Influence of the Surface Chemical Composition Differences between Zirconia and Titanium with the Similar Surface Structure and Roughness on Bone Formation

The osseointegration of zirconia (ZrO2) implants is still controversial. In this study, we aimed to make clear the influence of surface chemical composition, Ti or ZrO2, to osseointegration. First, a roughened Ti surface was prepared with a combination of large-grit sandblasting and acid treatment. Then, we applied molecular precursor solution containing Zr complex onto roughened Ti surface and can deposit thin ZrO2 film onto roughened Ti surface. We can change surface chemical composition from Ti to ZrO2 without changing the surface structure and roughness of roughened Ti. The tetragonal Zr was uniformly present on the ZrO2-coated Ti surface, and the surface of the ZrO2-coated Ti showed a higher apparent zeta potential than Ti. Ti and ZrO2-coated Ti rectangular plate implant was placed into the femur bone defect. After 2 and 4 weeks of implantation, histomorphometric observation revealed that the bone-to-implant contact ratio and the bone mass values for ZrO2-coated Ti implants inserted into the femur bone defects of the rats at 2 weeks were significantly higher than those for Ti implants (p < 0.05). It revealed that ZrO2 with a similar surface structure and roughness as that of roughened Ti promoted osteogenesis equivalent to or better than that of Ti in the early bone formation stage.


Introduction
Using titanium (Ti) as a dental implant material has advantages owing to its excellent mechanical properties, biocompatibility, and tight bonding to the bone tissue, which is known as osseointegration. However, aesthetic, and hypersensitive reactions including allergies to Ti implants have been reported [1][2][3]. An yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) was used as another option as a dental implant material [4]. However, the osseointegration of zirconia (ZrO 2 ) implants is controversial. Kohal et al. [5]. evaluated 65 one-piece ZrO 2 implants incorporated in humans for three years post-operation and reported that the survival rates of ZrO 2 implants were inferior to those of Ti implants. In contrast, Roehling et al. [6]. studied 2758 publications and reported that ZrO 2 and Ti implants showed similar soft and hard tissue integration capacities.
It is well known that surface structure and surface roughness is key to the osseointegration of dental implants [7]. The surface treatment of Ti implants by combining the large-grit and acid-etched procedures, which is generally named SLA, produced positive effects on the activation of blood platelets and cell migration, and clinical success rate [8][9][10][11]. Surface modifications of Y-TZP implants have been reported [12][13][14], and large-grit and acid etching procedures were also applied to Y-TZP implants [12][13][14][15]. However, Y-TZP is known to exhibit high mechanical strength and chemical stability [16], and achieving a similar surface Nanomaterials 2022, 12, 2478 2 of 15 structure and roughness on Y-TZP as that on Ti is difficult. Surface roughness is a popularly used parameter for evaluating the material's surface. Bormann et al. [17]. compared the bone tissue response to those of the micro-structured ZrO 2 and SLA-Ti implants. ZrO 2 implants were chemically treated with a hot solution of hydrofluoric acid. They reported that the mean roughness of the SLA-Ti implants (Sa = 1.19 µm) was approximately twice that of similar ZrO 2 implants (Sa = 0.63 µm).
Apart from surface structure and roughness, the surface chemical composition also affects the osseointegration of dental implants. It is not clear which factor, surface structure, roughness, or chemical composition will contribute more to osseointegration for ZrO 2 implants. It is presumed that this is the reason for the controversy regarding the osseointegration of ZrO 2 . To elucidate the osseointegration of ZrO 2 implants, it is important to evaluate the bone response of the ZrO 2 surface for a similar structure and roughness as that of Ti. If we can prepare a ZrO 2 surface with a similar structure and roughness as that of Ti, we can say something about the contribution of the surface chemical composition to the osseointegration for ZrO 2 implant.
The molecular precursor method is a surface modifying method, i.e., an arbitrary metal oxide film coating on the ceramic and metal surfaces achieved using a metal complex liquid [18][19][20]. A thin apatite film can be deposited onto the Ti surface using the Ca-EDTA complex precursor solution [21,22]. The thickness of the thin apatite film was in the range of 0.6-0.8 µm. The molecular precursor method is a wet process [23] and can only change the chemical composition of substrates without changing the surface structure and roughness because of the very thin film coating. A thin ZrO 2 film can be deposited on the Ti surface using the Zr-complex precursor solution. Therefore, we can achieve a similar surface structure and roughness of the ZrO 2 surface as roughened Ti implant surface.
In this study, we tried to distinguish the effects of the surface chemical composition, Ti or ZrO 2 , from those of the surface structure and roughness between Ti and ZrO 2 coated Ti implants in the osseointegration process. The ZrO 2 coating fabricated by the molecular precursor method changed the surface chemical composition from Ti to ZrO 2 without changing the surface structure and roughness of roughened SLA-Ti. The main purpose of this study is to reveal the effects of the difference in surface chemical composition on the bone response with respect to ZrO 2 and Ti with a similar surface structure and roughness. We can make clear the difference in bone responses between Ti and ZrO 2 surfaces. The effectiveness of the ZrO 2 surface on the bone response will be clarified in this study. To the best of our knowledge, this is the first trial to elucidate the effectiveness of the surface chemical composition of implant materials on a bone formation without considering surface structure and roughness.

Specimen Preparation
Two shapes of Ti samples (JIS2 type, 99.9% mass, Furuuchi Chemical, Tokyo, Japan), disks, and rectangular plates were used in this study as shown in Figure 1. Ti disks with 12.0 mm in diameter and 1.0 mm in thickness were used for surface observation, crystal structure and atom elements analyses, phosphate-buffered saline (PBS), and simulated body fluid (SBF) immersion experiments. Ti rectangular plates with dimensions of 1.0 mm × 10.0 mm × 20.0 mm were used for zeta potential measurements and those with dimensions of 2.0 mm × 3.0 mm × 1.0 mm were for animal implantation experiments. The number of disks and rectangular plates was three for each experiment in addition to AFM measurements (n = 4).
coating materials, we standardized the shape of the sample on a disk. So, disks were used for other experiments. Before ZrO2 coating, the surfaces of the specimens were polished with a #1200 waterproof paper under running water. Then, specimens were subjected to SLA treatment. Namely, sandblasting was performed perpendicular to the titanium surface from a distance of 20 mm with 180 μm alumina particles at a 0.6 N/mm 2 air pressure. Then, acid etching was performed on the blasted surface with a mixture of 36% hydrochloric acid (HCl) and a 96% sulfuric acid (H2SO4) solution for 3 min at 70 °C [24]. SLA-treated specimens were then ultrasonically cleaned in ethanol and distilled water for 20 min (ultrasonic cleaner; VS-100III; AS ONE, Osaka, Japan).
Ti disks and rectangular plates were coated with ZrO2 (ZrO2/Ti) using the molecular precursor method. A molecular precursor solution (25 mL) for the ZrO2 film (Zr complex ethanol solution; Zr ion concentration = 0.32 mmol/g; TFTECH, Tokyo, Japan) was poured on the Ti surface. It was then spin-coated using a spin coater (1H-D7; MIKASA, Tokyo, Japan) in the double step mode; first at 500 rpm for 5 s, and then at 2000 rpm for 30 s. Ti disks and rectangular specimens covered with precursor films were then heated at 550 °C in air for 30 min using a tubular furnace (EPKPO12-K; ISUZU, Niigata, Japan). A thin film coating of ZrO2 can be achieved on Ti disks and rectangular plates using this method.

Surface Characterization of the ZrO2 Coating
The surface structure of ZrO2/Ti was observed using scanning electron microscopy (SEM; SU1510; Hitachi High-Technologies, Tokyo, Japan). Au supper-coated specimens were observed at a 15 kV accelerating voltage.
The three-dimensional surface structure of ZrO2/Ti was observed using atomic force microscopy (AFM; Nanosurf Easyscan 2, Nanosurf AG; Liestal, Switzerland). The tapping mode was employed using a TapAL-G cantilever (Budget sensors, Bulgaria; resonance frequency = 190 kHz, spring constant = 48 N/m). The average arithmetic roughness of the three-dimensional surface (Sa) was calculated within the 1.0 μm × 1.0 μm, 2.5 μm × 2.5 μm, 5.0 μm × 5.0 μm, and 10.0 μm × 10.0 μm areas of the AFM images. The surface of roughened Ti was also observed using SEM and AFM by the same method.
X-ray diffraction was used to characterize the crystal structure of the coated ZrO2 thin film on Ti disks with a thin layer technique (XRD; attachment incidence angle θ = 0.3°, SmartLab; Rigaku, Tokyo, Japan). An X-ray source is Cu Kα which powers 45 kV × 200 mA. The presence of Ti or Zr atoms on Ti and ZrO2/Ti disks on each surface was confirmed using an electron probe micro analyzer (EPMA; JXA-8900R; JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV. Cross-sectional observation was also performed. The disks were then embedded in an epoxy resin. After curing the resin, the specimens were vertically cut through the middle using a cutting machine. A rectangular plate is better to be set into the cell of the apparatus for zeta potential measurements. Regarding the animal experiment, the bone defect is box-shaped, and a rectangular plate fits this shape of the bone defect. It is difficult to fit the disks to the bone defect. In our previous studies for surface observation or SBF immersion experiments for coating materials, we standardized the shape of the sample on a disk. So, disks were used for other experiments.
Before ZrO 2 coating, the surfaces of the specimens were polished with a #1200 waterproof paper under running water. Then, specimens were subjected to SLA treatment. Namely, sandblasting was performed perpendicular to the titanium surface from a distance of 20 mm with 180 µm alumina particles at a 0.6 N/mm 2 air pressure. Then, acid etching was performed on the blasted surface with a mixture of 36% hydrochloric acid (HCl) and a 96% sulfuric acid (H 2 SO 4 ) solution for 3 min at 70 • C [24]. SLA-treated specimens were then ultrasonically cleaned in ethanol and distilled water for 20 min (ultrasonic cleaner; VS-100III; AS ONE, Osaka, Japan).
Ti disks and rectangular plates were coated with ZrO 2 (ZrO 2 /Ti) using the molecular precursor method. A molecular precursor solution (25 mL) for the ZrO 2 film (Zr complex ethanol solution; Zr ion concentration = 0.32 mmol/g; TFTECH, Tokyo, Japan) was poured on the Ti surface. It was then spin-coated using a spin coater (1H-D7; MIKASA, Tokyo, Japan) in the double step mode; first at 500 rpm for 5 s, and then at 2000 rpm for 30 s. Ti disks and rectangular specimens covered with precursor films were then heated at 550 • C in air for 30 min using a tubular furnace (EPKPO12-K; ISUZU, Niigata, Japan). A thin film coating of ZrO 2 can be achieved on Ti disks and rectangular plates using this method.

Surface Characterization of the ZrO 2 Coating
The surface structure of ZrO 2 /Ti was observed using scanning electron microscopy (SEM; SU1510; Hitachi High-Technologies, Tokyo, Japan). Au supper-coated specimens were observed at a 15 kV accelerating voltage.
The three-dimensional surface structure of ZrO 2 /Ti was observed using atomic force microscopy (AFM; Nanosurf Easyscan 2, Nanosurf AG; Liestal, Switzerland). The tapping mode was employed using a TapAL-G cantilever (Budget sensors, Bulgaria; resonance frequency = 190 kHz, spring constant = 48 N/m). The average arithmetic roughness of the three-dimensional surface (Sa) was calculated within the 1.0 µm × 1.0 µm, 2.5 µm × 2.5 µm, 5.0 µm × 5.0 µm, and 10.0 µm × 10.0 µm areas of the AFM images. The surface of roughened Ti was also observed using SEM and AFM by the same method.
X-ray diffraction was used to characterize the crystal structure of the coated ZrO 2 thin film on Ti disks with a thin layer technique (XRD; attachment incidence angle θ = 0.3 • , SmartLab; Rigaku, Tokyo, Japan). An X-ray source is Cu Kα which powers 45 kV × 200 mA. The presence of Ti or Zr atoms on Ti and ZrO 2 /Ti disks on each surface was confirmed using an electron probe micro analyzer (EPMA; JXA-8900R; JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV. Cross-sectional observation was also performed. The disks were then embedded in an epoxy resin. After curing the resin, the specimens were vertically cut through the middle using a cutting machine.
The atom elements in the surface of Ti and ZrO 2 /Ti were analyzed by using X-ray photoelectron spectroscopy (XPS; AXIS-ULTRA; Kratos Analytical, Stretford, UK), with an X-ray source of Al Kα and a power of 15 kV × 10 mA.
Zeta potential is known to indicate the surface properties, especially charging behavior of the material surface. Next, we tried to determine the apparent zeta potentials using an electrokinetic analyzer (SurPASS 3; Anton Paar GmbH, Graz, Austria). For determining the zeta potential, an aqueous electrolyte solution (1.0 mM of KCl) was used at a pH level of 5.6. KCl solution passed through a thin slit channel formed by two Ti or ZrO 2 /Ti plates with identical surfaces of rectangular plate implant. The pressure-driven flow of the KCl solution gave the streaming current and potential. The zeta potential was then calculated according to the Helmholtz-Smoluchowski equation [25,26]. Furthermore, the pH-dependent curves of the zeta potential were plotted by measuring the zeta potential at any pH level adjusted by 0.05 M HCl and 0.1 M KOH. The isoelectric points of Ti and ZrO 2 /Ti were obtained from these curves. Measurements of apparent zeta potential at a pH level of 5.6 were performed three times. In these experiments, coefficient of variation was less than 3%, and it was concluded the measurements possessed high reliability. Thus, measurements of zeta potential at different pH were performed only once.
For determining the durability of ZrO 2 coating, the ZrO 2 /Ti disks were immersed in PBS solution (pH = 7.4). The volume of PBS solution was 20 mL. The specimens were stored in 20 mL of PBS in a polypropylene bottle for 90 days. After immersion, the specimens were dried in a desiccator. The morphological changes in ZrO 2 films were observed using SEM at an accelerating voltage of 15 kV after sputter-coating with Au.

SBF Immersion Experiments
As SBF, Hanks' balanced salt solution (HBSS) was prepared without organic species and adjusted pH = 7.4 [27]. Ti and ZrO 2 /Ti disks were immersed in 20 mL of HBSS at 37 • C in a polypropylene bottle. After immersing for 6 and 24 h, the excess HBSS on the disks was removed using soft paper, and the disks were immediately dried in a desiccator. The surfaces of the Ti and ZrO 2 /Ti disks were then observed using SEM at an accelerating voltage of 15 kV after sputter-coating with Au. The crystallographic structures of the precipitates on the Ti and ZrO 2 /Ti disks were analyzed using XRD (RINT-RAPID2 MM007; Rigaku, Tokyo, Japan), with an X-ray source of Cu-Kα at a power of 40 kV × 30 mA, and FT-IR (FT/IR-430; Jasco, Tokyo, Japan) using the KBr method. The precipitated products were detached from the Ti and ZrO 2 /Ti disks for FT-IR measurements.

Animal Implantation Experiment
The approval of the animal implantation experiment was obtained from the Animal Experimental Ethical Guidelines of the Tsurumi University, School of Dental Medicine (certificate nos. 19A042, 20A001, 21A009, and 22A005). Overall, 12 male Wistar rats were used for animal implantation experiments. Their weight was approximately 180 g and ages are 6 weeks. The rats were housed in pairs of two per cage at 20-25 • C in a 12 h circadian light rhythm environment. They feed on water and food ad libitum during the experiment.
Ti or ZrO 2 /Ti rectangular plate implant was placed in a femur bone defect [28,29], and each rat received one implant. Overall, 3 ZrO 2 /Ti and 3 Ti rectangular plate implants were inserted for 2-and 4-week implantation periods, respectively.
For general inhalation, anesthesia using a 3% isoflurane and oxygen mixture was used. The concentration of isoflurane was reduced to 2% during surgical manipulation. The hind limb was shaved and then operating field was disinfected. Next, operation field was locally anesthetized by the injection of xylocaine. The distal surface of the hind limb was longitudinally incised, and femur was exposed. The cortical bone defect was prepared through the cortex and the medulla. A very gentle surgical technique was employed and internal cooling with a physiological saline solution was continuously introduced during the defect preparation. The size of the defect was 1 mm × 2 mm. A rectangular plate implant was press fitted into the bone defect as shown in Figure 2. The muscle tissue and skin were separately sutured using non-absorbable. After surgery, antibiotics (benzyl penicillin G procaine, 3,000,000 U/kg) were subcutaneously injected. during the defect preparation. The size of the defect was 1 mm × 2 mm. A rectangular plate implant was press fitted into the bone defect as shown in Figure 2. The muscle tissue and skin were separately sutured using non-absorbable. After surgery, antibiotics (benzyl penicillin G procaine, 3,000,000 U/kg) were subcutaneously injected. The rats were euthanized at 2 and 4 weeks after implantation using carbon dioxide gas to harvest the rectangular plate implants and the surrounding femoral bone.
After removing the excess tissue, the specimens were fixed in a 10% neutral buffered formalin solution (pH 7.4). Then, tissue blocks including implants were dehydrated through a gradient series of ethanol. Subsequently, they were embedded in methyl methacrylate. A cutting-grinding technique was applied to make non-decalcified thin sections. Cutting of the tissue blocks was performed in a direction perpendicular to the axis of the implants. After cutting and grinding by EXAKT-Cutting Grinding System (BS-300CP band system and 400CS microgrinding system; EXAKT, Norderstedt, Germany), thin sections in which thickness was approximately 50-70 μm were obtained.
Thin sections were double stained with methylene blue and basic fuchsin. Histological and histomorphometrical evaluation was performed using a light microscope (Eclipse Ni, Nikon, Tokyo, Japan, magnifications of ×40 and ×100). Bone response towards rectangular plate implant was histologically evaluated. As a histomorphometric analysis, the bone-to-implant contact (BIC) ratio and the bone mass (BM) around the implant were measured within the regions of interest (ROI). The ROIs were determined in accordance with a previous study [30], as illustrated in Figure 3. The BIC ratio was defined as the percentage of the length of direct bone-implant contact along with the implant surface. BM was calculated as the percentage of newly formed bone around the implant. The BIC ratio and BM values were obtained by the image analysis (WinROOF, Visual System Division Mitani, Tokyo, Japan). The rats were euthanized at 2 and 4 weeks after implantation using carbon dioxide gas to harvest the rectangular plate implants and the surrounding femoral bone.
After removing the excess tissue, the specimens were fixed in a 10% neutral buffered formalin solution (pH 7.4). Then, tissue blocks including implants were dehydrated through a gradient series of ethanol. Subsequently, they were embedded in methyl methacrylate. A cutting-grinding technique was applied to make non-decalcified thin sections. Cutting of the tissue blocks was performed in a direction perpendicular to the axis of the implants. After cutting and grinding by EXAKT-Cutting Grinding System (BS-300CP band system and 400CS microgrinding system; EXAKT, Norderstedt, Germany), thin sections in which thickness was approximately 50-70 µm were obtained.
Thin sections were double stained with methylene blue and basic fuchsin. Histological and histomorphometrical evaluation was performed using a light microscope (Eclipse Ni, Nikon, Tokyo, Japan, magnifications of ×40 and ×100). Bone response towards rectangular plate implant was histologically evaluated. As a histomorphometric analysis, the bone-toimplant contact (BIC) ratio and the bone mass (BM) around the implant were measured within the regions of interest (ROI). The ROIs were determined in accordance with a previous study [30], as illustrated in Figure 3. The BIC ratio was defined as the percentage of the length of direct bone-implant contact along with the implant surface. BM was calculated as the percentage of newly formed bone around the implant. The BIC ratio and BM values were obtained by the image analysis (WinROOF, Visual System Division Mitani, Tokyo, Japan).

Statistical Analysis
All data were calculated with the help of SPSS for Windows (SPSS Statics 17.0; SPSS, Chicago, IL, USA). The results of the surface roughness from AFM measurements were compared using the student t-test. A p-value < 0.05 was considered to be significant. The results of the BIC ratio and BM values were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey's test for multiple comparisons (p < 0.05). Figure 4 shows the SEM profiles of the Ti and ZrO2/Ti disk surfaces. No difference was observed in the surface shapes between the Ti and ZrO2/Ti disks. Both surfaces showed micro-scale dimples (arrows) on the roughened surface, which were formed by irregular roughness. Additionally, the detachment and defect of the ZrO2 coating film were not observed.

Statistical Analysis
All data were calculated with the help of SPSS for Windows (SPSS Statics 17.0; SPSS, Chicago, IL, USA). The results of the surface roughness from AFM measurements were compared using the student t-test. A p-value < 0.05 was considered to be significant. The results of the BIC ratio and BM values were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey's test for multiple comparisons (p < 0.05). Figure 4 shows the SEM profiles of the Ti and ZrO 2 /Ti disk surfaces. No difference was observed in the surface shapes between the Ti and ZrO 2 /Ti disks. Both surfaces showed micro-scale dimples (arrows) on the roughened surface, which were formed by irregular roughness. Additionally, the detachment and defect of the ZrO 2 coating film were not observed.

Statistical Analysis
All data were calculated with the help of SPSS for Windows (SPSS Statics 17.0; SPSS, Chicago, IL, USA). The results of the surface roughness from AFM measurements were compared using the student t-test. A p-value < 0.05 was considered to be significant. The results of the BIC ratio and BM values were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey's test for multiple comparisons (p < 0.05). Figure 4 shows the SEM profiles of the Ti and ZrO2/Ti disk surfaces. No difference was observed in the surface shapes between the Ti and ZrO2/Ti disks. Both surfaces showed micro-scale dimples (arrows) on the roughened surface, which were formed by irregular roughness. Additionally, the detachment and defect of the ZrO2 coating film were not observed.    Figure 5 shows the AFM profiles of the Ti and ZrO 2 /Ti disk surfaces. The obtained three-dimensional structure was almost identical on both surfaces. Moreover, there was no significant difference in the arithmetic average roughness of the three-dimensional surface (p > 0.05) ( Table 1).  Values in brackets are SD. Same superscripts indicate no significant differences in each square areas between the Ti and ZrO2/Ti groups. Figure 6 shows the EPMA mapping of ZrO2/Ti disks. The presence of ZrO2 coating was confirmed by the element mapping of Zr. Cross-sectional observation also identified the presence of Zr in the coating film (arrows in Figure 6b).    Figure 6 shows the EPMA mapping of ZrO 2 /Ti disks. The presence of ZrO 2 coating was confirmed by the element mapping of Zr. Cross-sectional observation also identified the presence of Zr in the coating film (arrows in Figure 6b).  Values in brackets are SD. Same superscripts indicate no significant differences in each square areas between the Ti and ZrO2/Ti groups. Figure 6 shows the EPMA mapping of ZrO2/Ti disks. The presence of ZrO2 coating was confirmed by the element mapping of Zr. Cross-sectional observation also identified the presence of Zr in the coating film (arrows in Figure 6b).   Figure 7 shows the XPS results. The O1s peak was observed at 526.5 eV owing to the presence of TiO 2 and ZrO 2 . The peaks attributed to Zr3d 2/3 and Zr3d 5/2 were observed at 179 and 181 eV, respectively. The peaks of Ti2p 3/2 and Ti2p 1/2 of the Ti substrate were observed at 461 and 455 eV, respectively, because of the low thickness of the ZrO 2 layer. Thus, the presence of the ZrO 2 coating on the surface of Ti disks was confirmed.

Surface Characterization
Nanomaterials 2022, 12, x 8 of 15 Figure 7 shows the XPS results. The O1s peak was observed at 526.5 eV owing to the presence of TiO2 and ZrO2. The peaks attributed to Zr3d2/3 and Zr3d5/2 were observed at 179 and 181 eV, respectively. The peaks of Ti2p3/2 and Ti2p1/2 of the Ti substrate were observed at 461 and 455 eV, respectively, because of the low thickness of the ZrO2 layer. Thus, the presence of the ZrO2 coating on the surface of Ti disks was confirmed.   The apparent zeta potential of ZrO2/Ti was higher than that of Ti at a pH level of 5.6 ( Table 2). Figure 9 shows the pH-dependent curves of the zeta potential at different pH levels. The isoelectric points of Ti and ZrO2/Ti obtained from Figure 8 are also listed in Table 2. The isoelectric point of ZrO2/Ti was higher than that of Ti.   Figure 7 shows the XPS results. The O1s peak was observed at 526.5 eV owing to the presence of TiO2 and ZrO2. The peaks attributed to Zr3d2/3 and Zr3d5/2 were observed at 179 and 181 eV, respectively. The peaks of Ti2p3/2 and Ti2p1/2 of the Ti substrate were observed at 461 and 455 eV, respectively, because of the low thickness of the ZrO2 layer. Thus, the presence of the ZrO2 coating on the surface of Ti disks was confirmed.   The apparent zeta potential of ZrO2/Ti was higher than that of Ti at a pH level of 5.6 ( Table 2). Figure 9 shows the pH-dependent curves of the zeta potential at different pH levels. The isoelectric points of Ti and ZrO2/Ti obtained from Figure 8 are also listed in Table 2. The isoelectric point of ZrO2/Ti was higher than that of Ti. The apparent zeta potential of ZrO 2 /Ti was higher than that of Ti at a pH level of 5.6 ( Table 2). Figure 9 shows the pH-dependent curves of the zeta potential at different pH levels. The isoelectric points of Ti and ZrO 2 /Ti obtained from Figure 8 are also listed in Table 2. The isoelectric point of ZrO 2 /Ti was higher than that of Ti. Nanomaterials 2022, 12, x 9 of 15 Figure 9. pH-dependent curves of the apparent zeta potentials of Ti and ZrO2/Ti at any pH level. Values in brackets are SD. Figure 10 shows the SEM pictures of the surfaces of ZrO2/Ti disks after PBS immersion. No differences were observed in the surface morphologies of ZrO2/Ti disks. The formation of cracks and pinholes was not observed on the surface.  Figure 11 shows the SEM images of the surfaces of each specimen after 6 and 24 h HBSS immersion. Crystals were deposited on the surfaces of all specimens. However, the growth of crystals was more on the surfaces of ZrO2/Ti disks compared to that on the surfaces of Ti disks after 6 and 24 h of immersion.   Values in brackets are SD. Figure 10 shows the SEM pictures of the surfaces of ZrO2/Ti disks after PBS immersion. No differences were observed in the surface morphologies of ZrO2/Ti disks. The formation of cracks and pinholes was not observed on the surface.  Figure 11 shows the SEM images of the surfaces of each specimen after 6 and 24 h HBSS immersion. Crystals were deposited on the surfaces of all specimens. However, the growth of crystals was more on the surfaces of ZrO2/Ti disks compared to that on the surfaces of Ti disks after 6 and 24 h of immersion.  Figure 11 shows the SEM images of the surfaces of each specimen after 6 and 24 h HBSS immersion. Crystals were deposited on the surfaces of all specimens. However, the growth of crystals was more on the surfaces of ZrO 2 /Ti disks compared to that on the surfaces of Ti disks after 6 and 24 h of immersion. Nanomaterials 2022, 12, x 10 of 15  Figure 12 shows the XRD and FT-IR profiles of the precipitated crystals 24 h after immersion. The peaks of the XRD patterns derived from apatite structures were identified at approximately 23.0°, 26.0°, 28.5°, 32.0°, 33.5°, and 46.5°. The peaks of the FT-IR spectra derived from phosphate groups were detected in the ranges of 550-600 and 900-1200 cm −1 , whereas those from carbonyl groups were detected at approximately 1200 cm −1 . Therefore, the precipitated crystals were identified as carbonate-containing hydroxyapatite.   The peaks of the FT-IR spectra derived from phosphate groups were detected in the ranges of 550-600 and 900-1200 cm −1 , whereas those from carbonyl groups were detected at approximately 1200 cm −1 . Therefore, the precipitated crystals were identified as carbonate-containing hydroxyapatite.  Figure 12 shows the XRD and FT-IR profiles of the precipitated crystals 24 h after immersion. The peaks of the XRD patterns derived from apatite structures were identified at approximately 23.0°, 26.0°, 28.5°, 32.0°, 33.5°, and 46.5°. The peaks of the FT-IR spectra derived from phosphate groups were detected in the ranges of 550-600 and 900-1200 cm −1 , whereas those from carbonyl groups were detected at approximately 1200 cm −1 . Therefore, the precipitated crystals were identified as carbonate-containing hydroxyapatite.

Histological and Histomorphometric Evaluations
The rats remained in good health during the experiment. No clinical signs of inflammation or adverse tissue reactions were observed when the animals were euthanized, and all implants were still in situ. Figures 13 and 14 show the histological appearances of the ZrO 2 /Ti and Ti implants. No inflammatory cells' permeation was observed. New bone was formed around the rectangular plate implants at 2 and 4 weeks after implantation. The bone remodeling process and mature bone formation were observed. Bone-to-implant bonding was detected for the ZrO 2 /Ti and Ti implants. However, some gaps were recognized between the bone and the ZrO 2 /Ti and Ti implants. Overall, Ti implants showed more gaps between the bone and the implant surface.

Histological and Histomorphometric Evaluations
The rats remained in good health during the experiment. No clinical signs of inflammation or adverse tissue reactions were observed when the animals were euthanized, and all implants were still in situ. Figures 13 and 14 show the histological appearances of the ZrO2/Ti and Ti implants. No inflammatory cells' permeation was observed. New bone was formed around the rectangular plate implants at 2 and 4 weeks after implantation. The bone remodeling process and mature bone formation were observed. Bone-to-implant bonding was detected for the ZrO2/Ti and Ti implants. However, some gaps were recognized between the bone and the ZrO2/Ti and Ti implants. Overall, Ti implants showed more gaps between the bone and the implant surface.   Table 3 lists the results of the BIC ratio and BM values after implantation. The BIC ratio and BM values for ZrO2/Ti implants at 2 weeks were significantly higher than those for Ti implants (p < 0.05). At 4 weeks of implantation, there were no significant differences between the Ti and ZrO2/Ti implants in terms of the BIC ratio and BM values (p > 0.05). The BIC ratio and BM values for the Ti implants at 4 weeks were significantly higher than those for Ti implants at 2 weeks (p < 0.05). However, no significant differences were

Histological and Histomorphometric Evaluations
The rats remained in good health during the experiment. No clinical signs of inflammation or adverse tissue reactions were observed when the animals were euthanized, and all implants were still in situ. Figures 13 and 14 show the histological appearances of the ZrO2/Ti and Ti implants. No inflammatory cells' permeation was observed. New bone was formed around the rectangular plate implants at 2 and 4 weeks after implantation. The bone remodeling process and mature bone formation were observed. Bone-to-implant bonding was detected for the ZrO2/Ti and Ti implants. However, some gaps were recognized between the bone and the ZrO2/Ti and Ti implants. Overall, Ti implants showed more gaps between the bone and the implant surface.   Table 3 lists the results of the BIC ratio and BM values after implantation. The BIC ratio and BM values for ZrO2/Ti implants at 2 weeks were significantly higher than those for Ti implants (p < 0.05). At 4 weeks of implantation, there were no significant differences between the Ti and ZrO2/Ti implants in terms of the BIC ratio and BM values (p > 0.05). The BIC ratio and BM values for the Ti implants at 4 weeks were significantly higher than those for Ti implants at 2 weeks (p < 0.05). However, no significant differences were Figure 14. Histological appearances after 4 weeks of implantation into the femur bone defects of rats (magnification of ×40 and ×100). Table 3 lists the results of the BIC ratio and BM values after implantation. The BIC ratio and BM values for ZrO 2 /Ti implants at 2 weeks were significantly higher than those for Ti implants (p < 0.05). At 4 weeks of implantation, there were no significant differences between the Ti and ZrO 2 /Ti implants in terms of the BIC ratio and BM values (p > 0.05). The BIC ratio and BM values for the Ti implants at 4 weeks were significantly higher than those for Ti implants at 2 weeks (p < 0.05). However, no significant differences were observed between the BIC ratio and BM values of ZrO 2 /Ti implants after 2 and 4 weeks (p > 0.05). Values in brackets are SD. The same superscripts indicate no significant differences. Different small letters indicate significant differences between Ti and ZrO 2 /Ti in the same implantation period. Different capital letters indicate significant differences between the values obtained at 2 and 4 weeks in the same specimen.

Discussion
In this study, we prepared a ZrO 2 -coated Ti surface with a similar structure and roughness as that of a Ti surface using the molecular precursor method and evaluated the bone response toward ZrO 2 thin films. It was revealed that the ZrO 2 surface with the same structure and roughness as those of Ti promotes osteogenesis equivalent to or better than that of Ti in the early stages of bone formation.
The surface structure and roughness analysis of ZrO 2 /Ti using SEM and AFM confirmed that the surface appearances and roughness of Ti and ZrO 2 /Ti were almost similar. The molecular precursor method can change the surface chemical composition without changing the surface structure and roughness. EPMA showed that ZrO 2 films were uniformly observed on all parts of the substrate.
Some studies have been reported on the osseointegration of ZrO 2 dental implants in comparison with that of Ti implants [31,32]. Gahlert et al. [33]. investigated the BIC ratio and peri-implant bone density of ZrO 2 implants with a rough acid-etched surface structure and roughness and compared it with Ti-SLA implants in the maxilla of pigs. They reported that there was no difference in the bone responses of ZrO 2 implants and Ti-SLA controls. However, an unacceptable difference was observed in the surface structure and roughness of the ZrO 2 and Ti implants using confocal three-dimensional white light microscopy. The Sa values with roughened ZrO 2 implants were higher than those of Ti-SLA implants. Thus, a majority of the studies on bone responses have not focused on the same surface structure and roughness between the ZrO 2 and Ti specimens. This present study is the first study comparing the bone responses of the Ti and ZrO 2 implants with almost similar surface structure and roughness.
The XRD patterns of the ZrO 2 films on the Ti disk revealed that the deposited coating comprised monoclinic and tetragonal zirconia. The broad peaks in XRD and FR-IR are due to the low crystallinity of precipitated apatite. It is well-known that the crystal structure of ZrO 2 changes with temperature. Therefore, a stabilized agent, such as yttria, is often used to stabilize tetragonal zirconia at room temperature. However, the molecular precursor solution did not contain the stabilized agents. The presence of tetragonal zirconia in this study was due to the extreme thinness of the ZrO 2 films. The atoms in ZrO 2 films can easily move at low energies, which might reduce the phase transition temperature. This provided different results from the bulk of ZrO 2 .
Kokubo et al. [34] reviewed the history of SBF, the correlation of the ability of apatite to form on various materials in SBF with their in vivo bone bioactivities, and some examples of the development of novel bioactive materials based on apatite formation in SBF and concluded that examination of apatite formation on material in SBF is useful for predicting the in vivo bone bioactivity of a material. Thus, we first employed the SBF immersion experiments and then performed the animal experiments. As a result, SBF immersion experiments suggested that the surface property of ZrO 2 promotes osteogenesis equivalent to or better than that of Ti in the early stages of bone formation. This result corresponded with that observed in the animal experiments.
The animal study suggested that with the same surface structure and roughness as that of Ti rectangular plate implants, the ZrO 2 surface exhibited the promotion of new bone formation surrounding the implants. In this study, the apparent zeta potential measurement showed that the value of ZrO 2 /Ti was higher than that of Ti at any pH level. It is inferred that the electrostatic repulsion force of the ZrO 2 against bone proteins, such as osteocalcin and osteopontin, which are negatively charged, may be smaller than that of Ti. Thus, these reactions may promote bone formation on ZrO 2 .
In the animal experiments, rectangular plate implants were inserted into the femurs of rats, and 2-and 4-week implantation periods were evaluated because we intended to observe the early stage of bone formation. A significantly higher value in BIC and BM for ZrO 2 /Ti was observed. Albrektsson et al. [35] argued that osseointegration corresponded to approximately 60% bone contact for Ti implants. In this study, the BIC ratio of the Ti implant was 50-70%, whereas that of the ZrO 2 /Ti implant was approximately 75%. In other words, the BIC ratio of the ZrO 2 /Ti implant was expected to be above the limit prescribed by Albrektsson et al.
This is a new approach to distinguishing the effects of surface chemistry from those of surface structure and roughness between ZrO 2 and Ti in the osseointegration process. This study is basic research to evaluate the bone response of the ZrO 2 surface with the same structure and roughness as that of Ti for elucidating the osseointegration of ZrO 2 implants. In contrast, it was suggested that ZrO 2 -coated Ti implants also have a sufficient clinical value from the viewpoint of BIC ratio and BM value. Non-metallic implants have been reported to exhibit the risks of fracture [36]. Therefore, ZrO 2 is often used as implant abutments only clinically. However, ZrO 2 -coated Ti implants can resolve this fracture problem because of the better mechanical properties of Ti. Moreover, it is expected that a ZrO 2 film preventing corrosion on the Ti substrate will lead to a decrease in allergies and hypersensitivity problems. Two-step rotations for spin coating were determined according to a previous report by Sato et al. [18]. The first step of spin coating, 500 rpm (low speed), was the setting for spreading the precursor solution over the entire substrate. The second step, 2000 rpm (high speed), was aimed to blow away the excess coating precursor solution and achieve a film thickness of fewer than 1 µm.
In these experiments, we inserted the rectangular plates into the cortical limb bone of rats. This is the basic model for evaluating the bone response to surface-modified materials as previously reported [28][29][30]. In dental clinics, cylindrical or screw-type implants were normally used. We already performed the thin film coating on tooth-shaped or cylindrical implants in the molecular precursor method using the spin-coating technique [37,38]. Moreover, bone responses after the implantation into the jaw bone should be examined. We also developed a mandibular bone defect model in rats [15]. In the next series of our experiments, ZrO 2 coating was deposited on the cylindrical or screw-type implants and assessed the bone formation after the implantation of ZrO 2 coating implants into the bone defects in the maxilla or mandible.
According to the results of this study, it is suggested that the ZrO 2 surface with the same surface structure and roughness as that of Ti implants can promote bone formation. In other words, ZrO 2 implant surfaces are required to have the same roughness as that Ti implants despite the difficulty in processing. Moreover, sandblasting Y-TZP surfaces caused a problem of phase transformation [39]. Hirota et al. prepared a unique roughened surface of Y-TZP by nano-pulsed laser irradiation without phase transformation, thereby enhancing the bone response [28]. Thus, it is important to have efficient roughened ZrO 2 surfaces, such as those in Ti implants, without phase transformation to achieve ZrO 2 osseointegration.

Conclusions
In this study, we evaluated the bone response of the ZrO 2 surface with the same structure and roughness as those observed with Ti rectangular plate implants. The molecular precursor method, which can deposit any shape without changing the material surface structure and roughness, produced a thin ZrO 2 film on the rough Ti surface. It was revealed that ZrO 2 with a similar surface structure and roughness as that of roughened Ti rectangular plate implants promoted osteogenesis equivalent to or better than that of Ti in the early bone formation stage. We can make clear the effectiveness of the surface chemical composition of the ZrO 2 surface on a bone formation without considering the factors of surface structure and roughness. The osseointegration mechanism will be clarified by investigating the ZrO 2 surface chemical composition details.