Synthesis and Characterization of Carbonate Hydroxyapatite from Pinctada Maxima Shell with Short Aging Time for Bone Biomaterial Candidate

Biomaterial products for bone repair are needed to support accelerated tissue healing. This research aimed to synthesize and characterize carbonate hydroxyapatite (CHA) from Pinctada maxima (P. maxima) using the precipitation method with a short aging time, 15 min. CHA was dried with oven-dry at 100°C for 12 h (CHA A) and a furnace-dry at 1000°C for 2 h (CHA B). Short aging time succeeded in producing CHA B-type with lattice parameters and of CHA A is 9.382 and 6.964 , while 9.451 and 6.962 for CHA B. The high temperature treatment made the diffraction peak indicating CHA more detected and crystallinity increased to 97.87%. The appearance of C–O bond and diffraction peaks of CHA verified substituted carbonates hydroxyapatite.


Introduction
Bone is an important organ that supports the body and the attachment of muscles and organs. Bone consists of 50-70% inorganic mineral components, 20-40% organic, 5-10% water, and approximately 3% fat [1]. The main inorganic component of bone is the mineral hydroxyapatite (HA). HA has the chemical formula Ca10(PO4)6(OH)2 in the presence of small amounts of carbonate, magnesium, and phosphoric acid. Each HA unit cell has a hexagonal structure ( Fig. 1.a) [2][3][4].
The carbonate content in bone is about 2-8% depending on the individual's age [5]. Carbonate that is substituted in the HA structure is called Carbonate Hydroxyapatite (CHA). The chemical formula is generally written as (Ca10(PO4)6-x(CO3)x(OH)2 indicated that unite cell CHA consist of phosphorus, carbonate, and calcium [6].
The carbonate ion can substitute for HA in three types. CHA A-type is the carbonate ion substituted on the hydroxyl ion (OH − ), CHA B-type on the phosphate ion (PO 4 3− ), and CHA AB-type on the hydroxyl ion and phosphate ion [7]. CHA B-type is predominantly found in the body and is easier to synthesize because it can be carried out at low temperatures. CHA B-type a planar bivalent (CO 3 2− ) that replaced (PO 4 3− ) showed in Fig. 1.b. Figure 1. Chemical structure and unit cell of HA (a) and CHA B-type (b) [7].
P. maxima is a biogenic material candidate for the bone because it contains a lot of calcium [8]. The content of calcium carbonate in biogenic materials is quite significant, around 95-99% [5]. Calcium carbonate (CaCO 3 ) at high temperatures decomposed into calcium oxide (CaO) and carbon gas (CO 2 ), as shown in Eq. (1). CaO can be used to synthesize biomaterials such as HA and CHA. CHA can be synthesized from biogenic materials such as egg shells, animal bones, fish bones, and shells.
The precipitation method was used to synthesize CHA because it was easy to perform. Aging time is one of the important factors in the carbonate substitution process in the HA structure. Patty et. al., and Anggraini synthesized CHA from P. maxima uses the aging time of 24 h [9,10]. Heating at high temperatures makes forming to produce substituted carbonates.
In this study, the synthesis of CHA was developed using a short aging time to shorten the synthesis time. The time of 15 minutes was chosen because the CHA constituent particles have settled after experiencing precipitation. The rapid aging process accompanied by annealing at high temperatures is expected to produce the desired CHA. CHA with carbonate content can be used for bone biomaterial applications because its composition resembles the natural condition of bone.
Experimental Method. The experimental process began with preparing the raw material, P. maxima shells. P. maxima shell was decomposed into CaO referring to previous research [9,10]. CaO was used as a calcium source for CHA synthesis.
Preparation Sample. P. maxima shells were cleaned with water and soaked in acetone for 24 h. The raw were dried and cut into small pieces of approximately 1-2 cm. The pieces of shells were ball-milled and sieved. The raw powder was furnaced at 1000°C for 4 h and then sieved again using a 230 mesh (67 size particle) sieve to collect a homogeneous CaO. Synthesis of CHA. Diammonium hydrogen phosphate (NH4)2.HPO4 (7.05 g) was dissolved in 35 ml of distilled water and then stirred at 500 rpm for 30 min. Ammonium bicarbonate NH4HCO3 (4.48 g) was dissolved in 35 ml of distilled water and then stirred at 500 rpm for 30 min. The (NH4)2.HPO4 solution was titrated using burette to NH4HCO3 solution with rate of approximately 1 ml/min. CaO (5 g) decomposed from the P. maxima was dissolved in 50 ml of distilled water and then stirred at 500 rpm for 30 min. The titration result of (NH4)2.HPO4 and NH4HCO3 solution titrated using burette to Ca(OH)2 solution with rate of approximately 1 ml/min while stirring at 500 rpm in 80 o C.
The pH solution was maintained at 10 by adding 3% NaOH. After titration, the solution was stirred and heated for up to 30 min until homogenous. The solution was aged for 15 min then filtered. The supernatant was dried in the oven at 100 o C for 12 h (CHA A) to remove moisture and annealed in furnaces at 1000 o C for 2 h (CHA B) to form the CHA structure more complete.
Characterization. The samples were characterized using Fourier transform infrared (FTIR) (Thermo Scientific Nicolet iS10) at the LPPT UGM Unit 1 with a range of wave number of 650-4000 cm -1 to determine functional vibrational groups. X-ray diffraction (XRD) (Shimadzu, R600) with CuK 1.54060 Å was performed at the Chemistry Laboratory, FMIPA, UGM, to determine the crystal structure of CHA. Scanning electron microscope-energy dispersive X-ray (SEM-EDX) (JEOL, JSM-6510LA) was conducted at LPPT UGM to determine the morphology and content of CHA samples.

Results and Discussion
FTIR Spectra. The FTIR of CaO ( Fig. 2.b) decomposition of raw P. maxima showed a sharp peak in the 3639 cm -1 indicating the stretching vibration of the O-H bond [11]. Absorption also appears at  [12]. The high temperature treatment made P. maxima decomposed, so the O-H bonds are more specific due to the contribution of Ca(OH) compounds. The FTIR results of CHA showed absorption similar to previous studies ( Fig. 2.a) [9]. CHA ( Fig.  2.b) showed a sharp peak at 1013-1020 cm -1 region indicating vibration from P-O bond [13]. Absorbance at 1410 cm -1 and 872 cm -1 are stretching and bending vibrations of the C-O bond [14]. Absorbance also appeared at 3360 and 3640 cm -1 are characteristic vibrations of the O-H bond [5], [15].
The absorption indicating O-H in CHA A has a low absorption intensity at 3360 cm -1 while CHA B shifted to 3640 cm -1 with a higher and sharper intensity. This O-H bond contributes to the hydroxyl in the CHA structure. The presence of C-O absorption in CHA A indicates that oven-dry has succeeded in substituting carbonates into the CHA. After high temperature treatment (CHA B), O-H absorption sharped, then P-O bond intensity decreased. During high temperature treatment, the particles vibrated so that the carbonate could substitute the sample by replacing the phosphate position to form CHA B-type [5,13,16]. Presence absorbance of C-O, P-O, and O-H bonds like structure of CHA in Fig. 1.b indicated that CHA has successfully formed. The main diffraction peak formed in CHA shifted from the diffraction peak HA indicating the presence of carbonate ion substitution in the HA structure [13]. The high temperature heating process makes more diffraction peaks and the width between the diffraction peaks gets narrower. The narrower peak indicates the better CHA structure. The presence of carbonate substitution makes the peaks at (211) and (300) coincide each other. Table 1 the results from the XRD diffraction analysis data using data on the main diffraction peaks. CHA A has lattice parameters and of 9.382 Å and 6.964 Å, respectively, while CHA B is 9.451 Å and 6.962 Å. CHA A and CHA B have a larger lattice parameter than the reference HA, while the lattice parameter CHA A is smaller. This condition is appropriate with the characteristic of CHA Engineering Chemistry Vol. 3 B-type, where the lattice parameter is smaller while the parameter is larger than the HA reference [6,13].  The annealing process at 1000 o C for 2 h caused the particles forming the CHA to vibrate and integrate to form the CHA structure. During the annealing process, the carbonate ion undergoes substitution to replace the phosphate ion position. This situation makes the lattice parameter decrease while the lattice parameter increases so that the ratio parameter ( ) of CHA B to be closer to the value of the reference than CHA A.
High-temperature treatment makes the micro and nano grains of HA to be integrated and makes the particles experience compaction. This makes the crystallite size in CHA B increased to 45.09 nm, while the volume unit cell increased to 1615.644 g/cm 3 compared to CHA A. A low microstrain was detected at 0.006 o and 0.004 o , which indicated a very small crystal defect [11]. The crystallinity of furnace-dried CHA is higher than that of oven-dried CHA up to 97.87%.
The diffraction peaks indicated the presence of CHA with lattice parameters decreased and increased indicating that the synthesis was confirmed to CHA B-type. This result is strengthened by the FTIR result, which confirmed the presence of C-O, P-O, and O-H bonds. analysis previously which reported that after high temperature treatment, CHA is more detected and indicating carbonate substituted in the HA structure. The EDX results showed that all CHA samples contained calcium and phosphate. The molar ratio (Ca P ⁄ ) of CHA is presented in Table 2. The calcium and phosphate content in CHA A was higher than CHA B so ratio (Ca P ⁄ ) of CHA A is 2.15 while CHA B is 2.09. High temperature treatment causes the calcium and phosphate content decreased. This condition happened because the carbonate substituted in the phosphate site, so the phosphate content decreased then (Ca P ⁄ ) in CHA was higher than HA.
This result is also validated by the FTIR results of CHA which showed the absorption intensity of P-O bonds decreased while C-O bonds increased. The XRD results also showed lattice parameter decreased while lattice parameter increased indicating CHA B-type. The results of CHA B-type are similar to the results of the previous CHA which can be used for bone repair such scaffold in bone tissue engineering [9].

Summary
CHA synthesized from P. maxima with short aging time of 15 min succeeded has major diffraction peaks (002), (211), and (300) indicating the formation of a CHA structure. CHA A had lattice parameters and are 9.382 Å and 6.964 Å, while CHA B was 9.451 Å and 6.962 Å that indicating CHA B-type. The substituted carbonate was evident from the presence of FTIR absorption at 1410 cm -1 and 872 cm -1 indicating the C-O bond. CHA A has ratio (Ca P ⁄ ) is 2.15 while CHA B is 2.09 occurs because of the carbonate content in the CHA. The high temperature treatment increased crystallite size increased up to 45.09 nm and crystallinity up to 97.87%. This result indicates that synthetic CHA can be used for applications in biomaterials such as bone repair.