Evaluating hydrothermal synthesis of fluorapatite nanorods: pH and temperature

ABSTRACT In this study, the fluorapatite was synthesised by a hydrothermal technique in different pH and temperature using apricot tree gum surfactant. The fluorapatite was synthesised in different shapes such as spherical, Chrysanthemum flower and rod. The effect of two factors (pH and temperature) on the shape and dimension of synthesised fluorapatite was investigated through the full factorial design. An experimental strategy was developed based on the analysis of variance to create mathematical models for the shape and dimension of synthesised fluorapatite. Findings revealed that the pH of hydrothermal solution is more significant factor than temperature in terms of shape and dimension of the synthesised fluorapatite. It was illustrated that similar nanorods structure to the human tooth enamel can be achieved in pH of 10 and temperature of 70 °C The transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM) and energy-dispersive X-ray spectroscopy (EDS) spectroscopy were carried out for characterisation of synthesised fluorapatite.


Introduction
Hydroxyapatite (HA) with chemical formula of [Ca 10 (PO 4 ) 6 (OH) 2 ] is widely used for biomedical applications due to its structural and biological similarity to human bones [1,2]. It is known that HA has the ability to bond to the surrounding tissue [3,4]. Moreover, the hydroxyl group in HA can be substituted with fluoride to fabricate the fluorapatite (FHA). Generally, fluorapatite is a bioceramic and can be found in the tooth and bone. It is known that fluorapatite has more acid resistance than HA. The dissolution process of HA inside the body is related to the crystallinity and its chemical composition. Therefore, addition of fluoride to the structure of HA is a suitable way to decrease the dissolution rate of HA. In addition, fluorapatite has more crystallinity compared to the HA as well as more thermal and chemical stability. Fluorapatite is harder and process slightly better thermal stability than the HA. It is demonstrated that the most portion of human teeth and bones [5] are formed of fluorapatite and HA. Great stiffness, significant resistance to acid damage and suitable biocompatibility of fluorapatite make it potential candidates for biomedical applications. The application of fluorapatite has been mentioned for dentistry application such as crowns, inlays, dentin simulators, coatings and cements [5,6]. In addition to dentistry applications, fluorapatite is an important biomaterial in orthopaedic applications as bone regeneration [7,8]. It was reported that desirable length and diameter for synthesised FHA nanorods to be used as enamel of tooth are between 100-1000 nm and 33-65 nm, respectively [9]. Up to now, it has been reported that fluorapatite can be synthesised through different techniques including the sol-gel [10][11][12], wetchemical processing [4,13], solid-state reaction [14] and hydrothermal process [13,14]. Among these techniques, hydrothermal process is considered as a promising approach for synthesise of FHA crystals owing to the advantages such as high quality, chemical homogeneity and low cost of production in large-scale [15].
In our previous study, we reported on the hydrothermal synthesis of fluoridated HA nanorods using different surfactants such as ATG, ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) and sodium dodecyl sulphate (SDS) [16]. The feasibility formation of FHA nanorods in different sizes, crystallinities and shapes at low temperature of hydrothermal conditions was investigated. Findings revealed that synthesised FHA nanorods by ATG surfactant were larger in dimension as compared to the EDTA and SDS. In addition, results indicated that the size and morphology of synthesised FHA nanorods can be controlled by pH value and surfactant. In this study, an experimental strategy based on the full factorial design is performed using analysis of variance (ANOVA) to investigate the effect of pH and temperature on the shape and dimension of fluorapatite in the hydrothermal condition. Finally, a precision mathematical model is created based on the effective parameters and their interactions to predict the desirable shape and dimension of fluorapatite in the hydrothermal conditions.

Materials and methods
The HA and sodium fluoride were purchased from sigma Aldrich (i-Chem Solution-Malaysia). The nitric acid and ammonium hydroxide for adjusting the pH were purchased from Merck (Putaka Elite). Apricot tree gum (Prunus Armenia) was achieved from the city of Taleghan in Iran.
To synthesis fluorapatite, three suspensions with different pH were prepared and put in the different hydrothermal conditions similar to our previous work [16]. 105 mg of HA powder with 8 mg of sodium fluoride was mixed in 100 ml of distilled water. The HNO 3 was added to the suspensions until the HA and sodium fluoride powders dissolved (pH was around 2.3). Thereafter, the ATG surfactant was added to the solutions with the amount of 200 mg. Solutions were stirred for two hours and then the NH3 was added dropwise to each solution until the pH of 6, 8 and 10 is achieved. The solutions were placed in autoclave under 50, 70 and 90 8C at 1 atm hydrothermal condition.
The synthesised fluorapatite at different pH values and temperatures was then characterised by transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM) and energy-dispersive X-ray spectroscopy (EDS). Moreover, the mathematical analysis was performed to investigate the effect of hydrothermal condition on the synthesis of fluorapatite in terms of shape and dimension.
An experimental investigation of pH and temperature effect on the shape and dimension of synthesised fluorapatite was carried out. In this study, the pH and temperature were considered as two independent variables. The low, middle and high levels of these two factors are given in Table 1. The obtained data were investigated through full factorial design with two replications. There are three centre points with the level of zero for each factor.    Chrysanthemum flower-like and rods structure of fluorapatite. It can be seen in FESEM images that the synthesised fluorapatite is in nano scales but in dissimilar shapes and dimension for different pH and temperature. In addition, it is demonstrated that pH values have a great effect on the shape of synthesised fluorapatite. A summary of qualitative and quantitative data of experimental results can be seen in Table 2. It is revealed that by changing the temperature of hydrothermal procedure, the shape and defined dimension component (a D horizontal per vertical length) are effected. Moreover, the quantitative and qualitative statistical analyses of FESEM images (Table 2)

Analysis of variance (ANOVA) for shape
The ANOVA was applied to test for the significance of the regression model, the test for significance on individual model coefficients. The ANOVA table for the shape is   [19]. F-value of the curvature is 0.14 which implies that the curvature (as measured by difference between the average of the centre points and the average of the factorial points) in the design space is not significant relative to the noise. There is a 72.46% chance that a  'Curvature F-value' this large could occur due to noise. The regression model in term of coded factor obtained from data is presented in Equation 1: The half normal plots of the shape are shown in Figure 6. A check on the plots revealed that the factors A and B and also the interaction AB are significant and affect the model. Figure 7 illustrates the effect of factor pH and temperature and also their interaction on the shape. This figure indicates that the effect of pH is more significant than the effect of temperature on the shape. The counter plot and three-dimensional (3D) surface graphs are shown in Figure 8. These graphs reveal that by increasing the pH and temperature, the shape of synthesised fluorapatite has been modified from ¡1 (spherical) to 1 (rods). This means that the synthesised conditions (pH and temperature) are the effective factors on the morphology of fluorapatite crystals. These factors have a great effect on the crystal Figure 6. Half normal plot of shape. growth of the synthesised fluorapatite. Findings demonstrate that the pH is more effective than temperature in this study. It may be due to the release profile of surfactant that can be manipulated by changing the pH of solution. It is interpreted that the release amount of calcium ions can be adjusted and consequently influenced the growth of fluorapatite crystals.

Analysis of Variance (ANOVA) for dimension
The ANOVA table for the dimension is presented in  [20]. If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve your model. F-value of the curvature is 1.19 which implies that the curvature (as measured by difference between the average of the centre points and the average of the factorial points) in the design space is not significant relative to the noise. There is a 31.72% chance that a 'Curvature F-value' this large could occur due to noise. The regression model in term of coded factor obtained from data is presented in Equation 2 : Dimension D C 1:94 C 0:61 A C 0:36 B ¡ 0:012 AB The half normal plots of the shape are shown in Figure 9. A check on the plots revealed that the factors A and B and also the interaction AB are significant and affect the model. Figure 10 illustrates the effect of factor pH and temperature and also their interaction on the shape. This figure indicates that the effect of pH is more significant than the effect of temperature on the dimension. The counter plot and 3D surface graphs are shown in Figure 11. These graphs reveal that by increasing the pH and temperature, the dimension of synthesised fluorapatite has been modified from a D 0.9 to a D 5. Increasing the amount of a means the dimension of synthesised fluorapatite changed from symmetric shape (spherical) to the asymmetric shape (rod). It can be seen in Figures 1-3 that the shape of synthesised crystal was changed from the spherical to the Chrysanthemum flower-like and then rod shape. This is related to the release profile of the calcium ions by ATG surfactant as well as the degree of temperature. It is illustrated by increasing the temperatures of solutions from 50 to 90 8C, the shape of crystals tends to the rods structure. Moreover, when the pH values increased, the same process occurred.

Conclusion
In this study, the effect of pH and temperature on the dimension and shape of flouapatite through a hydrothermal process was investigated. It was revealed that the presence of ATG as surfactant in different pH and temperature values has remarkable effect on the shape and dimension of synthesised fluorapatite. The similar nanorods' structure to the human tooth enamel was achieved in pH of 10 and temperature of 70 8Cthrough the hydrothermal process. It was demonstrated that with the increase of pH, the shape of synthesised fluorapatite can be changed from the spherical to Chrysanthemum flower-like and then rod shape. Moreover, the raising of temperature in a constant pH values was found to change the shape and dimension of fluorapatite. The full factorial design mathematical models illustrate that pH is more effective than the temperature in terms of shape and dimension of synthesised flourapatie.