Inﬂuence of HAP on the Morpho-Structural Properties and Corrosion Resistance of ZrO 2 -Based Composites for Biomedical Applications

: In the present work, ZrO 2 -based composites were prepared by adding different amounts of antibacterial magnesium oxide and bioactive and biocompatible hydroxyapatite (HAP) to the inert zirconia. The composites were synthesized by the conventional ceramic processing route and morpho-structurally analyzed by X-ray powder diffraction (XRPD) and scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS). Two metallic dental alloys (i.e., Ni–Cr and Co–Cr) coated with a chitosan (Chit) membrane containing the prepared composites were exposed to aerated artiﬁcial saliva solutions of different pHs (i.e., 4.3, 5, 6) and the corrosion resistances were investigated by electrochemical impedance spectroscopy technique. The obtained results using the two investigated metallic dental alloys shown quasi-similar anticorrosive properties, having quasi-similar charge transfer resistance, when coated with different ZrO 2 -based composites. This behavior could be explained by the synergetic effect between the diffusion process through the Chit-composite layer and the roughness of the metallic electrode surface.


Introduction
Beside many biomaterials, hydroxyapatite (HAP), with chemical formula Ca 10 (PO 4 ) 6 (OH) 2 , continue to be extensively studied, being bioactive, highly osteoconductive, with high strength and modulus of elasticity, and high biocompatibility [1][2][3]. From the point of view of biocompatibility, it is a calcium phosphate (CaP) bioceramic similar to the human hard tissues in morphology and composition and seems to be the most suitable ceramic material for hard tissue replacement implants. Thermodynamically, HAP it is the most stable CaP compound under physiological conditions as pH, temperature, and composition of the body fluids. HAP ceramics does not exhibit any cytotoxic effects, shows excellent biocompatibility with hard tissues and also with skin and muscle tissues and can directly bond to the bone [4,5]. However, HAP presents some disadvantages, such as brittleness, low tensile strength and fracture toughness. Consequently, these disadvantages resulted in the limitation of usage for example in load-bearing orthopaedic implants. In the last years, there has been a lot of research aiming to fabricate more mechanically reliable bioactive ceramics, including, of course, the HAP materials [6][7][8][9][10]. Several reinforcements are used to enhance the properties of HAP such as TiO 2 , Al 2 O 3 , MgO or ZrO 2 [11][12][13][14][15]. Among these various additives zirconia (ZrO 2 ), which is a bioinert material, has been commonly used as reinforcement for many ceramics because of its high strength and fracture toughness. On the other hand, at high temperature (T > 1200 • C) HAP decomposes the anticorrosive properties of the prepared composites by in vitro preliminary tests, after immersion in artificial saliva of different pHs.
HAP was prepared by the precipitation method previously described [33][34][35]. Briefly, the diammonium hydrogen phosphate (0.09 mol L −1 ) and the ammonium 25% solution were slowly added to the calcium nitrate solution (0.15 mol L −1 ). The pH of the reaction mixture was adjusted with ammonia solution to 11, and the reaction temperature was maintained at room temperature. The reaction mixture was stirred for 22 h. The Ca/P mole ratio was maintained at 1.67. After the reaction was accomplished, the precipitate was washed with ethanol and filtered. The filtered material was dried at 90 • C for 6 h.
ZrO 2 -based composites were obtained by ceramic processing route [36]. The nominal compositions of the five prepared samples are presented in Table 1. The starting precursor powders were intimately grounded using an agate mortar and pestle. The resulted powders were mixed with 5% PVA and uniaxially cold pressed in a metallic dye into cylindrical pellets of 1 g with 10 mm in diameter at a pressure of 400 kgf using a Carver Inc. hydraulic press (Carver Inc, Wabash, IN, USA). Further, the pellets were sintered at 1600 • C using a Nabertherm LHT 04/16 furnace, under an air atmosphere with a heating and cooling rate of 5 • C/min and dwell time of 12 h at the maximum temperature. Afterward, all the prepared pellets were ground again to prepare the samples for XRPD, SEM/EDS, and electrochemical investigations. Due to the fact that the natural saliva is a complex matrix containing a great variety of compounds, and having changeable and unstable properties (during the day time or in the function of pH of food consumed), an artificial saliva which reproduced it, is actually impossible to obtain. Artificial saliva was prepared using the Mondelli receipt [37] containing appropriate quantities of the following salts purchased from Sigma Aldrich or "Reactivul" Bucuresti: NaCl (0.5 g/L), KCl (0.5 g/L), CaCl 2 * 2H 2 O (0.795 g/L), citric acide (0.0005 g/L), NaH 2 PO 4 * H 2 O (0.78 g/L), urea (1 g/L), (NH 4 ) 2 SO 4 (0.3 g/L) and NaHCO 3 (0.1 g/L). A diluted solution of H 3 PO 4 (Sigma Aldrich) was used for pH adjustment to the desired value.
Distilled water was used for preparing all solutions. All reagents having p.a. quality, were used without supplementary purification.

Preparation of the Composite based Modified Ni-Cr or Co-Cr Electrodes
The dental metallic alloys samples have a cylindrical form and were embedded into a Teflon holder, such that a 3 mm diameter circle surface could be investigated. Before using, the surface of the dental Ni-Cr or Co-Cr alloys electrode was thoroughly polished on alumina slurry (of successively gradations: 500, 1000 and 4000). In order to remove alumina particles and other possible contaminants, the electrodes were sonicated for 5 min in acetone, then well-rinsed with distilled water and dried.
The ZrO 2 and S1-S5 composites were immobilized onto metals alloy electrode surfaces by using a chitosan membrane. Thus, 1 mg of ZrO 2 or S1-S5 composites were dispersed in 1 ml of chitosan solution, and the suspension was sonicated for 2 h. The drop-casting method of immobilisation consists in the deposition of 5 µL of the obtained suspension onto the clean alloy electrode surface and kept for drying under a beaker during 2 h, at room temperature.

Investigation Methods
The prepared composites were morpho-structurally analyzed. Structural analyses were performed on S1-S5 composites using a Shimadzu XRD-6000 diffractometer operating at 40 kV, 30 mA with a monochromator of graphite for CuKα (λ = 1.54060 Å). The diffraction patterns were recorded in the 2θ range of 10-80 at a scan speed of 2 • /min. Scanning Electron Microscopy (SEM) analysis was performed using a Hitachi SU8230 (Tokyo, Japan) microscope. The electron microscope was coupled with an Aztec X-Max 1160 energy dispersive X-ray (EDX) detector (Oxford Instruments).
The electrochemical measurements were performed by using a PC controlled electrochemical analyser (AUTOLAB PGSTAT302NEcoChemie, Utrecht, Netherlands) controlled by GPES 4.7 software for cyclic voltammetry and open circuit potential measurements, and FRA 2.1 software for impedance measurements. An undivided three-electrodes cell was connected to the potentiostat and was equipped with the prepared modified alloys electrodes, i.e. Co-Cr/Chit-ZrO 2 , Co-Cr/Chit-Sx (where x = 1÷5) or Ni-Cr/Chit-ZrO 2 , Ni-Cr/Chit-Sx (where x = 1÷5), as working electrode, a Pt wire, as counter electrode, and a Ag/AgCl, KCl sat as reference electrode. The open circuit potential (OCP) was recorded during 3600 s. The corrosion tests were carried out after the stabilization of unmodified and modified electrodes, under open circuit conditions. Impedance spectra at unmodified and modified dental Ni-Cr and Co-Cr alloys interface were recorded at the OCP value, by using an AC signal with amplitude of ±10 mV and a frequency range from 100 kHz down to 0.1 Hz. Electrochemical impedance spectroscopy (EIS) data were plotted as Nyquist and Bode diagrams. The experimental impedance data were fitted by using an equivalent electric circuit and a ZSimpWin 3.21 software. The parameters values of the electric circuit components were subsequently correlated with the processes occurring at the modified electrode surface. The electrode investigations were carried out at three pH values, namely: pH 6 (simulating a normal body environment), pH 5 and pH 4.3 (digestion situation) [38].
The pH of the artificial saliva was adjusted using a glass combined electrode (HI 11310) connected to a pH-meter (type MV 870, Pracitronic, Germany), by addition of small volumes of diluted H 3 PO 4 . All experiments were performed in aerated solutions at ambient temperature and repeated three times, using for each experiment a new modified surface of metallic alloy electrode.

Morpho-Structural Characterization of Composites
In order to evaluate the structural properties of the prepared composites, XRPD analysis was performed. Figure 1 displays the XRPD patterns of the prepared composites, after sintering in the air at 1600 • C.
Analysis of powder pattern of the S1-S5 samples showed peaks which correspond to both t-ZrO 2 and m-ZrO 2 phases in different ratios. No peaks belonging to the MgO phases were observed at this Mg doping level, confirming the solubility of Mg in the ZrO 2 . Besides, small peaks which could be assigned to β-TCP phase were identified. However, no characteristic peaks corresponding to any other CaP-compounds, like HAP, TTCP (tetracalcium phosphate), CaZrO 3 or CaO were detected for S2-S5 samples. It is known that these crystal structure transformations are the reactions that generally occur when sintering ZrO 2 and HAP at a high temperature. At higher sintering temperatures (T > 1200 • C), HAP decomposed in the presence of ZrO 2 as follows [39]: where HAP decomposes into α/β -TCP with the appearance of the second phase of CaZrO 3 . The decomposition rate of HAP to α/β -TCP is improved with increasing the ZrO 2 concentration in the samples, zirconia works as a catalyst [39]. Lukič et al. have synthesized HAP nano powder in the presence of zirconium (Zr 4+ ) ions and they reported that the phase transformations could be tailored by adjusting zirconium content [40]. They showed that the complete phase transformation from HAP to β-TCP provoked stress-induced transformation of t-ZrO 2 to m-ZrO 2 . In our case, taking into account that the ZrO 2 /MgO mole ratio was maintained the same in the preparation of the composites, the zirconia phase transformation may be due to the HAP decomposition. were observed at this Mg doping level, confirming the solubility of Mg in the ZrO2. Besides, small peaks which could be assigned to β-TCP phase were identified. However, no characteristic peaks corresponding to any other CaP-compounds, like HAP, TTCP (tetracalcium phosphate), CaZrO3 or CaO were detected for S2-S5 samples. It is known that these crystal structure transformations are the reactions that generally occur when sintering ZrO2 and HAP at a high temperature. At higher sintering temperatures (T > 1200 °C), HAP decomposed in the presence of ZrO2 as follows [39]: where HAP decomposes into α/β -TCP with the appearance of the second phase of Ca-ZrO3. The decomposition rate of HAP to α/β -TCP is improved with increasing the ZrO2 concentration in the samples, zirconia works as a catalyst [39]. Lukič et al. have synthesized HAP nano powder in the presence of zirconium (Zr 4+ ) ions and they reported that the phase transformations could be tailored by adjusting zirconium content [40]. They showed that the complete phase transformation from HAP to β-TCP provoked stress-induced transformation of t-ZrO2 to m-ZrO2. In our case, taking into account that the ZrO2/MgO mole ratio was maintained the same in the preparation of the composites, the zirconia phase transformation may be due to the HAP decomposition. The crystallite sizes (D) of the composites were calculated based upon on the (101) diffraction peak's broadening in the XRD pattern using the Scherrer equation: where: D-crystallite size along (hkl) direction, β-full width half maximum (FWHM) of the most intense diffraction line, λ-wavelength of X-ray, θ-the Bragg angle [41]. The crystallite sizes estimated using the Scherrer equation are ranged in the nanometric domain, with values from 37.98 nm to 60.64 nm, for both m-and t-ZrO2 phases. The crystallite sizes (D) of the composites were calculated based upon on the (101) diffraction peak's broadening in the XRD pattern using the Scherrer equation: where: D-crystallite size along (hkl) direction, βfull width half maximum (FWHM) of the most intense diffraction line, λwavelength of X-ray, θthe Bragg angle [41]. The crystallite sizes estimated using the Scherrer equation are ranged in the nanometric domain, with values from 37.98 nm to 60.64 nm, for both mand t-ZrO 2 phases. SEM/EDS analyses were performed for morphological characterization and to detect chemical elemental composition on the surface of the S2-S5 composites. The surface morphology appears homogenous and consists of small grains (30-50 µm) as shown in Figure 2. Pores are also observed for all the studied composites. The results of EDS analysis corresponding to the same nominal cationic compositions highlight the simultaneous presence of the Zr, Mg, Ca, and P elements, and no others were systematically detected. Moreover, for the S5 composite, where XRPD analysis detected the presence of β-TCP as a secondary phase, a Ca/P mole ratio of 1.55 was found, which is close to the theoretical Ca/P mole ratio in β-TCP.

The Potential in Open Circuit (OCP)
The value of the potential in open circuit (OCP) is an indicator of the anticorrosive tendency of metal and has an irreversible character because the instability of the metalsolution interface. The OCP dependence on time exhibited that the potentials of all the studied electrode materials are on an ascending trend in the first part of the recording period (approx. first 35 min), after that they reaching the equilibrium values (results not shown). Consequently, due to the fact that the active surface is very small (0.0707 cm 2 ), an interval time period of 3600 s for obtaining the constant OCP values is considered to be optimal for reaching a quasi-equilibrium state at the electrode/solution interface [42]. The The value of the potential in open circuit (OCP) is an indicator of the anticorrosive tendency of metal and has an irreversible character because the instability of the metalsolution interface. The OCP dependence on time exhibited that the potentials of all the studied electrode materials are on an ascending trend in the first part of the recording period (approx. first 35 min), after that they reaching the equilibrium values (results not shown). Consequently, due to the fact that the active surface is very small (0.0707 cm 2 ), an interval time period of 3600 s for obtaining the constant OCP values is considered to be optimal for reaching a quasi-equilibrium state at the electrode/solution interface [42]. The OCP stabilized values for all electrodes in all studied pH values are summarized in Figure 3. As shown in the literature the anti-corrosion performance of a coating/metal system is generally predictable, if the OCP of coated metal is more positive than that of the bare metal [43]. This is confirmed by analyzing the results of Figure 3, where it can be observed that the OCP values at the bare surface are the smallest and the OCP values are spread on an approximatively 200 mV range. Also, it can be observed, in all cases and all pH values, the same tendency of increasing/decreasing of the OCP values during the exposure of the samples to artificial saliva, irrespective of metallic alloy supports. This behavior is usually be attributed to thickening of the passive film, which becomes more protective conferring to the alloy more anticorrosive properties [44]. Also, it can be observed that the presence of composite layer on the metallic dental alloy surface, increases the OCP values for the majority of cases, proving its complementary protective properties in acidic pH values of artificial saliva (i.e., 4.3 and 5). generally predictable, if the OCP of coated metal is more positive than that of the bare metal [43]. This is confirmed by analyzing the results of Figure 3, where it can be observed that the OCP values at the bare surface are the smallest and the OCP values are spread on an approximatively 200 mV range. Also, it can be observed, in all cases and all pH values, the same tendency of increasing/decreasing of the OCP values during the exposure of the samples to artificial saliva, irrespective of metallic alloy supports. This behavior is usually be attributed to thickening of the passive film, which becomes more protective conferring to the alloy more anticorrosive properties [44]. Also, it can be observed that the presence of composite layer on the metallic dental alloy surface, increases the OCP values for the majority of cases, proving its complementary protective properties in acidic pH values of artificial saliva (i.e., 4.3 and 5). In order to establish if a dental alloy will suffer corrosion under certain condition of the oral cavity, the OCP value determination is necessary, but not sufficient, because OCP In order to establish if a dental alloy will suffer corrosion under certain condition of the oral cavity, the OCP value determination is necessary, but not sufficient, because OCP is a thermodynamic measure which gives only information related to the Gibbs free energy of the electrochemical system [31,45].

EIS Measurements
Electrochemical impedance spectroscopy (EIS) is an ac complex technique used to evaluate the electrical behavior of a material, by modelling the real system in terms of an equivalent circuit containing resistive, capacitive and diffusive components.
In order to obtain further information on the corrosion resistance properties of the both studied metallic dental alloys surfaces unmodified and modified with a layer of ZrO 2 or Sx (where x = 1÷5) composite in chitosan, impedance measurements were recorded with the electrodes immersed in artificial saliva of pH 4.3, 5 and 6, after 3600 s for OCP stabilization.
The obtained impedance spectrum was fitted by using a non-linear regression method, and the values of the obtained parameters of two used equivalent circuit models (i.e., R s (Q dl R ct ), R s (Q layer R layer )(Q dl R ct )) ( Figure 4) were summarized in Tables 2 and 3 for all studied composite matrix/dental alloys interfaces and all pH, respectively. The significance of the components of the equivalent circuits is as following: R is the resistance corresponding to the uncompensated solution (R s ), to the composite matrix (R layer ) and to the charge transfer process (R ct ) respectively; Q is the constant phase element corresponding to the composite matrix (Q layer ) or double layer (Q dl ), respectively. As known, R ct is a quantitative parameter, which could be an indicator of corrosion resistance of the composite thin film acting as a barrier to electron flow (resistor) and provides a value of the degree of protection furnished by the composite thin film deposited on the metallic dental alloy interface [46,47].
Due to lack of surface homogeneity of surface polishing, the presence of the composite matrix coating and the distortions on the double-layer, the constant phase element (Q) instead of an "ideal" capacitor was incorporated into the equivalent circuit models, in order to appropriately fit the experimental data. The equivalent capacitance of the Q was modelled as a non-ideal capacitor containing the capacitance of the double layer C dl and a roughness factor n, using the formula Q = (C dl R ct ) 1/n /R ct [48]. The roughness factor n is a number placed in the range 0-1, and it gives information about surface roughness (i.e., n = 1 is the case of a perfectly smooth surface of an ideal capacitor, and n = 0 is the case of an ideal resistance, respectively) [46,47].
From the data presented in Tables 2 and 3 it can be observed that R s value doesn't changed considerably during the experiment, having values in the range of 15-60 ohm×cm 2 , because the factors affecting its value like the stability of exposed surface and ions concentration in artificial saliva solution doesn't changed dramatically in the studied pH range. When a charge transfer reaction occurring to a metallic surface can be neglected as in the case of corrosion process, the corresponding estimated impedance could be attributed to the impedance of inorganic/organic composite protective coatings [31,49]. In Figure 5A-F, the Nyquist plots exhibit a depressed, capacitive-like semicircle and a second linear region, one at high frequencies and another at intermediate and lower frequency values, respec-tively, for all studied interfaces. This behavior is due to the inorganic/organic composite protective coatings having corrosion protection properties.  Values ± relative standard errors (RSD) expressed in percent (%).
Analyzing the data from Tables 2 and 3, the following information could be observed. In the case of bare Ni-Cr or Co-Cr metallic alloys the charge transfer resistance is in the range of 12-70 kohm×cm 2 , proving that the oxide passive layer is a weak protective against corrosion in artificial saliva of pH 4.3-6. Moreover, the Chit-ZrO 2 composite matrix on both Ni-Cr and Co-Cr exhibit R ct values higher than that obtained for bare electrode (in the range 70-90 kohm×cm 2 ), irrespective of studied pH values, proving the beneficial effect of this coating. For all Chit-Sx (x = 1÷5) composite matrix deposited on both Ni-Cr or Co-Cr metallic supports at all studied pHs, the values of R ct are higher than those determinated at bare or Chit-ZrO 2 composite coating. Additionally, the R ct values higher than 100 kohm×cm 2 which can rich 450 kohm×cm 2 (for particular electrode configuration or pH) indicate that Chit-Sx (x = 1÷5) composite matrix has anticorrosive protective properties. The behavior could be correlated with SEM/EDS experiments and could be attributed either to the quasi similar compact aspect of the composites S2-S5, or to the presence of increasing amount of Ca.
It can be seen that the best resistance at corrosion shown the composites S3-S5 having an increased amount of Ca in their initial and final composition (see EDS results in Figure 2). Also, the small differences between the R ct values of the composites S3-S5 could be due to the similar behavior concerning the electrolyte diffusion through the composite layer, without reaching the metallic surface [50].
A roughness factor n = 0.56 ÷ 0.95 in all studied cases indicates that an oxide passive film is formed on both metallic interfaces, suggesting a deviation of their behavior from pure ideal capacitor comportment. For all fitted data presented in Tables 2 and 3, the Chisquare distribution test (χ 2 ) have values between 10 −2 ÷ 10 −4 , and the relative standard errors (RSD, expressed in %) are less than 20%, indicating excellent agreement between the experimental and fitted data, and proving the correct choice of electric elements in the equivalent circuit model [51].
In the corresponding Bode impedance plots ( Figure 6A-F), in the case of Chit-S1 inorganic/organic matrix deposited on both metallic dental alloys, immersed in artificial saliva irrespective of pH values, the total |Z| impedance values is lower than for all other tested composite (i.e., Chit-Sx, where x = 2÷5). The same small total |Z| impedance value is observed in the case of Chit-S5 deposited on Ni-Cr metallic support and of Chit-ZrO 2 deposited on Co-Cr metallic support. This means that the presence of increased weight of Ca in the Chit-S5 composite matrix (on Ni-Cr) act similarly with pure ZrO 2 (Chit-ZrO 2 coating on Co-Cr) or ZrO 2 -MgO (Chit-S1 coating on both Ni-Cr and Co-Cr) and its absence or presence in the coating matrix, in a certain weight, maybe prevent the Chit swelling or dissolution [52]. In a previously work [31], it was demonstrated that the roughness of the metallic Ni-Cr electrode surface is more significant than that of the Co-Cr metallic electrode surface, leading to a better immobilization of the composite layer. Moreover, this result indicates that there should be a minimum of Ca amount in the composite matrix from which the anticorrosive resistance of the protective layer become significant.
In the 0.1-10 5 Hz frequency range of the Bode diagrams ( Figure 6A-F) it can be observed that the slope of the impedance diagram was around -0.75 and the maximum phase angle values is between 60-80 • , in the case of all Chit-Sx (where x = 2÷5) matrices containing HAP in the initial synthesis mixture. Exception are the cases of Chit-S1, Chit-S5 (at both pH 4.3, 5) and Chit-ZrO 2 (pH 6) on Ni-Cr, and Chit-S1 and Chit-ZrO 2 (all pHs) on Co-Cr metallic supports, respectively, where the slope of the impedance was around −0.35 in the same frequency range (i.e., 0.1-1000 Hz) and the phase angle was around 90 • for frequencies greater than 10 3 Hz. This behavior suggests a possibly existence of a synergetic effect between the diffusion process through the Chit-composite layer and the roughness of the metallic surface of the electrode [31,50,52].

Conclusions
ZrO 2 -based composites, based on different amounts of antibacterial magnesium oxide and bioactive and biocompatible hydroxyapatite to the inert zirconia, were prepared by ceramic method at high temperature, and morpho-structurally characterized by XRPD and SEM/EDS. Moreover, HAP decomposition at this high sintering temperature could play an important role in the anticorrosive properties if these composites are used as coating materials.
Two metallic dental alloys (i.e., Ni-Cr and Co-Cr) coated with a chitosan membrane containing the prepared composites were exposed to aerated artificial saliva solutions of different pHs (i.e., 4.3, 5, 6) and the corrosion resistance was investigated by electrochemical impedance spectroscopy technique. However, from the fitted data (using R s (Q dl R ct ) or R s (Q layer R layer )(Q dl R ct ) electrical circuits) it was observed that the two investigated metallic dental alloys shown quasi-similar anticorrosive properties when coated with layers containing different ZrO 2 -based composites, probably due to the synergetic effect between the diffusion process through the Chit-composite layer and the roughness of the metallic electrode surface.
The findings obtained by an interdisciplinary approach, between materials science and electrochemistry, represent preliminary researches, and further studies need in order to elucidate the complex phenomena which take place.