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Cell adhesion study of the titanium alloys exposed to glow discharge
NIM B Beam Interactions with Materials & Atoms
Nuclear Instruments and Methods in Physics Research B 261 (2007) 624–626 www.elsevier.com/locate/nimb
Cell adhesion study of the titanium alloys exposed to glow discharge V. Abidzina
a,*
¨ zdal-Kurt c, B.H. Sen d, I. Tereshko a, , I. Deliloglu-Gu¨rhan b, F. O e f I. Elkin , S. Budak , C. Muntele f, D. Ila f
a Belarusian-Russian University, Mogilev, Belarus Ege University Faculty of Engineering, Bioengineering Department, Izmir, Turkey Celal Bayar University, Faculty of Science and Arts, Department of Biology, Manisa, Turkey d Ege University Faculty of Dentistry, Izmir, Turkey e ‘KAMA VT’ Research and Production Enterprise, Mogilev, Belarus f Center for Irradiation of Materials, Alabama A&M University, Normal, AL, USA b
c
Available online 24 April 2007
Abstract Titanium for biomedical application stems mainly from its advantageous bulk mechanical properties in combination with a high degree of biocompatibility that is largely attributable to their surface properties. This work is focused on the investigation of surface properties of treated titanium and cell adhesion to titanium treated in glow-discharge plasma. Pure titanium samples (grade 4) were exposed to low-energy ion irradiation in a specially constructed plasma generator, where materials were irradiated by ions of residual gases in vacuum. The ion energy was 1–10 keV. The irradiation dose was maintained at 1017 ions cm 2. The irradiation time varied from 5 to 60 min. Rutherford backscattering spectrometry (RBS) was used for surface studies. RBS showed the presence of iron on the titanium surface that occurred from the cathode of plasma generator. In vitro biocompatibility test have been carried out with model cell lines (L929 mouse fibroblasts) to demonstrate that low-energy ion irradiation can favorably influence the surface of titanium for biomedical application. Scanning electron microscopy (SEM) was the main tool to demonstrate the cell attachment properties. 2007 Elsevier B.V. All rights reserved. PACS: 81.65. b; 82.80.Yc; 61.82.Bg Keywords: Titanium biocompatibility; Cell adhesion
1. Introduction Titanium and its alloys are among the most utilized biomaterials and are still the materials of choice for many structural implantable device applications [1]. However, current titanium implants face long-term failure problems due to poor bonding to juxtaposed bone, severe stress shielding and generation of debris that may lead to bone * Corresponding author. Address: Belarusian-Russian University, Prospect Mira 43, 212005 Mogilev, Belarus. Tel.: +375 296 466821; fax: +375 222 225518. E-mail address: [email protected] (V. Abidzina).
0168-583X/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2007.04.165
cell death and perhaps eventual necrotic bone [2–5]. Improving the bioactivity of titanium implants, especially with respect to cells, is a major concern in the near and intermediate future. Surface properties such as wettability, chemical composition and topography govern the biocompatibility of titanium. Conventionally processed titanium currently used in the orthopedic and dental applications exhibits a microrough surface and is smooth at the nanoscale. Surface smoothness on the nanoscale has been shown to favor fibrous tissue encapsulation [4–6]. An approach to design the next-generation of implants has recently focused on creating unique nanotopography (or roughness) on the
V. Abidzina et al. / Nucl. Instr. and Meth. in Phys. Res. B 261 (2007) 624–626
625
implant surface, considering that natural bone consists of nanostructured materials like collagen and hydroxyapatite. Some researchers have achieved nano-roughness in titanium substrates by compacting small (nanometer) constituent particles and/or fibers [7,8]. However, nanometer metal particles can be expensive and unsafe to fabricate. For this reason, alternative methods of titanium surface treatment are desirable. The aim of this work is to investigate the influence of low-energy ion treatment of titanium on cell adhesion. 2. Experimental methods Titanium samples were cut into pieces (1 cm · 0.5 cm) for low-energy ion treatment and further RBS and cell attachment study. Samples were placed in a specially constructed plasma generator and were exposed to glow-discharge plasma by ions of the residual gases of the vacuum. The ion energy depended on the voltage in the plasmatron and did not exceed 1–10 keV. Irradiated fluence was 1017 ion cm 2. The temperature of the specimens was controlled during the irradiation process and did not exceed 343 K while the irradiation time varied from 5 to 60 min. Rutherford backscattering spectrometry (RBS) was used to study the changes after the irradiation. Cell adhesion to titanium samples was tested with L929 mouse connective tissue (fibroblasts-like cells). L929 cells were cultured in Dullbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Initial cell density was 5 · 105 cells/ml. The samples were placed into the sterile disposable 9 cm diameter tissue culture Petri dishes. 2 ml growth medium with cells were distributed into each Petri then incubated in the 5% CO2, 37 C incubator for 2 h. After that period, cultures were prepared for scanning electron microscopy (SEM).
The amount of cells per lm2 as well as measurement errors are presented in Table 1. Fig. 2 shows SEM images for control (a) and irradiated sample (b) for 5 min at 10 kV. Analysis of cell attachment for the irradiated samples in comparison with control one shows high confluence (attachment ratio) and better spreading. Calculated data indicate an increase in density of flattened cells as well as in cell amount in comparison with control sample. One can see according to Table 1 that best adhesion and the most prolific cell attachment correspond to the sample that was exposed to the irradiation for maximum time at minimum voltage. For this sample we observed less percentage of iron and thickness of the iron layer in comparison with others that were exposed to higher voltage plasma irradiation.
3. Results and discussion
4. Conclusions
RBS data for the irradiated sample show the presence of iron on the surface that occurred from high-carbon steel cathode as a result of secondary emission process (Fig. 1). Percentage of iron and thickness of the layer were calculated using RUMP simulation. These data are given in Table 1 as well as voltage and time of the irradiation.
We studied cell adhesion on titanium that was exposed to the irradiation in glow-discharge plasma. RBS analysis showed presence of iron on the sample surface which favorably influenced cell attachment that was confirmed by SEM. Cell adhesion study showed the dependence on the plasma irradiation characteristics.
Fig. 1. RBS spectrum of the irradiated sample for 5 min at 10 kV.
Table 1 Dependence of cell adhesion (column 4) and total cell attachment (column 6) and surface iron (column 3) on the plasma parameters Voltage (kV)
Time of irradiation (min)
Fe:Ti atomic ratio
Density of flattened cells per lm2
Percentage of flattened cells
Factor increase in amount of all cells in comparison with control sample
0.4 1.2 10 Control
60 30 5 0
0.0277:1 0.0560:1 0.0549:1 0:1
534 ± 20 413 ± 9 381 ± 15 26 ± 8
50.2 ± 2.0 43.7 ± 0.9 42.5 ± 1.7 4.4 ± 1.5
1.78 1.63 1.53 1
626
V. Abidzina et al. / Nucl. Instr. and Meth. in Phys. Res. B 261 (2007) 624–626
Fig. 2. SEM images of cell attachment on the control (a) and the (b) sample that was irradiated for 5 min at 10 kV.
Acknowledgements Research sponsored by the Center for Irradiation of Materials, Alabama A&M University and by National Science Foundation under Grant No. EPS-0447675. Authors are grateful to Gene Sheldon, ZAAP USA for providing with test samples. References [1] D.M. Brunette et al., Titanium in Medicine: Material Science, Surface Science, Engineering, Biological Responses and Medical Applications, first ed., Springer, Germany, 2001, p. 14.
[2] D. Busher et al., J. Biomed. Mater. Res. 45 (2) (1999) 75. [3] T.J. Webster, Nanostructured Materials, Academic Press, New York, 2001. [4] F.S. Kaplan et al., Orthopedic Basic Science, American Academic of Orthopedic Surgeons, Columbus, 1994, p. 460. [5] F.S. Kaplan et al., Orthopedic Basic Science, American Academic of Orthopedic Surgeons, Columbus, 1994, p. 127. [6] D.M. Brunette et al., Titanium in Medicine: Material Science, Surface Science, Engineering, Biological Responses and Medical Applications, first ed., Springer, Germany, 2001, p. 562. [7] T.J. Webster, J.U. Ejiofor, in: MRS Symposium Proceedings MM10.4, 2003. [8] T.J. Webster, J.U. Ejiofor, Biomaterials 25 (2004) 4731.
Nuclear Instruments and Methods in Physics Research B 261 (2007) 624–626 www.elsevier.com/locate/nimb
Cell adhesion study of the titanium alloys exposed to glow discharge V. Abidzina
a,*
¨ zdal-Kurt c, B.H. Sen d, I. Tereshko a, , I. Deliloglu-Gu¨rhan b, F. O e f I. Elkin , S. Budak , C. Muntele f, D. Ila f
a Belarusian-Russian University, Mogilev, Belarus Ege University Faculty of Engineering, Bioengineering Department, Izmir, Turkey Celal Bayar University, Faculty of Science and Arts, Department of Biology, Manisa, Turkey d Ege University Faculty of Dentistry, Izmir, Turkey e ‘KAMA VT’ Research and Production Enterprise, Mogilev, Belarus f Center for Irradiation of Materials, Alabama A&M University, Normal, AL, USA b
c
Available online 24 April 2007
Abstract Titanium for biomedical application stems mainly from its advantageous bulk mechanical properties in combination with a high degree of biocompatibility that is largely attributable to their surface properties. This work is focused on the investigation of surface properties of treated titanium and cell adhesion to titanium treated in glow-discharge plasma. Pure titanium samples (grade 4) were exposed to low-energy ion irradiation in a specially constructed plasma generator, where materials were irradiated by ions of residual gases in vacuum. The ion energy was 1–10 keV. The irradiation dose was maintained at 1017 ions cm 2. The irradiation time varied from 5 to 60 min. Rutherford backscattering spectrometry (RBS) was used for surface studies. RBS showed the presence of iron on the titanium surface that occurred from the cathode of plasma generator. In vitro biocompatibility test have been carried out with model cell lines (L929 mouse fibroblasts) to demonstrate that low-energy ion irradiation can favorably influence the surface of titanium for biomedical application. Scanning electron microscopy (SEM) was the main tool to demonstrate the cell attachment properties. 2007 Elsevier B.V. All rights reserved. PACS: 81.65. b; 82.80.Yc; 61.82.Bg Keywords: Titanium biocompatibility; Cell adhesion
1. Introduction Titanium and its alloys are among the most utilized biomaterials and are still the materials of choice for many structural implantable device applications [1]. However, current titanium implants face long-term failure problems due to poor bonding to juxtaposed bone, severe stress shielding and generation of debris that may lead to bone * Corresponding author. Address: Belarusian-Russian University, Prospect Mira 43, 212005 Mogilev, Belarus. Tel.: +375 296 466821; fax: +375 222 225518. E-mail address: [email protected] (V. Abidzina).
0168-583X/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2007.04.165
cell death and perhaps eventual necrotic bone [2–5]. Improving the bioactivity of titanium implants, especially with respect to cells, is a major concern in the near and intermediate future. Surface properties such as wettability, chemical composition and topography govern the biocompatibility of titanium. Conventionally processed titanium currently used in the orthopedic and dental applications exhibits a microrough surface and is smooth at the nanoscale. Surface smoothness on the nanoscale has been shown to favor fibrous tissue encapsulation [4–6]. An approach to design the next-generation of implants has recently focused on creating unique nanotopography (or roughness) on the
V. Abidzina et al. / Nucl. Instr. and Meth. in Phys. Res. B 261 (2007) 624–626
625
implant surface, considering that natural bone consists of nanostructured materials like collagen and hydroxyapatite. Some researchers have achieved nano-roughness in titanium substrates by compacting small (nanometer) constituent particles and/or fibers [7,8]. However, nanometer metal particles can be expensive and unsafe to fabricate. For this reason, alternative methods of titanium surface treatment are desirable. The aim of this work is to investigate the influence of low-energy ion treatment of titanium on cell adhesion. 2. Experimental methods Titanium samples were cut into pieces (1 cm · 0.5 cm) for low-energy ion treatment and further RBS and cell attachment study. Samples were placed in a specially constructed plasma generator and were exposed to glow-discharge plasma by ions of the residual gases of the vacuum. The ion energy depended on the voltage in the plasmatron and did not exceed 1–10 keV. Irradiated fluence was 1017 ion cm 2. The temperature of the specimens was controlled during the irradiation process and did not exceed 343 K while the irradiation time varied from 5 to 60 min. Rutherford backscattering spectrometry (RBS) was used to study the changes after the irradiation. Cell adhesion to titanium samples was tested with L929 mouse connective tissue (fibroblasts-like cells). L929 cells were cultured in Dullbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Initial cell density was 5 · 105 cells/ml. The samples were placed into the sterile disposable 9 cm diameter tissue culture Petri dishes. 2 ml growth medium with cells were distributed into each Petri then incubated in the 5% CO2, 37 C incubator for 2 h. After that period, cultures were prepared for scanning electron microscopy (SEM).
The amount of cells per lm2 as well as measurement errors are presented in Table 1. Fig. 2 shows SEM images for control (a) and irradiated sample (b) for 5 min at 10 kV. Analysis of cell attachment for the irradiated samples in comparison with control one shows high confluence (attachment ratio) and better spreading. Calculated data indicate an increase in density of flattened cells as well as in cell amount in comparison with control sample. One can see according to Table 1 that best adhesion and the most prolific cell attachment correspond to the sample that was exposed to the irradiation for maximum time at minimum voltage. For this sample we observed less percentage of iron and thickness of the iron layer in comparison with others that were exposed to higher voltage plasma irradiation.
3. Results and discussion
4. Conclusions
RBS data for the irradiated sample show the presence of iron on the surface that occurred from high-carbon steel cathode as a result of secondary emission process (Fig. 1). Percentage of iron and thickness of the layer were calculated using RUMP simulation. These data are given in Table 1 as well as voltage and time of the irradiation.
We studied cell adhesion on titanium that was exposed to the irradiation in glow-discharge plasma. RBS analysis showed presence of iron on the sample surface which favorably influenced cell attachment that was confirmed by SEM. Cell adhesion study showed the dependence on the plasma irradiation characteristics.
Fig. 1. RBS spectrum of the irradiated sample for 5 min at 10 kV.
Table 1 Dependence of cell adhesion (column 4) and total cell attachment (column 6) and surface iron (column 3) on the plasma parameters Voltage (kV)
Time of irradiation (min)
Fe:Ti atomic ratio
Density of flattened cells per lm2
Percentage of flattened cells
Factor increase in amount of all cells in comparison with control sample
0.4 1.2 10 Control
60 30 5 0
0.0277:1 0.0560:1 0.0549:1 0:1
534 ± 20 413 ± 9 381 ± 15 26 ± 8
50.2 ± 2.0 43.7 ± 0.9 42.5 ± 1.7 4.4 ± 1.5
1.78 1.63 1.53 1
626
V. Abidzina et al. / Nucl. Instr. and Meth. in Phys. Res. B 261 (2007) 624–626
Fig. 2. SEM images of cell attachment on the control (a) and the (b) sample that was irradiated for 5 min at 10 kV.
Acknowledgements Research sponsored by the Center for Irradiation of Materials, Alabama A&M University and by National Science Foundation under Grant No. EPS-0447675. Authors are grateful to Gene Sheldon, ZAAP USA for providing with test samples. References [1] D.M. Brunette et al., Titanium in Medicine: Material Science, Surface Science, Engineering, Biological Responses and Medical Applications, first ed., Springer, Germany, 2001, p. 14.
[2] D. Busher et al., J. Biomed. Mater. Res. 45 (2) (1999) 75. [3] T.J. Webster, Nanostructured Materials, Academic Press, New York, 2001. [4] F.S. Kaplan et al., Orthopedic Basic Science, American Academic of Orthopedic Surgeons, Columbus, 1994, p. 460. [5] F.S. Kaplan et al., Orthopedic Basic Science, American Academic of Orthopedic Surgeons, Columbus, 1994, p. 127. [6] D.M. Brunette et al., Titanium in Medicine: Material Science, Surface Science, Engineering, Biological Responses and Medical Applications, first ed., Springer, Germany, 2001, p. 562. [7] T.J. Webster, J.U. Ejiofor, in: MRS Symposium Proceedings MM10.4, 2003. [8] T.J. Webster, J.U. Ejiofor, Biomaterials 25 (2004) 4731.