on the surfaces (as depicted in Fig. 1), which markedly promoted the cell-material interaction [27]. 4. Conclusion For the first time, a novel and versatile route of fabricating TiO2 films on different polymer surfaces, even the high hydrophobic
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PTFE surface, has been realized via a simple LPD process. The formed TiO2 film plays a significant role in improving the biocompatibility of the polymer materials, demonstrating this simple approach has overcome the obstacles restricting the application of polymer in medical implanted materials and will be a promising surface modification methodology for its versatility and simple process. Acknowledgements The authors are grateful to the National Natural Science Foundation of China (Grant Nos. 50801065 and 20823008) and “Top Hundred Talents Program” of Chinese Academy of Sciences for financial support. References [1] K. Vallières, É. Petitclerc, G. Laroche, Macromol. Biosci. 7 (2007) 738. [2] W. Bonfield, M.D. Grynpas, A.E. Tully, J. Bowman, J. Abram, Biomaterials 2 (1981) 185. [3] J.M. Brynaert, M. Deldime, I. Dupont, J.L. Dewez, Y.J. Schneider, J. Colloid. Interf. Sci. 173 (1995) 236. [4] J.N. Meussdoerffer, H. Niederpriim, Chem. Zeitung 104 (1980) 45. [5] S. Ramakrishna, J. Mayer, E. Wintermantel, K.W. Leong, Comp. Sci. Technol. 61 (2001) 1189. [6] L. Yang, J. Chen, Y. Guo, Z. Zhang, Appl. Surf. Sci. 255 (2009) 4446. [7] V. Moby, C. Boura, H. Kerdjoudj, J.-C. Voegel, L. Marchal, D. Dumas, P. Schaaf, J.-F. Stoltz, P. Menu, Biomacromolecules 8 (2007) 2156. [8] K.-L. Ou, Y.-H. Shih, C.-F. Huang, C.-C. Chen, C.-M. Liu, Appl. Surf. Sci. 255 (2009) 2046. [9] L. Giorgetti, G. Bongiorno, A. Podestà, G. Berlanda, P.E. Scopelliti, R. Carbone, P. Milani, Langmuir 24 (2008) 11637. [10] M. Kemell, E. Färm, M. Ritala, M. Leskelä, Eur. Polym. J. 44 (2008) 3564. [11] G. Acharya, T. Kunitake, Langmuir 19 (2003) 2260. [12] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007) 426. [13] M. Guvendiren, P.B. Messersmith, K.R. Shull, Biomacromolecule 9 (2008) 122. [14] M.J. Sever, J.T. Weisser, J. Monahan, S. Srinivasan, J. Wilker, Angew. Chem. Int. Ed. 43 (2004) 448. [15] S. Deki, Y. Aoi, O. Hiroi, A. Kajinami, Chem. Lett. (1996) 433. [16] S. Deki, Y. Aoi, H. Yanagimoto, K. Ishii, K. Akamatsu, M. Mitzuhata, A. Kajinami, J. Mater. Chem. 6 (1997) 1879. [17] S. Deki, Y. Aoi, Y. Asaoka, A. Kajinami, M. Mizuhata, J. Mater. Chem. 7 (1997) 733. [18] H. Pizem, C.N. Sukenik, U. Sampathkumaran, A.K. McIlwain, M.R. De Guire, Chem. Mater. 14 (2002) 2476. [19] A. Razgon, C.N. Sukenik, J. Mater. Res. 20 (2005) 2544. [20] R.J. Collins, H. Shin, M.R. DeGuire, A.H. Heuer, C.N. Sukenik, Appl. Phys. Lett. 69 (1996) 860. [21] F. Zhang, S. Jin, Y. Mao, Z. Zheng, Y. Chen, X. Liu, Thin Solid Films 310 (1997) 29. [22] D. Huang, Z.-D. Xiao, J. -H. Gu, N.-P. Huang, C.W. Yuan, Thin Solid Films 305 (1997) 110. [23] Y. Masuda, N. Saito, R. Hoffmann, M.R. DeGuire, K. Koumoto, Sci. Technol. Adv. Mater. 4 (2003) 461. [24] H. Shin, R.J. Collins, M.R. De Guire, A.H. Heuer, C.N. Sukenik, J. Mater. Res. 10 (1995) 699. [25] X. Yang, Y. Chen, F. Yang, F. He, S. Zhao, Dent. Mater. 25 (2009) 473. [26] K. Mustafa, A. Wennerberg, K. Arvidson, E.B. Messelt, P. Haag, S. Karlsson, Clin. Oral Impl. Res. 19 (2008) 1178. [27] G.A. Crawford, N. Chawla, K. Das, S. Bose, A. Bandyopadhyay, Acta. Biomater. 3 (2007) 359.