Synthesis of Biphasic Calcium Phosphate and its Behaviour in Simulated Body Fluid

Hydroxyapatite [Ca10(PO4)6(OH)2, HA] is commonly used as bioactive materials in bone graft, orthopaedic application, dental implant etc. since its unique ability to bond to bone after implantation (Ellinge et al. 1986). The ability of bond to bone can be predicted by its capacity to form HA layer on the surface upon immersed in simulated body fluid (SBF) solution, of which the ion concentrations is similar to human plasma (Nery et al. 1990). SBF screening is commonly used to predict the ability to bond to bone since its simplicity and economic, before implant into a live animal. In the other hand, the β-tricalcium phosphate [β-Ca3(PO4)2, β-TCP] is known as biodegradable materials, but exposes its weak bioactivity (Petrov et al. 2001). So if we can combine HA and β-TCP with proper ration, we are also possible to control the advantage of biphasic calcium phosphate (BCP): high bioactivity and its fast biodegradation (Victoria & Gnanam 2002; Aslanidou et al. 2012).


Preparation of BCP Powder
BCP is prepared similar to the research of Nezahat Kivrak with the weight ratio of HA and β-TCP is 20/80 (Kivrak & Cuneyt Tas 1998).At first, (NH 4 ) 2 HPO 4 0.1128 M was dropped into Ca(NO 3 ) 2 .4H 2 O 0.4 M so that the weight ratio of HA/β-TCP is 20/80.The dropping rate was approximately at 3 ml/min, and chemical reaction was maintained at 40 o C, then adding NH 4 OH 0.1 M to adjust pH = 8.After finishing, it was filtered many times by distilled water to remove the bad smell of NH 4 OH solution.Then the aqueous suspension was transferred into the oven and dried at 120 o C for 8 h to obtain the powder, following the calcination at 1000 o C for 5 h.The synthetic flowchart is presented in Figure 1.

In vitro Experiment
To predict the bond to the bone ability of BCP, we soaked BCP powder in the SBF solution.The ion composition of SBF solution is similar to human plasma.SBF solution was prepared by NaCl, NaHCO 3 , KCl, K 2 HPO 4 .3H 2 O, MgCl 2 .6H 2 O and C a Cl 2 .They are dissolved in distilled water and buffered with (CH 2 OH) 3 CNH 2 and HCl (6N) to adjust the pH value at 7.4.This process is according to the Kokubo's method (Kokubo & Takadama 2006).The ratio (50 mg of material in 100 ml of SBF solution) was chosen in in vitro experiment.

Characterization of BCP Powder
To characterize physicochemical properties of biomaterial before and after soaking in SBF solution, some analysis of physico-chemical methods were used like X-ray diffractometer (XRD) and scanning electron microscopy (SEM).To evaluate the crystalline phase, constitution and component of BCP, XRD (Bruker D8 Advance) were employed.The XRD performed with a scan speed of 0.02 o /min and step time was 1 s/min.XRD data were acquired in the range of 2θ from 5 o to 65 o .SEM (Hitachi, Joel 5) was used to observe and evaluate the morphology of material before and after soaking in SBF solution, and determine the formation of the new apatite layer after soaking in SBF solution.

Characterization of Synthetic BCP Material
Figure 2 shows XRD patterns of synthetic BCP with HA standard and β-TCP standard.
The synthetic BCP had specific peaks of both HA and β-TCP and had not stranger peaks (Pramanik et al. 2007;Biqin et al. 2008;Mir et al. 2012).XRD pattern of BCP had the sharp peaks which showed that BCP had an excellent crystallinity.The obtained result confirmed that the synthetic BCP was a combination of two phases: HA and β-TCP and of complete purity.Figure 3 shows SEM images of β-TCP, HA, and BCP.The SEM micrograph showed two phase of HA and β-TCP with HA phase was the small particles that attached to bigger particles of β-TCP.This result confirmed the combination of HA and β-TCP in the structure of BCP.

In vitro test
Figure 4 regroups the XRD patterns of BCP after 0, 1, 5 and 10 days of soaking in SBF solution.After 1 day of soaking in SBF, there was a significant change before soaking.The β-TCP phase in BCP had crystallized while the HA phase had decomposed.But from 5 to 10 days the process carried out reversely.The HA phase had crystallized, and the β-TCP phase had decomposition.These phenomena confirmed the chemical interactions between BCP and SBF medium to conduit a new apatite layer on its surface.After a long time of soaking, β-TCP phase in BCP would have been completely decomposed and replaced by a new apatite layer.
Figure 5 shows SEM micrographs of BCP after 0, 1, 5 and 10 days of soaking in SBF solution.After one day of soaking, no phenomena appeared.After five days of soaking, a thin layer was coated on BCP surface like grass.After ten days, the new layer continued to develop.This confirmed the bioactivity of synthetic material.

CONCLUSIONS
Our research group succeeded to synthesise BCP powder via wet chemical precipitate method.Physicochemical characterization by XRD and FTIR confirmed the presence of two phases HA and β-TCP.
In vitro test to evaluate the bioactivity was carried out in the SBF solution.XRD and SEM analyses showed that the formation of a new apatite layer and the disappearance of β-TCP phase.This material could be used as biomaterials for bone replacement.

Date of submission: December 2016
Date of acceptance: January 2017

Figure 2 .
Figure 2. XRD patterns of synthetic BCP with HA standard and β-TCP standard.