Journal of Biosciences and Medicines, 2014, 2, 22-29
Published Online April 2014 in SciRes.
How to cite this paper: De Jesus, J., et al. (2014) In Vivo Evaluation of Titanium Implants Bioactivated by a Modified Koku-
bo’s Treatment. Journal of Biosciences and Medicines, 2, 22-29.
In Vivo Evaluation of Titanium Implants
Bioactivated by a Modified Kokubo’s
Jailson de Jesus, Enori Gemelli, Fábio Nery, Nelson H. A. Camargo
Materials Science and Engineering, Department of Mechanical Engineering, University of Santa Catarina State,
Campus Universitário, Joinville, Brazil
Received December 2013
In this study a modified Kokubo’s treatment (MKT) was used to bioactivate titanium implants. Ti-
tanium surfaces were alumina-blasted and etched in HCl/H2SO4 solution followed by treatment in
autoclave at 120˚C for 1, 3, 5 hour periods with chemical solutions containing 1, 2 and 3 M of NaOH.
The materials were characterized by surface angle measurements, roughness, field emission
scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and in vivo tests.
Statistical analysis by polynomial regression showed that solutions within 2.2 and 2.8 M NaOH
lead to surface contact angles close to zero degree after 1 h of treatment. XPS analysis carried out
on a sample treated 1 h in 2.2 M NaOH indicated the presence of a titanate film. This solution was
selected to treat titanium implants for in vivo tests. These tests were performed on sheep in three
types of separating surfaces: machined group (standard), SLA group (A) and MKT group (B) di-
vided into two time intervals of 3 and 6 weeks. The samples in group B displayed better results
compared to group A only after 6 weeks in terms of counter torque and bone implant contact
Titanium; Thermochemical Treatment; Wettability; In Vivo Test
1. Introduction
It is well known that a series of interactions occur between the surface of biomaterials and the biological envi-
ronment after they have been implanted into the human body [1]. The set of these interactions was called os-
seointegration, concept originally introduced by Brånemark et al. in 1977 [2]. The osseointegration is seen as a
close contact between bone and implant [3], and the importance of the implant surface properties for a success-
ful osseointegration was first pointed out by Albrektsson and co-workers [4]. The bone response, which means
rate, quantity and quality are related to implant surface properties [5]. The composition and charges are critical
for protein adsorption and cell attachment [6]. Hydrophilic surfaces seem to favor the interactions with biologi-
cal fluids and cells when compared to the hydrophobic ones [7] [8], and hydrophilicity is affected by the surface
J. de Jesus et al.
chemical composition. Titanium and titanium alloys are frequently exploited as implant materials, because of
their anticorrosive properties, mechanical properties, lightness and excellent biocompatibility of their surface
oxides [9]. However, titanium is a bioinert material, i.e ., the interface between titanium and host bone consists of
simple interlocking attachment, without physic-chemical bonding with bone [10] [11]. Surface treatments are
promising ways to improve titanium surface for dental implants in terms of chemical composition, energy level,
morphology, topography and roughness [12]. Alkali treatment using NaOH solution is a well-recognized treat-
ment to improve Ti implant surfaces for higher bone affinity. After treatment sodium titanate layer is produced
on Ti surfaces where TiO2 hydrogel acts as nucleation regions for calcium phosphate precipitation [13]. During
the alkali treatment, the passive oxide film is partially attacked by hydroxyl groups and dissolves into the alkali
solution while hydration of Ti metal proceeds simultaneously. Negatively charged hydrates such as
nH2O produced on the substrate surface combine with alkali ions from the aqueous solution to form an alkali ti-
tanate hydrogel layer [14]. A thermal treatment is usually carried out after alkali treatment. During the heating
the hydrogel layer is dehydrated and densified to form a stable amorphous or crystalline titanate layer, this im-
proves surface wettability of the implant [1]. Surface wettability influences implant osseointegration, since hy-
drophilic surfaces promote a conductive environment to bone formation, as evidenced by in vitro [15] and clini-
cal tests [16]. After examining osteoblast response to controlled surface chemistries, Zhao and co-workers found
that hydrophilic surfaces are osteogenic [8]. The Kokubo method is an effective treatment for bioactivation of
titanium surface. However, the treatment produces a thick surface layer which may delaminate during implanta-
tion. Also, it is not an attractive treatment from practical viewpoint because it is a time consuming method in-
volving chemical attack for a long period followed by a thermal treatment [17]. In this study a modified Kokubo
treatment (MKT) was used to bioactivate titanium surfaces. This treatment is faster and exhibited capability to
improve surface wettability and osseointegration of titanium implants.
2. Material and Methods
2.1. Surface Treatment
In this study solutions with varied amount of NaOH were used to treat titanium surfaces (Ti grade 4). The solu-
tions were prepared with reagent grades containing Ca, Na and Si. The same base solution was used in the three
situations, varying only the amount of NaOH, which was 1, 2 and 3 M NaOH. The samples were treated in these
solutions in an autoclave for 1 to 5 hours at 120˚C. Afterwards, the samples were rinsed with distilled water and
dried in air. Titanium grade 4 discs of 6 mm diameter and 3 mm in length were blasted with alumina powder
(290 µm) and etched in HCl/H2SO4 before the treatments. Three samples of each treatment were used for each
surface characterization.
2.2. Surface Characterization
The sample surfaces were examined by contact angle measurements, FE-SEM (JSM-6701 F, JEOL, Tokyo, Ja-
pan), XPS and roughness. Using the sessile drop technique, contact angle measurements were taken of three
samples of 6 mm in diameter of the as-treated materials. Liquid droplets of 2 μL of deionized water were depo-
sited onto the solid surfaces with a syringe. The contact angles were measured at room temperature 10 s after
depositing the drop, which was the estimated time required to reach equilibrium with the liquid in use. A photo
of the liquid droplet was taken with a digital camera and the contact angle was calculated by image analysis us-
ing ImageJ software. For statistical analyses of wettability a 32 factorial design was used, varying the amount of
NaOH (1, 2, 3 M) and the time of attack (1, 3 and 5 h). The experimental design was designed by Statistica 8.0
(StatSoft, Inc., 2007) software. The XPS analyses were performed with a Phoibos 100 spectrometer (SPECS,
Germany). The X-ray source was generated by MgKa (1253.6 eV), with 200 W power. The C1 s peak (284.6 eV)
was used as an internal standard to correct the peak shifts caused by the accumulation of surface charge on insu-
lating samples. The surface roughness was measured with a Mitutoyo SJ-201 profilometer.
2.3. In Vivo Tests
2.3.1. Selected Implants
Ninety six implants (diameter = 4.3 mm, length = 13 mm) of Ti grade 4 were included in this study. Sixty four
samples were blasted with Al2O3 powder with an average particle size of 290 µm and subsequently ultrasonical-
J. de Jesus et al.
ly cleaned in ethanol and water, dried in air, and etched with a solution of HCl/H2SO4. The specimens were then
divided into three equally sized groups (n = 32). The first group was machined (surface standard—ST), the second
group was SLA type (surface A) and the third group of blasted and acid etched implants from MKT (group B).
2.3.2. Surgical Procedures
The in vivo study comprised of 6 Finnish Dorset crossed-bred sheep (each weighing approximately 150 pounds)
approximately 1.5 years old. The sheep remained in the facility for 2 weeks prior to the surgical procedures. A
total of 96 implants were distributed among the experimental groups (n = 16 per group and time-point). On each
sheep, 8 implants were first placed in one side of the hip, and after 3 weeks the other side of the hip underwent
the same procedure (16 samples per animal). Prior to the experimental procedures, the surgical area was shaved
and aseptic procedures were done. The animals were then continuously monitored for heart rate, oxygen satura-
tion, respiratory rate, temperature, and tissue coloration prior to complete shaving of the intended surgical site
and the adjacent areas and application of a Poviodine solution. After monitoring, the animals were transferred
over to an automated system, and aseptically draped. Anesthesia was inducted with Sodium Pentothal (15 - 20
mg/kg) in Normasol solution in the jugular vein. Anesthesia was maintained with Isoforane (1.5% - 3%) in
O2/N2O (50/50). 500 mg Cefazolin was administered pre-op intravenously and then again post-op. General
anesthesia was maintained by the surgical staff of the vivarium. A circulating hot water blanket maintained body
temperature while under anesthesia. ECG, End Tidal CO2 and SpO2 were monitored during the procedure. Im-
plant placement was performed using drills prepared by the implant manufacturer.
2.3.3. Removal Torque and Histologic Analysis
The animals were euthanized after 6 weeks counting from the first implant surgery via anesthesia overdose, and
the 3 and 6 weeks samples were retrieved by sharp dissection. Half of the implants were biomechanically tested
(removal torque), while the remaining samples were referred to non-decalcified histology processing. In brief,
the retrieved samples were dehydrated in a graded series of ethanol and thereafter were infiltrated and embedded
in methylmethacrylate (MMA). The embedded resin blocks were then subjected to non-decalcified histologic
sectioning where the samples were first perpendicularly sectioned into slices with a slow speed precision di-
amond saw (Isomet 2000, Buehler Ltd., Lake Bluff, USA). After gluing the sections to a plexi glass with a light
curing acrylate-based adhesive (Technovit 7210 VLC adhesive, Heraeus Kulzer GMBH, Wehrheim, Germany),
the samples were grinded and polished under abundant water irrigation using a series of silicon carbide (SiC)
papers (400, 600, 800, 200) (Buehler Ltd., Lake Bluff, IL, USA) down to a final thickness of 30 µm. Thereafter,
the sections were then stained in 1% toluidine blue. The bone-to-implant contact (BIC) along the implant was
determined at ×50 - 200 magnification (Leica DM2500M, Leica Microsystems GmbH, Wetzlar, Germany) by
means of specialized computer software (Leica Application Suite, Leica Microsystems GmbH, Wetzlar, Ger-
many). All evaluations concerning removal torque testing, histologic preparation, and histomorphometry was
performed in a blinded manner.
2.3.4. Statistical Analyses
The effects of implant surface and time in vivo on removal torque and BIC were evaluated by a general linear
ANOVA model (NCSS LLC). Statistical significance was set at 5% (α = 0.05).
3. Results
3.1. Surface Characterization
In accordance with statistical analyses of contact angle measurements displayed in Figure 1, treatments for 1 h
with solutions containing 2.2 - 2.8 M NaOH lead to surface contact angles of approximately zero degree. Based
on these results, surface characterization and in vivo tests were performed with samples treated with 2.2 M
NaOH for one hour (group B).
Figure 2 is a representative image of water drop behavior on surfaces of the different groups. The contact an-
gles of group ST and A were found to be 63.10 ± 1,25˚ and 67.10 ± 2,30˚, respectively. As can be seen in Fig-
ure 2 the water drop was flat on the surface of group B, displaying a contact angle of zero degree. Figure 3
shows a sequence of water wettability on a Ti implant surface of group B. The water gradually rises as the sur-
face becomes wet.
J. de Jesus et al.
Figure 1. Contour plot of surface contact angle as a function of
NaOH concentration and attack time.
Figure 2. Surface contact angle of group ST, A and B.
Figure 3. Wettability test of a Ti implant from group
FE-SEM observations revealed that machined samples (group standard) presented grooves coming from ma-
chining tools (Figure 4(a)). The group A samples (type SLA) presented macrorough surfaces structure formed
after blasting and dual acid treatments (Figure 4(b)). After MKT treatment (group B), the implant surface
showed finely irregular nanoporous structure (Figure 4(c) and (d)). According to XPS results this structure is
composed of Ti, Na, Ca and Si (Figure 5).
As can be seen, the MKT treatment introduced elements which form bioactive phases/bonds. It is more likely
that titanium surface is covered by a sodium and calcium titanate thin layer containing silicium. This change in
morphology leads to a slight increase in roughness. The roughness of standard group was 0.20 ± 0.04 μm, for
group A was 1.93 ± 0.23 μm and for group B was 2.03 ± 0.13 μm.
3.2. Animal Model
In the animal model, no signs of inflammation or infection were observed during the post-operatory phases and
during sharp dissection following sacrifice.
J. de Jesus et al.
Figure 4. SEM images of different Ti surfaces (5 kx): (a) group St; (b)
group A; (c) and (d-150 kx) group B.
Figure 5. XPS binding energy of a sample from Group B.
3.3. Removal Torque
Both time in vivo and implant surface significantly affected removal torque values (both at p < 0.001). A signif-
icant increase in torque values were detected from 3 to 6 weeks in vivo when all surfaces were collapsed for
analysis (p < 0.001). When surface treatment was evaluated by collapsing both times in vivo, significantly higher
values were observed for the B surface group relative to the others (p < 0.001). A complete statistics summary
presenting the estimated means and standard error for all surfaces at both times in vivo is presented in Figure 6.
At first time in vivo A and B groups presented significantly higher removal torque values relative to standard
group. At second time in vivo B group presented significantly higher removal torque relative to other groups. At
6 weeks, all groups presented significantly higher removal torque values relative to control.
3.4. Histologic Observati on
Histologic observation presented bone formation in close contact to the implant surfaces for all groups at both
time-points (Figure 7). However, the content of the bone seemed to differ depending on the surfaces, where
higher degrees of interaction were observed for the experimental A and B surfaces relative to the standard
J. de Jesus et al.
Figure 6. Statistics summary presenting the removal torque
estimated means ± standard error for the different surfaces at
both times in vivo.
Figure 7. Representative histologic sections of all groups at 3
and 6 weeks in vivo.
as-machined control. At 6 weeks, the experimental B surface presented higher degrees of bone in contact to the
implant. At 6 weeks, the bone in contact to the implant for all groups seemed to be higher compared to 3 weeks
and the volume seemed to have increased compared to 3 weeks.
3.5. Histomorphometric Evalua tionBIC
The time in vivo significantly affected BIC values (p < 0.001). The highest BIC values at 3 weeks were experi-
mental A and B surface. At 6 weeks, the group B presented the highest value when compared to all other expe-
rimental groups. A significant increase in BIC values was detected from 3 to 6 weeks in vivo when all surfaces
were collapsed for analysis (p < 0.001). A complete statistics summary presenting the estimated means and
standard error for all surfaces at both times in vivo is presented in Figure 8.
4. Discussion
The topography at the micrometer scale obtained by blasting and acid etching treatments increased osseointegration
J. de Jesus et al.
Figure 8. Statistics summary presenting the BIC estimated means ±
standard error for the different surfaces at both times in vivo.
by increasing surface roughness. The roughness is an important factor for cell adhesion. This process takes place
by attachment of fibrins to the implant surfaces, which promote cell migration. After migration the implant sur-
face becomes colonized by osteogenic cells prior to bone matrix production [18].
However, the SLA surfaces do not exhibit the capability to exchange ions with physiological medium. Ions
exchanges are critical for a physic-chemical interaction of the implanted material with physiological medium.
The mechanism of Ca-P deposition on alkali and heat treated titanium was proposed by Kokubo et al. [19].
When exposed to the medium, bioactive titanium generally releases the Na+ ion from its sodium titanate surface
into the medium via exchange with H3O+ ions in the fluid, thereby forming many Ti-OH groups on its surface
[19]. As a result, the surface is negatively charged, and then reacts with Ca+2 cations from the fluid to form a
calcium titanate film. As the Ca+2 cations accumulate, the surface becomes positively charged, reacting with
phosphate anions to form a bone-like apatite [19]. Chen et al. [20] investigated the release of Na+ ions from
NaOH-treated titanium with and without a 1 h heat treatment at 600˚C. The specimens were immersed in a buf-
fer solution at biological temperature and pH and the release of Na+ ions into the buffer solution was measured
after 1, 3, 5 and 7 days. They found that the Na+ concentration increased sharply in the buffer solution, and that
the amount of this increase in NaOH treated titanium was higher than in NaOH heat-treated titanium. The Na+
ion concentration reached its highest dissolution level of 0.20 and 0.55 mM after 1 - 3 days in samples with and
without heat treatment, respectively. The sodium titanate appeared to become more stable after heating, hinder-
ing the release of Na+ ions. In this study the titanate film produced by MKT treatment is composed of sodium
and calcium. The presence of calcium in the film may be an advantage compared to the Kokubo treatment be-
cause it may accelerate apatite formation, since apatite formation depends on the formation of a calcium titanate
layer. The titanate layer produced by the MKT treatment is less stable than that formed by NaOH heat-treated at
600˚C. Thus, a higher ions exchange is expected for MKT treatment, which may accelerate apatite formation on
titanium implants. The in vivo results showed that the MKT treatment (group B) accelerated osseointegration
compared to the other groups. The hydrophilicity of group B is attributed to the formation of TiOH bonds on
the surface.
5. Conclusion
The MKT treatment was demonstrated to be efficient for bioactivation of titanium implants. A completely hy-
drophylic surface was achieved by 1 h treatment in autoclave at 120˚C with a solution containing 2.2 M NaOH.
The treatment produced a thin titanate layer composed of sodium, calcium and silicium. This microstructure
promoted physic-chemical interactions between biological medium and titanium surface. These interactions in-
creased cell proliferation and osseointegration as evidenced by the in vivo tests.
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