Production and Evaluation of Hydroxyapatite (HAp) Properties of Broiler’s Composite Bone (BCB) Waste at Different Sintering Temperatures

1Lab. of Animal By-Products Processing Technology, Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia 2Lab. of Meat and Egg Processing Technology, Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia Department of Periodontology, Faculty of Dentistry, Hasanuddin University, Makassar, Indonesia Department of Chemistry, Faculty of Mathematics and Natural Sciences, Hasanuddin University, Makassar, Indonesia Graduate of Animal Products Technology Study Program, Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia


Introduction
The waste products from poultry slaughterhouse (PSh) industry have remained a greater challenge (Enginuity worldwide LLC, 2017; Bodirsky et al., 2014;Khan and Ghouri, 2011) because of their susceptibility to deterioration (Centner, 2004). Bone constitutes is one of the most abundant solid wastes in PSh industry (Arshadi et al., 2015) and may transmit diseases. On the contrary, many beneficial applications of the bone were reported such as catalyst (Corro et al., 2016) and inexpensive organic fertilizer (Gousterova et al., 2008). Without further use, the bone waste might be a serious pollutant that contaminates air, water and soil, especially in PSh (Kazemi-Bonchenari et al., 2017). Bone contains a valuable component that needs to explore. It was reported to have a high content of calcium and phosphorus (Gopi et al., 2014). Its high mineral content may represent the adequacy of mineral supply contained in the feed (Onyango et al., 2003). As by-product, the bone is not only applicable for fabrication of feed, but also for environmentally friendly packaging material (Said et al., 2016;2011). The broiler's composite bone refers to bone in the whole body except claw and head.
In Indonesia, the broiler's composite bone (BCB) is abundant and needs a further exploration. Based on MA (2015), broiler population reached 1,528.33 million. If assumed 70% of the population is slaughtered, the number of slaughtered broiler per month is 127.26 million (70% ×1,528.33 million/12 months). The bone waste accounts for 20.85% of total body weight. With average weight of 1.5 kg per head, the potential bone waste reaches 39.83 million kg per month (20.85% ×191.04 million kg = 39.83 million kg).
In term of chemical components, the bone is rich in collagen and calcium (Ockerman and Hansen, 1999). Collagen extract is now widely used as an antiosteoporosis and anti-aging food supplement (English, 2011;English and Cass, 2001). Furthermore, duck beak bone was reported to be a promising source of of Hydroxyapatite HAp (Son et al., 2014). The collagen extract and HAp could be used as a composite material (Imanieh and Aghahosseini, 2013). Chest skin of broiler was also a good source of collagen, especially collagen type I (Bilgen et al., 1999). The extraction of collagen from bone produced HAp [Ca 10 (PO 4 ) 6 (OH) 2 ] by ±93% as by-product (Niakan et al., 2013;Gabriela et al., 2008). Previous studies reported that HAp could contribute to the bone regeneration (Kattimani et al., 2016) and serve as biomaterial in medicine science by means of HAp-collagen engineering (Shibata et al., 2005;Khiri et al., 2016;Heinz et al., 2010;Acharya et al., 2016). In dentistry, HAp was useful material for repairing and treating teeth and dental parts (Pepla et al., 2014;Görken et al., 2013).
The properties of HAp are highly depending on source and preparation conditions (such as heating temperature). The use of cattle's bone, shellfish and rock for fabrication of HAp has been studied (Khiri et al., 2016), whereas study pertaining broiler' bone composites for HAp production is rather scarce. This research was very important to increase the added value of BCB waste and reduce the pollution effect of PSh; thus, our study aimed to produce and evaluate the properties of HAp produced from broiler's composite bone at different sintering temperatures.

Methods
BCB waste (180 g) was cleaned from meat and fat, washed and then weighed. The HAp was produced by using three following stages, i.e., calcination, precipitation and sintering. The method was carried out according to Khiri et al. (2016).

Stage 1. Calcination
BCB was heated at 1000°C for 5 h using furnace, leading to removal of organic materials and conversion of CaCO 3 into CaO through elimination of CO 2 gasses (Equation 1). At this stage, the potential amount of waste was also determined. BCB (60 g) was transferred into 6 porcelain containers (10 g BCB per container). The Calcination Efficiency (CE) of the BCB was calculated as follow: CE = A/B ×100%, where A = weight (g) of BCB after calcination; B = weight (g) of BCB before calcination. XRD analysis was also performed to evaluate the resultant of calcination (using CaO as a standard) according to Joint Committee on Powder Diffraction Standard (JCPDS) 82-1691:

Stage 2. Precipitation
The calcined BCB was powdered using electrical grinder to obtain a particle size of 45 µm. The powder was used to prepare 1.0 M of calcium hydroxide (CaOH) 2 solution by mixing with aquadest (100 mL) at 40°C, as previously determined using a stoichiometric equation. The solution was stirred using a magnetic stirrer for 2 h to obtain a uniform mixture of Ca(OH) 2 . The reaction process occurs according to Equation 2: To produce HAp powder, Ca(OH) 2 suspension was added with 100 mL of phosphate acid (HPO 4 ) solution at a rate of 15-20 drops/min. Next, the solution was stirred with a magnetic stirrer and maintained at pH 8 by adding ammonium hydroxide (NH 4 OH) solution to complete the reaction then stored for 24 h at room temperature. The ratio of Ca:P was 1.67: 2. The white precipitate formed was then filtered using Whatman no 42 paper and dried for 5 h at 110°C. The results were weighed using analytic scales. The reaction process was presented in Equation 3:

Stage 3. Sintering
Sintering refers to heating process below melting point to form a new crystal phase as desired. This process aims to assist chemical reactions of materials making up both ceramic and metal. The HAp powder was sintered at a temperature level of 550°C, 600°C, 650°C, 700°C, 750°C, 800°C (50°C intervals) using a furnace for 1 h. After that, the HAp powder was cooled in a furnace at room temperature. The results were weighed to determine the efficiency. The Sintering Efficiency (SE) of BCB was calculated as follow: CE = A/B ×100%, where A = weight (g) of BCB after sintering, B = weight (g) of BCB before sintering. Furthermore, to know the characteristics of Ca 10 (PO 4 ) 6 (OH) 2 produced, analysis using XRD (Shimadzu 6000) was carried out. The results of sintering were also analyzed using a diffractometer at the diffraction angle (2θ) with scanning distance of 5°. The standard for characterization of HAp (Ca 10 (PO 4 ) 6 (OH) 2 ) product followed the Joint Committee Powder Diffraction Standard (JCPDS) 18-0272.

Experimental Design and Statistical Analysis
The pattern of X-Ray diffraction (diffractogram) was descriptively analyzed, while CE and SE parameters. The data were evaluated using Analysis of Variance (ANOVA). Significant difference between means was compared using Duncan's Multiple Range Test (DMRT) at 5% (Steel and Torrie, 1991).

Calcination Efficiency (CE)
The CE value is important to evaluate the effectiveness of the calcination process. High efficiency was expected in a production process. The CE of BCB at calcination condition of 1000°C for 5 h was presented in Table 1.
HAp, a type of bioceramic, has been widely used as an alternative material of artificial bone. It is selected for several biological reasons such as biocompatibility, bio-affinity, bioactivity, osteoconduction (Dubok, 2000), osteo-integration (Hench and Thompson, 2010) and osteo-induction (under certain conditions) (Weiner and Wagner, 1998). In composition, HAp was composed of calcium and phosphate ions, so there has been no reported adverse local or systemic toxicity. When HAp was applied to a new bone, it directly binds HAp through the apatite layer. This occurs because of the lack of calcium carbonate in bone implants (Dorozhkin, 2012;LeGeros, 2008).
The results showed that CE of BCB at heating temperature of 1000°C for 5 h to produce CaO was 41.99% (Atirah, 2017). This CE was lower than that reported by Putri (2012) using different sources of bone, i.e., chicken egg shell of (54.04±1.00), duck egg shell (55.03±1.30) and quail egg shell (53.19±3.00). The low CE can be caused by the insufficient temperature and processing time. The heating process eliminates all organic materials. Chicken bone shows a high crystallinity with single phase pattern. High crystallinity can increase the mechanical strength of the HAp. However, in excessive level of crystallinity, this may also complicate the absorption process in the body compared with other materials (Suzuki et al., 2005).
In recent years, the heating process was recommended to remove organic compounds (Lin et al., 1999). The crystalline phase composition of the sintered bone was similar to that of the natural bone of 93% HAp (Ca 10 (PO 4 ) 6 (OH) 2 ) of bone weight and about 7% βtricalcium phosphate (Ca 3 (PO4) 2 , β-TCP) (Martin, 2000). The heat treatment yields an interconnected porous structure (up to 70% porous). Therefore, it allows for faster bone growth (Lin et al., 1999). The ratio of Ca/P ratio also affects the mechanical properties of HAp. The greater the ratio of Ca/P results in the higher strength, but the maximum ratio is 1.67. The mechanical properties of HAp decreased at Ca/P ratio of >1.67 or <1.67. In recent studies, many researchers have tried to extract HAp from natural materials such as coral, shell, eggshell, cuttlefish shells, natural gypsum and natural calcite (Herliansyah et al., 2012). The raw materials were CaCO 3 or CaSO 4 . Some chemical reaction processes were performed to produce HAp. In our study, since BCB is from living beings, the production of HAp from BCS has certain advantages, including the reduction of polluted waste. Additionally, the BCB from Psh and meat processing industry is abundant and cheap. Thus, this suggests that the BCB may substitute the role of other natural ingredients.

X-Ray Diffraction of Calcined BCB
The diffraction profile of BCB was important to evaluate the potential content of HAp present in the sample. The standard used in this evaluation was Joint Committee on Powder Diffraction Standard (JCPDS) 82-1691 special for CaO. The diffractogram of BCB before and after calcination at 1000°C for 5 h was obtained by using X-Ray Diffraction (XRD). As presented in Fig. 1, diffractogram shows a similarity pattern to JCPDS 82-1691. This indicates a process of converting CaCO 3 into CaO by eliminating CO 2 gasses Fig. 1. Demonstrates that BCB can be used as a candidate of bone replacement material according to its corresponding crystallinity properties. The BCB crystallization can be controlled by using different levels of time and temperature when the preparation process begins (Ooi et al., 2007).
Different intensity of X-Ray absorption in BCB before and after calcination is presented in Table 2.  Based on Table 2 and Fig. 1, we found the 3 highest peaks of CaO crystallization present before and after calcination. The crystallinity degree of CaO as the main HAp constituent can be observed from its diffraction patterns at the peaks. The maximum intensity peak of BCB was found at 2θ: 32.5250°; 19,7200° and 21,1200° (before calcination) and 32,5187°; 33,5626° and 33,0000° (after calcination) (Atirah, 2017). The molarity of Na 3 PO 4 affects content of CaO. Higher level of CaO was responsible for higher purity degree of HAp. At molarity >5 M, this may damage the crystal structure of the material, leading to reduction of CaO concentration.

Sintering Efficiency (SE)
The formation of HAp crystal involves sintering process after precipitation. The standard for characterizing HAp was the Joint Committee on Powder Diffraction Standard (JCPDS) 18-0272. The SE value was presented in Fig. 2.  As depicted in Fig. 2, different sintering temperatures showed no significant effects on the amount of dried HAp precipitate (p>0.05). This suggests that sintering temperatures (550-800°C) could be performed to produce HAp precipitates. The amount of dry sintering precipitates did not affect the levels of HAp in the precipitates. Therefore, to know the amount of crystal content of HAp, characterization using XRD was required (Fig. 3). Figure 3 exhibits the diffractogram of sintered BCB at temperature variations of 550-800°C for 1 h. The results showed that sintering temperature of 700°C resulted in peak with the most optimum intensity, suggesting that this sintering condition could produce HAp crystallinity at optimum level. Powdered HAp has a great potential as the bone replacement. This is because it was easily absorbed by the bones and can be received by the body. There were two kinds of HAp powder, i.e., micro and nano sized HAp. Micro HAp has a small surface area and has a strong crystal bond, so that easily absorbed by the body. In addition, it can also increase biostability and strength. However, HAp with nanostructures has better density, strength and bioactive properties (Pepla et al., 2014).