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Study on bacteria adherence properties of Mo surface-modified layer in Ti6Al4V alloy prepared by plasma surface alloying technique
Applied Surface Science 255 (2008) 419–421
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Study on bacteria adherence properties of Mo surface-modified layer in Ti6Al4V alloy prepared by plasma surface alloying technique Fan Ailan, Qin Lin, Tian Linhai, Tang Bin * Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan 030024, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Mo surface-modified layer in Ti6Al4V alloy was prepared using plasma surface alloying technique. Microstructure of the modified layer was analyzed using X-ray photoelectron spectroscopy (XPS), rough-meter and GDA750 glow discharge optical emission spectrometer. Phase composition of the Mo surface-modified layer was characterized by D/max 2500 X-ray diffraction. Results show that the Mo surface-modified layers consist of pure Mo surface layer with h1 1 0i and h2 1 1i orientations and diffusion layer. Mo 3d, O 1s, C 1s and Ti 2p, O 1s, C 1s XPS spectra are recorded at topsurface in the Momodified layer and titanium substrate respectively. Because of the different roughness and microstructure, the Mo surface-modified layer can to some extent inhibit bacteria adherence. ß 2008 Elsevier B.V. All rights reserved.
Available online 4 July 2008 PACS: 96.12.Kz 52.77.j 79.60.i Keywords: Ti6Al4V Mo surface-modified layer Bacteria adherence
1. Introduction
2. Experimental
With the development of modern medical science, biomaterials play an important role in replacing human organs, tissues and modifying their functions. Among them, metallic biomaterials such as titanium and its alloys are being used widely in the fields of dental orthopaedic and cardiovascular surgery [1–7]. However, the applications of titanium and titanium alloys are limited due to low surface hardness and low wear resistance. Literatures [8–10] indicate that the Mo surface-modified layer in Ti6Al4V alloy with plasma surface alloying technique can effectively improve the tribological properties of titanium alloys. But there is less work concerning the bacteria adherence of the Mo surface-modified layer. However, bacterial infection after implant placement is a significant rising complication. The bacteria adherence is the firstline factor that leads to infections. It is necessary to study the bacteria adherence of the Mo surface-modified layer. In this paper, the Mo surface-modified layer in Ti6Al4V alloy was prepared using the plasma surface alloying technique. The bacteria adherence properties of the Mo surface-modified layer in Ti6Al4V alloy on the oral bacteria Streptococcus mutans was investigated and compared with that of Ti6Al4V alloy.
2.1. Material
* Corresponding author. Tel.: +86 351 6010540; fax: +86 351 6018760. E-mail address: [email protected] (T. Bin). 0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2008.06.180
Annealed Ti6Al4V alloy disks with a size of 16 mm 2 mm and a hardness of HK0.01 350 were polished to an average roughness of Ra < 0.06 0.01 mm. Mo surface-modified layer in Ti6Al4V alloy was prepared using LS-750 plasma surface alloying equipment [8]. The process parameters were as follows: the Ar gas pressure was 30– 40 Pa, the source voltage for supplying Mo elements was 1200 to 1250 V, the cathode (specimen) voltage was 550 to 600 V, the distance from the source target to the substrate sample was 15 mm, the process temperature was 830 8C and the process duration was 3 h. 2.2. Characterization test Phase composition of the Mo surface-modified layer was characterized by D/max 2500 X-ray diffraction with Cu Ka. The component distribution of the elements across the modified layer was analyzed using GDA750 glow-discharge optical emission spectroscope (GDOES). Valence conditions of the surface main alloy elements for the Mo surface-modified layer in Ti6Al4V alloy and Ti6Al4V alloy were characterized by X-ray photoelectron spectroscopy (XPS) using ESCALAB 250 spectrometer with Al Ka excitation. The binding energies obtained in XPS analysis were corrected by referencing the C 1s line to 284.6 eV. The surface roughness (Ra) of the Mo-modified layer and Ti6Al4V alloy were investigated by TR 240 rough-meter.
F. Ailan et al. / Applied Surface Science 255 (2008) 419–421
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Fig. 2. Component distribution of Mo-modified layer (wt.%). Fig. 1. X-ray diffraction pattern of Mo-modified layer.
2.3. Bacteria adherence
3.4. Bacteria adherence property
The bacteria adherence properties of the Mo surfacemodified layer in Ti6Al4V alloy and Ti6Al4V alloy were tested using streptococcus mutans strain ATCC#25175. Bacteria were precultured in nutrient broth at 37 8C for 24 h, then the Tris–HCl suspension was diluted to approximately 1.5 107 bacteria per ml with sterilized distilled water. Ti6Al4V alloy and surfacemodified specimens were put into sterilized glass bottles with 5 ml of bacterial suspension and were incubated at 37 8C for 1 h. After incubation, the samples were rinsed with distilled water six times, fixed in 3% glutaraldehyde at 4 8C for 30 min and stained with a 1% Acridine Orange solution for 30 min. Bacteria number and bacteria morphology on each sample were analyzed using a fluorescence microscope (OLYMPUS-BX51T32000) at 400 magnification.
Fluorescence microscope images of bacteria adherence on the Ti6Al4V alloy and the Mo-modified layer surface are shown in Fig. 4a and b respectively. The rounded region is the effective observing field of vision of the fluorescence microscope, and the small circles are the individual bacteria adherence on the specimen surfaces. Fig. 5 shows the number of bacteria adherence on the Ti6Al4V alloy and the Mo-modified layer. Figs. 4 and 5 show that the bacteria adherence on the Mo-modified layer is lesser than that on the Ti6Al4V alloy.
3. Results 3.1. Structure and element distributions X-ray diffraction spectrum of the Mo-modified layer indicates that the Mo-modified layer is composed of single phase Mo. The preferred orientation is h1 1 0i and h2 1 1i, as shown in Fig. 1. Fig. 2 demonstrates that a gradient distribution of elements (Ti, Al, V, and Mo) exist in the Mo-modified layer. Result shows that the modified layer is composed of 7-mm Mo coating layer and 13-mm Mo diffusion layer.
4. Discussion Adhesion of bacteria to solid surfaces is a complex of various mechanisms. It is considered that surface roughness and surface chemical composition of the materials are the principle factors influencing bacteria adherence [11]. Usually, there is more bacteria adherence on surfaces of higher Ra values than that of lower Ra values. But Quirynen et al. [12] considered that the total amount of adherent microorganism on the implant base had almost no distinct difference, when the surface roughness value (Ra) of the implant base was less than 0.2 mm. In the present study, the surface roughness values of Ti6Al4V alloy and the Mo-modified layer were less than 0.2 mm. So
3.2. X-ray photoelectron spectroscopy analysis Fig. 3 shows the XPS spectra of the samples. For the Ti6Al4V alloy, the main peaks turning up in the spectra include Ti 2p, C 1s and O 1s. For the Mo-modified layer, the main peaks turning up in the spectra include Mo 3d, C 1s and O 1s. In addition, the carbon contained in the sample surface may be contaminated. 3.3. Surface roughness The surface roughness measuring results show that the surface roughnesses (Ra) of polished Ti6Al4V alloy and the Mo-modified layer were 0.06 0.01 and 0.18 0.01 mm respectively. The surface roughness of the Mo-modified layer was greater than that of Ti6Al4V alloy.
Fig. 3. The XPS survey spectra for the surfaces of (a) Ti6Al4V alloy and (b) Momodified layer.
F. Ailan et al. / Applied Surface Science 255 (2008) 419–421
421
Fig. 4. Fluorescence microscope image of bacterial adhesion on (a) Ti6Al4V alloy and (b) Mo-modified layer surface.
demonstrated that the Mo-modified layer could inhibit the Streptococcus mutans adherence, compared with Ti6Al4V alloy. The surface roughness of the Mo-modified layer affect the bacterial attachment characteristics less than the chemical composition of the surface. Acknowledgments This work was supported by the National Natural Science Foundation of China (50501016, 50771070), the National HighTech Research and Development Program of China (863 Program) (2007AA03Z521), the Talented Persons in University of Shanxi program and Scientific Foundation for Returned Overseas Scholars of Shanxi (2006–27). References Fig. 5. The number of bacterial attachment on Ti6Al4V alloy and Mo-modified layer.
if the surface roughness of samples would have an influence, it may be less significant. Based on the result of the analysis, Mo on surface-modified layer may play a vital role in inhibiting the bacteria adherence. Accordingly, the observed bacteria adherence results of Ti6Al4V alloy and Mo-modified layer may be the consequence of the chemical composition of samples. 5. Conclusions The Mo-modified layer in Ti6Al4V alloy was obtained by the plasma surface alloying technique. The bacteria adherence tests
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
X.Y. Liu, P.K. Chu, C.X. Ding, Mater. Sci. Eng. R 47 (2004) 49–121. A. Hossain, S. Okawa, O. Miyakawa, Dent. Mater. 23 (2007) 186–192. K.Y. Chiu, M.H. Wong, F.T. Cheng, H.C. Man, Appl. Surf. Sci. 253 (2007) 6762–6768. C. Delgado-Alvarado, P.A. Sundaram, Acta Biomater. 2 (2006) 701–708. J.V. Lobato, N. Sooraj Hussain, C.M. Botelho, A.C. Maurı´cio, J.M. Lobato, M.A. Lopes, A. Afonso, N. Ali, J.D. Santos, Thin Solid Films 515 (2006) 279–284. W. Chrzanowski, J. Szewczenko, J. Tyrlik-Held, J. Mater. Process. Technol. 162–163 (2005) 163–168. H.Q. Tang, H.J. Feng, J.H. Zheng, J. Zhao, Surf. Coat. Technol. 201 (2007) 5633–5636. B. Tang, P.Q. Wu, X.Y. Li, A.L. Fan, Z. Xu, J.-P. Celis, Surf. Coat. Technol. 179 (2004) 333–339. D.X. Liu, B. Tang, X.D. Zhu, H. Chen, J.W. He, J.-P. Celis, Surf. Coat. Technol. 116–119 (1999) 234–238. L. Qin, L.H. Tian, A.L. Fan, B. Tang, Z. Xu, Surf. Coat. Technol. 201 (2007) 5282–5285. Y.L. Jeyachandran, Sa.K. Narayandass, D. Mangalaraj, C.Y. Bao, W. Li, Y.M. Liao, C.L. Zhang, L.Y. Xiao, W.C. Chen, Surf. Coat. Technol. 201 (2006) 3462–3474. M. Quirynen, H.C. van der Mei, C.M.L. Bollen, G.I. Geertsema-Doornbusch, H.J. Busscher, D. van Steenberghe, Colloid Surf. B: Biointerf. 2 (1994) 25–31.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Study on bacteria adherence properties of Mo surface-modified layer in Ti6Al4V alloy prepared by plasma surface alloying technique Fan Ailan, Qin Lin, Tian Linhai, Tang Bin * Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan 030024, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Mo surface-modified layer in Ti6Al4V alloy was prepared using plasma surface alloying technique. Microstructure of the modified layer was analyzed using X-ray photoelectron spectroscopy (XPS), rough-meter and GDA750 glow discharge optical emission spectrometer. Phase composition of the Mo surface-modified layer was characterized by D/max 2500 X-ray diffraction. Results show that the Mo surface-modified layers consist of pure Mo surface layer with h1 1 0i and h2 1 1i orientations and diffusion layer. Mo 3d, O 1s, C 1s and Ti 2p, O 1s, C 1s XPS spectra are recorded at topsurface in the Momodified layer and titanium substrate respectively. Because of the different roughness and microstructure, the Mo surface-modified layer can to some extent inhibit bacteria adherence. ß 2008 Elsevier B.V. All rights reserved.
Available online 4 July 2008 PACS: 96.12.Kz 52.77.j 79.60.i Keywords: Ti6Al4V Mo surface-modified layer Bacteria adherence
1. Introduction
2. Experimental
With the development of modern medical science, biomaterials play an important role in replacing human organs, tissues and modifying their functions. Among them, metallic biomaterials such as titanium and its alloys are being used widely in the fields of dental orthopaedic and cardiovascular surgery [1–7]. However, the applications of titanium and titanium alloys are limited due to low surface hardness and low wear resistance. Literatures [8–10] indicate that the Mo surface-modified layer in Ti6Al4V alloy with plasma surface alloying technique can effectively improve the tribological properties of titanium alloys. But there is less work concerning the bacteria adherence of the Mo surface-modified layer. However, bacterial infection after implant placement is a significant rising complication. The bacteria adherence is the firstline factor that leads to infections. It is necessary to study the bacteria adherence of the Mo surface-modified layer. In this paper, the Mo surface-modified layer in Ti6Al4V alloy was prepared using the plasma surface alloying technique. The bacteria adherence properties of the Mo surface-modified layer in Ti6Al4V alloy on the oral bacteria Streptococcus mutans was investigated and compared with that of Ti6Al4V alloy.
2.1. Material
* Corresponding author. Tel.: +86 351 6010540; fax: +86 351 6018760. E-mail address: [email protected] (T. Bin). 0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2008.06.180
Annealed Ti6Al4V alloy disks with a size of 16 mm 2 mm and a hardness of HK0.01 350 were polished to an average roughness of Ra < 0.06 0.01 mm. Mo surface-modified layer in Ti6Al4V alloy was prepared using LS-750 plasma surface alloying equipment [8]. The process parameters were as follows: the Ar gas pressure was 30– 40 Pa, the source voltage for supplying Mo elements was 1200 to 1250 V, the cathode (specimen) voltage was 550 to 600 V, the distance from the source target to the substrate sample was 15 mm, the process temperature was 830 8C and the process duration was 3 h. 2.2. Characterization test Phase composition of the Mo surface-modified layer was characterized by D/max 2500 X-ray diffraction with Cu Ka. The component distribution of the elements across the modified layer was analyzed using GDA750 glow-discharge optical emission spectroscope (GDOES). Valence conditions of the surface main alloy elements for the Mo surface-modified layer in Ti6Al4V alloy and Ti6Al4V alloy were characterized by X-ray photoelectron spectroscopy (XPS) using ESCALAB 250 spectrometer with Al Ka excitation. The binding energies obtained in XPS analysis were corrected by referencing the C 1s line to 284.6 eV. The surface roughness (Ra) of the Mo-modified layer and Ti6Al4V alloy were investigated by TR 240 rough-meter.
F. Ailan et al. / Applied Surface Science 255 (2008) 419–421
420
Fig. 2. Component distribution of Mo-modified layer (wt.%). Fig. 1. X-ray diffraction pattern of Mo-modified layer.
2.3. Bacteria adherence
3.4. Bacteria adherence property
The bacteria adherence properties of the Mo surfacemodified layer in Ti6Al4V alloy and Ti6Al4V alloy were tested using streptococcus mutans strain ATCC#25175. Bacteria were precultured in nutrient broth at 37 8C for 24 h, then the Tris–HCl suspension was diluted to approximately 1.5 107 bacteria per ml with sterilized distilled water. Ti6Al4V alloy and surfacemodified specimens were put into sterilized glass bottles with 5 ml of bacterial suspension and were incubated at 37 8C for 1 h. After incubation, the samples were rinsed with distilled water six times, fixed in 3% glutaraldehyde at 4 8C for 30 min and stained with a 1% Acridine Orange solution for 30 min. Bacteria number and bacteria morphology on each sample were analyzed using a fluorescence microscope (OLYMPUS-BX51T32000) at 400 magnification.
Fluorescence microscope images of bacteria adherence on the Ti6Al4V alloy and the Mo-modified layer surface are shown in Fig. 4a and b respectively. The rounded region is the effective observing field of vision of the fluorescence microscope, and the small circles are the individual bacteria adherence on the specimen surfaces. Fig. 5 shows the number of bacteria adherence on the Ti6Al4V alloy and the Mo-modified layer. Figs. 4 and 5 show that the bacteria adherence on the Mo-modified layer is lesser than that on the Ti6Al4V alloy.
3. Results 3.1. Structure and element distributions X-ray diffraction spectrum of the Mo-modified layer indicates that the Mo-modified layer is composed of single phase Mo. The preferred orientation is h1 1 0i and h2 1 1i, as shown in Fig. 1. Fig. 2 demonstrates that a gradient distribution of elements (Ti, Al, V, and Mo) exist in the Mo-modified layer. Result shows that the modified layer is composed of 7-mm Mo coating layer and 13-mm Mo diffusion layer.
4. Discussion Adhesion of bacteria to solid surfaces is a complex of various mechanisms. It is considered that surface roughness and surface chemical composition of the materials are the principle factors influencing bacteria adherence [11]. Usually, there is more bacteria adherence on surfaces of higher Ra values than that of lower Ra values. But Quirynen et al. [12] considered that the total amount of adherent microorganism on the implant base had almost no distinct difference, when the surface roughness value (Ra) of the implant base was less than 0.2 mm. In the present study, the surface roughness values of Ti6Al4V alloy and the Mo-modified layer were less than 0.2 mm. So
3.2. X-ray photoelectron spectroscopy analysis Fig. 3 shows the XPS spectra of the samples. For the Ti6Al4V alloy, the main peaks turning up in the spectra include Ti 2p, C 1s and O 1s. For the Mo-modified layer, the main peaks turning up in the spectra include Mo 3d, C 1s and O 1s. In addition, the carbon contained in the sample surface may be contaminated. 3.3. Surface roughness The surface roughness measuring results show that the surface roughnesses (Ra) of polished Ti6Al4V alloy and the Mo-modified layer were 0.06 0.01 and 0.18 0.01 mm respectively. The surface roughness of the Mo-modified layer was greater than that of Ti6Al4V alloy.
Fig. 3. The XPS survey spectra for the surfaces of (a) Ti6Al4V alloy and (b) Momodified layer.
F. Ailan et al. / Applied Surface Science 255 (2008) 419–421
421
Fig. 4. Fluorescence microscope image of bacterial adhesion on (a) Ti6Al4V alloy and (b) Mo-modified layer surface.
demonstrated that the Mo-modified layer could inhibit the Streptococcus mutans adherence, compared with Ti6Al4V alloy. The surface roughness of the Mo-modified layer affect the bacterial attachment characteristics less than the chemical composition of the surface. Acknowledgments This work was supported by the National Natural Science Foundation of China (50501016, 50771070), the National HighTech Research and Development Program of China (863 Program) (2007AA03Z521), the Talented Persons in University of Shanxi program and Scientific Foundation for Returned Overseas Scholars of Shanxi (2006–27). References Fig. 5. The number of bacterial attachment on Ti6Al4V alloy and Mo-modified layer.
if the surface roughness of samples would have an influence, it may be less significant. Based on the result of the analysis, Mo on surface-modified layer may play a vital role in inhibiting the bacteria adherence. Accordingly, the observed bacteria adherence results of Ti6Al4V alloy and Mo-modified layer may be the consequence of the chemical composition of samples. 5. Conclusions The Mo-modified layer in Ti6Al4V alloy was obtained by the plasma surface alloying technique. The bacteria adherence tests
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
X.Y. Liu, P.K. Chu, C.X. Ding, Mater. Sci. Eng. R 47 (2004) 49–121. A. Hossain, S. Okawa, O. Miyakawa, Dent. Mater. 23 (2007) 186–192. K.Y. Chiu, M.H. Wong, F.T. Cheng, H.C. Man, Appl. Surf. Sci. 253 (2007) 6762–6768. C. Delgado-Alvarado, P.A. Sundaram, Acta Biomater. 2 (2006) 701–708. J.V. Lobato, N. Sooraj Hussain, C.M. Botelho, A.C. Maurı´cio, J.M. Lobato, M.A. Lopes, A. Afonso, N. Ali, J.D. Santos, Thin Solid Films 515 (2006) 279–284. W. Chrzanowski, J. Szewczenko, J. Tyrlik-Held, J. Mater. Process. Technol. 162–163 (2005) 163–168. H.Q. Tang, H.J. Feng, J.H. Zheng, J. Zhao, Surf. Coat. Technol. 201 (2007) 5633–5636. B. Tang, P.Q. Wu, X.Y. Li, A.L. Fan, Z. Xu, J.-P. Celis, Surf. Coat. Technol. 179 (2004) 333–339. D.X. Liu, B. Tang, X.D. Zhu, H. Chen, J.W. He, J.-P. Celis, Surf. Coat. Technol. 116–119 (1999) 234–238. L. Qin, L.H. Tian, A.L. Fan, B. Tang, Z. Xu, Surf. Coat. Technol. 201 (2007) 5282–5285. Y.L. Jeyachandran, Sa.K. Narayandass, D. Mangalaraj, C.Y. Bao, W. Li, Y.M. Liao, C.L. Zhang, L.Y. Xiao, W.C. Chen, Surf. Coat. Technol. 201 (2006) 3462–3474. M. Quirynen, H.C. van der Mei, C.M.L. Bollen, G.I. Geertsema-Doornbusch, H.J. Busscher, D. van Steenberghe, Colloid Surf. B: Biointerf. 2 (1994) 25–31.