The aim of this study is to extract natural hydroxyapatite (HAP) from bovine bone. Hydrothermal method followed by calcination treatment at different temperatures is used in this current research. Bovine bone has potential for producing hydroxyapatite, a chief component present in bone and teeth of vertebrates. HAP is an excellent material used in bone restoration and tissue regeneration. Characterizations of the bovine bone natural HAP powder were done by scanning electron microscopy (SEM), Fourier transforms infrared (FTIR) and energy dispersive x-ray (EDX) spectroscopy. The FTIR result confirms the presence of phosphate (PO4-3), hydroxyl (OH)- and carbonate (CO3-2) groups in the powder. SEM displays the surface morphology whereas EDX provides calcium (Ca) to phosphorous (P) ratio in the HAP samples. The results of the EDX analysis indicate that the Ca/P ratio of HAP varies from 1.67 to 1.83. The extracted HAP material is found to be thermally stable up to 1100ÂºC

Abu Bakar, Sharifah Adzila

Bano, Nazia

Basri, Hatijah

Jikan, Suzi Salwah

Nuhu, Awwal Hussain

English

Journals of Universiti Tun Hussein Onn Malaysia (UTHM)

Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 *Corresponding author: suzi@uthm.edu.my  2017 UTHM Publisher. All right reserved. penerbit.uthm.edu.my/ojs/index.php/jst     Natural Hydroxyapatite Extracted From Bovine Bone   Nazia Bano¹, Suzi Salwah Jikan1*, Hatijah Basri1, Sharifah Adzila S. Abu Bakar2, Awwal Hussain Nuhu1  1Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia, Johor, Malaysia 2Faculty of Mechanical and Manufacturing Engineering, Universiti Tun Hussein Onn Malaysia, Johor, Malaysia  Received 30 September 2017; accepted 14 December 2017; available online 18 December 2017     1. Introduction  HAP is one of the most attractive and well-known biomaterials for teeth and bone tissues implant. Its composition and biological structure resemble to natural bone. The chief constituent of bone mineral is hydroxyapatite (HAP) with a chemical formula of  Ca10(PO4)6(OH)2 [1–4]. Natural HAP has all the distinctive features of biomaterials, such as bioactive, osteoconductive, non-inflammatory, biocompatible, non-toxic, and non- immunogenic assets. Moreover, the close resemblance of HAP with  calcium phosphates components of human bone provokes researchers to use it for different biomedical applications [5, 6]. It is considered that HAP plays a dynamic role for numerous applications [7] such as restoration of skull defects [8], restoration of huge area bone defect [9], bone tissue engineering scaffolds [10–12], removal of heavy metals and  drugs delivery [13]. In this study, bovine bones were used to extract natural HAP by hydrothermal followed by calcination at different temperatures. Through this extraction method, organic components in bones can be thermally decomposed and removed. Every single genome sign of disease can be removed and thus provides high biological safety factors [14]. Extraction of natural HAP from bovine femur bones is done at different calcination temperature and different particle sizes [15–17]. The use of bovine bone to generate hydroxyapatite will decrease the pollution effect of the waste and the subsequent conversion of the waste into an extremely valuable product. The process of converting bovine bone into HAP is an environmentally friendly process.  2.  Materials and Methods  The fresh cortical bone of mature bovine (2-3 years old) was used as raw material purchase from local market of Parit Raja Malaysia.  2.1 Bone samples preparation Bones were cleaned with water and soft parts inside the bones were manually detached from compact part manually by stiletto. Then the bones were cut into small pieces using cutter. The bones were boiled with water for 2 to 3 hours. Subsequently, the fluids in bone Abstract: The aim of this study is to extract natural hydroxyapatite (HAP) from bovine bone. Hydrothermal method followed by calcination treatment at different temperatures is used in this current research. Bovine bone has potential for producing hydroxyapatite, a chief component present in bone and teeth of vertebrates. HAP is an excellent material used in bone restoration and tissue regeneration. Characterizations of the bovine bone natural HAP powder were done by scanning electron microscopy (SEM), Fourier transforms infrared (FTIR) and energy dispersive x-ray (EDX) spectroscopy. The FTIR result confirms the presence of phosphate (PO4-3), hydroxyl (OH)- and carbonate (CO3-2) groups in the powder. SEM displays the surface morphology whereas EDX provides calcium (Ca) to phosphorous (P) ratio in the HAP samples. The results of the EDX analysis indicate that the Ca/P ratio of HAP varies from 1.67 to 1.83. The extracted HAP material is found to be thermally stable up to 1100ºC. Keyword: Hydroxyapatite, Bovine femur bone, Hydrothermal, Calcination Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 23  marrow and residual soft material were removed with knife and washed with distilled water several times. The bone fragments were dried in an oven at 80ºC for 72 hours (3-days) to denaturalize the protein. After that, dry fragments of bone were abridged to small particles by means of crusher. The crushed bone was subjected to ball milling process for 24 hours until the powder was fine enough. Bone powder was obtained by sieving. 25μm particle size of powdered bone sample was used in this study. Fig. 1 shows the flow chart of extraction of HAP from bovine bone.                             Fig. 1 Flow chart of extraction of HAP from bovine bone 2.2 Calcination of HAP powder Pure natural-HAP (mineral phase of the bone) free from fat and protein was achieved by calcination. The powder samples of HAP were calcined in a furnace at 600ºC, 700ºC, 800ºC, 900ºC, 1000ºC and 1100ºC, which named as HAP-600, HAP-700, HAP-800, HAP-900, HAP-1000, HAP-1000 and HAP-1100 respectively with 3 hours holding time at a heating and cooling rate of 5 ºC/min. Natural HAP was produced by decomposition of the organic phase. These temperatures were designated after a Thermo gravimetric analysis (TGA). The entire bone powder samples were cooled inside the furnace.  2.3 Characterizations of HAP powder  The characterizations of HAP before and after calcination were performed using SEM, FT-IR and EDX. FTIR was used to obtain information about the functional groups present in HAP powders in the range of 4000 cm-1 - 450 cm-1. Morphology of HAP particles were obtained by SEM (JSM 6490LA, Jeol, Japan) and the element compositions of the powder sample was determined by EDX.  3.  Results and Discussions  3.1 SEM analysis Fig. 2 shows the SEM micrographs of the HAP powder before and after calcination at different temperature. The particles are agglomerated in some parts and have irregular shapes including small spheres and rods as reported in literatures [18–20]. It is due to the van der Waals attraction between HAP particles during calcination [21].   Bovine cortical bone Ball milling for 24 h   Oven dried at 80ºC for 72 h Natural HAP (HAP) Cut into small pieces Boiled in water for 2-3 h   Crushing Calcination Characterizations  Raw- HAP  Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 24         Fig. 2 SEM micrographs of uncalcined  powder sample (raw-HAP) (a) and calcined samples at different temperature (b) HAP-600 (c),  HAP-700, (d) HAP-800, (e)  HAP-900,(f)  HAP-1000 and (g)  HAP-1100 respectively.       3.2 FTIR analysis  Fig. 3 reveals the FTIR spectra of uncalcined sample (raw-HAP) and calcined HAP at different temperatures. The FTIR spectra indicate the presence of phosphate (PO4-3), hydroxyl (OH-) and carbonate (CO3-2) groups. These spectra are more clearly seen in calcined HAP samples because the calcination process has destroyed the cross linked structure in the raw HAP bovine bone powder. In the spectra  of HAP, many bands matched  with the HAP reference spectrum and are in agreement with reported data on HAP [22, 23].    Fig. 3 FTIR spectra of extracted HAP powders of uncalcined sample (raw-HAP) and calcined samples at different temperature.  As shown in Fig. 3, the bands of raw HAP and calcined n-HAP are apparently different due to the alterations in their chemical bonds during calcination at different temperature. The color of raw HAP changes from yellowish white to Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 25  white after calcination. This color changes indicates the decomposition of organic substance which are collagen and proteins in calcined HAP bone powder [24, 25]. The FTIR band at 1637 cm-1 corresponds to Amide I of collagen [19] that is present in the raw HAP of bovine bone powder and is completely removed in calcined HAP samples. A strong and broad band due to symmetric stretching vibration that appears at 961.66 cm-1, 962.99 cm-1, and asymmetric stretching vibration bands at 1023cm-1 and 1092cm-1   in calcined HAP bone samples are associated with the phosphate group (PO4-3). The raw HAP bone powder exhibit a wide band near 3285.48 cm-1 which corresponds to the absorbed water molecule. Low intensity peaks of carbonate groups are identified in the FTIR spectra at 1460 cm-1 and 1520 cm-1 due to asymmetric stretching v3 of (CO3-2) groups. The sharp narrow band at 630.92 cm-1, 634.47 cm-1 and wide band at 3525.94 cm-1 are associated with hydroxyl group that verify the presence of HAP phase. In addition, after calcination most of the bands have largely increased in intensity due to the phosphate vibrations of hydroxyapatite as described by other authors [25, 26].   3.3 EDX analysis  The chemical compositions of HAP powder before and after calcination at different temperatures were evaluated by using EDX analyzer. Fig. 4 shows the EDX analysis of HAP powders prepared by calcination at different temperatures. The Ca/ P ratios of the extracted HAP samples were calculated and the ratios are 1.79, 1.79, 1.78, 1.81, 1.67, 1.75 and 1.83 for HAP-600, HAP-700, HAP-800, HAP-900, HAP-1000, HAP-11 00 for calcined samples and uncalcined sample (raw-HAP) respectively. The ratios are equivalent with previous study [25]. As the Ca/P weight ratio of the extracted HAP calcined at different temperatures did not show any significant difference, it can be concluded that Ca/P weight ratio does not depends on the calcination temperature. The higher Ca/P value of bovine bone derived HAP powder indicates that its chemical composition is close to the composition of human bone [27].The higher Ca/P values may be due to the occurrence of either calcium and phosphorus compounds other than hydroxyapatite or Ca compounds deprived of phosphorus [28]. In fact, Figueiredo  et al  articulated that natural HAP is non-stoichiometric having  Ca/P ratio greater than 1.67 [29].      Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 26       Fig. 4 EDX analysis of HAP powders prepared by calcination at different temperature (a) HAP-600, (b) HAP-700, (c) HAP-800 (d) HAP-900 (e) HAP-1000 (f) HAP-1100 and (g) uncalcined sample (raw-HAP) respectively.   5. Conclusion  The study confirms that HAP is able to be extracted from bovine bone wastes.  So, bovine bone wastes have great potential to be used in extracting the HAP deprived of any impurities as indicated by FTIR analysis. The methods involved hydrothermal followed by calcination of bovine bone powder at different temperature to remove its organic components thus the produced HAP which is suitable for the use in orthopedic and dental applications. Powder samples of HAP of bovine bone have close Ca/P molar ratio (1.67 - 1.83) to the stoichiometric HAP (1.67). The HAP extraction methodology is simple and low cost.  Acknowledgements  This study was partially supported by Center of Graduate Studies (CGS), Universiti Tun Hussein Onn Malaysia from the UTHM scholarship (UTHM/PS/500-14/8).    References [1] C. V. M. Rodrigues et al., (2003)“Characterization of a bovine collagen-hydroxyapatite composite scaffold for bone tissue engineering,” Biomaterials, vol. 24, no. 27, pp. 4987–4997. [2] H. Eslami, M. Solati-Hashjin, and M. Tahriri, (2009)“The comparison of powder characteristics and physicochemical, mechanical and biological properties between nanostructure ceramics of hydroxyapatite and fluoridated hydroxyapatite,” Mater. Sci. Eng. C, vol. 29, no. 4, pp. 1387–1398. [3] A. Rogina, M. Ivanković, and H. Ivanković, (2013)“Preparation and characterization of nano-hydroxyapatite within chitosan matrix,” Mater. Sci. Eng. C, vol. 33, no. 8, pp. 4539–4544. [4] W. L. Suchanek, P. Shuk, K. Byrappa, R. E. Riman, K. S. TenHuisen, and V. F. Janas, (2002)“Mechanochemical-hydrothermal synthesis of carbonated apatite powders at room temperature,” Biomaterials, vol. 23, no. 3, pp. 699–710. [5] G. Chen, T. Ushida, and T. Tateishi, (2001)“Development of biodegradable porous scaffolds for tissue engineering,” Mater. Sci. Eng. C, vol. 17, no. 1–2, pp. 63–69. [6] Y. Z. Wan et al., (2007)“Biomimetic synthesis of hydroxyapatite/bacterial cellulose nanocomposites for biomedical applications,” Mater. Sci. Eng. C, vol. 27, no. 4, pp. 855–864. [7] M. Aizawa et al., (2000)“Fabrication of Porous by Sintering Ceramics of Fibrous with Open Pores Hydroxyapatite Particles,” J. Ceram. Soc. Japan, vol. Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 27  108, no. 3, pp. 249–253. [8] P. F. Tang, G. Li, J. F. Wang, Q. J. Zheng, and Y. Wang, (2009)“Development, characterization, and validation of porous carbonated hydroxyapatite bone cement,” J. Biomed. Mater. Res. - Part B Appl. Biomater., vol. 90 B, no. 2, pp. 886–893. [9] Y. Liu et al., (2013)“Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects,” Biomaterials, vol. 34, no. 38, pp. 9998–10006. [10] M. B. Nair, S. Suresh Babu, H. K. Varma, and A. John, (2008)“A triphasic ceramic-coated porous hydroxyapatite for tissue engineering application,” Acta Biomater., vol. 4, no. 1, pp. 173–181. [11] S. V. Dorozhkin, (2009)“Calcium orthophosphates in nature, biology and medicine,” Materials (Basel)., vol. 2, no. 2, pp. 399–498. [12] M. J. Coughlin, J. S. Grimes, and M. P. Kennedy, (2006)“Coralline hydroxyapatite bone graft substitute in hindfoot surgery.,” Foot ankle Int., vol. 27, no. 1, pp. 19–22. [13] S. McGrellis., J.-N. Serafin, J. JeanJean, J.-L. Pastol, and M. Fedoroff, (2001)“Influence of the sorption protocol on the uptake of cadmium ions in calcium hydroxyapatite,” seperation Purif. Technol., vol. 24, no. 1, pp. 129–138. [14] E. Hosseinzadeh, M. Davarpanah, N. H. Nemati, and S. A. Tavakoli, (2014)“Fabrication of a hard tissue replacement using natural hydroxyapatite derived from bovine bones by thermal decomposition method,” Int. J. Organ Transplant. Med., vol. 5, no. 1, pp. 23–31. [15] C. F. Ramirez-Gutierrez, A. F. Palechor-Ocampo, S. M. Londoño-Restrepo, B. M. Millán-Malo, and M. E. Rodriguez-García, (2016)“Cooling rate effects on thermal, structural, and microstructural properties of bio-hydroxyapatite obtained from bovine bone.,” J. Biomed. Mater. Res. B. Appl. Biomater., vol. 104, no. 2, p. 339–344. [16] S. M. Londoño-Restrepo, C. F. Ramirez-Gutierrez, A. Del Real, E. Rubio-Rosas, and M. E. Rodriguez-García, (2016)“Study of bovine hydroxyapatite obtained by calcination at low heating rates and cooled in furnace air,” J. Mater. Sci., pp. 1–11. [17] S. S. Rahavi, O. Ghaderi, A. Monshi, and M. H. Fathi, (2017)“A comparative study on physicochemical properties of hydroxyapatite powders derived from natural and synthetic sources,” Russ. J. Non-Ferrous Met., vol. 58, no. 3, pp. 276–286. [18] W. Khoo, F. M. Nor, H. Ardhyananta, and D. Kurniawan, (2015)“Preparation of Natural Hydroxyapatite from Bovine Femur Bones Using Calcination at Various Temperatures,” Procedia Manuf., vol. 2, pp. 196–201. [19] N. A. M. Barakat et al., (2008)“Physiochemical characterizations of hydroxyapatite extracted from bovine bones by three different methods: Extraction of biologically desirable HAp,” Mater. Sci. Eng. C, vol. 28, no. 8, pp. 1381–1387. [20] I. Hilmi, M. Rinastiti, and M. K. Herliansyah, (2011)“Synthesis of hydroxyapatite from local bovine bones for biomedical application,” Proc. - Int. Conf. Instrumentation, Commun. Inf. Technol. Biomed. Eng. 2011, ICICI-BME 2011, no. November, pp. 409–411. [21] A. Ruksudjarit, K. Pengpat, G. Rujijanagul, and T. Tunkasiri, (2008)“Synthesis and characterization of nanocrystalline hydroxyapatite from natural bovine bone,” Curr. Appl. Phys., vol. 8, no. 3–4, pp. 270–272. [22] S. Jinawath, D. Pongkao, and M. Yoshimura, (2002)“Hydrothermal synthesis of hydroxyapatite from natural source,” J. Mater. Sci. Mater. Med., vol. 13, no. 5, pp. 491–494. [23] R. Murugan, T. S. S. Kumar, and K. P. Rao, (2002)“Fluorinated bovine hydroxyapatite: Preparation and characterization,” Mater. Lett., vol. 57, no. 2, pp. 429–433. [24] E. Kusrini, A. R. Pudjiastuti, S. Astutiningsih, and S. Harjanto, (2012)“Preparation of Hydroxyapatite from Bovine Bone by Combination Methods of Ultrasonic and Spray Drying,” Int. Conf. Chem. Bio-Chemical Environ. Sci. December 14-15, 2012 Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 28  Singapore Prep. [25] C. Y. Ooi, M. Hamdi, and S. Ramesh, (2007)“Properties of hydroxyapatite produced by annealing of bovine bone,” Ceram. Int., vol. 33, no. 7, pp. 1171–1177. [26] S. E. Etok et al., (2007)“Structural and chemical changes of thermally treated bone apatite,” J. Mater. Sci., vol. 42, no. 23, pp. 9807–9816. [27] E. Kusrini and M. Sontang, (2012)“Characterization of x-ray diffraction and electron spin resonance: Effects of sintering time and temperature on bovine hydroxyapatite,” Radiat. Phys. Chem., vol. 81, no. 2, pp. 118–125. [28] A. Doostmohammadi, A. Monshi, M. H. Fathi, and O. Braissant, (2011)“A comparative physico-chemical study of bioactive glass and bone-derived hydroxyapatite,” Ceram. Int., vol. 37, no. 5, pp. 1601–1607. [29] M. Figueiredo, A. Fernando, G. Martins, J. Freitas, F. Judas, and H. Figueiredo, (2010)“Effect of the calcination temperature on the composition and microstructure of hydroxyapatite derived from human and animal bone,” Ceram. Int., vol. 36, no. 8, pp. 2383–2393.     

Natural Hydroxyapatite Extracted From Bovine Bone

The aim of this study is to extract natural hydroxyapatite (HAP) from bovine bone. Hydrothermal method followed by calcination treatment at different temperatures is used in this current research. Bovine bone has potential for producing hydroxyapatite, a chief component present in bone and teeth of vertebrates. HAP is an excellent material used in bone restoration and tissue regeneration. Characterizations of the bovine bone natural HAP powder were done by scanning electron microscopy (SEM), Fourier transforms infrared (FTIR) and energy dispersive x-ray (EDX) spectroscopy. The FTIR result confirms the presence of phosphate (PO4-3), hydroxyl (OH)- and carbonate (CO3-2) groups in the powder. SEM displays the surface morphology whereas EDX provides calcium (Ca) to phosphorous (P) ratio in the HAP samples. The results of the EDX analysis indicate that the Ca/P ratio of HAP varies from 1.67 to 1.83. The extracted HAP material is found to be thermally stable up to 1100ºC

Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 
*Corresponding author: suzi@uthm.edu.my  
2017 UTHM Publisher. All right reserved. 
penerbit.uthm.edu.my/ojs/index.php/jst 
   
 
Natural Hydroxyapatite Extracted From Bovine Bone 
 
 
Nazia Bano¹, Suzi Salwah Jikan1*, Hatijah Basri1, 
Sharifah Adzila S. Abu Bakar2, Awwal Hussain Nuhu1 
 
1Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia, Johor, Malaysia 
2Faculty of Mechanical and Manufacturing Engineering, Universiti Tun Hussein Onn Malaysia, Johor, Malaysia 
 
Received 30 September 2017; accepted 14 December 2017; available online 18 December 2017 
 
 
 
 
1. Introduction 
 
HAP is one of the most attractive and 
well-known biomaterials for teeth and bone 
tissues implant. Its composition and biological 
structure resemble to natural bone. The chief 
constituent of bone mineral is hydroxyapatite 
(HAP) with a chemical formula of  
Ca10(PO4)6(OH)2 [1–4]. Natural HAP has all 
the distinctive features of biomaterials, such as 
bioactive, osteoconductive, non-inflammatory, 
biocompatible, non-toxic, and non- 
immunogenic assets. Moreover, the close 
resemblance of HAP with  calcium phosphates 
components of human bone provokes 
researchers to use it for different biomedical 
applications [5, 6]. It is considered that HAP 
plays a dynamic role for numerous 
applications [7] such as restoration of skull 
defects [8], restoration of huge area bone 
defect [9], bone tissue engineering scaffolds 
[10–12], removal of heavy metals and  drugs 
delivery [13]. In this study, bovine bones were 
used to extract natural HAP by hydrothermal 
followed by calcination at different 
temperatures. Through this extraction method, 
organic components in bones can be thermally 
decomposed and removed. Every single 
genome sign of disease can be removed and 
thus provides high biological safety factors 
[14]. Extraction of natural HAP from bovine 
femur bones is done at different calcination 
temperature and different particle sizes [15–
17]. The use of bovine bone to generate 
hydroxyapatite will decrease the pollution 
effect of the waste and the subsequent 
conversion of the waste into an extremely 
valuable product. The process of converting 
bovine bone into HAP is an environmentally 
friendly process. 
 
2.  Materials and Methods 
 
The fresh cortical bone of mature bovine 
(2-3 years old) was used as raw material 
purchase from local market of Parit Raja 
Malaysia. 
 
2.1 Bone samples preparation 
Bones were cleaned with water and soft 
parts inside the bones were manually detached 
from compact part manually by stiletto. Then 
the bones were cut into small pieces using 
cutter. The bones were boiled with water for 2 
to 3 hours. Subsequently, the fluids in bone 
Abstract: The aim of this study is to extract natural hydroxyapatite (HAP) from bovine bone. Hydrothermal 
method followed by calcination treatment at different temperatures is used in this current research. Bovine 
bone has potential for producing hydroxyapatite, a chief component present in bone and teeth of vertebrates. 
HAP is an excellent material used in bone restoration and tissue regeneration. Characterizations of the 
bovine bone natural HAP powder were done by scanning electron microscopy (SEM), Fourier transforms 
infrared (FTIR) and energy dispersive x-ray (EDX) spectroscopy. The FTIR result confirms the presence of 
phosphate (PO4-3), hydroxyl (OH)- and carbonate (CO3-2) groups in the powder. SEM displays the surface 
morphology whereas EDX provides calcium (Ca) to phosphorous (P) ratio in the HAP samples. The results 
of the EDX analysis indicate that the Ca/P ratio of HAP varies from 1.67 to 1.83. The extracted HAP 
material is found to be thermally stable up to 1100ºC. 
Keyword: Hydroxyapatite, Bovine femur bone, Hydrothermal, Calcination 
Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 
23 
 
marrow and residual soft material were 
removed with knife and washed with distilled 
water several times. The bone fragments were 
dried in an oven at 80ºC for 72 hours (3-days) 
to denaturalize the protein. After that, dry 
fragments of bone were abridged to small 
particles by means of crusher. The crushed 
bone was subjected to ball milling process for 
24 hours until the powder was fine enough. 
Bone powder was obtained by sieving. 25μm 
particle size of powdered bone sample was 
used in this study. Fig. 1 shows the flow chart 
of extraction of HAP from bovine bone. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
           
 
Fig. 1 Flow chart of extraction of HAP from 
bovine bone 
2.2 Calcination of HAP powder 
Pure natural-HAP (mineral phase of the 
bone) free from fat and protein was achieved 
by calcination. The powder samples of HAP 
were calcined in a furnace at 600ºC, 700ºC, 
800ºC, 900ºC, 1000ºC and 1100ºC, which 
named as HAP-600, HAP-700, HAP-800, 
HAP-900, HAP-1000, HAP-1000 and HAP-
1100 respectively with 3 hours holding time at 
a heating and cooling rate of 5 ºC/min. Natural 
HAP was produced by decomposition of the 
organic phase. These temperatures were 
designated after a Thermo gravimetric analysis 
(TGA). The entire bone powder samples were 
cooled inside the furnace. 
 
2.3 Characterizations of HAP powder 
 
The characterizations of HAP before and 
after calcination were performed using SEM, 
FT-IR and EDX. FTIR was used to obtain 
information about the functional groups 
present in HAP powders in the range of 4000 
cm-1 - 450 cm-1. Morphology of HAP particles 
were obtained by SEM (JSM 6490LA, Jeol, 
Japan) and the element compositions of the 
powder sample was determined by EDX. 
 
3.  Results and Discussions 
 
3.1 SEM analysis 
Fig. 2 shows the SEM micrographs of the 
HAP powder before and after calcination at 
different temperature. The particles are 
agglomerated in some parts and have irregular 
shapes including small spheres and rods as 
reported in literatures [18–20]. It is due to the 
van der Waals attraction between HAP 
particles during calcination [21]. 
 
 
Bovine cortical bone 
Ball milling for 24 h 
 
 
Oven dried at 80ºC for 72 h 
Natural HAP 
(HAP) 
Cut into small pieces 
Boiled in water for 2-3 h 
 
 
Crushing 
Calcination 
Characterizations 
 
Raw- HAP 
 
Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 
24 
 
 
 
 
 
 
 
 
Fig. 2 SEM micrographs of uncalcined  
powder sample (raw-HAP) (a) and calcined 
samples at different temperature (b) HAP-600 
(c),  HAP-700, (d) HAP-800, (e)  HAP-900,(f)  
HAP-1000 and (g)  HAP-1100 respectively.      
 
3.2 FTIR analysis 
 
Fig. 3 reveals the FTIR spectra of 
uncalcined sample (raw-HAP) and calcined 
HAP at different temperatures. The FTIR 
spectra indicate the presence of phosphate 
(PO4-3), hydroxyl (OH-) and carbonate (CO3-2) 
groups. These spectra are more clearly seen in 
calcined HAP samples because the calcination 
process has destroyed the cross linked 
structure in the raw HAP bovine bone powder. 
In the spectra  of HAP, many bands matched  
with the HAP reference spectrum and are in 
agreement with reported data on HAP [22, 23].  
 
 
Fig. 3 FTIR spectra of extracted HAP powders 
of uncalcined sample (raw-HAP) and calcined 
samples at different temperature. 
 
As shown in Fig. 3, the bands of raw HAP and 
calcined n-HAP are apparently different due to 
the alterations in their chemical bonds during 
calcination at different temperature. The color 
of raw HAP changes from yellowish white to 
Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 
25 
 
white after calcination. This color changes 
indicates the decomposition of organic 
substance which are collagen and proteins in 
calcined HAP bone powder [24, 25]. The 
FTIR band at 1637 cm-1 corresponds to Amide 
I of collagen [19] that is present in the raw 
HAP of bovine bone powder and is completely 
removed in calcined HAP samples. A strong 
and broad band due to symmetric stretching 
vibration that appears at 961.66 cm-1, 962.99 
cm-1, and asymmetric stretching vibration 
bands at 1023cm-1 and 1092cm-1   in calcined 
HAP bone samples are associated with the 
phosphate group (PO4-3). The raw HAP bone 
powder exhibit a wide band near 3285.48 cm-1 
which corresponds to the absorbed water 
molecule. Low intensity peaks of carbonate 
groups are identified in the FTIR spectra at 
1460 cm-1 and 1520 cm-1 due to asymmetric 
stretching v3 of (CO3-2) groups. The sharp 
narrow band at 630.92 cm-1, 634.47 cm-1 and 
wide band at 3525.94 cm-1 are associated with 
hydroxyl group that verify the presence of 
HAP phase. In addition, after calcination most 
of the bands have largely increased in intensity 
due to the phosphate vibrations of 
hydroxyapatite as described by other authors 
[25, 26].  
 
3.3 EDX analysis 
 
The chemical compositions of HAP 
powder before and after calcination at different 
temperatures were evaluated by using EDX 
analyzer. Fig. 4 shows the EDX analysis of 
HAP powders prepared by calcination at 
different temperatures. The Ca/ P ratios of the 
extracted HAP samples were calculated and 
the ratios are 1.79, 1.79, 1.78, 1.81, 1.67, 1.75 
and 1.83 for HAP-600, HAP-700, HAP-800, 
HAP-900, HAP-1000, HAP-11 00 for calcined 
samples and uncalcined sample (raw-HAP) 
respectively. The ratios are equivalent with 
previous study [25]. As the Ca/P weight ratio 
of the extracted HAP calcined at different 
temperatures did not show any significant 
difference, it can be concluded that Ca/P 
weight ratio does not depends on the 
calcination temperature. The higher Ca/P value 
of bovine bone derived HAP powder indicates 
that its chemical composition is close to the 
composition of human bone [27].The higher 
Ca/P values may be due to the occurrence of 
either calcium and phosphorus compounds 
other than hydroxyapatite or Ca compounds 
deprived of phosphorus [28]. In fact, 
Figueiredo  et al  articulated that natural HAP 
is non-stoichiometric having  Ca/P ratio 
greater than 1.67 [29].  
 
 
 
 
Journal of Science and Technology, Vol. 9 No. 2 (2017) p. 22-28 
26 
 
 
 
 
 
 Fig. 4 EDX analysis of HAP powders 
prepared by calcination at different 
temperature (a) HAP-600, (b) HAP-700, (c) 
HAP-800 (d) HAP-900 (e) HAP-1000 (f) 
HAP-1100 and (g) uncalcined sample (raw-
HAP) respectively.  
 
5. Conclusion 
 
The study confirms that HAP is able to be 
extracted from bovine bone wastes.  So, 
bovine bone wastes have great potential to be 
used in extracting the HAP deprived of any 
impurities as indicated by FTIR analysis. The 
methods involved hydrothermal followed by 
calcination of bovine bone powder at different 
temperature to remove its organic components 
thus the produced HAP which is suitable for 
the use in orthopedic and dental applications. 
Powder samples of HAP of bovine bone have 
close Ca/P molar ratio (1.67 - 1.83) to the 
stoichiometric HAP (1.67). The HAP 
extraction methodology is simple and low 
cost. 
 
Acknowledgements 
 
This study was partially supported by Center 
of Graduate Studies (CGS), Universiti Tun 
Hussein Onn Malaysia from the UTHM 
scholarship (UTHM/PS/500-14/8).   
 
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Natural Hydroxyapatite Extracted From Bovine Bone

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