Chapter 2 Literature Review
2.5 Ion implantation
Plasma immersion ion implantation (PIII) is a faster and more cost-effective modification that can obtain dense and pore-free films without adhesion problems (Conrad, Radtke et al. 1987;
Mandl, Krause et al. 2001). Ion implantation comprises high-vacuum technology that can be applied under controlled temperature conditions. The ions disrupt the surface of the material due to their high kinetic energy, penetrating and becoming implanted within its atomic network – a phenomenon that implies modifications in the most superficial layers of the material. The implanted zone forms an integrated part of the material, thus avoiding the risk of delamination associated with coating techniques. Furthermore, there is no material loss with such processes – a fact that affords advantages over material removal techniques. Ion implantation is clean, versatile, highly controllable and reproducible, and induces intrinsic modifications within the more superficial layers, while preserving the structure and characteristics of the background material.
Several in vitro and in vivo studies have been performed using this method based on ion implantation. Previously published reports have shown better results when using this treatment, with greater bone–implant contact (BIC) when compared to simple machine-turned
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titanium implants and diamond-like carbon (DLC)-coated implants (De Maeztu, Alava et al.
2003; De Maeztu, Braceras et al. 2008). Better results were also obtained over a short period of time in dogs with implants treated with ion implantation when compared to machine-turned titanium (De Maeztu, Braceras et al. 2007). Faster bone formation around implants treated using the ion implantation technique has been confirmed by other authors such as Bosetti et al.
(Bosetti, Masse et al. 2001), who also demonstrated an improvement in surface-treated material in terms of resistance to corrosion, fatigue and metal ion release.
S. Mändl et al. investigated oxygen PIII treated titanium implants using a well-established rat model (Mandl, Krause et al. 2001). They concluded that using oxygen PIII, the formation of dense rutile layers with rather good adhesion on titanium is possible. Comparing the osseointegration of different titanium alloys in rat femurs with and without PIII treatment, it was observed that plasma immersion ion implantation can further improve the osseointegration of treated implants (Mandl, Sader et al. 2002).
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Chapter 3 Materials and Methods
3.1 Materials and preparation
The grade II titanium substrates (Bio Tech One Inc., Taipei, Taiwan) used in these experiments were 1-mm-thick plates with a diameter of 14.5 mm. The titanium plates for the present study were polished using 600-grit SiC metallographic paper. After being polished, specimens were cleaned with methylethylketone solvent for 5 min, washed in distilled water for 20 min, acid pacified in 30% nitric acid for 30 min according to the American Standard Testing Materials (ASTM) procedure and rinsed again in ultrapure water for 20 min.
The plasma-oxidized samples were first cleaned in Ar plasma to remove the native surface layer (adsorbed contamination and impurities) and produce a repeatable starting condition for the subsequent oxidation procedure. The Ar plasma cleaning was immediately followed by different plasma treatments, using Ar and O2 as the process gases. Samples were cleaned by argon-plasma treatment in the reactor. Plasma cleaning was performed at a working time of 10 min, and an argon flow rate of 100 sccm, after the base pressure was reduced to below 50 mTorr. The argon plasma treatment powers were the same as oxygen plasma treatment powers, which were at three different conditions (1kw, 2kw, and 3kw), denoted as Ti-1, Ti-2, and Ti-3, respectively. After plasma treatment, samples were annealed for 10 minutes in the reactor.
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3.2 Samples analysis
To analyze the properties of treated titanium plates, the surface morphologies of the treated titanium plates were analyzed using an atomic force microscope (AFM, Nanosurf-Mobil S) with a Si probe. The AFM probe was scanned over an area of 5 m x 5 m, with 512 scans performed at a scanning rate of 1 Hz in the tapping mode. X-ray diffractometry (XRD, RIGAKU-2200) was used to identify the phases of the films in order to analyze the properties of titanium plates. Secondary ion mass spectroscopy (SIMS) was used to obtain the oxide thickness.
Surface wettability was evaluated by optical measurement of the static contact angle of water using a goniometer (KYOWR, CA-VP 150, Japan) at room temperature. For each measurement, a 5 l droplet of distilled water was applied to the sample surface. The measurements were taken using a FTA-32 video contact angle system (First Ten Angstrom Inc., USA).
3.3 Cell culture
The cell culture experiments used MG-63 cells. Plasma treatment and control titanium plates were placed, modified side up, in 24 well culture plates. Then the titanium plates seeded at 5
× 104 cells/well in suspension on all titanium plates. After incubation, for all experiments, cells were cultured in different periods (12 hrs, 1 day, 3 days, 5 days, 7 days, and 9 days).
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3.4 Glutaraldehyde- OsO4 primary fixation
Cells were fixed with a 3% Glutaraldehyde/0.2M PBS solution for 15 min and washed with 0.1M PBS (5 min, 3 times). Then fixed with OsO4 solution for 30 min and washed with 0.1M PBS solution (5 min, 3 times). Samples were dehydrated by gradient alcohol, and then sputter coated with gold, to be examined with scanning electron microscopy (SEM; Hitachi S4200).
3.5 Cell activity assay
Put the samples in 24 well culture plates, and seeded the MG-63 cell onto the surface of the specimens at a density of 5 × 104 cells/cm2. After 4 hours cultured, washed with PBS (0.1M，
pH7.2), then added 500μl medium and 50 μl MTT （3-[4,5-dimethylthiazol-2-yl] -2,5- diphenyltetrazolium bromide）. Cells were incubated at 37℃ in an atmosphere containing 5%
CO2. After 4 hours, the active cells formed the purple crystalline of formazan salt, then added the 10% SDS/ 0.01M HCl，500μl/ well. Take the culture overnight, and then the optical densities were measured using ELISA reader (ELISA reader; Csbiotech Anthos-2020) at 595 nm. Repeated the experiments for five times, and the mean results were compared with control group. Differences were considered significant for p<0.05.
Alkaline phosphatase activity of cell lysates was performed using extraction buffer, including 2mM (MgCl2) and 1% TritonX-10, to dissolve cells. Place the samples in -20℃, 30 minutes then move to room temperature for 30 minutes, the procedures were preformed twice. The use of pipet tip helps the mechanical damage of cells, so that the cell membrane was perforated and the destruction was complete. Put 0.05 ml of samples in the 96 well plates, then added
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0.05ml AB mixture (ALP buffer and phosphatase substrate solution) at 37℃ for 30 minutes reaction. The optical densities were measured using ELISA reader (ELISA reader; Csbiotech Anthos-2020) at 405 nm.
3.6 Hemocompatibility of the samples
The biocompatibility tests also use the blood to observe the clotting time, blood adhesion, and platelet adhesion. The optical densities were measured using ELISA reader to test the fibrinogen for plasma protein assay, CD 61 for platelet adhesion assay, and P-selectin for platelet activation assay. The details of experiments could be seen at Fig. 3-1-a, 3-1-b, 3-1-c, 3-1-d.
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Chapter 4 Results
4.1 Atomic Force Microscope (AFM) surface analysis
Table 4.1 shows the surface roughness parameters of the control and the investigated samples.
Under 5 m resolution, the Rms and the Ra values for the control sample were 0.51 nm and 0.36 nm respectively, which were lower than other treated samples. Under 1 m resolution, the Rms and the Ra values for the control sample were 6.05 nm and 4.73 nm respectively, which were larger than other treated samples. Besides, the Rms and the Ra values for the treated samples were observed to be between 2-3 nm under 5 m and 1m resolution.
Average roughness of Ti\O
2 samples under 1, 2, and 3 KW ion power treated were showed in Fig4.1. The average roughness for Ti-1 was larger than other treated groups, especially the Ra values. There was no significant difference in Ra values between Ti-2 and Ti-3 groups.
4.2 Ti\O
2 Raman analysis
Raman analysis of Ti\O
2 samples under 1, 2, and 3 KW ion power treatments were shown in Fig 4.2. No differences in peaks among the investigated groups were observed.
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4.3 Ti\O
2 contact angle analysis
As shown in Fig 4.3, the larger of the mean contact angle was observed when the ion power was increased to treat the titanium disks. Ti-1 group showed the better hydrophilic properties when compared to the other groups.
4.4 Ti\O
2 XRD analysis
Fig 4.4 shows typical XRD patterns. The XRD patterns show that all of the Ti\O
2 groups were alpha Ti based crystal structure. No other crystal structure peaks were observed. And ion power treatment did not change the crystal structure of Ti disk.
4.5 The young’s modulus and the hardness of different Ti\O
2 samples
Fig 4.5.1 showed the young’s modulus of the Ti\O
2 samples. The results revealed no significant difference between all the Ti\O
2 samples. Besides, Ti-1 group showed the lowest value of hardness. The hardness of the Ti\O
2 samples increased with the increase of ion power (Fig 4.5.2).
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4.7 MTT values of MG-63 cells on different Ti\O
2 samples the lowest MTT values than other Ti\O
2 groups.
4.8 ALP activity of MG-63 cells on different Ti\O
2 samples
As shown in Fig 4.8, ALP activity of MG-63 cells was lower for all the Ti\O
2 groups at 12 hours and 1 day of culture compared to the control group. However, there was no significant difference in ALP activity between control and all the Ti\O
2 groups at 3 and 7 days of culture.
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4.9 Clotting time assay on different Ti\O
2 samples
The results of clotting time assay demonstrated titanium treated by O2 ion power could promote blood coagulation (Fig 4.9). After 20 minutes, the mean optical density of solution from the treated disks was lower for all the Ti\O
2 groups compared to the control groups.
However, there was no significant difference after 30 and 40 minutes.
4.10 Fibrinogen adhesion assay on different Ti\O
2 samples
The amount of fibrinogen adhesion was shown in Fig 4.10. The Ti\O
2 samples treated with 1 KW ion power revealed higher values of optical density than other Ti\O
2 and control groups. However, the differences between all the Ti\O
2 groups and the control group did not significant.
4.11 Platelet activation assay on different Ti\O
2 samples
The results of sP-selectin were shown in Fig 4.11. There were no significant different between Ti\O
2 samples treated with 1, 2, 3 KW ion power and the control group.
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4.12 Platelet adhesion assay on different Ti\O
2 samples
Fig 4.12 shows the platelet adhesion assay of the control and experiment samples. The results of Ti\O
2 samples treated with 1 and 3 KW ion power were observed higher than the control group. However, The Ti\O
2 groups treated with 2 KW ion power showed the least optical density. The differences between all the Ti\O
2 groups and control group did not significant.
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Chapter 5 Discussion
Titanium-based alloy is the most common material of implants. It is investigated that the air exposure of the titanium surface results in extremely rapid oxide formation, which contributes to its excellent biocompatibility (Kasemo 1983). Numerous surface modifications have been carried out to improve the biological activity and promote osseointegration by thickening the oxide layer (Larsson, Thomsen et al. 1994), increasing surface roughness (Martin, Schwartz et al. 1995) or increasing surface energy (Rupp, Scheideler et al. 2006). The major part of the study was performed using oxygen ion implantation method. The physical analysis results of Raman and XRD showed the main composition was titanium oxide. The hardness and young’s modulus were almost the same, except that Ti-1 group showed lower value of hardness than other groups. It seems that oxygen ion implantation power did not significantly alter mechanical properties of titanium. It was contrast to the results of Yang et al. (Yang, Wang et al. 2011), who found that OPIII-treated Ti surfaces possessed higher surface hardness and young’s modulus, lower I
corr and I
pass in SBP solution and better cell adhesion and spreading morphologies. On the other hand, it is worth noting that the TiO2 coatings of 40 nm grains possess significantly lower stiffness than the other groups of 20 and 80 nm grains (Yang, Oh et al. 2006). Therefore, it may indicate that the surface roughness between 3-8 nm, its mechanical properties, such as hardness and young's modulus are not significantly affected.
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Most studies investigated surface roughness in micro- and submicro-scale. Surface roughness in nano-scale could also play an important role in osteoblast differentiation and tissue regeneration because it directly corresponds to the sizes of proteins and cell membrane receptors (Gittens, McLachlan et al. 2011). Thus, nanostructure may have a potential opportunity for faster healing times and improved implant osseointegration in vivo. In this study, the Ra values of Ti\O
2 surface roughness were observed in nano-scale and were between 2-3 nm. However, as the ion power increased, the average surface roughness did not increase gradually. Yang et al. found that after O-PIII treatment, the surface topography and roughness of Ti specimens was not distinctly changed (Yang, Wang et al. 2011). This may implied that O-PIII treatment had no influence on surface topography and roughness of Ti specimens. In the present study, average roughness of Ti-1 group revealed the highest value than other groups. The difference between Ti-2 and Ti-3 groups did not significant. It can be suggest that ion power above 2 KW did not affect surface roughness.
To quantify the number of live cells on each surface treatment, we carried out an evaluation of the cellular viability by using an MTT test. This test makes it possible to quantify the mitochondrial activity and, as a consequence, to measure survival or the cellular proliferation.
In addition, alkaline phosphatase activity is an early marker of osteoblast differentiation and relates to the matrix mineralized production. It was reported that increases in surface roughness lead to enhanced osteoblast differentiation and local factor production in vitro (Kieswetter, Schwartz et al. 1996; Raines, Olivares-Navarrete et al. 2010). In this study, it was found that the average roughness for Ti-1 group were larger than other treated groups. At 9 days of culture, higher ALP activity was observed for all the Ti\O
2 groups compared to the control groups. This results is consistent with the findings of Zhao et al. (Zhao, Schwartz et al.
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2005) and Lincks and Batzer et al. (Batzer, Liu et al. 1998; Lincks, Boyan et al. 1998). Both the cellular alkaline phosphatase and cell layer alkaline phosphatase increased as the surface roughness increased. However, the results of other experiments also showed that on the rougher surfaces, cell number was decreased. In this study, Ti-1 group revealed the greater MTT values than other Ti\O
2 groups. On the other hand, Kim et al. investigated the effects of the process parameters of Ti alloy substrate on MG-63 osteoblast-like cell proliferation. It was found that as the surface roughness increases, the proliferation of osteoblast-like cell also increases (Kim, Jang et al. 2004). In the study of Webster et al., osteoblast proliferation was significantly greater on nano-phase (materials with grain sizes less than 100 nm) titanium than on conventional ones after 3 and 5 days. Moreover, the synthesis of ALP was greater on nano-phase titanium after 21 and 28 days (Webster, Ergun et al. 2000). It seems that rougher surface in this study not only affected cell differentiation but also proliferation. Furthermore, other studies found that as the increase of surface roughness, the bone-to-implant contact increased in vivo (Buser, Schenk et al. 1991; Park, Heo et al. 2007). It was suggested that rougher titanium surface may improve cell activity and implant osseointegration.
As a surface begins to contact with biological tissues, water molecules first reach the surface.
Hence, surface wettability may play a major role in adsorption of proteins onto the surface, as well as cell adhesion. It was reported that hydrophilic titanium surfaces have a significant influence on cell differentiation and growth factor production. In addition, animal experiments have pointed out that hydrophilic surfaces improve early stages of soft tissue and hard tissue integration of titanium implants (Schwarz, Wieland et al. 2009). In this study, it was found that with the decreased of ion power, the measurements of contact angle decreased. The values of the control and treated surface groups were in the range of 70 to 100 degree, as
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similar as the results of Zhu et al. (Zhu, Chen et al. 2004) , namely, all the surfaces were hydrophilic surfaces. When the MG-63 osteoblast-like cells were cultured on the Ti-1 plates for 9 days, the cell viability increased significantly (Fig 4.7). The results indicated that the hydrophilic surfaces have positive effects on cell viability. Hemocompatibility can be determined by measuring the adhesion of blood platelets, the sP-selectine expression, and of the blood clotting time on the samples. In the present study, the blood clotting time reduced after 20 minutes for all the Ti\O
2 groups. However, the measurements of platelets adhesion, activation and fibrinogen expressions revealed that all the Ti\O
2 groups and the control groups had no significant differences. In the experiment of Maitz et al., surface roughness below 50 nm has minor effects on the blood compatibility (Maitz, Pham et al. 2003). It may be the explanations of the results in this study.
Once the MG-63 cells attached on the surface, the spherical cells started to spread. During cell spreading, the shape of cells is changed and the cellular skeleton is reorganized. In the study of Zhu et al. (Zhu, Chen et al. 2004), the fully spread cells present flatten and extension of plasma membrane to all sides. The morphologies of cells were polygonal shape. It is reported that surface texture of the Ti substrate can affect the expression of fibronectin and vitronectin integrin receptors, modify their clustering or aggregation, and therefore determine variations in shape and spreading of cells (Degasne, Basle et al. 1999). On the rougher surface, cells were more likely to adhere to the surface and were found to project more small elongated processes, presenting a more elongated morphology. However, with the increase of culture time, cell number increased and became confluent on both rough and smooth surfaces (Zhu, Chen et al. 2004; Kim, Kim et al. 2005; Simon, Lagneau et al. 2005; Advincula, Rahemtulla et al. 2006). In the present study, cells grew well on the titanium surfaces. It was obviously to
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observe osteoblasts adhered to all surfaces by means of thin cytoplasmic digitations or filopodia. The morphology of cells started to extend after 1 day. After 9 days of culture, cells became confluent to cover the surfaces. Different condition of treated power did not affect cell spreading significantly.
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Chapter 6 Conclusions
The biocompatibility of titanium is closely related with the surface oxide film. The improvement of the biological activity of titanium with plasma immersion ion implantation has been investigated. However, the effects of oxygen ion implantation on the titanium were still lack. In this study, the titanium plates were implanted with oxygen at different power conditions and subsequently analyzed for surface morphologies and phase composition. Also, the study investigated the biocompatibility of surface modification on titanium-based alloys.
The results revealed that different ion power treatments contributed to different surface roughness. In the oxygen ion implantation groups, titanium alloy treated with 1 KW ion power showed larger surface roughness than other groups. It also showed greater MTT values and ALP activity. It was suggested that with oxygen ion implantation treatment, titanium-based alloys could maintain or improved the physical properties and increase both cell differentiation and proliferation. In addition, the hemocompatibility of modified titanium alloys could maintain as untreated titanium alloys. Therefore, titanium modified by oxygen ion implantation may not only maintain its properties but also improve cell activity.
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Table 2.1 Chemical compositions of titanium and its alloys (ASTM) Grade I Grade II Grade III Grade IV
O (%) 0.18 0.25 0.35 0.40
N (%) 0.03 0.03 0.05 0.05
C (%) 0.10 0.10 0.10 0.10
H (%) 0.015 0.015 0.015 0.015
Fe (%) 0.20 0.30 0.30 0.40
Ti : 99.0-99.5%
Table 2.2 Mechanical and physical property of titanium metal (ASTM) Material Ultimate tensile
strength (MPa)
Proportional limit (MPa)
Elongation (%)
Density (g/cm3)
Elastic modulus
(GPa)
Grade I 240 170 24 4.5 100
Grade II 345 235 20 4.5 100
Grade III 450 380 18 4.5 100
Grade IV 550 483 15 4.5 100
Cortical bone 140 130 1 0.7 18
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Table 4.1 Surface roughness parameters of the control and the investigated Ti\O2 samples.
Sample
5 m resolution Rms (nm)
5 m resolution
Ra (nm)
1 m resolution Rms (nm)
1 m resolution
Ra (nm)
Control 0.51 0.36 6.05 4.73
Ti-1 3.5 2.67 3.88 3.02
Ti-2 3.13 2.47 3.07 2.41
Ti-3 3.08 2.35 3.3 2.52
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Figure 3.1-a Clotting time analysis experimental procedures
Figure 3-1-b Fibrinogen assay experimental procedures
Figure 3-1-b Fibrinogen assay experimental procedures