Synthesis and Properties of Novel Calcia-Stabilized Zirconia (Ca-SZ) with Nano Calcium Oxide Derived from Cockle Shells and Commercial Source for Dental Application

Featured Application: The Ca-SZ fabricated in the current study may be used as framework for construction of dental crown and bridgework. Abstract: Various oxides are used to stabilize zirconium oxide (ZrO 2 ), but their superior hardness causes wear of the machining tool. Calcia-doped zirconia has been studied but reports on properties suitable for dental application are lacking. Therefore, this study aimed to fabricate and characterize zirconia stabilized by calcium oxide (CaO) derived from cockle shells and compare it with zirconia stabilized by commercial CaO, sintered at di ﬀ erent temperatures. In this study, 176 pressed pellets of zirconia mixed with CaO either derived from cockle shells or commercial CaO were sintered between 1200 and 1500 ◦ C to produce calcia-doped zirconia. Characterizations were made with SEM and XRD. Specimens were subjected to density, compressive and ﬂexural strength, and Vickers hardness testing. Data were analyzed using the independent t -test and one-way ANOVA. XRD revealed the zirconia was stabilized into tetragonal and cubic phases (Ca-SZ). Ca-SZ cockle shells (CS) and Ca-SZ commercial (CC) have average particle sizes of 267 nm and 272 nm, respectively, with similar surface roughness. At 1400 ◦ C sintering temperature, ﬂexural strengths were 1165 and 1152 MPa, compressive strengths were 4914 and 4913 MPa, and Vickers hardness were 977 and 960 MPa for Ca-SZ(CS) and Ca-SZ(CC), respectively. Both Ca-SZ materials showed no signiﬁcant di ﬀ erence in most properties ( p < 0.05) when sintered at di ﬀ erent temperatures. The fully sintered Ca-SZ is less hard compared to the ceria-stabilized tetragonal zirconia polycrystal (Ce-TZP) available on the market. Thus, Ca-SZ may be used as an alternative to the current zirconia available on the market for dental application.


Introduction
Zirconia-based ceramics have been used as dental implants [1] in addition to framework underneath the brittle veneering porcelain in the fabrication of fixed dental prostheses, such as will have the benefit of waste removal (recycling), but at the same time produce added value to the cockle shell waste.
Therefore, the purpose of this study was to synthesize and investigate the effect of different sintering temperatures on the physical and mechanical properties of novel calcia-stabilized zirconia (Ca-SZ) with the CaO derived from cockle shells and compare it to the Ca-SZ stabilized by commercial CaO.

Extraction of Calcium Oxide Cockle Shells
Local cockle shells were collected from a local beach, Pantai Bachok. The cockle shells were washed, pulverized and sieved to form a uniform sized cockle shell powder of 63 µm. The cockle shell powder was dissolved in hydrochloric acid to produce calcium chloride (CaCl 2 ), which was then titrated with ethanol and potassium carbonate to form insoluble calcium carbonate (CaCO 3 ). This was followed by calcination of CaCO 3 at 750 • C for 30 min to produce spherical, nano-CaO powder, which was used in the current study.

Fabrication of CaO Stabilized Zirconia Pellet
To prepare the zirconia specimen pellet, 9.2 g of zirconium oxide (92%) was mixed with 0.8 g of calcium oxide (8%), either CaO(CS) or CaO(CC) (8%). The mixed powder was placed in a plastic bottle containing ceramic balls, which was then placed on an electric mixer for 30 min to allow for proper mixing. Then, the inner surface of a stainless steel mold with the dimension of 32 mm in diameter and 6 mm in height was isolated with silicone wax-based separating medium. The mixed powder was placed in the stainless steel mold, and then pressed using a hydraulic press (Atlas TM Manual 15 T and 25 T Hydraulic Press, Specac, Kent, Orpington, UK) under 5 tons (60 Mpa) pressure. A total of 176 pressed zirconia specimen pellets were then divided into four groups according to the different sintering temperatures. Each group was sintered in a furnace (Linn High Therm Furnace VMK-1800) for two hours at four different temperatures (1200, 1300, 1400, and 1500 • C) to produce pellet blanks of calcia-stabilized zirconia (Ca-SZ) with either CaO derived from cockle shells (CS) or commercial CaO (CC), which from here on will be called Ca-SZ(CS) and Ca-SZ(CC), respectively. An example of the sintered zirconia pellet blank is shown in Figure 1.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 of 14 only will have the benefit of waste removal (recycling), but at the same time produce added value to the cockle shell waste. Therefore, the purpose of this study was to synthesize and investigate the effect of different sintering temperatures on the physical and mechanical properties of novel calcia-stabilized zirconia (Ca-SZ) with the CaO derived from cockle shells and compare it to the Ca-SZ stabilized by commercial CaO.

Extraction of Calcium Oxide Cockle Shells
Local cockle shells were collected from a local beach, Pantai Bachok. The cockle shells were washed, pulverized and sieved to form a uniform sized cockle shell powder of 63 µm. The cockle shell powder was dissolved in hydrochloric acid to produce calcium chloride (CaCl2), which was then titrated with ethanol and potassium carbonate to form insoluble calcium carbonate (CaCO3). This was followed by calcination of CaCO3 at 750 °C for 30 min to produce spherical, nano-CaO powder, which was used in the current study.

Fabrication of CaO Stabilized Zirconia Pellet
To prepare the zirconia specimen pellet, 9.2 g of zirconium oxide (92%) was mixed with 0.8 g of calcium oxide (8%), either CaO(CS) or CaO(CC) (8%). The mixed powder was placed in a plastic bottle containing ceramic balls, which was then placed on an electric mixer for 30 min to allow for proper mixing. Then, the inner surface of a stainless steel mold with the dimension of 32 mm in diameter and 6 mm in height was isolated with silicone wax-based separating medium. The mixed powder was placed in the stainless steel mold, and then pressed using a hydraulic press (Atlas TM Manual 15 T and 25 T Hydraulic Press, Specac, Kent, Orpington, UK) under 5 tons (60 Mpa) pressure. A total of 176 pressed zirconia specimen pellets were then divided into four groups according to the different sintering temperatures. Each group was sintered in a furnace (Linn High Therm Furnace VMK-1800) for two hours at four different temperatures (1200, 1300, 1400, and 1500 °C) to produce pellet blanks of calcia-stabilized zirconia (Ca-SZ) with either CaO derived from cockle shells (CS) or commercial CaO (CC), which from here on will be called Ca-SZ(CS) and Ca-SZ(CC), respectively. An example of the sintered zirconia pellet blank is shown in Figure 1.

Physical and Mechanical Tests of Ca-SZ(CS) and Ca-SZ(CC) Sintered at Different Temperatures
Eight specimens from both Ca-SZ(CS) and Ca-SZ(CC) groups sintered at different temperatures were analyzed with an X-ray diffractometer (XRD) (Shimadzu XRD-6000 powder diffractometer) using Cu Kα radiation (λ = 1.5406 Å, 40 kV at 160 mA) at a scan rate of 0.05 • 2θ s −1 .
Another eight specimens, from each group sintered at four different temperatures, were analyzed using a scanning electron microscope (SEM) (LEICA EM SCD005, Bannockburn, IL, USA). Specimens were then polished using an electric polishing machine (EXAKT 400CS, AW110, Oklahoma City, OK, USA) with 300 mm Schleifscheiben sandpaper discs (Hermes Schleifmittel GmbH & Co. KG, Hamburg, Germany) at different grit sizes (P500, P800, P1000 and P1200) for 15 min for each grit, at 5000 rpm. Water was used to prevent the overheating of the samples. The sandpaper was changed for every sample. After that, the polished specimens were then re-analyzed using the SEM machine.
Forty specimens from both groups, sintered at four different temperatures (n = 5), were weighed on a precision balance using Mettler Toledo DA110 toploading electrical balance (Tokyo, Japan), before and after immersion in double distilled water (DDW), for actual and suspended weights, respectively. Archimedes' principle was used to measure the density of sintered specimens. The following formula was used to calculate the density [18].
where, ρ is the calculated density (g/cm 3 ) and ρ w is the density of the water (g/cm 3 ). Forty specimens with dimensions of 25 mm × 2 mm × 2 mm were measured with vernier calipers and cut using a micro motor handpiece driven at 30,000 rpm for a flexural strength test (n = 5). Another 40 specimens with dimensions of 6 mm height × 4 mm diameter (International Standards Organization (ISO) no. 9917 (2000)) were prepared for a compressive strength test (n = 5). Another 40 specimens measuring 30 mm × 10 mm × 2.5 mm (International Standards Organization (ISO) Specification no. 1567) were prepared for the Vickers hardness test (n = 5). The cut specimens were polished using the same protocol mentioned above. The flexural strength and compressive strength of the specimens were tested using the universal testing machine (Shimadzu, Model: AGX plus 20 kN, Kyoto, Japan) at the crosshead speed of 0.75 mm/min. The formula used for calculating the flexural strength and compressive strength was: Flexural strength = 3PL/2bd 2 where, P is the maximum force applied, L is the span length, b is the width, d is the thickness; Compressive strength = 4P/πd 2 where P stands for maximum force applied and d is the specimen diameter. For measuring hardness, a load of 1000 g = 9.81 newtons earth was placed on the top surface of the polished surface of the specimens by the Vickers hardness indenter.
Statistical Analyses The one-way ANOVA test was used to analyze the means of flexural, compressive, Vickers hardness and density of the materials sintered at different temperatures, with p < 0.05 considered as significant. After checking the equal variance, the post-hoc Tukey test was used for multiple comparisons between the groups. The independent t test was used to analyze the mechanical and physical strengths between Ca-SZ(CS) and Ca-SZ(CC), with p < 0.05 considered significant.

XRD Analyses
The phase identification of the prepared Ca-SZ using XRD are shown in Figure 2A

SEM Analyses
The SEM analyses indicated that the average particle size of Ca-SZ derived from cockle shells and commercial nano-CaO are 64 nm and 110 nm, respectively, before sintering ( Figure 3). The SEM also show the uniform and even distribution of the particles.

SEM Analyses
The SEM analyses indicated that the average particle size of Ca-SZ derived from cockle shells and commercial nano-CaO are 64 nm and 110 nm, respectively, before sintering ( Figure 3). The SEM also show the uniform and even distribution of the particles.

Density
The densities of the prepared Ca-SZ(CS) and Ca-SZ(CC) are shown in Figure 8. As the sintering temperature increased, the density of the Ca-SZ increased up to 1400 °C and levelled off as the

Density
The densities of the prepared Ca-SZ(CS) and Ca-SZ(CC) are shown in Figure 8. As the sintering temperature increased, the density of the Ca-SZ increased up to 1400 • C and levelled off as the sintering temperature further increased to 1500 • C. The independent t-test showed that the mean density of Ca-SZ(CS) was not significantly different from Ca-SZ(CC) when the samples were sintered at different temperatures with p > 0.05, except when samples were sintered at 1500 • C. One-way ANOVA analysis revealed that there was a significant difference in the density of the Ca-SZ from both groups between different sintering temperatures (p < 0.05). Further post-hoc Tukey analysis revealed that the density of both materials at 1200 • C were significantly lower compared to other temperatures with p < 0.05.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 9 of 14 sintering temperature further increased to 1500 °C. The independent t-test showed that the mean density of Ca-SZ(CS) was not significantly different from Ca-SZ(CC) when the samples were sintered at different temperatures with p > 0.05, except when samples were sintered at 1500 °C. One-way ANOVA analysis revealed that there was a significant difference in the density of the Ca-SZ from both groups between different sintering temperatures (p < 0.05). Further post-hoc Tukey analysis revealed that the density of both materials at 1200 °C were significantly lower compared to other temperatures with p < 0.05.  Figure 9 shows the increasing trend of flexural strength in both materials with increasing temperature. The independent t-test showed no significant difference in the flexural strengths  Figure 9 shows the increasing trend of flexural strength in both materials with increasing temperature. The independent t-test showed no significant difference in the flexural strengths between both groups of Ca-SZ when sintered at different temperatures with p < 0.05, apart from when the samples were sintered at 1300 • C. The highest flexural strength was observed in Ca-SZ sintered at 1400 • C for Ca-SZ(CS) and Ca-SZ(CC), with the values of 1165 and 1152 MPa, respectively. One-way ANOVA analysis revealed that there was a significant difference in the flexural strength of Ca-SZ(CS) and Ca-SZ(CC) between different sintering temperatures with p < 0.05. Further post-hoc Tukey test revealed that the flexural strength value of Ca-SZ sintered at 1200 • C was significantly lower compared to other temperatures with p < 0.05.  Figure 9 shows the increasing trend of flexural strength in both materials with increasing temperature. The independent t-test showed no significant difference in the flexural strengths between both groups of Ca-SZ when sintered at different temperatures with p < 0.05, apart from when the samples were sintered at 1300 °C. The highest flexural strength was observed in Ca-SZ sintered at 1400 °C for Ca-SZ(CS) and Ca-SZ(CC), with the values of 1165 and 1152 MPa, respectively. Oneway ANOVA analysis revealed that there was a significant difference in the flexural strength of Ca-SZ(CS) and Ca-SZ(CC) between different sintering temperatures with p < 0.05. Further post-hoc Tukey test revealed that the flexural strength value of Ca-SZ sintered at 1200 °C was significantly lower compared to other temperatures with p < 0.05.  Figure 10 shows the increase in compressive strength of both groups of Ca-SZ, as the sintering temperature increases. One-way ANOVA analysis revealed that there was a significant difference in the compressive strength of Ca-SZ(CS) and Ca-SZ(CC) between different sintering temperatures (p < 0.05). Further post-hoc Tukey test revealed that the compressive strength of both Ca-SZ groups sintered at temperature 1200 • C was significantly lower compared to other temperature with p < 0.05. The independent t-test showed that there were no significant differences in the compressive strength of Ca-SZ(CS) and Ca-SZ(CC) sintered at different temperature p < 0.05, with the exception of 1200 • C.  Figure 10 shows the increase in compressive strength of both groups of Ca-SZ, as the sintering temperature increases. One-way ANOVA analysis revealed that there was a significant difference in the compressive strength of Ca-SZ(CS) and Ca-SZ(CC) between different sintering temperatures (p < 0.05). Further post-hoc Tukey test revealed that the compressive strength of both Ca-SZ groups sintered at temperature 1200 °C was significantly lower compared to other temperature with p < 0.05. The independent t-test showed that there were no significant differences in the compressive strength of Ca-SZ(CS) and Ca-SZ(CC) sintered at different temperature p < 0.05, with the exception of 1200 °C.  Figure 11 illustrates the Vickers hardness values of both Ca-SZ groups. The hardness increased with the increase in sintering temperature, reaching their maximum at 1500 °C in both Ca-SZ(CS) and Ca-SZ(CC). One-way ANOVA analysis revealed that there was a significant difference in the Vickers  Figure 11 illustrates the Vickers hardness values of both Ca-SZ groups. The hardness increased with the increase in sintering temperature, reaching their maximum at 1500 • C in both Ca-SZ(CS) and Ca-SZ(CC). One-way ANOVA analysis revealed that there was a significant difference in the Vickers hardness values of Ca-SZ(CS) and Ca-SZ(CC) between different sintering temperatures (p < 0.05). Further post-hoc Tukey test revealed that the hardness of the samples sintered at 1200 • C was significantly lower compared to other temperatures (p < 0.05). The independent t test revealed there was a significant difference in the Vickers hardness values between Ca-SZ(CS) and Ca-SZ(CC) at all temperatures (p < 0.05), except the group that was sintered at 1400 • C.

Discussion
Several studies have worked on stabilizing zirconia with CaO [19][20][21][22] and on obtaining CaO from cockle shells [23][24][25][26][27]. However, to the authors' knowledge, there has not been any work on the use of CaO obtained from cockle shells to stabilize zirconia for dental application.
In the current study, XRD analysis showed that the ZrO2 was successfully stabilized with both the nano-CaO derived from cockle shells and also commercial nano-CaO (Ca-SZ). Different sintering temperatures (1200 °C, 1300 °C, 1400 °C, and 1500 °C) were used to prepare Ca-SZ pellets. Physical and mechanical properties of Ca-SZ(CS) were compared with Ca-SZ(CC).
The morphological micrographs of Ca-SZ were characterized by SEM in order to deduce its surface structure and the average particle size. The particle sizes of Ca-PSZ(CS) and Ca-SZ(CC) obtained in the current study were 267 nm and 272 nm, respectively. The particle size of CaO derived from cockle shells ranged from 22 to 70 nm [17] and the particle size of the commercial CaO was <160 nm as compared to the 0.6-µm grain size of yttria used in a previous study [28]. The smaller particle size of the CaO derived from cockle shells might provide a larger surface area for CaO to be uniformly dispersed on the surface of ZrO2 and therefore may enhance the mechanical properties of the material [29], hence making the material more resistant to degradation or transformation [28]. The application of external heat during sintering enables the Ca-PSZ particle to be closely packed, with pores or voids removed, leading to shrinkage of the materials and forming more compact structures [18]. The voids and spaces shown on the polished surface of both Ca-PSZ pellets when sintered at lower temperatures suggests insufficient heat energy supplied in order to densely pack the materials, leading to an irregular surface upon polishing. Ca-PSZ(CS) sintered at 1400 °C showed the glossiest appearance with minimum surface crack, in addition to the smooth texture, which is indicative of sufficient and optimum heat energy during the sintering of the material.
The density of the materials is inter-correlated with the porosity of the microstructure. The incorporation of the Ca particle in ZrO2 solid solution was probably the main reason the density of the prepared material increased for both groups. The increase in density of the prepared materials as the sintering temperature increased was in agreement with other researchers [18,30,31]. The slight enhancement of the density of Ca-SZ(CS) was in accordance with the SEM image in Figure 6, showing a compact structure with smooth morphology. However, the decrement of the density of Ca-PSZ for

Discussion
Several studies have worked on stabilizing zirconia with CaO [19][20][21][22] and on obtaining CaO from cockle shells [23][24][25][26][27]. However, to the authors' knowledge, there has not been any work on the use of CaO obtained from cockle shells to stabilize zirconia for dental application.
In the current study, XRD analysis showed that the ZrO 2 was successfully stabilized with both the nano-CaO derived from cockle shells and also commercial nano-CaO (Ca-SZ). Different sintering temperatures (1200 • C, 1300 • C, 1400 • C, and 1500 • C) were used to prepare Ca-SZ pellets. Physical and mechanical properties of Ca-SZ(CS) were compared with Ca-SZ(CC).
The morphological micrographs of Ca-SZ were characterized by SEM in order to deduce its surface structure and the average particle size. The particle sizes of Ca-PSZ(CS) and Ca-SZ(CC) obtained in the current study were 267 nm and 272 nm, respectively. The particle size of CaO derived from cockle shells ranged from 22 to 70 nm [17] and the particle size of the commercial CaO was <160 nm as compared to the 0.6-µm grain size of yttria used in a previous study [28]. The smaller particle size of the CaO derived from cockle shells might provide a larger surface area for CaO to be uniformly dispersed on the surface of ZrO 2 and therefore may enhance the mechanical properties of the material [29], hence making the material more resistant to degradation or transformation [28]. The application of external heat during sintering enables the Ca-PSZ particle to be closely packed, with pores or voids removed, leading to shrinkage of the materials and forming more compact structures [18]. The voids and spaces shown on the polished surface of both Ca-PSZ pellets when sintered at lower temperatures suggests insufficient heat energy supplied in order to densely pack the materials, leading to an irregular surface upon polishing. Ca-PSZ(CS) sintered at 1400 • C showed the glossiest appearance with minimum surface crack, in addition to the smooth texture, which is indicative of sufficient and optimum heat energy during the sintering of the material.
The density of the materials is inter-correlated with the porosity of the microstructure. The incorporation of the Ca particle in ZrO 2 solid solution was probably the main reason the density of the prepared material increased for both groups. The increase in density of the prepared materials as the sintering temperature increased was in agreement with other researchers [18,30,31]. The slight enhancement of the density of Ca-SZ(CS) was in accordance with the SEM image in Figure 6, showing a compact structure with smooth morphology. However, the decrement of the density of Ca-PSZ for both specimens over 1400 • C was probably due to surface diffusion phenomenon as previously reported [32,33].
The highest flexural strength was observed in Ca-SZ(CS) and Ca-SZ(CC) sintered at 1400 • C with the values of 1165 and 1152 MPa, respectively. These values were higher than another study, which reported the flexural strength value of 800-1000 MPa [3]. The higher flexural strength of the Ca-SZ in the present study was in accord with Stawarczyk et al. who reported that the optimal sintering temperature for the third generation of ZrO 2 is in between 1400 • C and 1550 • C [34]. Therefore, the high flexural strength of Ca-SZ enables the material to afford restorations with less susceptibility to fracture, thus may be suitable for dental application.
The highest compressive strength was achieved when the Ca-SZ was sintered at 1400 • C, with similar values of 4914 and 4913 MPa for Ca-PSZ(CS) and Ca-SZ(CC), respectively. The current result is in agreement with a previous report by Hu et al. [35]. The shrinkage and distance of the particles depend on the strength of the cohesion between the particles of the material, as well as the shape and size of the particles. The more particles form close to a spherical shape, the more likely they are to bind the particles together leading to high compressive strength [35]. The higher compressive strength in the current study was probably due to more particles forming close to their spherical shape, which further enhanced the bonding, in addition to the densification of the material.
The mean Vickers hardness of Ca-SZ(CS) and Ca-SZ(CC) was 977 MPa and 960 MPa, respectively, which are lower in comparison to the Vickers hardness of Ce-TZP, which was around 1200 MPa [35,36]. Turon-Vinas et al. [29] developed pure 10 mol% ceria-stabilized tetragonal zirconia polycrystal (10Ce-TZP) with hardness value of 6.2 GPa. Tavor-Vargas et al. reinforced ceria-calcia stabilized zirconia with alumina, and reported hardness of 10 GPa-13 GPa [22]. Thus, it can be seen in the current study that the zirconia stabilized with calcia produced lower Vickers hardness when compared to the zirconia stabilized using other oxides, which suggests that it is easier to prepare using cutting tools with minimal wear.
In general, all mechanical properties show optimum values when the specimens were sintered at 1400 • C. At 1200 • C, the heat was not sufficient to fully sinter the zirconia and the presence of pores and voids lead to its lower density, flexural, compressive and hardness values. Whereas, at 1500 • C, the zirconia was overheated, showing lower mechanical properties.
Other properties that are important to dental zirconia should be tested in future research. These results are limited to in-vitro study. Further in-vivo animal studies are needed to test tissue response to the current material, followed by clinical trials. Currently the material is produced in a small quantity. More work is needed for upscaling to prepare for possibility of future commercialization.

Conclusions
The zirconia was successfully stabilized with nano-CaO derived from cockle shells and from commercial source into tetragonal and cubic phase (Ca-SZ) using a physical mixing technique. The physical and mechanical properties of the material were at their optimum when the pellets were sintered at 1400 • C. The physical and mechanical properties of the fabricated Ca-SZ with nano-CaO derived from cockle shell were comparable to the Ca-SZ stabilized by commercial nano-CaO. The flexural, compressive strength are also comparable with zirconia stabilized by other oxides, namely, Y-TZP. The fully sintered Ca-SZ is less hard compared to Ce-TZP, which makes it easier to mill. From the study, it can be concluded that the experimental Ca-PSZ with the CaO derived from cockle shells may be used as an alternative to the current zirconia available on the market for dental application.