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Incorporation of cytochrome C with thin calcium phosphate film formed by electron-beam evaporation
Surface & Coatings Technology 202 (2008) 5742–5745
Contents lists available at ScienceDirect
Surface & Coatings Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s u r f c o a t
Incorporation of cytochrome C with thin calcium phosphate film formed by electron-beam evaporation Yan Li a, In-Seop Lee b,⁎, Fu-Zhai Cui a, Zeng Lin b, Jong-Chul Park c, Sung-Min Chung d a
Advanced Materials Laboratory, Department of Materials Science & Engineering, Tsinghua University, Beijing 100084, China Institute of Physics & Applied Physics, and Atomic-scale Surface Science Research Center, Yonsei University, Seou1 120-749, South Korea Department of Medical Engineering, College of Medicine, Yonsei University, Seoul 120-749, South Korea d Implantium Implant Institute, Seoul 135-879, South Korea b c
A R T I C L E
I N F O
Available online 14 June 2008 Keywords: Calcium phosphate Cytochrome C DPBS e-beam evaporation Cyt C-apatite composite layer
A B S T R A C T Surface properties play a major role to determine the biocompatibility that is the ability of a material to perform with an appropriate host response in a specific application because biomaterials contact and interact with biological systems. Various methods have been studied to improve the surface properties and farther the osseous intergradation of bone implants. Cytochrome C (cyt C) was immobilized on the thin calcium phosphate films formed by electron-beam evaporation in the form of cyt C-apatite composite layer. The newly formed apatite layer was observed by scanning electron microscopy, and X-ray photoelectron spectroscopy confirmed that cyt C existed in the newly formed layer. Cyt C released from the cyt C-apatite composite layer for at least 10 days in the physiological salt solution. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Due to their good biocompatibility and superior mechanical properties, titanium and its alloys are widely used as biomedical implant materials, such as artificial tooth roots or joint prosthesis. Calcium phosphate layer is commonly coated on the surface of metallic implants using various coating methods for fast fixation, such as plasma spraying technique [1], ion-beam sputter deposition [2], pulsed laser deposition [3], and ion-beam-assisted deposition (IBAD) [4–6]. Biomaterials incorporated with biologically active proteins are promising in the field of biomedical engineering and tissue engineering. Researchers have been seeking for various methods to produce substrates carrying with proteins, such as physical absorption, electrostatic self-assembly, and chemical crosslink [7–10]. Firmly and densely immobilized proteins improve cell attachment to an implant, hence, the final tissue-implant adhesion. So far, protein has been firmly immobilized in a protein-apatite composite layer on various substrates by immersing biomaterials in supersaturated calcium phosphate solutions contain proteins [11–15]. Basic fibroblast growth factor (bFGF or FGF-2) has crucial properties in bone formation and the wound-healing process. For a sustained release of FGF-2, different scaffolds including hydroxyapatite and biodegradable polymers were used as the carrier of FGF-2 [16–20].
⁎ Corresponding author. Institute of Physics & Applied Physics, and Atomic-scale Surface Science Research Center, Yonsei University, 134 Shinchon-dong, Seodaemoongu, Seoul 120-749, South Korea. Tel.: +82 2 2123 4806; fax: +82 2 313 3537. E-mail address: [email protected] (I.-S. Lee). 0257-8972/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2008.06.082
Since the molecular weight and the isoelectric point of cytochrome C (cyt C, 12.3 kDa, pI = 10) are almost the same as those of FGF-2 (18 kDa, pI = 9.6), cyt C can be used as a dummy protein for FGF-2. Sogo et al. [14] has incorporated cyt C, in the homogeneously bone-like apatite layer by immersing hydroxyapatite disk in supersaturated calcium phosphates solution containing cyt C. In the present study, cytochrome C (cyt C) was incorporated with the thin calcium phosphate films formed on titanium by electron-beam evaporation during the formation of cyt C-apatite composite layer in Dulbecco's Phosphate buffered saline (DPBS) solution containing cyt C. And, the cyt C-apatite composite layer was evaluated. 2. Materials and methods 2.1. Preparation of samples Commercially pure Ti disks (10 mm in diameter and 2 mm in thickness) with different surface treatments were used as substrates. Thin calcium phosphate films were deposited on the substrates to a thickness of 500 nm by electron-beam evaporation. Heat treatments were conduced after the deposition. Samples were heated to different temperatures with the heating rate of 5 °C/min, and held for 1 h, and then cooled naturally in furnace. Three groups of samples with different surface finish were obtained. They were calcium phosphate coating on machined surface, heated to 700 °C in air (M700), calcium phosphate coating on anodized surface, heated to 450 °C in air (A450), and calcium phosphate coating on anodized surface, heat to 350 °C in vacuum (A350). Details of the anodized procedures and deposition employed in this study have been described previously [4,21].
Y. Li et al. / Surface & Coatings Technology 202 (2008) 5742–5745
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Fig. 3. X-ray photoelectron spectra of sample M700 (a) and sample M700 after immersion into DPBSC solution (b). Fig. 1. Amount of cyt C incorporated with each disk of different samples.
2.2. Immersion in solutions Dissolving Dulbecco's Phosphate buffered saline (Calcium/Magnesium free, Gibcobrl Life Technologies, USA) and reagent-grade CaCl2 (100 mg/L) in ultra-pure water to prepare the DPBS solution. Cyt C (Sigma Chemicals, USA. 40 μg/mL) was added to the DPBS solution to prepare the DPBSC solution. The samples with different surface finish were immersed in 2.0 mL of the DPBSC solution at 37 °C for 2 days.
For release test, the samples incorporated with cyt C were washed with a physiological salt solution twice and then immersed in 1 mL of physiological salt solution, stand at 37 °C for up to 10 days in a water bath. The physiological salt solution was prepared by dissolving reagent-grade NaCl (142 mM) in ultra-pure water and buffering to PH 7.4 at a temperature of 37 °C using TRIS (50 mM) and 1 M HCl. Before use, all the solutions were sterilized by filtration using a membrane with a pore size of 0.22 μm.
Fig. 2. SEM micrographs of surfaces of different samples before and after immersion in DPBSC solution for 2 days at 37 °C. (a) M700, before immersion. (b) and (c) M700, after immersion. (d) A450, before immersion. (e) and (f) A450, after immersion. (g) A350, before immersion. (h) and (i) A350, after immersion. The scales in (a), (b), (d), (e), (g), and (h) are 30 μm in length, and in (c), (f) and (i) are 3 μm in length.
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Y. Li et al. / Surface & Coatings Technology 202 (2008) 5742–5745
the other two groups, sample A350 has loaded the maximum amount of cyt C, which is 4.546 μg per disk. 3.2. SEM observation Fig. 2 shows the morphology of the three groups of samples before and after incubation in DPBSC solution for 2 days at 37 °C. Once the samples were immersed in the DPBSC solutions, flake like crystals completely covered the surfaces of the samples. The morphologies of the crystals exhibited only negligible differences between sample A450 and sample A350, while the crystals formed on sample M700 grow larger. 3.3. XPS analyses
Fig. 4. Release curve of cytochrome C incorporated with different samples.
After immersion in the DPBSC solution, nitrogen, which exited only in cyt C among the reagents used in the present study, was detected on all the three groups of samples as shown in Fig. 3. Considering the protein quantitative assay based on the BCA method and SEM observations, it is confirmed that a thin new apatite layer incorporated with cyt C was formed on all the three groups of samples.
2.3. Analysis of samples and solutions 3.4. Release test For surface analyses, the samples were washed with ultra-pure water twice and dried at room temperature after immersion. The surfaces of the samples were observed by scanning electron microscopy (SEM, S-4200, Hitachi, Japan). Additionally, the surfaces were analyzed using X-ray photoelectron spectroscopy (XPS, PHI 5700) with Al Kα X-rays. The photoelectron take-off angle was set at 45° for XPS. The cyt C concentration of the DPBSC solution after immersion of the samples was measured using the BCA (Micro BCA™ protein assay kit, Pierce Biotechnology Inc, USA) method. Before test, 0.1 mL of 0.1 M HCl solution was supplemented to dissolve any calcium phosphate precipitate dispersed in the solution. The amounts of the released cyt C in physiological salt solution were also analyzed by the BCA method.
Cyt C was slowly released from the apatite layer newly formed on the surfaces of the samples when immersed in a physiological salt solution. The release continued for at least 10 days (Fig. 4). According to the release curves of M700 and A450, there is a burst release within 1 day, and the amount of released cyt C almost reached to one-half of that released for 10 days. And after that, the release rate remained slowly. Whereas the release rate of cyt C for Sample A350 remained rather constant throughout the whole period of the 10 days. Fig. 5 shows the changes in morphology of the calcium phosphate layer incorporating cyt C after 3-day immersion in the physiological salt solution. The changes indicated the dissolution of the apatite layer.
3. Results
4. Discussion
3.1. Amount of cyt C incorporated with samples
Recently, soaking the implants in supersaturated calcium phosphate solutions containing some biologically active molecules at physiological temperatures has been developed to form the coprecipitate of active molecules and apatite on the surface of the implants [11–14]. When implanted, these loaded molecules will release in the body and be of
The concentrations of cyt C in the DPBSC solutions decreased after immersion all the three groups of samples. The amount of immobilized cyt C on each disk of different samples was shown in Fig. 1. Compared to
Fig. 5. SEM micrographs of surfaces of different samples incorporated with cyt C after immersion in the physiological salt solution for 3 days at 37 °C. (a) and (d) M700. (b) and (e) A450. (c) and (f) A350. The scales in (a), (b) and (c) are 30 μm in length, in (d), (e) and (f) are 3 μm in length.
Y. Li et al. / Surface & Coatings Technology 202 (2008) 5742–5745
great value for the intergradation of the implants with the host tissue. Our results suggested that cyt C was successfully immobilized in a cyt C-apatite composite layer formed on the surface of the sample by the use of DPBS solution. Although the structure of the newly formed calcium phosphate layer is yet to be identified, both powder X-ray diffraction and thin-film X-ray diffraction analyses failed to identify the calcium phosphate phase due to the overlapping of diffraction peaks of the newly formed apatite layer and those of the as-deposited calcium phosphate, and also the precise mechanism of formation of the composite layer should be investigated in future, the surface changes of the samples can be explained as follows. After immersing in the DPBSC solution, a small quantity of calcium phosphate is deposited onto the surface of the sample. This calcium phosphate should be apatite nuclei or precursors of apatite such as amorphous calcium phosphate. Because the DPBSC solutions are supersaturated with respect to apatite, and apatite has lower solubility than any other calcium phosphates in aqueous media around PH 7.40, the nuclei or precursors of apatite spontaneously grow into an apatite layer. Simultaneously, the incorporation of cyt C into the growing apatite proceeded to produce a cyt Capatite composite layer on the surface of the sample. The amount of cyt C incorporated with sample A350 was the maximum. It is apparent that the surface area of anodized sample is larger than that of machined sample because of roughness. That's why the amount of cyt C incorporated with anodized sample was larger than that with machined sample. In addition, to heat in vacuum, the structure of the calcium phosphate deposited on the Ti disk by electron-beam evaporation was less steady than to heat in the air because of the less of oxygen. So when immersing into DPBSC solution, the dissolution of calcium phosphate deposited on the samples heated in vacuum was easier than on the samples heated in the air. Dissolution and precipitation occur simultaneously in the solution. At the mean time of the dissolution of deposited calcium phosphate, apatite precipitated on the surface of the samples. The easier the dissolution occurs, the more calcium and phosphate ions would be in the solution, the more cyt C-apatite composite would further form on the surface of the samples. So that sample A350 has incorporated with the largest amount of cyt C. The immobility of cyt C in cyt C-apatite layer has advantages over that of cyt C absorbed to substrates, which can be confirmed by the release test that the release of cyt C continued at least 10 days. In addition, the incorporation of cyt C in the apatite layer was homogeneously since cyt C was released constantly. Other bioactive protein moleculars should also be incorporated with the substrate using the similar method, especially for FGF-2 which has the similar isoelectric point and molecular weight with cyt C.
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5. Conclusions Cyt C-apatite composite layer was successfully formed on thin calcium phosphate film when immersed in DPBSC solution, and Sample A350 loaded with the maximum amount of cyt C. The release of cyt C from the cyt C-apatite composite layer continued at least 10 days. Similar results were expected in the case of FGF-2. Acknowledgements This work was supported by a grant (code #: 08K1501-01220) from Center for Nanostructured Materials Technology under 21st Century Frontier R&D Program of the Ministry of Science and Technology, Korea. References [1] Y.C. Tsui, C. Doyle, T.W. Clyne, Biomaterials 19 (1998) 2015. [2] C.X. Wang, Z.Q. Chen, M. Wang, J. Mater. Sci.-Mater. Med. 13 (2002) 247. [3] Q.H. Bao, C.Z. Chen, D.G. Wang, T.Q. Lei, J.M. Liu, Mater. Sci. Eng. A-Struct. Mater. Prop. Microstruct. Process. 429 (2006) 25. [4] J.M. Choi, H.E. Kim, I.S. Lee, Biomaterials 21 (2000) 469. [5] I.S. Lee, C.N. Whang, H.E. Kim, J.C. Park, J.H. Song, S.R. Kim, Mater. Sci. Eng. C-Biomimetic Supramol. Syst. 22 (2002) 15. [6] F.Z. Cui, Z.S. Luo, Q.L. Feng, J. Mater. Sci.-Mater. Med. 8 (403) (1997). [7] Y.Z. Yang, R. Cavin, J.L. Ong, J. Biomed. Mater. Res. Part A 67A (2003) 344. [8] M. Gilbert, C.M. Giachelli, P.S. Stayton, J. Biomed. Mater. Res. Part A 67A (2003) 69. [9] H.G. Zhu, J. Ji, J.C. Shen, Biomacromolecules 5 (2004) 1933. [10] W.M. Tian, C.L. Zhang, S.P. Hou, X. Yu, F.Z. Cui, Q.Y. Xu, S.L. Sheng, H. Cui, H.D. Li, J. Control. Release 102 (2005) 13. [11] A. Oyane, M. Uchida, A. Ito, J. Biomed. Mater. Res. Part A 72A (2005) 168. [12] Y.L. Liu, P. Layrolle, J. de Bruijn, C. van Blitterswijk, K. de Groot, J. Biomed. Mater. Res. 57 (2001) 327. [13] M. Uchida, A. Oyane, H.M. Kim, T. Kokubo, A. Ito, Adv. Mater. 16 (2004) 1071. [14] Y. Sogo, A. Ito, K. Fukasawa, N. Kondo, Y. Ishikawa, N. Ichinose, A. Yamazaki, Curr. Appl. Phys. 5 (2005) 526. [15] G. Daculsi, P. Pilet, M. Cottrel, G. Guicheux, J. Biomed. Mater. Res. 47 (1999) 228. [16] X.E. Dereka, C.E. Markopoulou, A. Mamalis, E. Pepelassi, I.A. Vrotsos, Clin. Oral Implant. Res. 17 (2006) 554. [17] X. Huang, D.S. Yang, W.Q. Yan, Z.L. Shi, J. Feng, Y.B. Gao, W.J. Weng, S.G. Yan, Biomaterials 28 (2007) 3091. [18] H. Igai, Y. Yamamoto, S.S. Chang, M. Yamamoto, Y. Tabata, H. Yokomise, J. Thorac. Cardiovasc. Surg. 134 (2007) 170. [19] M.S. Park, S.S. Kim, S.W. Cho, C.Y. Choi, B.S. Kim, J. Biomed. Mater. Res. Part B 79B (2006) 353. [20] G. Zellin, A. Linde, Bone 26 (2000) 161. [21] J.M. Choi, Y.M. Kong, S. Kim, H.E. Kim, C.S. Kwang, I.S. Lee, J. Mater. Res. 14 (1999) 2980.
Contents lists available at ScienceDirect
Surface & Coatings Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s u r f c o a t
Incorporation of cytochrome C with thin calcium phosphate film formed by electron-beam evaporation Yan Li a, In-Seop Lee b,⁎, Fu-Zhai Cui a, Zeng Lin b, Jong-Chul Park c, Sung-Min Chung d a
Advanced Materials Laboratory, Department of Materials Science & Engineering, Tsinghua University, Beijing 100084, China Institute of Physics & Applied Physics, and Atomic-scale Surface Science Research Center, Yonsei University, Seou1 120-749, South Korea Department of Medical Engineering, College of Medicine, Yonsei University, Seoul 120-749, South Korea d Implantium Implant Institute, Seoul 135-879, South Korea b c
A R T I C L E
I N F O
Available online 14 June 2008 Keywords: Calcium phosphate Cytochrome C DPBS e-beam evaporation Cyt C-apatite composite layer
A B S T R A C T Surface properties play a major role to determine the biocompatibility that is the ability of a material to perform with an appropriate host response in a specific application because biomaterials contact and interact with biological systems. Various methods have been studied to improve the surface properties and farther the osseous intergradation of bone implants. Cytochrome C (cyt C) was immobilized on the thin calcium phosphate films formed by electron-beam evaporation in the form of cyt C-apatite composite layer. The newly formed apatite layer was observed by scanning electron microscopy, and X-ray photoelectron spectroscopy confirmed that cyt C existed in the newly formed layer. Cyt C released from the cyt C-apatite composite layer for at least 10 days in the physiological salt solution. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Due to their good biocompatibility and superior mechanical properties, titanium and its alloys are widely used as biomedical implant materials, such as artificial tooth roots or joint prosthesis. Calcium phosphate layer is commonly coated on the surface of metallic implants using various coating methods for fast fixation, such as plasma spraying technique [1], ion-beam sputter deposition [2], pulsed laser deposition [3], and ion-beam-assisted deposition (IBAD) [4–6]. Biomaterials incorporated with biologically active proteins are promising in the field of biomedical engineering and tissue engineering. Researchers have been seeking for various methods to produce substrates carrying with proteins, such as physical absorption, electrostatic self-assembly, and chemical crosslink [7–10]. Firmly and densely immobilized proteins improve cell attachment to an implant, hence, the final tissue-implant adhesion. So far, protein has been firmly immobilized in a protein-apatite composite layer on various substrates by immersing biomaterials in supersaturated calcium phosphate solutions contain proteins [11–15]. Basic fibroblast growth factor (bFGF or FGF-2) has crucial properties in bone formation and the wound-healing process. For a sustained release of FGF-2, different scaffolds including hydroxyapatite and biodegradable polymers were used as the carrier of FGF-2 [16–20].
⁎ Corresponding author. Institute of Physics & Applied Physics, and Atomic-scale Surface Science Research Center, Yonsei University, 134 Shinchon-dong, Seodaemoongu, Seoul 120-749, South Korea. Tel.: +82 2 2123 4806; fax: +82 2 313 3537. E-mail address: [email protected] (I.-S. Lee). 0257-8972/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2008.06.082
Since the molecular weight and the isoelectric point of cytochrome C (cyt C, 12.3 kDa, pI = 10) are almost the same as those of FGF-2 (18 kDa, pI = 9.6), cyt C can be used as a dummy protein for FGF-2. Sogo et al. [14] has incorporated cyt C, in the homogeneously bone-like apatite layer by immersing hydroxyapatite disk in supersaturated calcium phosphates solution containing cyt C. In the present study, cytochrome C (cyt C) was incorporated with the thin calcium phosphate films formed on titanium by electron-beam evaporation during the formation of cyt C-apatite composite layer in Dulbecco's Phosphate buffered saline (DPBS) solution containing cyt C. And, the cyt C-apatite composite layer was evaluated. 2. Materials and methods 2.1. Preparation of samples Commercially pure Ti disks (10 mm in diameter and 2 mm in thickness) with different surface treatments were used as substrates. Thin calcium phosphate films were deposited on the substrates to a thickness of 500 nm by electron-beam evaporation. Heat treatments were conduced after the deposition. Samples were heated to different temperatures with the heating rate of 5 °C/min, and held for 1 h, and then cooled naturally in furnace. Three groups of samples with different surface finish were obtained. They were calcium phosphate coating on machined surface, heated to 700 °C in air (M700), calcium phosphate coating on anodized surface, heated to 450 °C in air (A450), and calcium phosphate coating on anodized surface, heat to 350 °C in vacuum (A350). Details of the anodized procedures and deposition employed in this study have been described previously [4,21].
Y. Li et al. / Surface & Coatings Technology 202 (2008) 5742–5745
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Fig. 3. X-ray photoelectron spectra of sample M700 (a) and sample M700 after immersion into DPBSC solution (b). Fig. 1. Amount of cyt C incorporated with each disk of different samples.
2.2. Immersion in solutions Dissolving Dulbecco's Phosphate buffered saline (Calcium/Magnesium free, Gibcobrl Life Technologies, USA) and reagent-grade CaCl2 (100 mg/L) in ultra-pure water to prepare the DPBS solution. Cyt C (Sigma Chemicals, USA. 40 μg/mL) was added to the DPBS solution to prepare the DPBSC solution. The samples with different surface finish were immersed in 2.0 mL of the DPBSC solution at 37 °C for 2 days.
For release test, the samples incorporated with cyt C were washed with a physiological salt solution twice and then immersed in 1 mL of physiological salt solution, stand at 37 °C for up to 10 days in a water bath. The physiological salt solution was prepared by dissolving reagent-grade NaCl (142 mM) in ultra-pure water and buffering to PH 7.4 at a temperature of 37 °C using TRIS (50 mM) and 1 M HCl. Before use, all the solutions were sterilized by filtration using a membrane with a pore size of 0.22 μm.
Fig. 2. SEM micrographs of surfaces of different samples before and after immersion in DPBSC solution for 2 days at 37 °C. (a) M700, before immersion. (b) and (c) M700, after immersion. (d) A450, before immersion. (e) and (f) A450, after immersion. (g) A350, before immersion. (h) and (i) A350, after immersion. The scales in (a), (b), (d), (e), (g), and (h) are 30 μm in length, and in (c), (f) and (i) are 3 μm in length.
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Y. Li et al. / Surface & Coatings Technology 202 (2008) 5742–5745
the other two groups, sample A350 has loaded the maximum amount of cyt C, which is 4.546 μg per disk. 3.2. SEM observation Fig. 2 shows the morphology of the three groups of samples before and after incubation in DPBSC solution for 2 days at 37 °C. Once the samples were immersed in the DPBSC solutions, flake like crystals completely covered the surfaces of the samples. The morphologies of the crystals exhibited only negligible differences between sample A450 and sample A350, while the crystals formed on sample M700 grow larger. 3.3. XPS analyses
Fig. 4. Release curve of cytochrome C incorporated with different samples.
After immersion in the DPBSC solution, nitrogen, which exited only in cyt C among the reagents used in the present study, was detected on all the three groups of samples as shown in Fig. 3. Considering the protein quantitative assay based on the BCA method and SEM observations, it is confirmed that a thin new apatite layer incorporated with cyt C was formed on all the three groups of samples.
2.3. Analysis of samples and solutions 3.4. Release test For surface analyses, the samples were washed with ultra-pure water twice and dried at room temperature after immersion. The surfaces of the samples were observed by scanning electron microscopy (SEM, S-4200, Hitachi, Japan). Additionally, the surfaces were analyzed using X-ray photoelectron spectroscopy (XPS, PHI 5700) with Al Kα X-rays. The photoelectron take-off angle was set at 45° for XPS. The cyt C concentration of the DPBSC solution after immersion of the samples was measured using the BCA (Micro BCA™ protein assay kit, Pierce Biotechnology Inc, USA) method. Before test, 0.1 mL of 0.1 M HCl solution was supplemented to dissolve any calcium phosphate precipitate dispersed in the solution. The amounts of the released cyt C in physiological salt solution were also analyzed by the BCA method.
Cyt C was slowly released from the apatite layer newly formed on the surfaces of the samples when immersed in a physiological salt solution. The release continued for at least 10 days (Fig. 4). According to the release curves of M700 and A450, there is a burst release within 1 day, and the amount of released cyt C almost reached to one-half of that released for 10 days. And after that, the release rate remained slowly. Whereas the release rate of cyt C for Sample A350 remained rather constant throughout the whole period of the 10 days. Fig. 5 shows the changes in morphology of the calcium phosphate layer incorporating cyt C after 3-day immersion in the physiological salt solution. The changes indicated the dissolution of the apatite layer.
3. Results
4. Discussion
3.1. Amount of cyt C incorporated with samples
Recently, soaking the implants in supersaturated calcium phosphate solutions containing some biologically active molecules at physiological temperatures has been developed to form the coprecipitate of active molecules and apatite on the surface of the implants [11–14]. When implanted, these loaded molecules will release in the body and be of
The concentrations of cyt C in the DPBSC solutions decreased after immersion all the three groups of samples. The amount of immobilized cyt C on each disk of different samples was shown in Fig. 1. Compared to
Fig. 5. SEM micrographs of surfaces of different samples incorporated with cyt C after immersion in the physiological salt solution for 3 days at 37 °C. (a) and (d) M700. (b) and (e) A450. (c) and (f) A350. The scales in (a), (b) and (c) are 30 μm in length, in (d), (e) and (f) are 3 μm in length.
Y. Li et al. / Surface & Coatings Technology 202 (2008) 5742–5745
great value for the intergradation of the implants with the host tissue. Our results suggested that cyt C was successfully immobilized in a cyt C-apatite composite layer formed on the surface of the sample by the use of DPBS solution. Although the structure of the newly formed calcium phosphate layer is yet to be identified, both powder X-ray diffraction and thin-film X-ray diffraction analyses failed to identify the calcium phosphate phase due to the overlapping of diffraction peaks of the newly formed apatite layer and those of the as-deposited calcium phosphate, and also the precise mechanism of formation of the composite layer should be investigated in future, the surface changes of the samples can be explained as follows. After immersing in the DPBSC solution, a small quantity of calcium phosphate is deposited onto the surface of the sample. This calcium phosphate should be apatite nuclei or precursors of apatite such as amorphous calcium phosphate. Because the DPBSC solutions are supersaturated with respect to apatite, and apatite has lower solubility than any other calcium phosphates in aqueous media around PH 7.40, the nuclei or precursors of apatite spontaneously grow into an apatite layer. Simultaneously, the incorporation of cyt C into the growing apatite proceeded to produce a cyt Capatite composite layer on the surface of the sample. The amount of cyt C incorporated with sample A350 was the maximum. It is apparent that the surface area of anodized sample is larger than that of machined sample because of roughness. That's why the amount of cyt C incorporated with anodized sample was larger than that with machined sample. In addition, to heat in vacuum, the structure of the calcium phosphate deposited on the Ti disk by electron-beam evaporation was less steady than to heat in the air because of the less of oxygen. So when immersing into DPBSC solution, the dissolution of calcium phosphate deposited on the samples heated in vacuum was easier than on the samples heated in the air. Dissolution and precipitation occur simultaneously in the solution. At the mean time of the dissolution of deposited calcium phosphate, apatite precipitated on the surface of the samples. The easier the dissolution occurs, the more calcium and phosphate ions would be in the solution, the more cyt C-apatite composite would further form on the surface of the samples. So that sample A350 has incorporated with the largest amount of cyt C. The immobility of cyt C in cyt C-apatite layer has advantages over that of cyt C absorbed to substrates, which can be confirmed by the release test that the release of cyt C continued at least 10 days. In addition, the incorporation of cyt C in the apatite layer was homogeneously since cyt C was released constantly. Other bioactive protein moleculars should also be incorporated with the substrate using the similar method, especially for FGF-2 which has the similar isoelectric point and molecular weight with cyt C.
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5. Conclusions Cyt C-apatite composite layer was successfully formed on thin calcium phosphate film when immersed in DPBSC solution, and Sample A350 loaded with the maximum amount of cyt C. The release of cyt C from the cyt C-apatite composite layer continued at least 10 days. Similar results were expected in the case of FGF-2. Acknowledgements This work was supported by a grant (code #: 08K1501-01220) from Center for Nanostructured Materials Technology under 21st Century Frontier R&D Program of the Ministry of Science and Technology, Korea. References [1] Y.C. Tsui, C. Doyle, T.W. Clyne, Biomaterials 19 (1998) 2015. [2] C.X. Wang, Z.Q. Chen, M. Wang, J. Mater. Sci.-Mater. Med. 13 (2002) 247. [3] Q.H. Bao, C.Z. Chen, D.G. Wang, T.Q. Lei, J.M. Liu, Mater. Sci. Eng. A-Struct. Mater. Prop. Microstruct. Process. 429 (2006) 25. [4] J.M. Choi, H.E. Kim, I.S. Lee, Biomaterials 21 (2000) 469. [5] I.S. Lee, C.N. Whang, H.E. Kim, J.C. Park, J.H. Song, S.R. Kim, Mater. Sci. Eng. C-Biomimetic Supramol. Syst. 22 (2002) 15. [6] F.Z. Cui, Z.S. Luo, Q.L. Feng, J. Mater. Sci.-Mater. Med. 8 (403) (1997). [7] Y.Z. Yang, R. Cavin, J.L. Ong, J. Biomed. Mater. Res. Part A 67A (2003) 344. [8] M. Gilbert, C.M. Giachelli, P.S. Stayton, J. Biomed. Mater. Res. Part A 67A (2003) 69. [9] H.G. Zhu, J. Ji, J.C. Shen, Biomacromolecules 5 (2004) 1933. [10] W.M. Tian, C.L. Zhang, S.P. Hou, X. Yu, F.Z. Cui, Q.Y. Xu, S.L. Sheng, H. Cui, H.D. Li, J. Control. Release 102 (2005) 13. [11] A. Oyane, M. Uchida, A. Ito, J. Biomed. Mater. Res. Part A 72A (2005) 168. [12] Y.L. Liu, P. Layrolle, J. de Bruijn, C. van Blitterswijk, K. de Groot, J. Biomed. Mater. Res. 57 (2001) 327. [13] M. Uchida, A. Oyane, H.M. Kim, T. Kokubo, A. Ito, Adv. Mater. 16 (2004) 1071. [14] Y. Sogo, A. Ito, K. Fukasawa, N. Kondo, Y. Ishikawa, N. Ichinose, A. Yamazaki, Curr. Appl. Phys. 5 (2005) 526. [15] G. Daculsi, P. Pilet, M. Cottrel, G. Guicheux, J. Biomed. Mater. Res. 47 (1999) 228. [16] X.E. Dereka, C.E. Markopoulou, A. Mamalis, E. Pepelassi, I.A. Vrotsos, Clin. Oral Implant. Res. 17 (2006) 554. [17] X. Huang, D.S. Yang, W.Q. Yan, Z.L. Shi, J. Feng, Y.B. Gao, W.J. Weng, S.G. Yan, Biomaterials 28 (2007) 3091. [18] H. Igai, Y. Yamamoto, S.S. Chang, M. Yamamoto, Y. Tabata, H. Yokomise, J. Thorac. Cardiovasc. Surg. 134 (2007) 170. [19] M.S. Park, S.S. Kim, S.W. Cho, C.Y. Choi, B.S. Kim, J. Biomed. Mater. Res. Part B 79B (2006) 353. [20] G. Zellin, A. Linde, Bone 26 (2000) 161. [21] J.M. Choi, Y.M. Kong, S. Kim, H.E. Kim, C.S. Kwang, I.S. Lee, J. Mater. Res. 14 (1999) 2980.