Quantitative Analysis of Apatite Formation on Titanium and Zirconia in a Simulated Body Fluid Solution Using the Quartz Crystal Microbalance Method

The bone-bonding ability of a material is evaluated by examining apatite formation on its surface in simulated body fluid (SBF). Partially stabilized zirconia (ZrO2) is currently attractive as an alternative to titanium (Ti) implants; however, no quantitative analysis of apatite formation between Ti and ZrO2 in SBF has been reported. In the present study, we quantitatively evaluated apatite formation onto Ti or ZrO2 in SBF using the 27MHz quartz crystal microbalance method (QCM). In the QCM measurements, apatite formation was detected as a frequency decrease in the Ti or ZrO2 sensor. Frequency decreases were observed at around 1 hour for Ti and at around 2 hours for the ZrO2 sensor after the injection of SBF. This revealed that the Ti sensor showed faster apatite formation than ZrO2. There was no significant difference in the amounts of apatite formation between the Ti and ZrO2 sensors after 24 hours of apatite formation in SBF. In conclusion, the present quantitative study using QCM revealed that apatite formation on the Ti surface in the SBF was obviously faster than that on the ZrO2 surface. Faster apatite formation may predict faster initiation of bone formation on Ti compared with ZrO2.


Introduction
For evaluating the biocompatibility or tissue responses of biomaterials in hard tissue, in vitro simulation methods using simulated body fluid have been postulated in addition to animal experiments and/or cell assays.Kokubo [1] reported that the essential requirement for a biomaterial to bind to living bone is the formation of bone-like apatite on its surface when implanted in the living body.They proposed that in vivo apatite formation could be reproduced after the immersion of a material in a SBF, in which ion concentrations are nearly equal to human blood plasma [1].There are numerous studies on the effectiveness of SBF immersion experiments by Kokubo and his colleagues, and the in vivo bioactivity, namely, osteoconductivity, of biomaterials is precisely mirrored by the in vitro apatite-forming ability in SBF [2][3][4].In addition to Kokubo's SBF, Hanawa et al. [5,6] proposed Hanks' balanced salt solution (HBSS) without organic species at pH 7.4 as an SBF and found apatite formation on a Ti surface in HBSS.Our group [7][8][9][10] examined apatite deposition on the surface of modified Ti materials, such as thin-apatite-coated or DNA-coated Ti materials, after immersion in SBF and found that better apatite deposition on the biomaterials in HBSS corresponded with better in vivo bone formation.Thus, an SBF immersion experiment is useful for evaluating biocompatibility or tissue responses of biomaterials for hard tissue, which can reduce the number of animals used and the duration of animal experiments.
Recently, yttria-stabilized tetragonal zirconia polycrystal, which is known as partially stabilized zirconia (ZrO 2 ), attracted attention as an alternative to titanium as an implant material that overcomes the shortcomings of titanium implants such as the dark grayish color [11] and metal sensitivity [12].There are various results regarding bone response to ZrO 2 implants.Some report that ZrO 2 ceramic implants show similar or better behavior than Ti implants in bone response [13][14][15].In contrast, it was reported that the removal torque of Ti implants was significantly higher than that of ZrO 2 after implantation into the femurs of rabbits and that the bone formation of ZrO 2 seemed to proceed more slowly than roughened Ti [16,17].However, there are no quantitative analyses for apatite formation between Ti and ZrO 2 in SBF.
The quartz crystal microbalance (QCM) technique is a straightforward method for detecting the adsorption or precipitation of biomolecules onto a material surface by measuring differences in the oscillating frequency of the quartz cell [18].The adsorption or precipitation of biomolecules onto the surface of the oscillating quartz crystal causes the oscillation frequency to decrease in relation to the amount of biomolecules bound to the crystal surface.In the QCM measurements, apatite formation was detected as a frequency decrease in Ti or ZrO 2 sensor in SBF.
There are a few reports on monitoring apatite formation on a material in SBF using the QCM method.Tanahashi et al. [19] reported on apatite formation on gold sensors in SBF.They assessed the process of apatite formation in two steps, namely, the initial nucleation step with glass powder and the crystal growth step in the absence of glass powder using QCM.Other QCM studies suggested that negatively charged surfaces of a self-assembled monolayer or nanocrystalline TiO 2 coating induced apatite formation in SBF [20][21][22].
In the present study, we aimed to investigate apatite formation onto Ti or ZrO 2 quantitatively in SBF using a 27 MHz QCM, which enabled measurements with high sensitivity and low noise [23,24].The Ti and ZrO 2 sensor were characterized by contact angle measurement and X-ray photoelectron spectroscope (XPS) analysis, and the obtained apatite crystals were identified by scanning electron microscopy (SEM) and XPS.The null hypothesis tested was that the difference in the materials Ti and ZrO 2 did not influence apatite formation in SBF.

QCM Apparatus and Sensors.
A 27-MHz QCM (AT cut shear mode, AFFINIX QN, ULVAC Co., Ltd., Tokyo, Japan) with a 500 L cell was used.The temperature was maintained at 25 ± 1 ∘ C, and the SBF solution in the cells was not stirred during the measurements.
Ti and ZrO 2 sensors were used.Each sensor was prepared by the sputter coating of each material on an Au electrode [25].Briefly, Ti or zirconium disks (Quartz 4N, ULVAC, Inc., Kanagawa, Japan) were used as a target, and the deposition of each material was performed using sputtering deposition equipment (CS200, ULVAC, Inc., Kanagawa, Japan).Ti sputtering was performed under argon gas, and zirconium sputtering under oxygen gas to deposit ZrO 2 .The film thickness of Ti and ZrO 2 sensor was measured by the step profiler (Dektak 150, Veeco Instruments, Inc., USA) and the surface roughness of each sensor was measured by atomic force microscope (AFM: Nanosurf Easyscan 2, Nanosurf, AG, Switzerland) according to the previous report [25].AFM measurements were carried out by the tapping mode using tapping mode silicon probes (Tap190AL-G, force contact 48 N/m, Budget sensors, Bulgaria) with resonance frequencies of approximately 190 kHz in an area with 2 × 2 m 2 .Each sensor was irradiated by ultraviolet radiation (BioForce Nanosciences Holdings Inc., USA) for 20 minutes before QCM measurement.

Measurements of Apatite Precipitation by QCM Method within SBF Solution.
Hanks' balanced salt solution (HBSS) without organic species was prepared as the SBF.The ion concentrations of HBSS are as follows: Na + : 142, K + : 5.81, Mg 2+ : 0.811, Ca 2+ : 1.26, Cl − : 145, HCO 3 − : 4.17, HPO 4 2− : 0.778, and SO 4 2− : 0.811 mmol/L.A cell with a Ti or ZrO 2 sensor was set in the QCM apparatus.After stabilization of the frequency, a 500 L SBF solution was injected into the cell.The frequency decrease was monitored until 24 hours after injection of the SBF solution.The amount of apatite formation onto each surface at 24 hours after SBF injection was calculated by Sauerbrey's equation [26].A frequency decrease of 1 Hz corresponds to a mass change of 0.61 ± 0.1 ng/cm 2 on the sensor in a 27 MHz QCM system.By curve fitting for the Δ curve against time, the apparent reaction rate, Kobs, in the following equation was obtained.Δ ∞ is the frequency shift at infinite time.Three runs of QCM measurements were performed during a 24-hour period.
Before the SBF experiment, the solubility of Ti and ZrO 2 was checked by detecting frequency change in ultrapure water instead of SBF and no dissolution of Ti and ZrO 2 during the 24 hours was confirmed.

Contact Angle Measurements of Ti and ZrO 2 Sensors.
The contact angles of Ti and ZrO 2 sensor surfaces with respect to double-distilled water were measured using a contact angle meter (DMe-201, Kyowa Interface Science Co., Ltd., Tokyo, Japan) after the ultraviolet irradiation of each sensor.The water drop volume was maintained at 0.5 L, and three measurements of 15 seconds each were made for each surface type.The measurements were performed at the same room temperature and humidity.

SEM Observation.
After 24 hours of apatite formation in SBF, each sensor was cleaned with distilled water and was naturally dried in the atmosphere.After ion coating with gold, the morphology of the crystals was observed using a SEM (JSM-5600LV, JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV.

XPS Analysis.
Surface elemental analyses of each sensor before and after apatite formation were performed using an X-ray photoelectron spectroscope (XPS, KRATOS AXIS Nova, Shimadzu Corp., Kyoto, Japan) equipped with an Alk X-ray source operated at 300 W. After 24 hours of apatite formation in SBF, each sensor was cleaned with distilled water and was naturally dried in the atmosphere.The Ti2p, Advances in Materials Science and Engineering 3 The same letters denote no significant difference ( > 0.05).
Zr3d, and O1s peaks of each sensor were analyzed before apatite formation, and the Ca2p, P2p, and O1s peaks of apatite crystals on each sensor were analyzed after apatite formation.
2.6.Statistical Analyses.Significant differences were determined using statistical analysis software (GraphPad Prism, GraphPad Software Inc., San Diego, CA, USA).Statistical significance was set at  < 0.05.Nonpaired -test was employed to compare data obtained from contact angle measurements and in QCM measurements.

3.1.
Characterization of QCM Sensors.The film thickness of Ti and ZrO 2 sensor was approximately 150 and 110 nm, respectively, and the surface roughness of each sensor was approximately 4∼5 nm [25].The contact angles of the Ti and ZrO 2 sensors are shown in Table 1.There was no significant difference in the contact angle between the Ti and ZrO 2 sensors ( > 0.05), and both showed hydrophilic surfaces.XPS analyses of the Ti and ZrO 2 sensors are shown in Figure 1.
Wide spectra indicated the presence of Ti and Zr3d for Ti and ZrO 2 sensor as shown in Figures 1(a) and 1(d), respectively.O1s peak was identified for both Ti and ZrO 2 sensors.Higher resolution Ti2p spectrum showed the presence of Ti2p 3/2 at 459.0 eV and Ti2p 1/2 peak at 464.8 eV [27].The O1s peak at 530.6 eV and 532.1 eV as shown in Figure 1(c) corresponded to the bulk oxygen in titanium dioxide and basic terminal OH (Ti-OH), respectively [28,29].For the ZrO 2 sensor, higher resolution spectrum identified the presence of Zr3d 5/2 at 182.1 eV and Zr3d 3/2 at 184.5 eV as shown in Figure 1(d) [30].Higher resolution O1s spectrum showed two O1s peaks as shown in Figure 1(f).O1s peak at 530.4 eV could be attributed to the bulk oxygen in ZrO 2 and that at 531.8 eV may be the oxygen of Zr-OH [31].

QCM Measurements.
The frequency curves for the Ti and ZrO 2 sensors are shown in Figure 2. No apparent frequency decrease was observed at the initial stage.Distinct frequency decrease was observed at around 1 hour for the Ti sensor and at around 2 hours for the ZrO 2 sensor, after the injection of SBF (arrow in Figure 2).Thus, the frequency curves were separated into two stages: the stable initial stage and the apatite formation stage.Table 2 lists the time for the stable initial stage before frequency decrease (apatite formation).The ZrO 2 sensor showed a significantly longer time for the initial stage than the Ti sensor ( < 0.05).The amounts of apatite formation on Ti and ZrO 2 24 hours after SBF injection are shown in The different letters denote significant difference ( < 0.05).
Figure 3.There was no significant difference in the amounts of apatite formation between the Ti and ZrO 2 sensors ( > 0.05).Figure 4 shows the apparent reaction rate for apatite formation during the apatite-forming stage until 24 hours.No significant difference existed between the Ti and ZrO 2 sensors ( > 0.05).

SEM Observation of Apatite Crystals.
Figure 5 shows the SEM pictures of apatite crystals on each sensor after 24 hours of SBF immersion.It was confirmed that apatite crystals were randomly precipitated on each sensor (Figures 5(a) and 5(c)).Apatite crystals with a diameter of approximately 500 nm were sparsely observed on the Ti and ZrO 2 sensors (Figures 5(b) and 5(d)).Both showed similar morphologies of apatite crystals.
The Ca/P atomic ratio of apatite crystals obtained by XPS measurement was approximately 1.46, which was relatively smaller than that of stoichiometric hydroxyapatite.

Discussion
In the present study, we evaluated apatite formation on Ti and ZrO 2 surfaces in SBF solution using the QCM method.The study revealed that apatite formation on Ti was significantly faster than that on ZrO 2 .Therefore, the null hypothesis was rejected.
The surface charge of a substrate plays an important role in the reaction of the substrate in an aqueous solution.Some researchers prepared negatively and positively charged surfaces using the SAM technique and monitored apatite formation in an SBF solution using the QCM method [20,21].They found that negatively charged surfaces with PO 4 H 2 or COOH groups showed more progressed apatite formation than positively charged surfaces with NH 2 groups.The apatite formation was initiated by the calcium ion adsorption on negatively charged surfaces, and consequently phosphate ions bound to the adsorbed calcium ions.With the increase in immersion time, a large amount of calcium ions and phosphate ions was attracted to the surface to form apatite crystals.
In our previous study, the apparent zeta potentials of Ti and ZrO 2 at pH = 7.4, which were measured by the streaming  potential technique, were reported to be approximately −87 and −43 mV, respectively [25].This means that both Ti and ZrO 2 surfaces were negatively charged, and Ti was more negatively charged than ZrO 2 .Thus, it is presumed that the faster apatite formation on Ti was due to the more negatively charged surface.The present results accorded with previous reports [20][21][22]; namely, a higher number of calcium ions in the SBF were initially attracted to the more negatively charged Ti surface.Subsequently, phosphate ions and the calcium ions in the SBF combined, and apatite nuclei formed.Faster apatite formation may predict faster initiation of bone formation.The amounts of apatite formation between Ti and ZrO 2 were not significantly different for the 24 hours of SBF immersion.The difference in surface charge only influenced the initial nucleation stage not the apatite formation stage.Besides apatite formation, protein behaviors such as adsorption, proliferation, and differentiation influence the achievement of bone formation.Kusakawa et al. [34] studied protein adsorption onto Ti and ZrO 2 using the QCM method.It was reported that Ti showed greater adsorption of fibronectin, cell adhesive protein, and albumin, a cell adhesion-inhibiting protein.Monitoring apatite formation and protein behaviors is needed to analyze the level of bone formation.

Conclusion
This paper examined apatite formation on Ti and ZrO 2 in SBF using the 27 MHz QCM method.The obtained apatite crystals were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscope (XPS) analysis.It was found that the apatite formation on the Ti surface in the SBF was clearly faster than that on the ZrO 2 surface.This was due to the more negatively charged Ti surface compared with ZrO 2 .No significant difference existed in the amounts of apatite formation, the size of apatite crystals, and the Ca/P atomic ratio of apatite crystals between the Ti and ZrO 2 sensors.Faster apatite formation may predict faster initiation of bone formation in Ti compared with ZrO 2 .

Figure 1 :Figure 2 : 2 Figure 3 : 1 )Figure 4 :
Figure 1: XPS analysis of Ti and ZrO 2 sensors.(a) XPS spectrum of the Ti sensor, (b) higher resolution XPS spectrum of Ti2p of the Ti sensor, (c) higher resolution XPS spectrum of O1s of the Ti sensor, (d) XPS spectrum of the ZrO 2 sensor, (e) higher resolution XPS spectrum of Zr3d of the ZrO sensor, and (f) higher resolution XPS spectrum of O1s of the ZrO 2 sensor.

Figure 5 :
Figure 5: SEM pictures of apatite crystals on each sensor after 24 hours of SBF immersion.(a, c) Apatite crystals on Ti sensor with lower and higher magnification.(b, d) Apatite crystals on ZrO 2 sensor with lower and higher magnification.

Table 2 :
Time at which frequency began to decrease (mean ± SD  = 3).