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Investigation of mechanical properties of natural hydroxyapatite samples prepared by cold isostatic pressing method
Accepted Manuscript Investigation of mechanical properties of natural hydroxyapatite samples prepared by cold isostatic pressing method Fatemeh Heidari, Mehdi Razavi, Mehrorang Ghaedi, Maryam Forooghi, Mohammadreza Tahriri, Lobat Tayebi PII:
S0925-8388(16)33189-9
DOI:
10.1016/j.jallcom.2016.10.081
Reference:
JALCOM 39248
To appear in:
Journal of Alloys and Compounds
Received Date: 19 August 2016 Revised Date:
3 October 2016
Accepted Date: 10 October 2016
Please cite this article as: F. Heidari, M. Razavi, M. Ghaedi, M. Forooghi, M. Tahriri, L. Tayebi, Investigation of mechanical properties of natural hydroxyapatite samples prepared by cold isostatic pressing method, Journal of Alloys and Compounds (2016), doi: 10.1016/j.jallcom.2016.10.081. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Investigation of mechanical properties of natural hydroxyapatite samples prepared by cold isostatic pressing method
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Fatemeh Heidari1, Mehdi Razavi2,3, Mehrorang Ghaedi4, Maryam Forooghi1, Mohammadreza Tahriri5, Lobat Tayebi5,6* 1
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Department of Materials Science and Engineering, School of Engineering, Yasouj University, Yasuj, 75918-74934, Iran 2 BCAST, Institute of Materials and Manufacturing, Brunel University London, Uxbridge, London UB8 3PH, UK 3 Brunel Institute for Bioengineering, Brunel University London, Uxbridge, London UB8 3PH, UK 4 Department of Chemistry, School of Basic science, Yasouj University, Yasuj, 75918-74934, Iran 5 Marquette University School of Dentistry, Milwaukee, WI, 53233, USA Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, UK
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* Corresponding Author:
Abstract
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[email protected]
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Hydroxyapatite (HA) has been broadly employed for bone treatment applications. In the present research, HA was extracted from hen, goat and cattle bone. Samples were prepared by cold isostatic pressing method (CIP) and sintered at 1100, 1200 and 1300 °C. In order to evaluate the characteristics of the produced HA, X-ray diffraction (XRD), X-ray fluorescence (XRF), Fourier Transform spectroscopy (FTIR), scanning electron microcopy (SEM) and energy dispersive spectroscopy (EDS) analyses were carried out on it. Hardness and compression tests were examined before and after sintering. Also, the morphology of 1
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the samples at different sintering temperatures was studied by SEM imaging. The obtained experimental results ascertained that with increasing the sintering temperature up to 1300 °C, relative density and hardness of the samples increased. Finally, compressive strength of
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the sintered sample at 1200 °C was determined as maximum value.
Keywords: Hydroxyapatite, Cold isostatic press, Sintering, Mechanical properties
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1. Introduction
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Hydroxyapatite (HA) with the chemical formula of Ca10(PO4)6(OH)2 similar to the bone and teeth mineral structure has been widely used for hard tissue repair as a highly biocompatible biomaterial (1-12). Synthesizing the HA has been carried out by chemical processes and extraction from natural sources such as bovine bone (13), fish bone (14), coral (15) and egg shells (16-18). HA extracted from natural materials is different from synthetic
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type. Natural HA (NHA) has carbonate, citrate groups and usually a little magnesium, potassium, strontium and sodium in its chemical structure (19). The ratio of calcium to
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phosphorus in NHA is usually more than that of the synthetic state. Due to the all mentioned differences, NHA is more appropriate for medical applications (20). By converting cow bone
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ash to HA, up to about 1 kg HA has acquired from 1.6 kg of cortical bone (13). Studies show that the sintered density of HA without pressure is below 80%. Hot-
pressing is able to achieve densification at much lower temperatures than pressureless sintering (21). One method of forming hydroxyapatite powder is cold isostatic pressing method. So far, sintering after shaping has been done at 900 to 1350°C (22). In order to improve the uniformity in density and obtain ceramics with homogeneous microstructure,
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CIP is worth to be used due to its isotropic pressure (23). Also it has been used to manufacture the personalized traditional ceramic products with fewer pores (24). This paper compares the production efficiency of HA synthesized from hens, goats
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and cattle. The highest efficiency of the HA powders was selected as an optimum sample and then cold isostatic pressed and sintered at 1100, 1200 and 1300 °C. Finally, we
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evaluated the mechanical properties of the sintered NHA.
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2. Materials and methods 2.1. Material preparation
Three animals bone including cattle, goat and hen was provided. HA preparation method from the bones was performed in accordance with Heidari et al. (25). Briefly, bones
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were boiled in water to remove the meat and bones fat. Then, they were burned in the air by direct flame so that organic materials of bones were removed and converted to coal. Among three types of bones used in this research including cattle, goat and hen, fully
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burning of bovine cortical bone took more time (approximately 2.5 h) than hen (20 min) and goat (1 h) bones. The produced coals were converted to powder by quern. Three types of
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powders were heated in furnace at 800 °C for 2 h. Finally, after this process, black powders were changed to the white powders (25). Samples were cold isostatic pressed at 250 MPa in the form of circular disc (16 mm
diameter and 4 mm height) for hardness test and cylinder with 10 mm diameter and 10 mm height for compression test. The sintering was conducted in air atmosphere at different
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temperatures varying from 1100 to 1300 °C for 2.5 h in a box furnace at a heating rate of 5 °C/min.
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2.2. Weight changes
To determine the weight changes of bone char when it is converted to HA, char powder was weighted at ambient temperature and after heat treatment in furnace at
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800 °C. The percentage of weight changes was evaluated according to Eq. 1:
(1)
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ΔW=(W2-W1)/W1*100
Where W2 and W1 are weight of the samples after and before sintering.
2.3. XRD analysis
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To investigate the phase composition of extracted HA powder, X ray diffraction (XRD: Bruker-AXS-D8-Discover) analysis was carried out before and after sintering the HA. This system works with voltage and current setting of 40 kV and 40 mA, respectively with
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parallel graphite monochromator and uses CuKα radiation (1.5406 Å). For qualitative
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analysis, XRD diagrams were recorded in the interval 20 to 70° with a step size of 0.02°.
2.4. FTIR analysis
To identify the existence of organic species and also the degree of probable
dehydroxylation of HA during the heat treatment, Fourier transform infrared (FTIR: Shimadzu 8300) analysis was performed. The measurements were carried out in the transmission mode in the mid-infrared range with wave numbers from 400 cm−1 to 4000
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2.5. XRF analysis
A Philips, PW 2400 X-ray fluorescence spectrometer was used to obtain elemental chemical composition of the extracted powder. In order to prepare samples for XRF analysis,
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the powder was poured into a special die and compacted in a pressing machine with 8
thickness.
2.6. Scanning electron microscopy
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tonnes load to prepare a disk shaped specimen with 30 mm in diameter and 5 mm in
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HA powder was milled by zirconia cup and balls. The weight ratio of powder to balls was 1:10 and balls diameter was 10 mm. In order to prevent sticking of powder to balls and their agglomeration, 0.2 wt% steareic acid was added to balls and powder blend. Extracted
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HA was milled 4 h with 300 rpm rotational speed. Finally, a scanning electron microscope equipped with energy dispersive spectroscopy (EDS) was used to examine the morphology of
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the powder and prepared samples before and after sintering and also the existed elements in the produced HA.
2.7. Mechanical testing Mechanical characteristics of the samples was evaluated with two tests including compressive and hardness tests. The compressive tests were performed on cylindrical 5
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samples (10 mm in diameter and 10 mm in length) using a universal testing machine (zwick, material prufung, 1446–60) with 10 kN load cell. The crosshead speed was 0.5 mm/min. Disc shape samples was selected for microhardness test (16 mm diameter and 4 mm height). The
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microhardness (Hv) of the polished sintered samples was determined via the Vickers indentation (MHV1000Z) using an applied load of 200g with a dwell time of 10s. The surface area under the stress-strain curves were calculated and considered as the fracture
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toughness of the samples.
3. Results and discussion
3.1. Extraction of hydroxyapatite from bone
The char which had been produced from burning of bones has black color (see Fig.
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1a). This black color changes to white after heating at 800 °C for 2.5 h (see Fig. 1b). This phenomenon shows that organic material in char has been removed at this heating temperature. Fig. 1c shows SEM micrograph of the HA particles and according to this figure,
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the size of the particles is under 500 nm and their shape is spherical. Finally, EDS graph is depicted in Fig. 1d and this analysis confirms the presence of the Ca, P, O and C elements in
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the prepared hydroxyapatite.
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Figure 1. (a) Bone ash after burning in air, (b) Bone ash after heating in furnace at 800 °C for 2.5 h, (c) SEM micrograph of HA after milling and (d) SEM-EDX analysis of HA powder
3.2. Weight changes
All three types of the HA powders were weighted before entering to and after
existing from furnace. Weight changes percentage of the powder has been shown in Fig. 2 for HA extracted from bovine, goat and hen.
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Bovine Goat Hen
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in
e
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∆W%
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Figure 2. Weight changes percent of the three type of HA powders from ambient temperature to 800 °C
As shown in Fig. 2, ΔW% of the HA extracted from bovine bone is the lowest which indicated efficiency of producing HA from cortical bovine bone was more than goat and hen.
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For this reason, in this research, HA extracted from bovine bone was selected as an optimum sample for further studies due to the highest efficiency.
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3.3. XRD analysis
Fig. 3 shows the XRD pattern of the HA extracted from bovine cortical bone (a) and
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XRD patterns of isostatic pressed and sintered at 1100°C (b), 1200°C (c) and 1300°C (d). The XRD pattern of these samples shows the following broad diffraction peaks at
26.172, 28.25, 29.1, 31.9, 32.35, 33, 34.2, 39.96, 46.85, 48.25, 50.65, 51.4, 52.35, 63.1 and 53.38°. All reflections are characteristics of the hexagonal phase of hydroxyapatite [Ca10(PO4)6(OH)2] according to the standard data (JCPDS No. 74-0566) (26). The obtained patterns are in agreement with Kim et al. (21) and Heidari et al. (25, 27).
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These mentioned angles are observed in the XRD pattern confirming that the powder extracted from cortical bovine bone is HA (see Fig. 3). Cuccu et al. (28) and Farzin et al. (29) indicated that the appearance of the new peaks in the XRD patterns in different heat
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temperature can related to β-TCP and α-TCP phases (28). Whereas Figs. 3 b-d do not show any new peaks in the XRD patterns. Therefore, Fig. 3b-d displays that sintering at 1100°C, 1200°C and 1300°C has not caused the conversion of HA to TCP phases.
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This observation is not consistent with other published papers, which have described
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that decomposition of HA starts at about 1200/1300°C (30-32). The difference in result in the present study could in part be ascribed to the relatively high humidity content present in the sintering atmosphere and the nature of the prepared powder. It is reported that the high humidity content slows down decomposition rate by forestalling dehydration of the OH
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group from the HA lattice (33).
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Figure 3. XRD pattern of (a) Bone ash powder which was heat treated at 800°C for 2 h, (b) Cold isostatic pressed and sintered HA at 1100°C, (c) 1200°C and (d) 1300°C
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Decreasing the intensity in Fig. 3 b-d can be due to undeveloped grain growth in the samples sintered at 1100°C, 1200°C and 1300°C (34).
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3.4. FTIR analysis
The FTIR spectrum of the sample which was heat treated at 800 °C is in good agreement with the spectrum reported by Bahrololoom et al. (13) for natural HA material
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(see Fig. 4). In this figure, the stretching band at 3419.17 cm−1 and libration band at
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632.54 cm−1 originate from OH− groups. The bands located at 470.54, 570.83, 601.7, 1051, and 1091.5 cm−1 originate by PO43- ions is weaker than the strong P-O stretching vibration due to hydroxyapatite stoichiometry. The bands at 1415.7, and 1459.85 cm−1 originate from CO32- ions. Carbonate ions are a common impurity in both synthetic and natural
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hydroxyapatite (35). The intensity of the O-H stretching vibration in Frequency bands
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observed at 2365 cm−1 is characteristics of free CO2 (22).
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OHCO2
CO32-
OH-
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PO43-
OH-
PO43-
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PO43-
PO43-
PO43-
3.5. XRF analysis
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Figure 4. Fourier transforms infrared analysis of bone ash which was treated at 800°C for 2 h
To examine the HA elements, XRF analysis was utilized. The chemical composition of
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the HA in oxide form is shown in Table 1. As seen, calcium and phosphorous are the main
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components. Also, magnesium element and some traces of aluminum (Al), silicon (Si), sulfur (S), chlorine (Cl), zinc (Zn) and strontium (Sr) are observed. The natural trace element available in the natural HA (i.e. K, Na, Mg, Si and Sr) were incorporated in the crystalline structure of synthesized HA makes the material more biocompatible to human bone (36, 37). As seen in Table 1, the concentrations of CaO and P2O5 are 51.51 wt.% and 44.68 wt.%, respectively.
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The elemental chemical analysis of the natural hydroxyapatite extracted from bovine bone by the Merck Chemicals (Endobon) has also been reported by Joschek et al. (37) and their investigation resulted in identifying numerous elements. This multitude of elements is
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not astonishing to the well accepted since ion exchange can take place in the apatite component of bone. The ionic components of hydroxyapatite, i.e. Ca2+, OH− and PO43−can be readily exchanged by other ions. It is obvious that the composition of the trace elements
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varies considerably in bone depending on some biological factors such as nutrition, sexuality,
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type and age of animal (38). That’s reason why the amount of Mg and Sr elements in the research of Bahrololoom (13) and Haberko (36) was different with our research.
Table 1. Composition of the NHA, heat treated at 800 °C, in oxide form obtained from XRF analysis
Concentration (wt%)
Na2O
<0.1
MgO
1.63
Al2O3
0.24
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Compounds
SiO2
0.49
P2O5
44.68
SO3
0.25
CaO
51.51
ZnO
<0.05
SrO
0.13
Cl
<0.05
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3.6. SEM observations Morphology of the samples after sintering at 1100°C, 1200°C and 1300°C is shown in Fig. 5. Since, the isostatic pressure required for the preparation of all samples was 250 MPa
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and initial condition of the HA powder was the same for all of the samples so that morphological changes are due to the changes that occur in sintering temperature. Fig. 5 indicate a more densely packed microstructure with increasing sintering temperature from Hydroxyapatite particles also joint together with increasing
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1100 up to 1300 °C.
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temperature so that interface between particles is not detectable. Also, Fig. 5a shows the re-crystallization process from melt phase. Grains are fine but they do not joint together. Fig. 5b shows grain growth of re-crystalized phase and porosities are trapped between large
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grains. Fig. 5c shows that grain growth as well and pore density and pore size has decreased.
Figure 5. SEM micrographs of the natural HA sintered at (a) 1100 °C, (b) 1200 °C, and (c) 1300 °C
3.7. Mechanical properties The mechanical properties of the HA were evaluated in terms of bulk density, hardness, compressive strength and toughness as a function of sintering temperature as shown in Fig. 6. Fig. 6a shows the size of the samples has decreased with increasing the 14
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sintering temperature. A steady increase in density was observed with an increase in sintering temperature up to 1300 °C (Fig. 6 (c)). Confirming well with a densely packed microstructure of sintered HA with increasing sintering temperature as can be observed in
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Fig. 5. The relative density increased from 12.44% at 1100 °C to a maximum of 30.3% at 1300
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°C.
Figure 6. (a) Disk samples for micro-hardness tests, (b) Cylindrical samples for compressive tests. The effect of sintering temperature on the (c) relative density, (d) Vickers hardness and compressive strength and (e) fracture toughness of the natural HA (NHA)
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Fig. 6d shows the microhardness of the sintered natural HA. The hardness increased with increasing the sintering temperature and the maximum hardness was 3.7 GPa at sintering temperature of 1300 °C. In fact, hardness increased with decreasing pores size
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(see Fig. 5 b and c).
Ramesh et al. (22) studied on the HA extracted from eggshells. The maximum Vickers hardness which they measured was 5.62 GPa at sintering temperature of 1250 °C.
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Wang et al. (39) worked on the nanocrystalline HA and they concluded that the
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hardness of HA increases with decreasing grain size. Also, they found the hardness of HA with grain size of 67 nm was 5.1 GPa while with grain size of 625 nm was 4.3 GPa. Khandan et al. (40) worked on the natural HA extracted from bovine bone and concluded the microhardness of the natural HA with the average size of 40 nm at sintering
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temperature of 1350°C is 2.652 GPa. In our study, the average size of the HA grains was about 500 nm, but with isostatic pressure of 250 MPa for sample preparation, the hardness of the samples was more than the value that has been reported by other researchers.
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Fig. 6d shows that the maximum compressive strength is 0.44 MPa for sintered sample at 1200 °C. Compressive strength for sintered sample at 1300 ° C was lower than that
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of the 1200 °C. Since this sample was harder, it was more brittle than the sample which had been sintered at 1200 °C. As shown in Fig. 5a, the grains are fine but they haven’t been jointed together. Therefore, the sample which was sintered at 1100 °C had lesser compressive strength compared with other samples. Shariful Islam et al. (41) investigated the compressive strength of the synthetic HA and compressive strength of it was lesser than 0.05 MPa at sintering temperatures of 1100 and 1200 °C. Adding the bio-glass and chitosan 16
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to HA can increase its compressive strength as has been observed in other published papers (27, 42). Fig. 6e shows the fracture toughness of the sintered natural HA. As it can be
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observed in this figure, the maximum fracture toughness is 0.59 MPa for sintered sample at 1200 °C. Fracture toughness for sintered sample at 1300 ° C was lower than that of the 1200
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4. Conclusion
In this research, HA was extracted from hen, goat and cattle bone and then was cold isostatic pressed and sintered at 1100, 1200 and 1300 °C. The efficiency of producing of HA extracted from cattle was more than hen and goat. The relative density and hardness
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increased with increasing the sintering temperature and also compressive strength was maximum for the sample sintered at 1200 °C.
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5. Acknowledgement
The authors are thankful for the support from the Research Committee of Yasouj
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University (Grant No. GRYU-89111604 to F. Heidari).
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35. Markovic M, Fowler BO, Tung MS. Preparation and comprehensive characterization of a calcium hydroxyapatite reference material. Journal of research of the National Institute of Standards and Technology. 2004;109(6):553. 36. Haberko K, Bućko MM, Brzezińska-Miecznik J, Haberko M, Mozgawa W, Panz T, et al. Natural hydroxyapatite—its behaviour during heat treatment. Journal of the European Ceramic Society. 2006;26(4):537-42. 37. Joschek S, Nies B, Krotz R, Göpferich A. Chemical and physicochemical characterization of porous hydroxyapatite ceramics made of natural bone. Biomaterials. 2000;21(16):1645-58. 38. Göller G, Oktar FN, Agathopoulos S, Tulyaganov D, Ferreira JMF, Kayali E, et al., editors. The influence of sintering temperature on mechanical and microstructural properties of bovine hydroxyapatite. Key Engineering Materials; 2005: Trans Tech Publ. 39. Wang J, Shaw LL. Nanocrystalline hydroxyapatite with simultaneous enhancements in hardness and toughness. Biomaterials. 2009;30(34):6565-72. 40. Khandan A, Abdellahi M, Ozada N, Ghayour H. Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating. Journal of the Taiwan Institute of Chemical Engineers. 2016;60:538-46. 41. Islam MS, Todo M. Effects of sintering temperature on the compressive mechanical properties of collagen/hydroxyapatite composite scaffolds for bone tissue engineering. Materials Letters. 2016;173:231-4. 42. Ghomi H, Fathi M, Edris H. Effect of the composition of hydroxyapatite/bioactive glass nanocomposite foams on their bioactivity and mechanical properties. Materials Research Bulletin. 2012;47(11):3523-32.
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Highlights:
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The efficiency of producing of HA extracted from cattle was more than hen and goat. With increasing the sintering temperature up to 1300 °C, relative density and hardness of the samples increased. Compressive strength was maximum for the sample that sintered at 1200 °C
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S0925-8388(16)33189-9
DOI:
10.1016/j.jallcom.2016.10.081
Reference:
JALCOM 39248
To appear in:
Journal of Alloys and Compounds
Received Date: 19 August 2016 Revised Date:
3 October 2016
Accepted Date: 10 October 2016
Please cite this article as: F. Heidari, M. Razavi, M. Ghaedi, M. Forooghi, M. Tahriri, L. Tayebi, Investigation of mechanical properties of natural hydroxyapatite samples prepared by cold isostatic pressing method, Journal of Alloys and Compounds (2016), doi: 10.1016/j.jallcom.2016.10.081. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Investigation of mechanical properties of natural hydroxyapatite samples prepared by cold isostatic pressing method
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Fatemeh Heidari1, Mehdi Razavi2,3, Mehrorang Ghaedi4, Maryam Forooghi1, Mohammadreza Tahriri5, Lobat Tayebi5,6* 1
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Department of Materials Science and Engineering, School of Engineering, Yasouj University, Yasuj, 75918-74934, Iran 2 BCAST, Institute of Materials and Manufacturing, Brunel University London, Uxbridge, London UB8 3PH, UK 3 Brunel Institute for Bioengineering, Brunel University London, Uxbridge, London UB8 3PH, UK 4 Department of Chemistry, School of Basic science, Yasouj University, Yasuj, 75918-74934, Iran 5 Marquette University School of Dentistry, Milwaukee, WI, 53233, USA Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, UK
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* Corresponding Author:
Abstract
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[email protected]
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Hydroxyapatite (HA) has been broadly employed for bone treatment applications. In the present research, HA was extracted from hen, goat and cattle bone. Samples were prepared by cold isostatic pressing method (CIP) and sintered at 1100, 1200 and 1300 °C. In order to evaluate the characteristics of the produced HA, X-ray diffraction (XRD), X-ray fluorescence (XRF), Fourier Transform spectroscopy (FTIR), scanning electron microcopy (SEM) and energy dispersive spectroscopy (EDS) analyses were carried out on it. Hardness and compression tests were examined before and after sintering. Also, the morphology of 1
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the samples at different sintering temperatures was studied by SEM imaging. The obtained experimental results ascertained that with increasing the sintering temperature up to 1300 °C, relative density and hardness of the samples increased. Finally, compressive strength of
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the sintered sample at 1200 °C was determined as maximum value.
Keywords: Hydroxyapatite, Cold isostatic press, Sintering, Mechanical properties
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1. Introduction
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Hydroxyapatite (HA) with the chemical formula of Ca10(PO4)6(OH)2 similar to the bone and teeth mineral structure has been widely used for hard tissue repair as a highly biocompatible biomaterial (1-12). Synthesizing the HA has been carried out by chemical processes and extraction from natural sources such as bovine bone (13), fish bone (14), coral (15) and egg shells (16-18). HA extracted from natural materials is different from synthetic
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type. Natural HA (NHA) has carbonate, citrate groups and usually a little magnesium, potassium, strontium and sodium in its chemical structure (19). The ratio of calcium to
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phosphorus in NHA is usually more than that of the synthetic state. Due to the all mentioned differences, NHA is more appropriate for medical applications (20). By converting cow bone
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ash to HA, up to about 1 kg HA has acquired from 1.6 kg of cortical bone (13). Studies show that the sintered density of HA without pressure is below 80%. Hot-
pressing is able to achieve densification at much lower temperatures than pressureless sintering (21). One method of forming hydroxyapatite powder is cold isostatic pressing method. So far, sintering after shaping has been done at 900 to 1350°C (22). In order to improve the uniformity in density and obtain ceramics with homogeneous microstructure,
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CIP is worth to be used due to its isotropic pressure (23). Also it has been used to manufacture the personalized traditional ceramic products with fewer pores (24). This paper compares the production efficiency of HA synthesized from hens, goats
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and cattle. The highest efficiency of the HA powders was selected as an optimum sample and then cold isostatic pressed and sintered at 1100, 1200 and 1300 °C. Finally, we
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evaluated the mechanical properties of the sintered NHA.
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2. Materials and methods 2.1. Material preparation
Three animals bone including cattle, goat and hen was provided. HA preparation method from the bones was performed in accordance with Heidari et al. (25). Briefly, bones
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were boiled in water to remove the meat and bones fat. Then, they were burned in the air by direct flame so that organic materials of bones were removed and converted to coal. Among three types of bones used in this research including cattle, goat and hen, fully
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burning of bovine cortical bone took more time (approximately 2.5 h) than hen (20 min) and goat (1 h) bones. The produced coals were converted to powder by quern. Three types of
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powders were heated in furnace at 800 °C for 2 h. Finally, after this process, black powders were changed to the white powders (25). Samples were cold isostatic pressed at 250 MPa in the form of circular disc (16 mm
diameter and 4 mm height) for hardness test and cylinder with 10 mm diameter and 10 mm height for compression test. The sintering was conducted in air atmosphere at different
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temperatures varying from 1100 to 1300 °C for 2.5 h in a box furnace at a heating rate of 5 °C/min.
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2.2. Weight changes
To determine the weight changes of bone char when it is converted to HA, char powder was weighted at ambient temperature and after heat treatment in furnace at
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800 °C. The percentage of weight changes was evaluated according to Eq. 1:
(1)
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ΔW=(W2-W1)/W1*100
Where W2 and W1 are weight of the samples after and before sintering.
2.3. XRD analysis
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To investigate the phase composition of extracted HA powder, X ray diffraction (XRD: Bruker-AXS-D8-Discover) analysis was carried out before and after sintering the HA. This system works with voltage and current setting of 40 kV and 40 mA, respectively with
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parallel graphite monochromator and uses CuKα radiation (1.5406 Å). For qualitative
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analysis, XRD diagrams were recorded in the interval 20 to 70° with a step size of 0.02°.
2.4. FTIR analysis
To identify the existence of organic species and also the degree of probable
dehydroxylation of HA during the heat treatment, Fourier transform infrared (FTIR: Shimadzu 8300) analysis was performed. The measurements were carried out in the transmission mode in the mid-infrared range with wave numbers from 400 cm−1 to 4000
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2.5. XRF analysis
A Philips, PW 2400 X-ray fluorescence spectrometer was used to obtain elemental chemical composition of the extracted powder. In order to prepare samples for XRF analysis,
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the powder was poured into a special die and compacted in a pressing machine with 8
thickness.
2.6. Scanning electron microscopy
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tonnes load to prepare a disk shaped specimen with 30 mm in diameter and 5 mm in
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HA powder was milled by zirconia cup and balls. The weight ratio of powder to balls was 1:10 and balls diameter was 10 mm. In order to prevent sticking of powder to balls and their agglomeration, 0.2 wt% steareic acid was added to balls and powder blend. Extracted
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HA was milled 4 h with 300 rpm rotational speed. Finally, a scanning electron microscope equipped with energy dispersive spectroscopy (EDS) was used to examine the morphology of
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the powder and prepared samples before and after sintering and also the existed elements in the produced HA.
2.7. Mechanical testing Mechanical characteristics of the samples was evaluated with two tests including compressive and hardness tests. The compressive tests were performed on cylindrical 5
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samples (10 mm in diameter and 10 mm in length) using a universal testing machine (zwick, material prufung, 1446–60) with 10 kN load cell. The crosshead speed was 0.5 mm/min. Disc shape samples was selected for microhardness test (16 mm diameter and 4 mm height). The
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microhardness (Hv) of the polished sintered samples was determined via the Vickers indentation (MHV1000Z) using an applied load of 200g with a dwell time of 10s. The surface area under the stress-strain curves were calculated and considered as the fracture
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toughness of the samples.
3. Results and discussion
3.1. Extraction of hydroxyapatite from bone
The char which had been produced from burning of bones has black color (see Fig.
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1a). This black color changes to white after heating at 800 °C for 2.5 h (see Fig. 1b). This phenomenon shows that organic material in char has been removed at this heating temperature. Fig. 1c shows SEM micrograph of the HA particles and according to this figure,
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the size of the particles is under 500 nm and their shape is spherical. Finally, EDS graph is depicted in Fig. 1d and this analysis confirms the presence of the Ca, P, O and C elements in
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the prepared hydroxyapatite.
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Figure 1. (a) Bone ash after burning in air, (b) Bone ash after heating in furnace at 800 °C for 2.5 h, (c) SEM micrograph of HA after milling and (d) SEM-EDX analysis of HA powder
3.2. Weight changes
All three types of the HA powders were weighted before entering to and after
existing from furnace. Weight changes percentage of the powder has been shown in Fig. 2 for HA extracted from bovine, goat and hen.
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Bovine Goat Hen
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t oa
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in
e
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∆W%
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Figure 2. Weight changes percent of the three type of HA powders from ambient temperature to 800 °C
As shown in Fig. 2, ΔW% of the HA extracted from bovine bone is the lowest which indicated efficiency of producing HA from cortical bovine bone was more than goat and hen.
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For this reason, in this research, HA extracted from bovine bone was selected as an optimum sample for further studies due to the highest efficiency.
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3.3. XRD analysis
Fig. 3 shows the XRD pattern of the HA extracted from bovine cortical bone (a) and
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XRD patterns of isostatic pressed and sintered at 1100°C (b), 1200°C (c) and 1300°C (d). The XRD pattern of these samples shows the following broad diffraction peaks at
26.172, 28.25, 29.1, 31.9, 32.35, 33, 34.2, 39.96, 46.85, 48.25, 50.65, 51.4, 52.35, 63.1 and 53.38°. All reflections are characteristics of the hexagonal phase of hydroxyapatite [Ca10(PO4)6(OH)2] according to the standard data (JCPDS No. 74-0566) (26). The obtained patterns are in agreement with Kim et al. (21) and Heidari et al. (25, 27).
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These mentioned angles are observed in the XRD pattern confirming that the powder extracted from cortical bovine bone is HA (see Fig. 3). Cuccu et al. (28) and Farzin et al. (29) indicated that the appearance of the new peaks in the XRD patterns in different heat
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temperature can related to β-TCP and α-TCP phases (28). Whereas Figs. 3 b-d do not show any new peaks in the XRD patterns. Therefore, Fig. 3b-d displays that sintering at 1100°C, 1200°C and 1300°C has not caused the conversion of HA to TCP phases.
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This observation is not consistent with other published papers, which have described
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that decomposition of HA starts at about 1200/1300°C (30-32). The difference in result in the present study could in part be ascribed to the relatively high humidity content present in the sintering atmosphere and the nature of the prepared powder. It is reported that the high humidity content slows down decomposition rate by forestalling dehydration of the OH
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group from the HA lattice (33).
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Figure 3. XRD pattern of (a) Bone ash powder which was heat treated at 800°C for 2 h, (b) Cold isostatic pressed and sintered HA at 1100°C, (c) 1200°C and (d) 1300°C
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Decreasing the intensity in Fig. 3 b-d can be due to undeveloped grain growth in the samples sintered at 1100°C, 1200°C and 1300°C (34).
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3.4. FTIR analysis
The FTIR spectrum of the sample which was heat treated at 800 °C is in good agreement with the spectrum reported by Bahrololoom et al. (13) for natural HA material
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(see Fig. 4). In this figure, the stretching band at 3419.17 cm−1 and libration band at
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632.54 cm−1 originate from OH− groups. The bands located at 470.54, 570.83, 601.7, 1051, and 1091.5 cm−1 originate by PO43- ions is weaker than the strong P-O stretching vibration due to hydroxyapatite stoichiometry. The bands at 1415.7, and 1459.85 cm−1 originate from CO32- ions. Carbonate ions are a common impurity in both synthetic and natural
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hydroxyapatite (35). The intensity of the O-H stretching vibration in Frequency bands
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observed at 2365 cm−1 is characteristics of free CO2 (22).
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OHCO2
CO32-
OH-
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PO43-
OH-
PO43-
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PO43-
PO43-
PO43-
3.5. XRF analysis
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Figure 4. Fourier transforms infrared analysis of bone ash which was treated at 800°C for 2 h
To examine the HA elements, XRF analysis was utilized. The chemical composition of
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the HA in oxide form is shown in Table 1. As seen, calcium and phosphorous are the main
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components. Also, magnesium element and some traces of aluminum (Al), silicon (Si), sulfur (S), chlorine (Cl), zinc (Zn) and strontium (Sr) are observed. The natural trace element available in the natural HA (i.e. K, Na, Mg, Si and Sr) were incorporated in the crystalline structure of synthesized HA makes the material more biocompatible to human bone (36, 37). As seen in Table 1, the concentrations of CaO and P2O5 are 51.51 wt.% and 44.68 wt.%, respectively.
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The elemental chemical analysis of the natural hydroxyapatite extracted from bovine bone by the Merck Chemicals (Endobon) has also been reported by Joschek et al. (37) and their investigation resulted in identifying numerous elements. This multitude of elements is
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not astonishing to the well accepted since ion exchange can take place in the apatite component of bone. The ionic components of hydroxyapatite, i.e. Ca2+, OH− and PO43−can be readily exchanged by other ions. It is obvious that the composition of the trace elements
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varies considerably in bone depending on some biological factors such as nutrition, sexuality,
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type and age of animal (38). That’s reason why the amount of Mg and Sr elements in the research of Bahrololoom (13) and Haberko (36) was different with our research.
Table 1. Composition of the NHA, heat treated at 800 °C, in oxide form obtained from XRF analysis
Concentration (wt%)
Na2O
<0.1
MgO
1.63
Al2O3
0.24
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Compounds
SiO2
0.49
P2O5
44.68
SO3
0.25
CaO
51.51
ZnO
<0.05
SrO
0.13
Cl
<0.05
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3.6. SEM observations Morphology of the samples after sintering at 1100°C, 1200°C and 1300°C is shown in Fig. 5. Since, the isostatic pressure required for the preparation of all samples was 250 MPa
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and initial condition of the HA powder was the same for all of the samples so that morphological changes are due to the changes that occur in sintering temperature. Fig. 5 indicate a more densely packed microstructure with increasing sintering temperature from Hydroxyapatite particles also joint together with increasing
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1100 up to 1300 °C.
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temperature so that interface between particles is not detectable. Also, Fig. 5a shows the re-crystallization process from melt phase. Grains are fine but they do not joint together. Fig. 5b shows grain growth of re-crystalized phase and porosities are trapped between large
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grains. Fig. 5c shows that grain growth as well and pore density and pore size has decreased.
Figure 5. SEM micrographs of the natural HA sintered at (a) 1100 °C, (b) 1200 °C, and (c) 1300 °C
3.7. Mechanical properties The mechanical properties of the HA were evaluated in terms of bulk density, hardness, compressive strength and toughness as a function of sintering temperature as shown in Fig. 6. Fig. 6a shows the size of the samples has decreased with increasing the 14
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sintering temperature. A steady increase in density was observed with an increase in sintering temperature up to 1300 °C (Fig. 6 (c)). Confirming well with a densely packed microstructure of sintered HA with increasing sintering temperature as can be observed in
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Fig. 5. The relative density increased from 12.44% at 1100 °C to a maximum of 30.3% at 1300
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°C.
Figure 6. (a) Disk samples for micro-hardness tests, (b) Cylindrical samples for compressive tests. The effect of sintering temperature on the (c) relative density, (d) Vickers hardness and compressive strength and (e) fracture toughness of the natural HA (NHA)
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Fig. 6d shows the microhardness of the sintered natural HA. The hardness increased with increasing the sintering temperature and the maximum hardness was 3.7 GPa at sintering temperature of 1300 °C. In fact, hardness increased with decreasing pores size
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(see Fig. 5 b and c).
Ramesh et al. (22) studied on the HA extracted from eggshells. The maximum Vickers hardness which they measured was 5.62 GPa at sintering temperature of 1250 °C.
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Wang et al. (39) worked on the nanocrystalline HA and they concluded that the
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hardness of HA increases with decreasing grain size. Also, they found the hardness of HA with grain size of 67 nm was 5.1 GPa while with grain size of 625 nm was 4.3 GPa. Khandan et al. (40) worked on the natural HA extracted from bovine bone and concluded the microhardness of the natural HA with the average size of 40 nm at sintering
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temperature of 1350°C is 2.652 GPa. In our study, the average size of the HA grains was about 500 nm, but with isostatic pressure of 250 MPa for sample preparation, the hardness of the samples was more than the value that has been reported by other researchers.
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Fig. 6d shows that the maximum compressive strength is 0.44 MPa for sintered sample at 1200 °C. Compressive strength for sintered sample at 1300 ° C was lower than that
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of the 1200 °C. Since this sample was harder, it was more brittle than the sample which had been sintered at 1200 °C. As shown in Fig. 5a, the grains are fine but they haven’t been jointed together. Therefore, the sample which was sintered at 1100 °C had lesser compressive strength compared with other samples. Shariful Islam et al. (41) investigated the compressive strength of the synthetic HA and compressive strength of it was lesser than 0.05 MPa at sintering temperatures of 1100 and 1200 °C. Adding the bio-glass and chitosan 16
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to HA can increase its compressive strength as has been observed in other published papers (27, 42). Fig. 6e shows the fracture toughness of the sintered natural HA. As it can be
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observed in this figure, the maximum fracture toughness is 0.59 MPa for sintered sample at 1200 °C. Fracture toughness for sintered sample at 1300 ° C was lower than that of the 1200
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4. Conclusion
In this research, HA was extracted from hen, goat and cattle bone and then was cold isostatic pressed and sintered at 1100, 1200 and 1300 °C. The efficiency of producing of HA extracted from cattle was more than hen and goat. The relative density and hardness
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increased with increasing the sintering temperature and also compressive strength was maximum for the sample sintered at 1200 °C.
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5. Acknowledgement
The authors are thankful for the support from the Research Committee of Yasouj
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University (Grant No. GRYU-89111604 to F. Heidari).
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The efficiency of producing of HA extracted from cattle was more than hen and goat. With increasing the sintering temperature up to 1300 °C, relative density and hardness of the samples increased. Compressive strength was maximum for the sample that sintered at 1200 °C
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