Good Biocompatibility and Sintering Properties of Zirconia Nanoparticles Synthesized via Vapor-phase Hydrolysis

ZrO2 nanoparticles were synthesized by a vapor-phase hydrolysis process, and characterized in terms of crystalline structures, hardness and microstructures by X-ray diffraction, Vickers hardness test method, and atomic force microscopy (AFM) measurements. Moreover, in vitro cytotoxicity evaluation and hemolysis assay showed that the nanoparticles possessed good biocompatibility. Hardness investigations and AFM measurements indicated that both the sintering temperature and compression force played an important role in determining the physical behaviors (hardness, roughness and density) of flakes of the ZrO2 nanoparticles. When ZrO2 nanoparticles synthesized at 500 °C were pressed into flakes under 6 MPa and sintered at 1400 °C, the resulting flakes exhibited an optimal combination of hardness (534.58 gf·mm−2), roughness (0.07 μm) and density (4.41 g·cm−3). As the Vickers hardness value of human bones is of 315~535 gf·mm−2 and the density of adult femuris about 1.3~1.7 g·cm−3, the experimental results showed that the ZrO2 flakes were comparable to human bones with a higher density. As a result, the synthesized ZrO2 NPs may be useful for biomedical applications, especially for bone repair and replacement in future.

bones, teeth and joints due to their good biocompatibility, osseointegration, and bioinertness 20 . In fact, because of nontoxicity to the surrounding tissues, implants based on ZrO 2 nanoparticles (NPs) have been utilized for clinical total hip replacements, and as a prevalent biomaterial in prosthetic dentistry and dental implantology 5,13,16,21,22 . This is largely ascribed to the good physical performance of sintered ZrO 2 devices, in terms of hardness, density, roughness and stability.
In this paper, ZrO 2 NPs were synthesized by using a simple vapor-phase hydrolysis process at controlled temperatures, and utilized to prepare nanoflakes by compression and sintering. By a systematic variation of the compression force and sintering temperature, the hardness and density of the resulting ZrO 2 nanoflakes were maximized whereas the roughness was minimized. The physical properties such as hardness, roughness as well as density of the sintered ZrO 2 nanoflakes were better than those of the human bone. Furthermore, in vitro cytotoxicity and hemolysis evaluation showed that the ZrO 2 NPs possessed good biocompatibility. These findings suggest great potential of the ZrO 2 NPs as a biomcompatible material for medical implants for bone tissue engineering because they meet the demand of high physical properties of artificial hard tissues.

Results
Structures of ZrO 2 NPs. The structures of the obtained ZrO 2 NPs were first characterized by X-ray diffraction (XRD) measurements. Figure 1 shows the XRD patterns of ZrO 2 NPs synthesized at different temperatures (400, 500 and 600 °C). A series of well-defined peaks can be identified at 2θ = 30.2, 35.0, 50.4, 60.0 and 62.7°, which were ascribed to the diffractions of the (101), (110), (200), (211), and (202) crystalline planes of cubic phase ZrO 2 (JCPDS card no.   21 , respectively. In addition, the asymmetric line shape of the peaks at 35.0, 50.4 and 60.0° suggested the formation of a tetragonal phase. The shoulder at 2θ = 34.5° was the diffraction of the (002) crystalline plane of tetragonal phase ZrO 2 (JCPDS card no. , and those at 2θ = 50.2° and 59.1° corresponding to the diffractions of the (112) and (103) crystalline planes 19 . Furthermore, from Fig. 1, it can be seen that the crystallinity of the ZrO 2 NPs increased with increasing synthesis temperature from 400 °C to 600 °C. SEM Analysis. Further structural insights were obtained in SEM measurements. From Supporting Figure S1, it can be seen that the ZrO 2 NPs are mostly in the range of 15 to 65 nm in diameter. Statistical analysis based on more than 50 particles showed that the average diameter of the nanoparticles decreased with increasing synthesis temperature, 40 nm at 400 °C, 35 nm at 500 °C, and 30 nm at 600 °C, as manifested in the core size histograms (Supporting Figure S2).
In Vitro Cytotoxicity. Interestingly, the resulting ZrO 2 NPs were found to exhibit low cytotoxicity, as manifested in in vitro studies with human umbilical vein endothelial cells lines (HUVEC). Experimentally, ZrO 2 NPs were dispersed under sonication at varied concentration (up to 1 mg·mL −1 ) into dulbecco's modified eagle medium (DMEM) and added to the HUVEC cell culture. The in vitro cytotoxicity of the ZrO 2 NPs in HUVEC cells was evaluated by CCK-8 assay. Control experiments were also carried out by dispersing the ZrO 2 NPs in deionized water, and phosphate buffer saline (PBS) (Fig. 2). From Fig. 3, it can be seen that the ZrO 2 NPs (up to 500 μ g·mL −1 ) exerted virtually no effect on cell viability after co-incubation for 24 h. For example, the HUVEC cells retained 92% of viability even at the concentration of 500 μ g·mL −1 of ZrO 2 NPs synthesized at 400, 500, or 600 °C.
Hemolysis of the ZrO 2 NPs was also evaluated by incubating the NPs with red blood cells (RBCs) for 4 h. It can be seen that the hemolytic percentages of RBCs were lower than 3.6% for the NPs synthesized at 400, 500, and 600 °C even at the concentration as high as 800 μ g·mL −1 , implying that these NPs had a negligible hemolytic activity (Fig. 4a-c). Therefore, it can be concluded that the ZrO 2 NPs exhibit good biocompatibility and thus can act as a promising bio-ceramic materials for prosthetic dentistry and dental implantology 37,38 . Physical Hardness. With such remarkable cytocompatibility and hemocompatibility, ZrO 2 NPs-based materials may be viable candidates for biomedical applications. Thus, the ZrO 2 NPs were pressed into nanoflakes and subjected to sintering at elevated temperatures. Significantly, the obtained nanoflakes exhibited remarkable Physical characteristics. First, Vickers hardness tests were performed to examine the materials hardness, which was quantified by the peak load (P max ) and projected contact area (A), = H P A max . The results were listed in Table 1. It can be seen that for ZrO 2 NPs prepared at 400 °C, at the same sintering temperature, there is a maximum hardness that varies with the compression force (Supporting Figure S3); and at the same compression force, there is also a maximum hardness that varies with the sintering temperature. The maximum hardness (603.25 gf·mm −2 ) could be found at the compression force of 3 MPa and sintering temperature of 1200 °C. Similarly, for the ZrO 2 NPs prepared at 500 and 600 °C, the maximum hardness can be identified at 3 MPa and 1200 °C, and 3 MPa and 1400 °C, respectively.
As is known, when the ZrO 2 NPs are pressed into flakes, at low compression forces the flakes are likely crack-free such that the hardness increases with increasing compression force; however, at too high a compression force, (micro) cracks would start to form in the flakes, leading to reduced hardness. Likewise, at relatively   low sintering temperatures, thermal stress in the particle cores was minimal and the flake hardness increased with increasing sintering temperature; in contrast, thermal stress became increasingly significant during high-temperature calcinations which led to the formation of cracks and hence reduced hardness 39,40 . Taken together, these results suggest that both the compression force and sintering temperature play an important role in the determination of the hardness of the flakes, and the maximum hardness may be manipulated by sintering temperature and compression force.
Surface Roughness. The surface morphologies of the ZrO 2 nanoflakes were then analyzed by AFM measurements. From the AFM topographs in Fig. 5, the root mean square (rms) roughness of the ZrO 2 nanoflakes was evaluated as a function of sintering temperature and compression force 41 . Experimentally, the rms roughness was quantified by taking an average of five data points measured over an area of 4 μ m × 4 μ m ( Table 2). It can be seen that under the same compression force, the surface roughness of the flakes diminished with increasing sintering temperature (Supporting Figure S4).
From Table 2, it can also be seen that at the same sintering temperature, the roughness of the flakes remained almost invariant with the compression force. Yet, at a constant compression force, the roughness of the flakes decreased with increasing sintering temperature, consistent with the results in Fig. 5. Interestingly, at the same sintering temperature, the ZrO 2 surface roughness remained almost constant (Fig. 6), independent of the compression force. With increasing sintering temperature, the ZrO 2 NPs tended to agglomerate and the surface roughness decreased accordingly. It has been reported that the surface properties play a critical role in the stability and function of bone-rebuilding materials 39 . Thus, changing the sintering temperature to reduce flakes roughness may enhance the biomedical applications of the ZrO 2 flakes. Flake Density. Density is another important parameter in the assessment of materials for biomedical implants. The density of the ZrO 2 nanoflakes was quantitatively evaluated by using a precision electronic hydrometer 42,43 , and the results are summarized in Table 3.
One can see that the density of the flakes varied with the sintering temperature and compression force, ranging from 2 to 7 g·cm −3 , much higher than that of adult femurs (1.3~1.7 g·cm −3 ) 44,45 .
From the above studies, one can see that when ZrO 2 NPs synthesized at 500 °C were pressed into flakes under the compression force of 6 MPa and sintered at 1400 °C, the resulting flakes exhibited an optimal combination of hardness (534.58 gf·mm −2 ), roughness (0.07 μ m) and density (4.41 g·cm −3 ), considering that the highest hardness value for human bones is between 4 to 5 gf·mm −2 , corresponding to Vickers hardness between 315 to 535 gf·mm −2 , whereas the density of adult femurs of 1.3~1.7 g·cm −3 [44][45][46] . At this optimal point, the flakes hardness is comparable to that of the human skeleton while the density is far larger. Thus, the ZrO 2 flakes may be used as biological materials for hip replacements, rosthetic dentistry and dental implantology. This is being pursued in ongoing studies.

Discussion
ZrO 2 NPs were synthesized by a simple vapor-phase hydrolysis process 47 . Vickers hardness investigation indicated that both the sintering temperature and compression force played an important role in the determination of the hardness of the ZrO 2 flakes. AFM studies showed that the surface roughness of the ZrO 2 flakes gradually decreased with increasing sintering temperature. In addition, the density of the ZrO 2 flakes was also determined within the context of sintering temperature and compression force. With a systematic variation of these two parameters, hardness and density of the ZrO 2 nanoflakes were maximized and roughness was minimized simultaneously 48 and sintering at 1400 °C, the resulting flakes exhibited an optimal combination of hardness (534.58 gf·mm −2 ), roughness (0.07 μ m) and density (4.41 g·cm −3 ).
In conclusion, the experimental results show that both the sintering temperature and compression force played an important role in determining the physical behaviors (hardness, roughness, and density) of ZrO 2 flakes. Thus, by changing compression force and sintering temperature, the hardness parameters of the ZrO 2 flakes can be adjusted and comparable to those of human bones, along with a higher density. More importantly, the in vitro cytotoxicity and hemolysis evaluation shows that the ZrO 2 NPs have good biocompatibility. Therefore, it is believed the ZrO 2 NPs have promising application for bone tissue engineering and regenerative medicine.  Methods Material Preparation. ZrO 2 NPs were synthesized by a vapor-phase hydrolysis procedure and the experimental apparatus had been reported in a previous study 47 . In brief, the reactor was made of two glass tubes that were externally heated in a vertical furnace. ZrCl 4 was sublimated at 350 °C and carried by a N 2 gas (99.9%) into the reaction chamber via the nozzle. Water vapor was introduced into the reaction chamber from around the nozzle by dry air. These two gas streams were mixed rapidly, reacted, and formed ZrO 2 NPs at atmospheric pressure. Then, the aerosol was cooled by a water jacket tube and filtered at the exit of the reactor for analysis, followed by washing. Three samples of ZrO 2 NPs were synthesized at different temperatures (400, 500 and 600 °C) and pressed into flakes at different compression force (2, 3, 6, 10, 14 and 18 MPa), which were then subjected to sintering for 6 h at 800, 1000, 1200 and 1400 °C, respectively.    (100 μg·mL −1 Hyclone) at 37 °C in amoisturized 5% CO 2 incubator. The viability of the treated cells was measured by the Cell Counting Kit-8 (CCK-8) assay. Firstly, HUVEC cells were seeded into a 96-well cell culture plates at the densities of 2 × 10 4 cells/well, in 100 μ L of a complete culture medium at 37 °C for 24 h. Afterward, the ZrO 2 NPs dispersions were diluted with a fresh medium to the desired concentrations and then added to each well to replace the original culture medium. After another 24 h, the culture medium was removed and replaced by 10 μL of CCK-8 in serum-free media. After incubation for 2 h at 37 °C, the optical density of each well was read at 450 nm on a microplate reader (Spectra Max M2MDC, USA) 37,50-54 . Hemolysis Assay of ZrO 2 NPs were carried out as follows. Kunming mice (5 weeks old) were purchased from Vital River (Beijing, China) and the whole blood was obtained from the mice. All the animals experiments were performed in accordance with the guideline and regulation for the care and use of laboratory animals of Ministry of Science and Technology of People's Republic of China's requirements. The Animal Study Committee of the Ministry of Science and Technology of the People's Republic of China has approved the experiments. Briefly, mice blood were centrifuged and diluted 10 times with PBS to obtain red blood cells (RBCs). Then, 0.2 mL of diluted RBCs were added to 0.8 mL of PBS containing ZrO 2 NPs at various concentrations (15,30,60,120,250, 500, and 800 μ g·mL −1 ), 0.8 mL of distilled water (positive control), and 0.8 ML of PBS (negative control), respectively. After that, the mixtures were kept at room temperature for 4 h, before they were centrifuged at 12000 rpm for 3 min. Absorbance of the supernatants was measured by a UV-vis spectrophotometer and hemolysis percentage of RBCs was calculated based on the absorbance at 541 nm using equation (1) where A sample , A 0% and A 100% are the absorbance of the supernatant of the samples, the negative control, and positive control, respectively.

Materials Characterizations.
The composition, morphologies of the nanoparticles prepared above were characterized by X-ray powder diffraction (XRD, Bruker D8, Cu Kα radiation, λ = 1.54 Å). The size distribution and morphology of the nanoparticles were examined by scanning electron microscopy (SEM, HITACHI, S4800, 15 kV) studies. The hardness of the flakes was evaluated by using a Vickers hardness instrument (UHL Microhardness Testers VMHT. VMH-001) equipped with a cube-corner diamond tip, where the sintered flakes were cleaned by a stream of high-purity nitrogen. To minimize substrate contributions, the indentation experiments were performed in load control, with the load of 50 gf. The loading and unloading speed was 5 × 10 −6 m·s −1 , and the time under the peak load was 15 s 48,49,55-58 . Ten random tests were performed for each sample to evaluate the material hardness. The surface morphology was analyzed using AFM and the rms roughness was obtained for the ZrO 2 flakes. The rms roughness data were obtained over an area of 4 μ m × 4 μ m at five different points of the sample and averaged 57,59,60 . The density of the flakes was characterized with a precision electronic hydrometer (DH-120M) by equation (2),