Abstract
Titanium and its alloys have been used in dentistry to due their excellent corrosion resistance and biocompatibility. However, titanium coating is bioinert material and it cannot chemically bond to bone tissue. The purpose of this work was evaluating the bioactivity of Ti-7,5Mo alloy after chemical treatment using H2SO4 / H2O2 and soaking in SBF. Samples were chemically treated at room temperature for 4 h with a solution consisting of equal volumes of concentrated H2SO4 (200 ml) and 30% aqueous solution H2O2 (200 ml). The oxidized samples were rinsed with distilled water and were heat treated at 600°C for 1h in a electrical furnace in air. Then, all samples were immersed in SBF (Simulated Body Fluid) for 7 and 14 days to form a calcium phosphate (Ca/P) coating on the surface. Surfaces were characterized by using SEM, AFM and contact angle. The results indicated that calcium phosphate (Ca/P) was formed on surface of Ti-7.5Mo experimental alloy.
titanium alloys; chemical treatment; calcium phosphate
1 Introduction
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have been studied for biomedical applications.
Several studies have shown the use of chemical oxidation to create nanopatterns on
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. Some researchers have examined techniques of coating the
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films on titanium induced by osteoblast-like cell culture and the influence of
an H2O2 pretreatment. Journal of Biomedical Materials Research. Part B, Applied
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reported that in a hydrogen
peroxide solution, a titania gel layer is formed on the titanium surface, and this
titania gel helps to form an apatite layer in simulated body fluid due to the
increase of the number of hydroxyl groups to take place on
H2O2 treated titanium surfaces.
Acid etching, including hydrochloric, sulfuric, and fluoridric acid, and their use in
mixtures is of particular interest because it appears to have the potential to
enhance osseointegration considerably without any addition of material to the
implant surface. Moreover, acid etching may produce microstructural features on a
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By immersion of materials based Ti in a solution of concentrated sulfuric acid
(H2SO4) and aqueous hydrogen peroxide
(H2O2), it was possible to create a network of nanopits
reproducible on the surface, that has been shown to have beneficial effects on both
initial and subsequent osteogenesis in vitro. Therefore, using
H2SO4/H2O2 was possible to create
metal nanotexture surfaces by etching and simultaneous oxidation of the surface in a
controlled manner1313. Oliveira PT and Nanci A. Nanotexturing of titanium-based
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14. Yi J-H, Bernard C, Variola F, Zalzal SF, Wuest JD, Rosei F, et
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chemical oxidation of titanium. Surface Science. 2006; 600(19):4613-4621.
http://dx.doi.org/10.1016/j.susc.2006.07.053.
http://dx.doi.org/10.1016/j.susc.2006.07...
15. Bagno A and Di Bello C. Surface treatments and roughness
properties of Ti-based biomaterials. Journal of Materials Science. Materials in
Medicine. 2004; 15(9):935-949.
http://dx.doi.org/10.1023/B:JMSM.0000042679.28493.7f.
PMid:15448401
http://dx.doi.org/10.1023/B:JMSM.0000042...
16. Zhu X, Chen J, Scheideler L, Reichl R and Geis-Gerstorfer J.
Effects of topography and composition of titanium surface oxides on osteoblast
responses. Biomaterials. 2004; 25(18):4087-4103.
http://dx.doi.org/10.1016/j.biomaterials.2003.11.011.
PMid:15046900
http://dx.doi.org/10.1016/j.biomaterials...
17. Pan J, Liao H, Leygraf C, Thierry D and Li J. Variation of oxide
films on titanium induced by osteoblast-like cell culture and the influence of
an H2O2 pretreatment. Journal of Biomedical Materials Research. Part B, Applied
Biomaterials. 1998; 40(2):244-256.
http://dx.doi.org/10.1002/(SICI)1097-4636(199805)40:2<244::AID-JBM9>3.0.CO;2-L.
PMid:9549619
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18. Takeuchi M, Abe Y, Yoshida Y, Nakayama Y, Okazaki M and Akagawa
Y. Acid pretreatment of titanium implants. Biomaterials. 2003; 24(10):1821-1827.
http://dx.doi.org/10.1016/S0142-9612(02)00576-8. PMid:12593964
http://dx.doi.org/10.1016/S0142-9612(02)...
19. Taborelli M, Jobin M, François P, Vaudaux P, Tonetti M,
Szmukler-Moncler S, et al. Influence of surface treatments developed for oral
implants on the physical and biological properties of titanium. (I) Surface
characterization. Clinical Oral Implants Research. 1997; 8(3):208-216.
http://dx.doi.org/10.1034/j.1600-0501.1997.080307.x.
PMid:9586465
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demonstrated that
chemical treatment with a mixture of H2SO4 and
H2O2 yields free Ti oxide surfaces and alters the surface
topography at the micro and nanoscale. In their study, SEM analysis revealed that
the etching treatment produced a microstructured Ti surface characterized by
micropits in the range 5-20 mm, with no changes at the nanoscale. Although the
mechanism that generated the microstructured surface needs to be elucidated, a
likely explanation for the absence of typical nanoscale features is that the
handling of the implants after machining generates conditions that favoring
deoxidation2121. Nanci A, Wuest JD, Peru L, Brunet P, Sharma V, Zalzal S, et al.
Chemical modification of titanium surfaces for covalent attachment of biological
molecules. Journal of Biomedical Materials Research. Part B, Applied
Biomaterials. 1998; 40(2):324-335.
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.
Wang et al.2222. Wang XX, Hayakawa S, Tsuru K and Osaka A. Bioactive titania-gel
layers formed by chemical treatment of Ti substrate with a H2O2/HCl solution.
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showed that the
amorphous titania gel coatings could be produced by the chemical treatment of
titanium with the H2O2/HCl solution. This titania gel layers
exhibited a similar behavior with respect to the apatite deposition: a certain
thickness and a subsequent heat treatment was necessary for apatite to deposit. The
minimum thickness of the titania gel layer and the optimal temperature of heat
treatment were about 0.2 μm and 400-500°C, respectively.
Tavares et al.2323. Tavares MG, Oliveira PT, Nanci A, Hawthorne AC, Rosa AL and
Xavier SP. Treatment of a commercial, machined surface titanium implant with
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demonstrated that
microtopography can be created using a simple reduction/oxidation treatment with a
mixture of H2SO4/H2O2 on certain
machined implant surfaces of Ti and that this surface is interesting because it
supports contact osteogenesis and generates ratios similar to those obtained with
the current most efficient implant surfaces. Thus, the surface treatment applied may
represent a simple and cost-effective approach to improve the performance of
conventional screw-shaped Ti implant.
Lee et al.2424. Lee MH, Park S, Min KS, Ahn SG, Park JM, Song KY, et al.
Evaluation of in vitro and in vivo tests for Surface-Modified Titanium by
H2SO4 and HO Treatment. 22Metals
and Materials International. 2007; 13(2):109-115.
http://dx.doi.org/10.1007/BF03027560.
http://dx.doi.org/10.1007/BF03027560...
produced activity in
the surface of commercial pure Ti treated on the surface with a mixed solution
containing 97% H2SO4 and 30%H2O2 at the
ratio of 1:1 (vol. %) at 40°C for 1 h, and subsequently heat-treated at 400°C for 1
h. All the specimens were immersed in HBSS with pH 7.4 at 36.5°C for 15 d, and
examined its effect on biocompatibility. An amorphous titania gel layer was formed
on the titanium surface after the titanium specimen was treated with a solution of
H2SO4 and H2O2. The average of
roughness was 2.175 μm after chemical surface treatment. The amorphous titania was
subsequently transformed into anatase by heat treatment at 400°C for 1 h.
The purpose of this work was evaluated calcium phosphate coating produced on Ti-7,5Mo alloy after chemical treatment using H2SO4 / H2O2 and soaking in SBFx5.
2 Material and Methods
2.1 Specimen preparation
The Ti-7.5Mo alloy was produced from sheets of commercially pure titanium (99.9%) and molybdenum (99.9%). Samples were first melted in an arc furnace under an argon atmosphere. The ingots were then homogenized under vacuum at 1100°C for 86.4 ks to eliminate chemical segregation. The resulting samples were finally cold-worked by swaging, producing a 13-mm rod.
Bars of this alloy were machined using a CNC lathe ZIL (CENTUR 30S, ROMY, BR) with a rotation speed of 1000 rpm to obtain grooved surfaces. Samples were prepared by cutting out discs (10 mm in diameter and 4 mm in thickness). Median roughness (Ra) was measured with a roughness meter to be 2.5 μm. These samples were ultrasonically cleaned with distilled water and acetone for 15 min and air-dried prior to surface treatment. Machined samples were used as the control group and were not subjected to further surface treatment.
2.2 Acid etching
To evaluate the bioactivity of acid etching of surface, samples were treated with
a mixture consisting of equal volumes of H2SO4 and 30%
aqueous H2O2 for 4 h at room temperature under continuous
agitation, using a methodology proposed by De Oliveira et al.1313. Oliveira PT and Nanci A. Nanotexturing of titanium-based
surfaces upregulates expression of bone sialoprotein and osteopontin by cultured
osteogenic cells. Biomaterials. 2004; 25(3):403-413.
http://dx.doi.org/10.1016/S0142-9612(03)00539-8. PMid:14585688
http://dx.doi.org/10.1016/S0142-9612(03)...
. After acid etching, the
specimens were washed with distilled water, dried at 40°C for 24 h, heat-treated
at 600°C in an electric furnace under an air atmosphere for 1 h with the
temperature increasing at a rate of 5°C/min, and then cooled in the furnace.
2.3 Calcium phosphate deposition
SBFx5 solution proposed by Barrère et al.2525. Barrère F, Snel MM, Van Blitterswijk CA, Groot K and Layrolle P.
Nano-scale study of the nucleation and growth of calcium phosphate coating on
titanium implants. Biomaterials. 2004; 25(14):2901-2910.
http://dx.doi.org/10.1016/j.biomaterials.2003.09.063.
PMid:14962569
http://dx.doi.org/10.1016/j.biomaterials...
was prepared by dissolving the chemical reagents
NaCl (40.0 g), MgCl2.6H2O (1.52 g),
CaCl2.2H2O (1.84 g),
Na2HPO4.2H2O (0.89 g) and NaHCO3
(1.76 g) in 1000 ml of distilled water and buffering to pH 7.4 with
tris-hydroxymethyl aminomethane and hydrochloric acid at 36.5°C. All the
chemical reagents used in the preparation of SBFx5 were from Merck. The pH of
the SBFx5 before and after incubation was analyzed by an electrolyte-type pH
meter.
Specimens were soaked in SBFx5 solutions for 7 and 14 days to form a calcium phosphate (Ca/P) layer on the sample surface. The solution was refreshed every 48 h to maintain the ionic composition. After soaking, samples were removed and rinsed in distilled water, followed by drying at room temperature for 24 h.
2.4 Characterization: SEM, AFM and contact angle
Surfaces were evaluated using a scanning electron microscope (SEM, LEO 1450 VP, Zeiss, Germany) after acid etching, heat treatment and soaking in SBFx5.
The AFM analysis was performed with the use of a Veeco Nanoscope V atomic force microscope in air. During the analysis, the microscope was operated in contact mode and using a Si3N4 V-shape cantilever (scanning rate of 0.5 Hz). In such an operational mode, the microscope feedback system was regulated to keep the distance between the microscope tip and the surfaces constant during the scanning of the sample, and while scanning the z movement performed by the piezoelectric ceramic was recorded.
The wettability and surface energy were evaluated by water contact angle measurements. The contact angle was obtained by using the sessile drop method on a standard Rame-Hart goniometer, model 200. A microliter syringe pump was attached to a small needle in an XYZ manipulator to enable a drop to be slowly increased and decreased in size. The shape of the drop was recorded by a digital camera and the contact angles were measured from the images. The volume of each drop was 2 μl and the average value of at least 5 drops was calculated.
3 Results and Discussion
The results of the present study show that treatment of a machined surface Ti-7.5Mo with a mixture of H2SO4 and H2O2 altered the surface topography.
SEM analysis revealed that the acid etching treatment generated a nanostructured Ti surface characterized by nanopits in the range 200-500 nm (Figure 1b), whereas control samples exhibited a smooth surface patterned with parallel oriented shallow grooves created by the machining process (Figure 1a). The micrograph shows large grain with a smooth area of the transgranula surface due to corrosion.
Surface morphology: (a) machined control; (b) acid etching and heat treatment; (c) acid etching and soaking in SBF for 7 days; (d) acid etching and soaking in SBF for 14 days.
The initial period, may represent the time required to dissolve the passive oxide film and expose the metallic Ti to the acid. The acid etching of Ti in concentrated H2SO4 involves the following reactions (Equations 1, 2, 3):
Microtopography can be created using a simple reduction/oxidation treatment with a
mixture of H2SO4/H2O2 on machined
surfaces. This surface is interesting because it supports contact osteogenesis2626. Chen MF, Yang XJ, Liu Y, Zhu SL, Cui ZD and Man HC. Study on the
formation of an apatite layer on NiTi shape memory alloy using a chemical
treatment method. Surface and Coatings Technology. 2003; 173(2-3):229-234.
http://dx.doi.org/10.1016/S0257-8972(03)00733-3.
http://dx.doi.org/10.1016/S0257-8972(03)...
,2727. Huang YH, Xiropaidis AV, Sorensen RG, Albandar JM, Hall J and
Wikesjö UM. Bone formation at titanium porous oxide (TiUnite) oral implants in
type IV bone. Clinical Oral Implants Research. 2005; 16(1):105-111.
http://dx.doi.org/10.1111/j.1600-0501.2004.01086.x.
PMid:15642037
http://dx.doi.org/10.1111/j.1600-0501.20...
.
Titanium is known to dissolve in H2O2 solution as follows
(Equation 4)2828. Kakihana M, Tada M, Shiro M, Petrykin V, Osada M and Nakamura Y.
Structure and Stability of Water Soluble
(NH4)8[Ti4(CH4).
6O74(O2)4]8H2OInorganic
Chemistry. 2001; 40(5):891-894.
http://dx.doi.org/10.1021/ic001098l.
http://dx.doi.org/10.1021/ic001098l...
:
The dissolved titanium is thought to precipitate as titanium oxide or titanium
hydroxide under low pH conditions. Therefore, a chemical treatment with
H2SO4 acid and H2O2 aqueous solution
was performed, and an anatase type TiO2 film with very low crystallinity
(TiO2 gel) was obtained on the Ti surfaces. The details of this
chemical treatment were reported in a previous paper2929. Ueda M, Kinoshita T, Ikeda M and Ogawa M. Photo-induced
formation of hydroxyapatite on TiO synthesized by a chemical-hydrothermal
treatment. 2Materials Science and Engineering. 2009; 29(7):2246-2249.
http://dx.doi.org/10.1016/j.msec.2009.05.008.
http://dx.doi.org/10.1016/j.msec.2009.05...
.
Figure 1c shows specimen treated with acid etching, heat treatment and soaking in SBF for 7 days and we can observe the formation of the calcium phosphates with a round pattern on the surface of the specimen. Therefore, the surface was covered by numerous spherical particles precipitated from fluids.
With the increase of immersion time, after 14 days, the nucleation and growth of the particles continued. As a result, more and more globular particles were deposited on the surface layer and precipitation became thicker and denser (Figure 1d).
The AFM images (Figure 2) show in details the changing on surface morphology when comparing the machined control surface (Figure 2a) with the samples with acid etching (Figure 2b).
Surface morphology: (a) machined control; (b) acid etching and heat treatment; (c) acid etching and soaking in SBF for 7 days; (d) acid etching and soaking in SBF for 14 days.
From the images it can be seen that the Ca-P deposits present a nanosized and round morphology. Figure 2c presents smaller crystallites compared to Figure 2d
The measured contact angles (°) with the distilled water and diiodomethane droplets on the surfaces of samples are listed in Table 1. These results were used for the calculation of surface energy.
The contact angle of distilled water on Ti surface depends on the functional present groups. After oxidation in H2SO4/H2O2, a significant decrease in contact angle was observed, however, an increase of surface energy was obtained.
The surface energy of biomaterials is one of the most important surface properties
(such as morphology and chemical composition) determining interactions between
biomaterials and the surrounding biological environment. A material with a high
surface energy can become involved in more interactions in aqueous solution and
consequently will be more hydrophilic. The biological interactions between the
biomaterial surface and a biological medium are closely associated with
hydrophilicity of the surface2626. Chen MF, Yang XJ, Liu Y, Zhu SL, Cui ZD and Man HC. Study on the
formation of an apatite layer on NiTi shape memory alloy using a chemical
treatment method. Surface and Coatings Technology. 2003; 173(2-3):229-234.
http://dx.doi.org/10.1016/S0257-8972(03)00733-3.
http://dx.doi.org/10.1016/S0257-8972(03)...
.
Contact angle results showed that the contact angle of samples decreased from 74.82 °
(control group) to less than 24.07° (SBF 14 days) upon the growth of an calcium
phosphate layer, whereas the surface energy increased from 47.10 mJ m–2
(control group) to 79.16 mJ m–2 (SBF 14 days). This increase in surface
energy can be attributed to the increase in surface area caused by deposits of
calcium phosphate, also, due to decrease in size of the particles of calcium
phosphate3030. Wang XX, Hayakawa S, Tsuru K and Osaka A. A comparative study of
in vitro apatite deposition on heat-, H(2)O(2)-, and NaOH-treated titanium
surfaces. Journal of Biomedical Materials Research. 2001; 54(2):172-178.
http://dx.doi.org/10.1002/1097-4636(200102)54:2<172::AID-JBM3>3.0.CO;2-#.
PMid:11093176
http://dx.doi.org/10.1002/1097-4636(2001...
. This result was
confirmed in the current investigation (Table
1).
The wettability of the surface (hydrophobic or hydrophilic) has a profound influence on the behavior of cells during the process of osteointegration, a process that begins when the implant is in contact with blood. According to Elias et al.3131. Elias CN, Lima JHC, Valiev R and Meyers MA. Biomedical applications of titanium and its alloys. Biological Materials Science. 2008; 60:46-49., the adsorption and adhesion behavior of proteins on an implant surface is dependent on the implant surface properties. On hydrophobic surfaces, traces of antibodies reduce cellular adsorption. On hydrophilic surfaces, traces of thrombin and prothrombin are predominant, and cellular adsorption is enhanced. Therefore, to promote the proliferation of human osteoblasts, it is necessary to increase the surface area of the implant, which consequently increases the wettability of the surface. This increased wettability results in enhanced proliferation of cells, indicating the importance of hydrophilicity for applications such as dental implants.
4 Conclusions
It was observed that the surface topography produced by acid etching, which provides submicron structures, had an important role in the formation of calcium phosphate coating consisting of submicron structures, which is in agreement with literature reports stating that the morphology of the film follows that of the substrate.
It was possible to show that the Ti-7.5Mo alloy with a roughness of 2.5 um produced films of calcium phosphate with rounded structures (average diameter of approximately 200 nm).
Chemical treatments with H2SO4/H2O2 produced nanometer-sized pits with 200 nm in diameter and after biomimetic treatment it can be seen that the Ca-P deposits present a nanosize and round morphology.
In conclusion, nanotopography can be created using a simple reduction/oxidation treatment with a mixture of sulfuric acid/hydrogen peroxide.
Acknowledgments
The authors acknowledge financial support received from FAPESP (Project 2013/08200-9) and CAPES.
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Publication Dates
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Publication in this collection
Jan-Feb 2015
History
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Received
22 Mar 2012 -
Reviewed
11 Sept 2014