Chapter 3 Materials and Methods
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.
23
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.
24
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
25
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
26
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.
27
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.
28
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
30
Figure 3.1-a Clotting time analysis experimental procedures
Figure 3-1-b Fibrinogen assay experimental procedures PRPdiluted
31
Figure 3-1-c sP-selectin assay experimental procedures
Figure 3-1-d CD 61 assay experimental procedures Mixed 15 μl
32
Ti-1 Ti-2 Ti-3
0 2 4 6 8 10 12 14 16
Average roughness (nm)
Samples
Ra Rms
Fig 4.1 Average surface roughness of Ti\O
2 samples under 1, 2, and 3 KW ion power treatments.
500 1000 1500 2000 2500 3000 3500 4000
Intersity (arb. unit)
Raman Shift (cm-1)
Ti-1 Ti-2 Ti-3
Fig 4.2 Raman analysis of Ti\O
2 samples under 1, 2, and 3 KW ion power treated
33
Ti-1 Ti-2 Ti-3
0 20 40 60 80 100
Contact Angle ()
Samples
Fig 4.3 Average contact angle of different Ti\O
2 samples
20 40 60 80 100
Intensity (arb. unit)
2 (degree)
Ti-1
Ti-2
Ti-3
Fig 4.4 XRD patterns.
34
Ti-1 Ti-2 Ti-3
0 20 40 60 80 100 120 140
Young's modulus (GPa)
Samples
Fig 4.5.1 The young’s modulus of different Ti\O
2 samples
Ti-1 Ti-2 Ti-3
0 100 200 300 400 500 600
Hardness (Hv)
Samples Fig 4.5.2 The hardness of different Ti\O
2 samples
35
(a) Bright field (BF) (b) Dark field (DF)
Fig 4.6.1 OM images of MG-63 cells cultured for 3 days on Ti-1 titanium disk
(a) Bright field (BF) (b) Dark field (DF)
Fig 4.6.2 OM images of MG-63 cells cultured for 3 days on Ti-2 titanium disk
36
(a) Bright field (BF) (b) Dark field (DF)
Fig 4.6.3 OM images of MG-63 cells cultured for 3 days on Ti-3 titanium disk
(a) Bright field (BF) (b) Dark field (DF)
Fig 4.6.4 OM images of MG-63 cells cultured for 7 days on Ti-1 titanium disk
37
(a) Bright field (BF) (b) Dark field (DF).
Fig 4.6.5 OM images of MG-63 cells cultured for 7 days on Ti-2 titanium disk
(a) Bright field (BF) (b) Dark field (DF).
Fig 4.6.6 OM images of MG-63 cells cultured for 7 days on Ti-3 titanium disk
38
Fig 4.7 MTT values of MG-63 cells on different Ti\O
2 samples
Fig 4.8 ALP activity of MG-63 cells on different Ti\O
2 samples
39
Fig 4.9 Clotting time assay on different Ti\O
2 samples
Fig 4.10 Fibrinogen adhesion assay on different Ti\O
2 samples
40
Fig 4.11 sP-selectin expression assay on different Ti\O
2 samples
Fig 4.12 Platelet adhesion assay (CD-61) on different Ti\O
2 samples
41
References
Advincula, M. C., F. G. Rahemtulla, et al. (2006). "Osteoblast adhesion and matrix
mineralization on sol-gel-derived titanium oxide." Biomaterials 27(10): 2201-2212.
Albrektsson, T., P. I. Branemark, et al. (1981). "Osseointegrated titanium implants.
Requirements for ensuring a long-lasting, direct bone-to-implant anchorage in man."
Acta Orthop Scand 52(2): 155-170.
Batzer, R., Y. Liu, et al. (1998). "Prostaglandins mediate the effects of titanium surface roughness on MG63 osteoblast-like cells and alter cell responsiveness to 1 alpha,25-(OH)2D3." J Biomed Mater Res 41(3): 489-496.
Binon, P. P., D. J. Weir, et al. (1992). "Surface analysis of an original Branemark implant and three related clones." Int J Oral Maxillofac Implants 7(2): 168-175.
Bobyn, J. D., R. M. Pilliar, et al. (1980). "The optimum pore size for the fixation of
porous-surfaced metal implants by the ingrowth of bone." Clin Orthop Relat Res(150):
263-270.
Bosetti, M., A. Masse, et al. (2001). "In vivo evaluation of bone tissue behavior on ion implanted surfaces." Journal of Materials Science-Materials in Medicine 12(5):
431-435.
Bowers, K. T., J. C. Keller, et al. (1992). "Optimization of surface micromorphology for enhanced osteoblast responses in vitro." Int J Oral Maxillofac Implants 7(3): 302-310.
Boyan, B. D., R. Batzer, et al. (1998). "Titanium surface roughness alters responsiveness of MG63 osteoblast-like cells to 1 alpha,25-(OH)2D3." J Biomed Mater Res 39(1):
77-85.
Boyan, B. D., S. Lossdorfer, et al. (2003). "Osteoblasts generate an osteogenic
microenvironment when grown on surfaces with rough microtopographies." Eur Cell Mater 6: 22-27.
Branemark, P.-I., G. A. Zarb, et al. (1985). Tissue-integrated prostheses : osseointegration in clinical dentistry. Chicago ; London, Quintessence Publishing.
Branemark, P. I., B. O. Hansson, et al. (1977). "Osseointegrated implants in the treatment of the edentulous jaw. Experience from a 10-year period." Scand J Plast Reconstr Surg Suppl 16: 1-132.
Buser, D., R. K. Schenk, et al. (1991). "Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs." J Biomed Mater Res 25(7): 889-902.
42
Conrad, J. R., J. L. Radtke, et al. (1987). "Plasma Source Ion-Implantation Technique for Surface Modification of Materials." Journal of Applied Physics 62(11): 4591-4596.
De Maeztu, M. A., J. I. Alava, et al. (2003). "Ion implantation: surface treatment for
improving the bone integration of titanium and Ti6Al4V dental implants." Clin Oral Implants Res 14(1): 57-62.
De Maeztu, M. A., I. Braceras, et al. (2008). "Improvement of osseointegration of titanium dental implant surfaces modified with CO ions: a comparative histomorphometric study in beagle dogs." Int J Oral Maxillofac Surg 37(5): 441-447.
De Maeztu, M. A., I. Braceras, et al. (2007). "Histomorphometric study of ion implantation and diamond-like carbon as dental implant surface treatments in beagle dogs."
International Journal of Oral & Maxillofacial Implants 22(2): 273-279.
Degasne, I., M. F. Basle, et al. (1999). "Effects of roughness, fibronectin and vitronectin on attachment, spreading, and proliferation of human osteoblast-like cells (Saos-2) on titanium surfaces." Calcif Tissue Int 64(6): 499-507.
Doundoulakis, J. H. (1987). "Surface analysis of titanium after sterilization: role in implant-tissue interface and bioadhesion." J Prosthet Dent 58(4): 471-478.
Gittens, R. A., T. McLachlan, et al. (2011). "The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation."
Biomaterials 32(13): 3395-3403.
Hulbert, S. F., S. J. Morrison, et al. (1972). "Tissue reaction to three ceramics of porous and non-porous structures." J Biomed Mater Res 6(5): 347-374.
Kasemo, B. (1983). "Biocompatibility of titanium implants: surface science aspects." J Prosthet Dent 49(6): 832-837.
Keller, J. C., C. M. Stanford, et al. (1994). "Characterizations of titanium implant surfaces.
III." J Biomed Mater Res 28(8): 939-946.
Kieswetter, K., Z. Schwartz, et al. (1996). "Surface roughness modulates the local production of growth factors and cytokines by osteoblast-like MG-63 cells." J Biomed Mater Res 32(1): 55-63.
Kim, H. J., S. H. Kim, et al. (2005). "Varying Ti-6Al-4V surface roughness induces different early morphologic and molecular responses in MG63 osteoblast-like cells." J Biomed Mater Res A 74(3): 366-373.
Kim, H. K., J. W. Jang, et al. (2004). "Surface modification of implant materials and its effect on attachment and proliferation of bone cells." J Mater Sci Mater Med 15(7): 825-830.
Krennmair, G., R. Seemann, et al. (2010). "Clinical outcome of root-shaped dental implants of various diameters: 5-year results." Int J Oral Maxillofac Implants 25(2): 357-366.
Larsson, C., P. Thomsen, et al. (1996). "Bone response to surface-modified titanium implants:
studies on the early tissue response to machined and electropolished implants with
43
different oxide thicknesses." Biomaterials 17(6): 605-616.
Larsson, C., P. Thomsen, et al. (1994). "Bone response to surface modified titanium implants:
studies on electropolished implants with different oxide thicknesses and morphology."
Biomaterials 15(13): 1062-1074.
Lausmaa, J. (1996). "Surface spectroscopic characterization of titanium implant materials."
Journal of Electron Spectroscopy and Related Phenomena 81(3): 343-361.
Lincks, J., B. D. Boyan, et al. (1998). "Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition." Biomaterials 19(23): 2219-2232.
Lossdorfer, S., Z. Schwartz, et al. (2004). "Microrough implant surface topographies increase osteogenesis by reducing osteoclast formation and activity." J Biomed Mater Res A 70(3): 361-369.
Lu, J. X., B. Flautre, et al. (1999). "Role of interconnections in porous bioceramics on bone recolonization in vitro and in vivo." J Mater Sci Mater Med 10(2): 111-120.
Ma, W., J. H. Wei, et al. (2008). "Histological evaluation and surface componential analysis of modified micro-arc oxidation-treated titanium implants." J Biomed Mater Res B Appl Biomater 86(1): 162-169.
Machnee, C. H., W. C. Wagner, et al. (1993). "Identification of oxide layers of commercially pure titanium in response to cleaning procedures." Int J Oral Maxillofac Implants 8(5):
529-533.
Maitz, M. F., M. T. Pham, et al. (2003). "Blood compatibility of titanium oxides with various crystal structure and element doping." Journal of Biomaterials Applications 17(4):
Maitz, M. F., M. T. Pham, et al. (2003). "Blood compatibility of titanium oxides with various crystal structure and element doping." Journal of Biomaterials Applications 17(4):