Introduction

In the past few years, calcium phosphate-based materials have been used in many applications in bone regeneration and replacement, teeth pores filling, drug delivery, enhancement of bone growth and in tissue engineering due to their bioactivity and biocompatibility (Navarro et al. 2008; Viswanath and Ravishankar 2008; Rey et al. 2007). Different phases of calcium phosphate nanoparticles (CaP) with different sizes and shapes can be suitable for different purposes (Sørensen and Madsen 2000; Jadalannagari et al. 2011; Oliveira et al. 2007; Ahmed et al. 2008). Several researchers under different preparation conditions studied the synthesis of calcium phosphate extensively (Nilsson et al. 2002; Ren et al. 2012; Kundu et al. 2010a). By varying temperature, pH and initial reagent concentrations, one can obtain different calcium phosphate phases (Kundu et al. 2010b).

It is well known that, hydroxyapatite (HAP: Ca10(PO4)6(OH)2) with a hexagonal crystal structure, is one member of the ‘apatite’ family (Tas 2000; Badraoui et al. 2009). In osteology, calcium is one of the most important inorganic ions, in addition to phosphate given the key component of the mineralized bone matrix. It has a central role in maintaining calcium homeostasis that is very essential for the normal physiological functions (Blair et al. 2007; Peacock 2010). Bone diseases arise when homeostasis is not maintained. Within bone matrix, calcium is important at the whole tissue level, both as a structural component of the mineralized matrix and as a signal transduction molecule for eliciting cellular responses. Owing to its closed structural resemblance to bones and teeth, HAP has attracted much attention as a substitute material for damaged teeth or bones over the last years (Suchanek and Yoshimura 1997; Hench 1991; Ohura et al. 1996).

Calcium hydrogen phosphate dihydrate (DCPD: CaHPO4 ·2H2O), known as brushite, can be used alone or combined with other materials in the regeneration of bone and various surgical sites in animal models, because it has the ability to be resorbed under physiological conditions (Tamimi et al. 2012; Hench 1998). Therefore, this study is important for bio-mineralization phenomena (Singh et al. 2010). Brushite is also important as soil phosphate fertilizer; it is relatively water soluble as compared with HAP (Pattanayak et al. 2007). It can be used for supplying plant implants with the necessary amount of calcium phosphate (Singh et al. 2010). The physical properties of calcium phosphate ceramics vary strongly according to particle shape, particle size, distribution and agglomeration (Ferreira et al. 2003).

The objective of this work is the preparation of both brushite and hydroxyapatite nanoparticles by simple co-precipitation method to study deeply how the preparation conditions could represent crucial parameters to control calcium phosphate physico-chemical properties. Tuning the properties of such bioceramics could generally improve their use in different biomedical applications owing to their biodegradability, biocompatibility as mentioned above.

Materials and methods

All chemicals were of analytical grade form used without further purification. Disodium hydrogen phosphate (Na2HPO4, Merck) and calcium chloride (CaCl2 ·2H2O, Merck) were used as the starting materials. A diluted solution of ammonium hydroxide (NH4OH, Merck) and HCl were used as adjusting reagents for pH values.

The flowchart of samples preparation is shown in Fig. 1. Two different groups of samples were prepared using the same procedure, namely co-precipitation method. The first one is concerned with the Ca/P ratio (1:1) and pH values of 4, 5 and 6 adjusted using NH4OH and/or HCl. The second group in which Ca/P ratio is (5:3) and the pH values were adjusted at 7, 9 and 12 using NH4OH droplets.

Fig. 1
figure 1

Flowchart of prepared brushite and hydroxyapatite powder

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The solutions in both cases were continuously stirred for 24 h (Table 1). The pH value of the suspension was continuously adjusted and maintained at the above-mentioned values during precipitation. The obtained solutions were placed in Teflon flasks and tightly sealed, then aged at room temperature for 24 h to allow precipitation. White precipitated powder was filtered thoroughly and washed several times with double distilled water and finally dried at 50–60 °C for 12 h.

Table 1 Values of Ca/P, pH values and compound after 24 h reaction time
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Powder X-ray diffraction analyses were carried out using (analytical-x′ pertpro with Cu kα1 target, λ = 1.5404 Å, 45 kV, 40 mA, The Netherlands) to identify the phase composition and crystallinity of the calcium phosphate compounds. FT-IR spectrometer (Perkin–Elmer system 2000) was used for recording FTIR spectra in the range of 4000–400 cm−1. Thermo-gravimetric analysis (TGA) was carried out from room temperature up to 1200 °C in a DTG-60H SHIMADZU analyzer using an air flow rate of 100 mL/min and a heating rate of 10 °C/min. The surface morphology was studied using scanning electron microscope (SEM) model LEO-1530. The particle size and shape were investigated by transmission electron microscope (TEM) model JEOL/JME—2100.

Results and discussion

Structural analysis

X-ray diffraction (XRD)

The sample with low pH value 4 did not precipitate and was completely clear during the experiment. XRD patterns for the samples of the first group synthesized at pH 5 and 6 represent brushite with single phase as shown in Fig. 2a and agree well with the ICDD card no. 01-072-0713. These patterns show that the peaks of brushite at pH 5 have higher intensities than those at pH 6. The crystallinity of these samples was found to decrease with increasing pH value as reported in Table 2.

Fig. 2
figure 2

a XRD pattern of calcium phosphate at pH 5, 6, b XRD pattern of calcium phosphate at pH 7, 9, 12

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Table 2 Variation of crystallite size, particle size, shape, bulk density, theoretical density and relative porosity
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Figure 2b illustrates XRD patterns for the samples of the second group prepared at pH values 7, 9 and 12, which demonstrate that the pH value takes the upper hand in the formation of HAP. Phase analysis was identified using ICDD card no. 04-007-2837 for HAP. It is clearly noted that the sample with higher pH has sharper diffraction peaks (larger crystallite size) than other samples. These results indicate that HAP at pH 12 has larger crystallite size as shown in Fig. 2b. The broad pattern of the samples was attributed to the formation of poor crystalline HAP, which leads to higher dissolution compared with well-crystallized HAP. Moreover, it was reported that the crystallinity is a crucial factor that affects the biological behavior (Uehiraa et al. 2013; Dorozhkin 2010; Fulmer et al. 2002). Hong et al. have demonstrated that osteoblasts exhibited a high cellular activity such as adhesion on the low-crystallinity HAP thin film (Hong et al. 2003). The gradual increase in the intensity of HAP peaks with increasing the pH value indicates further nucleation growth of the hexagonal nanocrystals.

Crystallite size was calculated for HAP powder using Scherrer’s formula and reported in Table 2. The crystallite size of the HAP crystals was 5, 15, 16 nm for pH values of 7, 9 and 12, respectively, while in the case of brushite phase, the crystal size was 55 and 41 nm at pH 5, 6, respectively. The lattice parameters for HAP were a = 9.399 Å and c = 6.890 Å at pH = 7. The unit cell volume was 527.19 (Å)3, 527.44 (Å)3 and 526.249 (Å)3 for the pH values 7, 9 and 12, respectively, which agree well with that reported with ICDD card no. 04-007-2837. The theoretical density D x was determined using the formula:

$$D_{x} = \frac{ZM}{NV}$$
(1)

where M is the molecular weight of the sample, N is Avogadro’s number, Z is the number of molecules per unit cell and V is the unit cell volume calculated from XRD data (Ahmed et al. 2003; Cullity 1978).

The results in Table 2 show that the above investigated samples possess a considerable amount of apparent porosity (39–53 %) as calculated from

$$P = \left( {1 - \frac{D}{{D_{x} }}} \right) \times 100\,\%$$
(2)

where P is the porosity, and D and D x are the experimental and theoretical densities, respectively (Ahmed et al. 2003).

FTIR analysis

The structure of the samples prepared at pH values of 5 and 6 was investigated by FTIR spectra, as shown in Fig. 3a and presented in Table 3. From the figure, there is a neglected shift between pH 5 and 6. The absorption bands at 661–527 and 1218–989 cm−1 are due to ν 4 bending vibration of P–O–P mode. The vibrations at 871 and 791 cm−1 are attributed to the P–O–H vibrations for inorganic Ca–P component (Mandel and Tas 2010). The four bands, 871, 989, 1064 cm−1 and 1132 cm−1 are originating to the (HPO4 2−) stretching in both dicalcium phosphates, i.e.; monotite (DCPA) and brushite. A band at 989 cm−1 is assigned from the P–O(H) symmetric stretching vibration of PO4 3− group (Singh et al. 2010; Maity et al. 2011). Moreover, band near to 1064 cm−1 is reffered to the ν 3 stretching mode of the PO4 3− group. A band around 1132 cm−1 was attributed to the ν′ 6 and ν″ 6 of HPO4 2− ions in brushite (Maity et al. 2011).

Fig. 3
figure 3

FTIR spectra of calcium phosphate: a pH 5, 6, b pH 7, 9, 12

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Table 3 Characteristic transmittance infrared bands of brushite and hydroxyapatite at different pH values
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Further investigation of the FTIR spectra revealed that the band at 1649 cm−1 was ascribed to the bending mode of H2O. The bands at 3166, 3287, 3489 and 3542 cm−1 corresponded to stretching vibration of water [ν s (O–H)] (Singh et al. 2010; Ahmed et al. 2014). The bands between 3000 and 3600 cm−1 show the presence of DCPD but not in its anhydrous form DCPA (Singh et al. 2010; Zou et al. 2012).

In the spectrum shown in Fig. 3b for the samples prepared at pH (7, 9, 12), the absorption bands at 3435 and 1606 cm−1 were assigned to the absorbed water (Markovic et al. 2011). The bands at 1424 and 844 cm−1 were attributed to the vibrations of CO3 2− groups absorbed from the ambient atmosphere (Alobeedallah et al. 2011). The presence of carbonate results from the ambient CO2 dissolved during the crystallization process, whereas HAP crystals are very susceptible to CO2. It is observable that all samples are classified as B-type (where CO3 2− substitutes phosphate position) (Ahmed et al. 2014). It should be mentioned that carbonate substitution has a critical role in the crystal structure, stability and biological activity (Morales et al. 2013; Minh et al. 2013).

The appearance of a band at 1270 cm−1, where CO3 2− possibly replaces PO 3−4 , confirms the identity of carbonate (Asep and Sopyan 2009). The characteristic bands of PO4 3− appeared at 566, 604 and 1032 cm−1 where the bands at 566 and 604 cm−1 were assigned to the vibration of the O–P–O mode (Markovic et al. 2011). It is observed that the intensities of the bands were increased by increasing the pH value, as a result of the improvement of the crystallinity of the investigated samples. These results are also in good agreement with those mentioned in XRD results Fig. 2b. There is little shift with different pH values, and the assigned bands are reported in Table 3.

Microstructural and morphological features

TEM

Figure 4a, b shows the transmission electron micrograph of the brushite nanoparticles at pH 5. The particles are formed in spherical shapes, with sizes ranging from 50 to 90 nm. With further increase in the magnification, each particle looks like a silk ball with yarns linking between them. These silk filaments have different lengths with a diameter around 2–6 nm. TEM micrograph of brushite at pH 6 shown in Fig. 4c, d reveals that the particles are formed in a fiber network with lengths from 100 to 150 nm. The corresponding selected area electron diffraction SAED pattern shows that the particles are polycrystalline and have a preferred orientation. This special morphology of these samples facilitates their easy attachment to biological cells.

Fig. 4
figure 4

TEM micrographs of brushite phase: a, b pH 5, c, d pH 6

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Figure 5a shows the TEM micrograph of nano-HAP at pH 7. The HAP nanoparticles were uniform in both morphology and size. The sample appears as needle shape crystals, with a diameter around 5–10 nm with length 150–200 nm. SAED is shown in the inset of Fig. 4a and reveals diffraction rings indicating twin crystal structure and demonstrates that the needles are polycrystalline rings with a preferred direction (100) as indicated by XRD.

Fig. 5
figure 5

TEM micrographs of hydroxyapatite phase: a pH 7, b, c pH 9, d pH 12

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TEM of HAP at pH 9 together with the SAED was demonstrated in Fig. 5b, c. The micrograph shows nanorods with diameters ranging from 20 to 30 nm with a wide range of lengths starting from 200 to 300 nm. The rod shape becomes clear and uniform by increasing the magnification with a constant aspect ratio Fig. 5c.

HAP with these dimensions (~20 nm) resembles to the basic building blocks of enamel rods. In addition, it has been observed that HAP nanoparticles can be self-combined to form enamel-like structures in vitro and it could be adsorbed onto the enamel surface strongly. Therefore, it could be used to improve the repair of the enamel surface (Chen et al. 2005; Liu et al. 1998).

Figure 5d illustrates the TEM micrographs of HAP at pH 12 together with SAED, which appeared as rice shape with diameters in the range of 10–20 nm in length does not exceed 60 nm. It is clear that the particle size becomes shorter, which is attributed to the isotropic crystal growth at higher pH values. On the contrary, the nanoparticles synthesized at low pH have more complicated shapes (Liu et al. 1998).

SEM

Figure 6a illustrates the morphology of the nanobrushite sample at pH 5 which appears as homogenous surface with irregular particles, a small amount of apparent porosity. This irregularity appears on the surface as roughness, which could improve the osseointegration as well as the biocompatibility leading to the positive effect on inflammatory reactions (Morales et al. 2013; Müller 2010).

Fig. 6
figure 6

SEM micrograph of brushite phase: a pH 5, b, c pH 6

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For the sample prepared at pH 6; Fig. 6b, c, the particles appeared as a bundle of fibers arranged in a certain direction with irregular pores (24.71 %) as shown in Table 2. The morphological feature of the samples with pH 7 and 12 was demonstrated by SEM as shown in Fig. 7a–d. The nanoparticles appeared as micro fibers with diameters in ranges of 5–10 μm and length exceeding 900 μm at pH 7 as in Fig. 7a, b. Figure 7c, d reveals agglomerated grains with various sizes from 0.5 to 10 μm of pH 12.

Fig. 7
figure 7

SEM micrograph of hydroxyapatite phase: a, b pH 7, c, d pH 12

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Thermal analysis

TGA

Thermo-gravimetric analysis was carried out with the temperature range 30 to 1200 °C in the air at a heating rate 10 °C/min. Figure 8 shows the TGA of brushite at pH 5 and HAP at pH 9 and 12. For all the samples, the analysis of weight loss is observed in three stages. The first one was related to the water removal from the precipitated powders; the second and third loss ones resulted from the decomposition of organic components in the raw materials (Fulmer et al. 2002). The third stage illustrates the thermal stability of the samples from 550 to 1200 °C. Total weight loss of the brushite at pH 5, HAP at pH 9 and 12 was 21.15, 6.91 and 6.283 %, respectively, at the end of the experiment at 1200 °C. This emphasizes the hypotheses that the higher pH values lead to higher thermal stability which is in good agreement with the reported work (Ahmed et al. 2014). The advantage of thermal analysis is to specify the working temperature range at which the bioceramics are applicable. Another benefit is to determine the exact temperature of phase transformation.

Fig. 8
figure 8

TGA of brushite phase at pH 5, HAP at pH 9 and 12

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Conclusion

Brushite and HAP nanoparticles were successfully obtained from the homogeneous precipitation as perfect mono dispersed particles with sizes ranging from 50 to 200 nm. The nano-HAP particles will be extensively investigated for future use in biomedical applications such as bioactive ceramics for bone substituting materials and tissue scaffold. When regulating the preparation conditions, mainly pH and Ca/P ratio, different micro structures could be tailored to achieve fiber network, silk ball, short and long nanorods.