Shynthesis and Characterizations of Calcium Hydroxyapatite Derived from Crabs Shells (Portunus pelagicus) and Its Potency in Safeguard against to Dental Demineralizations

Crab's shells of Portunus pelagicus species were used as raw materials for synthesis of hydroxyapatite were used for protection against demineralization of teeth. Calcination was conducted to crab's shells of Portunus pelagicus at temperature of 1000°C for 5 hours. The results of calcination was reacted with (NH4)2HPO4, then dried at 110°C for 5 hours. Sintering was conducted to results of precipitated dried with temperature variations 400–1000°C for a hour each variation of temperature then characterized by X-ray diffractometer and FTIR in order to obtain the optimum formation temperature of hydroxyapatite is 800°C. The hydroxyapatite is then tested its effectiveness in protection against tooth demineralization using acetate buffer pH 5.0 with 1 M acetic acid concentration with the addition of hydroxyapatite and time variation of immersion. The results showed that the rate of tooth demineralization in acetate buffer decreased significantly with the provision of hydroxyapatite into a solution where the addition of the magnitude of hydroxyapatite is greater decrease in the rate of tooth demineralization.


Introduction
Hydroxyapatite biomaterials are materials that are very widely used in several health purposes, including as a source of calcium for the manufacture of toothpaste and as an important material in the formation/bone repair. The chemical properties of hydroxyapatite are bioactive and compatible with the adjacent bone and teeth. Hydroxyapatite is a calcium phosphate ceramic that is totally biocompatible and nontoxic and becomes an integral part of living bone and teeth tissue [1][2][3][4][5][6][7][8]. So it is important that these materials are produced independently. The raw material for the production of hydroxyapatite biomaterial is very easily available and abundant in Indonesia. The production process was easy and the cost is also relatively inexpensive if done on a large scale. Among the abundant raw materials are the shells of crabs, which are one of Indonesia's main export commodities. Export of Crabs commodity by Indonesia amounted to 604.215-625.000 tons/year without the shell form, while domestic consumption is expected to be so much more, as in Makassar, carrying 292.5 tons exported in the form of crab without shell with the main export destination being Singapore [9]. If the mass of crab shells 25-50% of the total mass, it can be estimated that in 2012 produced the shells of crabs around 151,053.75 to 302,107.5 tons in Indonesia and 73.125 to 146.25 tons in Makassar (a region of Indonesia), there was just from crabs production were exported. This value is of course even more if the crab's consumptions in the country are also taken into account. This suggests the existence of crab shells is abundant in Indonesia, including in Makassar. As known in Indonesia, the shells of crabs have not been used, so it will only be a waste disturbing environment [7,8].
The crystals structure of hydroxyapatite will be better by using CaO as a precursor of calcium. However, the use of these compounds also produces carbonate apatite [Ca 10 (PO 4 ) 6 CO 3 ] in a fairly large percentage. This is because the calcination process cannot completely eliminate carbon dioxide (CO 2 ) in CaCO 3 so that there can be reaction with the precursor phosphate. However, the carbonate apatite heating at a temperature of 700 ∘ -900 ∘ C for 2-5 hours, followed by washing using distilled water, the carbonate apatite can be hydroxyapatite [11][12][13]. The instrument were used in this research were glass apparatus, Ohaus analytical balance, petri dish, porcelain cup, Buchner flask, Buchner funnel, vacuum pump Sargent-Welch Co. Model 1400, magnetic stirrer, magnetic bar, hotplate Idealife, pH meter, Furnace Thermolyse 6000-Barnstead, dessicator, thermometers, Spnisosfd oven, stopwatch, Shimadzu X-ray diffractometer (XRD) Model 6000, X-ray Fluorescence (XRF) Shimadzu, Fourier Transform Infra-Red (FTIR) Prestige-21 Shimadzu, Scanning Electron Microscopy combined with the ability to generate localized chemical information (SEM-EDXA) Variant, and UV-VIS Shimadzu model 6105. This is because the carbonate ion compounds are able to inhibit the crystallization process Ca 10 (PO 4 ) 6 (OH) 2 so that the results will be dominated by an amorphous phase [14].
The calcinations temperatures of CaCO 3 range from 900 ∘ to 1200 ∘ C. If the CaCO 3 burned at temperature calcinations, decomposition reaction of CaCO 3 into CaO will occur and CO 2 emissions are dominant and will be issued as a result of the combustion reaction [13][14][15]

Synthesis of Hydroxyapatite from Waste Shell Crab Portunus pelagicus.
Crabs shells Portunus pelagicus waste was cleaned with distilled water and dried at room temperature. Furthermore, to transform crabs shells into CaCO 3 and then into CaO, calcinations were performed on the samples at 1000 ∘ C temperature for 5 hours at a rate of temperature rise 5 ∘ C/minute. The containment of calcium (Ca) was determined by using XRF. Calcium oxide (CaO) obtaining dominated as result of calcinations and then made suspensions in 100 mL of distilled water with a calcium concentration of 0.3 M. The suspensions reacted by dropwise with a 100 mL 0.2 M of (NH 4 ) 2 HPO 4 solution through the coprecipitation method, at temperatures around 40 ∘ C while the solution was stirred for 2-5 hours. The precipitation allowed stand overnight or 24 hours at room temperature, and the precipitate is filtered with a Whatman filter paper number 40 and dried at 110 ∘ C for 5 hours. The pure hydroxyapatite obtained by sintering to the dried precipitate at various temperatures of 500 ∘ -900 ∘ C for 4 hours [16,17]. The results washed with distilled water and then dried at a temperature of 110 ∘ C. The characterization of the compounds was performed by using X-ray diffraction (XRD), FTIR, and SEM-EDXA.

The Teeth Demineralization
Tested. The proven in vitro demineralization of teeth was conducted through evaluating the concentration of phosphate in solution by using the UV-Vis spectroscopy. The effectiveness of hydroxyapatite to protection against teeth demineralization was tested in acetate buffer pH 5.0 solutions, with 1 M of acetic acid concentration with the addition of hydroxyapatite in varying concentrations and immersion time [18]. Each of 5 beakers was filled with 300 mL of acetate buffer pH 5.0 with acetic acid concentration of 1 M. An acetate buffer was left without adding anything as a comparison. An acetate buffer was then added 10 ppm of NaF left without addition of Ca 10 (PO 4 ) 6 (OH) 2 . Other three pieces beaker of acetate buffer containing 10 ppm of NaF are adding of Ca 10 (PO 4 ) 6 (OH) 2 with variation concentration of 25 ppm, 50 ppm, and 100 ppm. The cleaned tooth samples were immersed in each of 5 beakers of solutions. The immersed times of tooth samples in solution are 3, 6, 9, 24, and 48 hours, respectively. Furthermore, the phosphate concentration in each solution was measured by using UV-Vis spectrophotometer with the wavelength for phosphate ( maks) being 432 nm.   Identification by FT-IR as shown in Figure 2 showed there is a reduction process of -CO 3 groups and some of IR-spectra were missing after calcinations. This shows the elimination of CO 2 and organic components occurred [19]. The elimination of -CO 3 groups and organic components can also be seen from the data of mass reduction of sample calcinations. Mass reduction during the calcinations process is 56.35% on average. This means that the efficiency of calcium compounds produced by 43.64%.

Results and Discussion
The determinations of calcium contained in sample was conducted by using X-ray fluorescence, where obtained calcium is 66.62% after calcinations. These results are then used to calculate the stoichiometry in determining the number of results calcinations which is needed to react with (NH 4 ) 2 HPO 4 as the precursor phosphate.

Precipitation with Phosphate Precursors.
The precipitation reactions aiming to produce Ca 10 (PO 4 ) 6 (OH) 2 used phosphate, (NH 4 ) 2 HPO 4 , as the precursor and then reacted with CaO as calcinations results. Side results Ca 10 (PO 4 ) 6 CO 3 also occur as a product of reaction of (NH 4 ) 2 HPO 4 and CaCO 3 presence in the calcinations results.
Dried precipitate further sintered on temperature variations of 400 ∘ -1000 ∘ C for 2 hours; it is intended to determine the optimum temperature where the Ca 10 (PO 4 ) 6 (OH) 2 is formed, furthermore characterized by using XRD, FT-IR, and SEM-EDXA. Figure 3. The diffractograms of each sintering results compounds indicate that the temperature is closely related to the formation of crystals. This is due to the nature of the vibrating atoms moving faster in higher temperatures [19].

Characterization of Sintering Results by XRD. XRD Diffractograms of compounds results presented in
The optimum temperature formation of hydroxyapatite was determined by calculation of the probability of the sample phase from the XRD results analysis according to JCPDS standard data, which, JCPDS; 24-0033 is standard data for Ca 10 (PO 4 ) 6 (OH) 2 ; 09-0169 for -Ca 3 (PO 4 ) 2 ; 29-0359 for -Ca 3 (PO 4 ) 2 , 35-0180 -Ca 3 (PO 4 ) 2 ; 35-0180 for Ca 10 (PO 4 ) 6 CO 3 (OH) 2 and 19-0272 standard data for Ca 10 (PO 4 ) 6 CO 3 (OH) 2 . Figures 3 and 4 showed that the temperature is closely associated with the formation of hydroxyapatite phase. In the both graphs it can be seen that the maximum intensity of the phase formation of hydroxyapatite has been found by sintering at temperature of 800 ∘ C, it means the optimum temperature of hydroxyapatite formation is 800 ∘ C, and then this result will be used for another application.
The formations of hydroxyapatite phase had been dominated at 800 ∘ C confirmed by percentage probability sample phase ( Figure 5   respectively. However, also there is still presence of a phase Ca 10 (PO 4 ) 6 CO 3 and Ca 10 (PO 4 ) 6 CO 3 (OH) 2 with a range of 11.84% and 4.43%, respectively, which indicates the presence of carbonates. All data coming from the calculations of the XRD spectrum used its software. The X-ray diffractograms of Ca 10 (PO 4 ) 6 (OH) 2 synthesized at a 800 ∘ C temperature sintering can be seen in Figure 6, where the peaks of HA are symbolized by the peak of the crystal Ca 10 (PO 4 ) 6 (OH) 2 , while peak -TKF is symbolized crystalline peaks of -Ca 3 (PO 4 ) 2 , the symbol -TKF for crystalline -Ca 3 (PO 4 ) 2 , AKA for Ca 10 (PO 4 ) 6 CO 3 ,  and AKB for Ca 10 (PO 4 ) 6 CO 3 (OH) 2 . The highest intensity peak at 31.2572 deg corresponding to crystalline -Ca 3 (PO 4 ) 2 is seen, the second highest peak intensity is 31.7783, and the third highest peak intensity is 28.0565 crystals suitable for Ca 10 (PO 4 ) 6 (OH) 2 .

Characterization of Ca-Hydroxyapatite with FT-IR.
FTIR results showed that the sintering temperature variation affects the absorption band shapes which generally all sintering results showed absorption band of -OH, absorption band 1, 2, 3, and 4 of PO 4 3− , and CO 3 2− groups. Infrared spectra in Figure 7 show the -OH groups at 633 cm −1 which are characteristic of hydroxyapatite [17] appearing on the sintering temperatures of 400-1000 ∘ C. Additionally spectrum also showed higher sintering temperature causing the sharper peaks phosphate group (PO 4 3− ) because the nature of the vibrating atoms moves faster at higher temperatures [19]. The presence of the phosphate group indicates the formation of hydroxyapatite in the precipitates. and Ca 10 (PO 4 ) 6 CO 3 (OH) 2 which has not been transformed into Ca 10 (PO 4 ) 6 (OH) 2 during the sintering process.

Characterization of Ca-Hydroxyapatite
Sintered at 800 ∘ C with SEM-EDXA. The results of characterization by SEM-EDXA shown in Figure 9(a) show that the size of hydroxyapatite formed from the synthesis tends to be small and only a few are large. While Figure 9(b) shows that the surface of hydroxyapatite is smooth and nonporous, this shows that hydroxyapatite which has synthesis from crab shells can function well as inhibiting tooth demineralization [13]. While in Figure 10 the EDXA spectrum shows the composition of the synthesis yield was dominated by oxygen (O) up to 59.52%, calcium (Ca) up to 23.76%, and phosphorus (P) up to 13.32%. The composition confirmed the composition of hydroxyapatite. It can be concluded that the synthesis results can be achieved to target.   to the acidic conditions by releasing Ca 2+ and PO 4 3− ions.
Demineralization of tooth causing increased levels of Ca 2+ and PO 4 3− in saliva in direct contact with the tooth. In vitro, the rate of tooth demineralization can be observed through the concentrations of Ca 2+ and PO 4 3− ions in solutions where the tooth was soaked each unit of time. Therefore, increase of PO 4 3− ions concentrations in solution a soaked gear can be one of indicators to measure the rate of tooth demineralization. Figure 11 shows the relationship between the soaking time of teeth versus the increase of the ion PO 4 3− levels in solution where the tooth was soaked as well; it appears that with the increasing addition of Ca 10 (PO 4 ) 6 (OH) 2 into the acetate buffer equals to the rate of demineralization decrease. It is can be altered by the amount of PO 4 3− ions in solutions; however the addition of Ca 10 (PO 4 ) 6 (OH) 2 ions showed lower amount of PO 4 3− ions compared to solutions without the addition of Ca 10 (PO 4 ) 6 (OH) 2 . This proves that the Ca 10 (PO 4 ) 6 (OH) 2 were synthesized from the crab shell effective for protection against tooth demineralization.
The decrease in the rate of tooth demineralization with the addition of Ca 10 (PO 4 ) 6 (OH) 2 can also be observed through analyzing the tooth mass reduction in the fifth variation of acetate buffer solution as shown in Table 1. The greater concentrations of Ca 10 (PO 4 ) 6 (OH) 2 in the acetate buffer solution were teeth immersed exhibit the smaller mass of teeth in the solution [19]. Table 1 shows the relationship between tooth mass and the addition of hydroxyapatite.