journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
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Research Paper
Preparation and evaluation of cerium oxide-bovine hydroxyapatite composites for biomedical engineering applications O. Gunduza,b,n, C. Godec, Z. Ahmadd, H. Go¨kc¸ee,f, M. Yetmezg, C. Kalkandelenh,i, Y.M. Sahinj, F.N. Oktarb,k,nn a
Department of Metallurgy and Materials Engineering, Faculty of Technology, Marmara University, Goztepe Campus, Istanbul, 34722, Turkey b Centre for Nanotechnology & Biomaterials Applied and Research at Marmara University, Istanbul, Goztepe, Campus, 34722, Turkey c School of Technical Related Professions, Pamukkale University, Denizli, Turkey d Leicester School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK e Metallurgical and Materials Engineering Deptartment, Istanbul Technical University, Istanbul, Turkey f Prof. Dr. Adnan Tekin Material Sciences and Production Technologies Applied Research Center, Istanbul Technical Univ., Istanbul, Turkey g Mechanical Engineering Deptartment, Faculty of Engineering, Bulent Ecevit University, Zonguldak, Turkey h Vocational School of Technical Sciences, Biomedical Devices Technology Deptartment, Istanbul University, Istanbul, Turkey i Biomedical Engineering Ph.D. Program, Institute of Sciences, Istanbul University, Istanbul, Turkey j Department of Biomedical Engineering, Faculty of Engineering and Architecture, Istanbul Arel University, Istanbul, Turkey k Department of Bioengineering, Faculty of Engineering, Marmara University, Istanbul, Turkey
ar t ic l e in f o
abs tra ct
Article history:
The fabrication and characterization of bovine hydroxyapatite (BHA) and cerium oxide
Received 18 November 2013
(CeO2) composites are presented. CeO2 (at varying concentrations 1, 5 and 10 wt%) were
Received in revised form
added to calcinated BHA powder. The resulting mixtures were shaped into green
11 March 2014
cylindrical samples by powder pressing (350 MPa) followed by sintering in air (1000–
Accepted 13 March 2014
1300 1C for 4 h). Density, Vickers microhardness (HV), compression strength, scanning
Available online 5 April 2014
electron microscopy (SEM) and X-ray diffraction (XRD) studies were performed on the
Keywords:
products. The sintering behavior, microstructural characteristics and mechanical proper-
Sintering Microstructure Mechanical properties
ties were evaluated. Differences in the sintering temperature (for 1 wt% CeO2 composites) between 1200 and 1300 1C, show a 3.3% increase in the microhardness (564 and 582.75 HV, respectively). Composites prepared at 1300 1C demonstrate the greatest compression strength with comparable results for 5 and 10 wt% CeO2 content (106 and 107 MPa) which
n Corresponding author at: Department of Metallurgy and Materials Engineering, Faculty of Technology, Marmara University, Goztepe Campus, Istanbul, 34722, Turkey. nn Corresponding author at: Centre for Nanotechnology & Biomaterials Applied and Research at Marmara University, Istanbul, Goztepe, Campus, 34722, Turkey. E-mail addresses: [email protected] (O. Gunduz), [email protected] (F.N. Oktar).
http://dx.doi.org/10.1016/j.jmbbm.2014.03.004 1751-6161/& 2014 Elsevier Ltd. All rights reserved.
journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
71
Bovine hydroxyapatite
are significantly better than those for 1 wt% and those that do not include any CeO2 (90 and
Cerium oxide
below 60 MPa, respectively). The results obtained suggest optimal parameters to be used in
Bioceramics
preparation of BHA and CeO2 composites, while also highlighting the potential of such materials in several biomedical engineering applications. & 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
It is estimated that 280,000 hip, 700,000 vertebral and 250,000 wrist fractures cost US $10 billion per year. This has led to a significant increase in demand within the surgical and biomaterial market. Globally, procedures involving bone grafts or bone substitutes, fluctuate around 4,000,000 per annum (Brydone et al., 2010). One of the main reasons for this is due to the increase in life expectancy, which comes with the natural aging process. For example, the life expectancy of 27 EU countries continues to project an increase in this, with the share of the population aged around 65 years and over rising from 17% in 2010 to 30% in 2060, and those aged 80 and over rising from 5% to 12% over the same period (Eurostat, 2011; Kalache et al., 2007). With such predictions in mind, an increase in the biomaterial and surgical markets can also be expected, as the number of people prone to bone fracture will also increase. Surgically, autografts and allografts (regarded as the golden standard) are accepted as standard treatments; they are sometimes accompanied by problems of donor site scarcity, rejection by the immune system, resorption and transfer of pathogens (Gao et al., 2006). Due to the scarcity of golden standards, biomaterials research has led to the development of new materials, which offer properties similar to those of bone. One synthetic material, hydroxyapatite (HA) [Ca10 (PO4)6 (OH)2], is perhaps one of the most utilized biomaterial in this regard (Gao et al., 2006). The approximate composition of this synthetic material is comparable to that of bone (Silva and Lameiras, 2000; Valerio et al., 2004; Oktar et al., 2001) and teeth (Silva and Lameiras, 2000; Oktar and Altintas, 1998) (naturally occurring HA). However, it exhibits low fracture toughness due to its lack of strength and high brittleness (Silva and Lameiras, 2000; Sampaio et al., 2005; Valerio et al., 2004; Gunduz et al., 2008), thereby providing an obstacle to its application as a standalone biomedical device that must withstand high loads (Silva and Lameiras, 2000). To improve such mechanical properties of HA materials (i.e., to increase their fracture toughness), incorporation of metallic materials, ceramic oxides, whiskers or even fibers have been suggested (Salman et al., 2009; Erkmen et al., 2007). In this study, CeO2 – a rare earth oxide was used. CeO2 is generally used for stabilizing the tetragonal polycrystalline structure of zirconia (Yousefpour et al., 2011). The potential use of rare-earth elements in the development of bone tissue has been unclear up to now (Ivanchenko et al., 2009). There are also some studies with lanthanum oxide and its potential use as a reinforcement material for HA (Pazarlioglu et al., 2011; Oktar et al., 2006). Some groups are also addressing other rare-earth elements such as cerium and what potential this might have in reduction of
dental enamel demineralization (Feng and Liao, 2005) and its antibacterial abilities in preventing caries (Yingguang et al., 2007). It is also known that CeO2 is used for improving the sintering of glass ceramics and strength and thermal stability (Ivanchenko et al., 2009). Cerium has also been used as a luminophor agent in the composition of ceramic powders, which may also be beneficial in dental applications. When a dental ceramic is not fluorescent, it tends to have an appearance of reduced vitality, presenting a grayish appearance (Volpato and Fredel, 2010). The aim of this study is to improve mechanical properties of bovine derived hydroxyapatite (BHA)–CeO2 composites by using different sintering temperatures in order to assess their microstructural and mechanical properties for high loadbearing biomedical engineering applications.
2.
Materials and methods
2.1.
Materials
Naturally occurring apatite (approximate composition of synthetic apatite) was obtained from fresh bovine bones. The shaft segment of femoral bovine bones was obtained (epiphyseal components were excluded due to excessive organic residues) from an international abattoir (CarrefourSA Erenkoy, Istanbul, Turkey). CeO2 powder (99.5% purity, REO) was obtained from Alfa Aesar, USA. This displayed an average particle size range between 0.07–0.1 mm and a surface area of 12 m2/g. Sieves were purchased from Horizontal Sieve Shaker AS 400 Retsch, Haan, Germany.
2.2.
Preparation of CeO2–BHA composites
The specimens were dissected and the medulla osseo components of the samples were removed by manual abrasion. The bone samples were irrigated with copious amounts of tap water and were then deproteinized using an alkali solution (NaOH, 1%) (Goller and Oktar, 2002). Following deproteinization, BHA samples were washed thoroughly again (using copious tap water), dried and calcined at 850 1C (Goller et al., 2006; Oktar et al., 2006) to remove any prions (i.e., Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy and BSE) (Goller et al., 2004; Ozyegin et al., 2004). The samples were then crushed first in a ceramic mortar and then with a ball grinder for 4 h (Planetary Ball Mill PM 200 Retsch, Haan, Germany). The resulting BHA powder was sieved (100 mm) and was subsequently mixed with 1, 5 and 10 wt% of CeO2 content separately. Each mixture was then ball grinded for further 4 h. According to British Standard 7253 (Salman et al., 2009), the prepared powder mixtures
journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
were pressed between hardened steel dies under the pressure of 350 MPa. The cylindrical green composite compacts were sintered for 4 h at different temperatures (i.e., 1000, 1100, 1200 and 1300 1C) (Nabertherm HT 16/17, Lilienthal, Germany). For density and microhardness (HV) measurements, data was obtained using ten samples. For compression testing, four cylindrical test samples were used. For statistical analysis (and quality assurance) an independent group t-test and ANOVA were used (WINKS SDA, Version 6.0.91, TexaSoft, Houston, Texas, at 5%). This was to ensure comparison and reproducibility.
120 0 wt.% Compression Strength (MPa)
72
100 80
1 wt.% 5 wt.% 10 wt.%
60
Reference [26]
40 20 0 1000
1100
1200
1300
Temperature ( 0C)
CeO2–BHA composite characterization
Fig. 1 – Comparison of maximum compression strength results versus sintering temperature.
The mechanical properties were investigated using a universal tensile testing machine DVT (Devotrans Inc., Istanbul, Turkey). Loading rate of all compression tests was 2 mm/min. Microhardness (HV) testing was completed at 200 g load with 20 s of dwell time (Shimadzu HMV-2, Kyoto, Japan). The density of sintered samples was measured using an Archimedes method. Scanning electron microscopy (SEM JEOL 590, Tokyo, Japan), EDS analysis (JEOL 590), and X-ray diffraction tests (XRD, D8 Advance, Bruker-AXS, Germany) were performed to characterize microstructure and phases.
3.
400 300
1 wt.% 5 wt.% 10 wt.% Reference [26]
200 100
1100
1200
1300
Temperature (0C)
Table 1 shows the densities of various samples prepared using different concentrations of CeO2 along with the sintering temperature. For all samples as the sintering temperature of each concentration is increased so does the density. In all cases when the sintering temperature is increased from 1000 to 1300 1C, the density is increased by 40–45%. For each specific sintering temperature the density of each sample concentration is comparable. Fig. 1 shows the compression strength of various BHA composites (0, 1, 5 and 10 wt% CeO2) prepared at different temperatures (1000, 1100, 1200 and 1300 1C). The maximum value for compression strength was obtained for samples prepared at 1300 1C in the order 5 wt% (107 MPa), 10 wt% (106 MPa) and finally 1 wt% CeO2 (92 MPa). Although there is a little difference between 5 and 10 wt% CeO2 samples (o1%), the addition of 1 wt% CeO2 results in compression strengths that are significantly higher than samples containing no CeO2 by over 30 MPa. Previous studies focusing on the use of other materials to form BHA composites have demonstrated Table 1 – Influence of CeO2 content (1, 5 and 10 wt%) on density of CeO2–BHA composites at different temperatures.
1000 1100 1200 1300
0 wt.%
500
0 1000
Results and discussions
Temperature T (1C)
600
Microhardness (HV)
2.3.
Density ρ (g/cm3) 0 wt. %
1 wt %
5 wt %
10 wt %
1.922 2.144 2.717 2.762
1.992 2.143 2.746 2.832
2.022 2.188 2.786 2.869
2.090 2.249 2.712 2.879
Fig. 2 – Comparison of Vickers microhardness results versus sintering temperature.
similar findings. Gunduz et al. show compression strengths of 133 and 104 MPa for BHA–CIG samples (1200 1C for 10 wt% and 1300 1C for 5 wt%, respectively) (Gunduz et al., 2009). However the same study demonstrated relatively poor compression strengths of 6.9 MPa for 10 wt% CIG–BHA composites (1300 1C). Comparatively, under the same conditions, this study shows compression strengths of 106 MPa at 1300 1C for 10 wt% content (Gunduz et al., 2009). Oktar et al. have added reduced quantities of lanthanum oxide to BHA (0.25, 0.50, 1 and 2 wt%) to prepare composite structures. The best compression result was 88 MPa which was obtained at 1300 1C for 2 wt% La2O3 content (Oktar et al., 2006). Direct comparison of 1 wt% La2O3 content in BHA composites (88 MPa) shows that the CeO2 composite demonstrates slightly better compression values 93 MPa. The best results for microhardness (Fig. 2) were obtained at 1 wt% CeO2 content at 1300 and 1200 1C (582.75 and 564 HV, respectively). By comparison this is also relative to the density values at the respective temperatures (2.832 and 2.746 g/cm3), indicating that the microhardness is also affected by density, although this is not the only factor. A value of 548.25 HV was obtained for samples prepared at 1200 1C using 5 wt% CeO2 content. The microhardness values obtained in this study are superior compared to those demonstrated in a recent study utilizing inert glass (CIG) addition to HA content (Gunduz et al., 2009). The best microhardness value obtained for CIG–BHA composites in the study was 507 HV, which is appreciably lower than the
journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
highest values reported in this study. It is clearly known that CeO2 is rare earth oxide and there are limited studies in the previous literature relating to it (and other rare earth oxides) as a composite with HA. In general, when comparing microhardness and compression values of composite BHA and BHA alone, (Fig. 1 and Fig. 2) the reinforcement role of CeO2 becomes extremely evident and valuable. Fig. 3 shows electron micrographs of various BHA samples prepared. Perfect grain growth of 1300 1C sintered pure BHA samples were notable at both low and high magnifications (Fig. 3 1a and b). Samples containing 1 wt% CeO2 (Fig. 3 2a and b) content clearly show the presence of CeO2 particles,
73
which on initial observation range in size from a few to approximately 10 μm (seen here as white debris). However, under higher magnification these structures are also found to be present as smaller particles (0.1–0.5 mm). An increase in CeO2 from 1 to 5 and 10 wt% becomes clearly evident from micrographic analysis (Figs. 3 3a and b, 4a and b). The quantity of particles becomes more abundant, although at the higher concentration (10 wt%) clustering of particles is more pronounced. As with the lower concentration samples the grain growth is clearly evident for all other samples but the grain boundaries become less obvious and appear as blurred intermediate lines as the concentration of CeO2 is increased (i.e. at
Fig. 3 – Electron micrographs showing (a) low x500 and (b) high x3000 magnifications of (1) 0 (2) 1 (3) 5 and (4) 10 wt.% CeO2 as sintered (1300 1C) BSA composites.
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journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
Fig. 4 – EDS analysis for 1 wt% CeO2 composites (1300 1C) (a) at point A and (b) at point B.
5 wt%). At the maximum experimental concentration (10 wt%) the grain borders are diminished and become difficult to locate which suggests the formation of a glassy phase. From the micrographs, samples containing 5wt% CeO2 demonstrate the
best compromise between quantity and uniform distribution (reduced clustering). Referring to the compression values obtained for 5 and 10 wt% CeO2 samples (107 and 106 MPa at 1300 1C, respectively) demonstrates a small difference between
journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
the two. Previous studies demonstrate the presence of relatively high glassy phase formations at critical concentrations which result in reduced compression values when compared to composites prepared from lower concentrations (e.g. machineable fluorapatite glass–BHA at 1300 1C and 44.29 MPa (Gunduz et al., 2008), 10 wt% of inert glass–BHA at 1300 1C and 6.86 MPa (Gunduz et al., 2009)). Lower concentration samples of BHA composites (1 wt% CeO2) were assessed using Energy-dispersive X-ray spectroscopy (EDS) by selecting two points on surfaces A and B (Fig. 4). At point A, no CeO2 was detected using EDS. The standard elements from BHA component was detected (P, Ca, O). However, when performing analysis closer to a particulate cluster on the sample, Ce is detected alongside the aforementioned elements – confirming the presence of CeO2. This suggests that CeO2 found within this composite material is not sufficiently distributed and filled throughout the structure due to low concentration. Although the clustering of CeO2 becomes more pronounced in the samples as a function of weight percent, SEM images also demonstrate that the distribution of clusters vary in size. This suggests that increasing the concentration of CeO2 will also increase its distribution within the sample relatively. However, this does not insure complete uniform distribution but includes small well distributed clusters. Quantifying these clusters becomes difficult (size, number of particles, height, density etc.), especially on miniature scale and the nature of sample boundary selection (when compared to 1 wt%). Variation in cluster size and distribution (based on CeO2 content) was similar for random areas selected under the SEM. XRD patterns of selected samples are shown in Fig. 5. Different CeO2 reinforcement concentrations (1, 5 and 10 wt %) and sintering temperatures (1000 and 1300 1C) are shown. Fig. 5a and b shows the XRD patterns of HA-1 wt% CeO2 composite samples, exhibiting strong diffraction peaks belonging to the hydroxyapatite (JCPDS card number: 98005-2689) and whitlockite (JCPDS card number: 98-000-2071) stable phases. Furthermore, whitlockite phase amount decreases sharply with increasing sintering temperature. Fig. 5c and d shows the XRD patterns of HA-5 wt% CeO2 composite samples. It is also obvious that HA, whitlockite, cerium oxide (JCPDS card number: 98-006-3146) stable phases and calcium cerium phosphate (JCPDS card number: 98-03-8252) intermetallic phase peaks were observed. Also, whitlockite phase amount decrease gradually with increasing sintering temperature and increase in calcium cerium phosphate peak height after sintering process. HA10 wt% CeO2 composite samples consisting of diffraction peaks belonging to HA, whitlockite, cerium oxide and calcium cerium phosphate phases depending upon sintering temperature changing between 1000 and 1300 1C are shown in Fig. 5e and f. Although any new phase was not observed in the samples for 5 wt% CeO2–BHA. It is indicated that XRD pattern shown in Fig. 5e and f, revealed the reactions that occurred during sintering and resulted in the formation of intermetallic compounds which were totally in accordance with the phase transformations, whitlockite phase amount decreased and increased in calcium cerium phosphate phase with increasing sintering temperature as shown in Fig. 5c and d.
75
Fig. 5 – X-ray diffractograms of BHA composites doped with (a) 1 wt% of CeO2 at 1000 1C, (b) 1 wt% of CeO2 at 1300 1C, (c) 5 wt% of CeO2 at 1000 1C, (d) 5 wt% of CeO2 at 1300 1C, (e) 10 wt% of CeO2 at 1000 1C and (f) 10 wt% of CeO2 at 1300 1C. X-ray diffraction studies were applied for 1000 and 1300 1C sintered samples of 1 wt%, 5 wt% and 10 wt% of CeO2 content. For 1 wt% of CeO2 content at X-ray diffraction pattern at 1000 1C sintered sample, CeO2 and calcium cerium phosphate picks are hard to differentiate. On the other hand, due to the low content of CeO2, CeO2 and calcium cerium phosphate picks are a little bit easier to differentiate so that X-ray diffractograms are much more trustable than EDS. It can be said that the major picks for 1 wt% of CeO2 content at 1000– 1300 1C are BHA picks and with some minor whitlockite picks, especially whitlockite a biodegradable ceramic (Klein 1985) and also with some ß-tricalcium phosphate (ß-TCP) picks (Vicente et al., 1996). Regarding 5 wt% and 10 wt% CeO2 at 1000–1300 1C, the major picks were BHA and whitlockite picks. For these compositions (i.e., 5 or 10 wt%), it is notable that CeO2 picks were much more dominate when compared with 1 wt% of CeO2 content.
4.
Conclusion
The preparation and characterization of CeO2–BHA composites have demonstrated the potential of a novel biomaterial composite. Changes in microhardness and compression strength could be varied due to the concentration of CeO2
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journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
and sintering temperature. Optimal preparation conditions have been determined for both of these properties based on the current system, demonstrating results, which are comparable to other (HA) BHA-rare earth composites previously researched for biomedical engineering applications.
Acknowledgments This study was carried out mainly with the equipment furnished (between 2007–2008) to Marmara University with the support of the Turkish Republic Government Planning Organization in the framework of the project 2003K120810 “Manufacturing and Characterization of Electro-Conductive Bioceramics”.
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Research Paper
Preparation and evaluation of cerium oxide-bovine hydroxyapatite composites for biomedical engineering applications O. Gunduza,b,n, C. Godec, Z. Ahmadd, H. Go¨kc¸ee,f, M. Yetmezg, C. Kalkandelenh,i, Y.M. Sahinj, F.N. Oktarb,k,nn a
Department of Metallurgy and Materials Engineering, Faculty of Technology, Marmara University, Goztepe Campus, Istanbul, 34722, Turkey b Centre for Nanotechnology & Biomaterials Applied and Research at Marmara University, Istanbul, Goztepe, Campus, 34722, Turkey c School of Technical Related Professions, Pamukkale University, Denizli, Turkey d Leicester School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK e Metallurgical and Materials Engineering Deptartment, Istanbul Technical University, Istanbul, Turkey f Prof. Dr. Adnan Tekin Material Sciences and Production Technologies Applied Research Center, Istanbul Technical Univ., Istanbul, Turkey g Mechanical Engineering Deptartment, Faculty of Engineering, Bulent Ecevit University, Zonguldak, Turkey h Vocational School of Technical Sciences, Biomedical Devices Technology Deptartment, Istanbul University, Istanbul, Turkey i Biomedical Engineering Ph.D. Program, Institute of Sciences, Istanbul University, Istanbul, Turkey j Department of Biomedical Engineering, Faculty of Engineering and Architecture, Istanbul Arel University, Istanbul, Turkey k Department of Bioengineering, Faculty of Engineering, Marmara University, Istanbul, Turkey
ar t ic l e in f o
abs tra ct
Article history:
The fabrication and characterization of bovine hydroxyapatite (BHA) and cerium oxide
Received 18 November 2013
(CeO2) composites are presented. CeO2 (at varying concentrations 1, 5 and 10 wt%) were
Received in revised form
added to calcinated BHA powder. The resulting mixtures were shaped into green
11 March 2014
cylindrical samples by powder pressing (350 MPa) followed by sintering in air (1000–
Accepted 13 March 2014
1300 1C for 4 h). Density, Vickers microhardness (HV), compression strength, scanning
Available online 5 April 2014
electron microscopy (SEM) and X-ray diffraction (XRD) studies were performed on the
Keywords:
products. The sintering behavior, microstructural characteristics and mechanical proper-
Sintering Microstructure Mechanical properties
ties were evaluated. Differences in the sintering temperature (for 1 wt% CeO2 composites) between 1200 and 1300 1C, show a 3.3% increase in the microhardness (564 and 582.75 HV, respectively). Composites prepared at 1300 1C demonstrate the greatest compression strength with comparable results for 5 and 10 wt% CeO2 content (106 and 107 MPa) which
n Corresponding author at: Department of Metallurgy and Materials Engineering, Faculty of Technology, Marmara University, Goztepe Campus, Istanbul, 34722, Turkey. nn Corresponding author at: Centre for Nanotechnology & Biomaterials Applied and Research at Marmara University, Istanbul, Goztepe, Campus, 34722, Turkey. E-mail addresses: [email protected] (O. Gunduz), [email protected] (F.N. Oktar).
http://dx.doi.org/10.1016/j.jmbbm.2014.03.004 1751-6161/& 2014 Elsevier Ltd. All rights reserved.
journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
71
Bovine hydroxyapatite
are significantly better than those for 1 wt% and those that do not include any CeO2 (90 and
Cerium oxide
below 60 MPa, respectively). The results obtained suggest optimal parameters to be used in
Bioceramics
preparation of BHA and CeO2 composites, while also highlighting the potential of such materials in several biomedical engineering applications. & 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
It is estimated that 280,000 hip, 700,000 vertebral and 250,000 wrist fractures cost US $10 billion per year. This has led to a significant increase in demand within the surgical and biomaterial market. Globally, procedures involving bone grafts or bone substitutes, fluctuate around 4,000,000 per annum (Brydone et al., 2010). One of the main reasons for this is due to the increase in life expectancy, which comes with the natural aging process. For example, the life expectancy of 27 EU countries continues to project an increase in this, with the share of the population aged around 65 years and over rising from 17% in 2010 to 30% in 2060, and those aged 80 and over rising from 5% to 12% over the same period (Eurostat, 2011; Kalache et al., 2007). With such predictions in mind, an increase in the biomaterial and surgical markets can also be expected, as the number of people prone to bone fracture will also increase. Surgically, autografts and allografts (regarded as the golden standard) are accepted as standard treatments; they are sometimes accompanied by problems of donor site scarcity, rejection by the immune system, resorption and transfer of pathogens (Gao et al., 2006). Due to the scarcity of golden standards, biomaterials research has led to the development of new materials, which offer properties similar to those of bone. One synthetic material, hydroxyapatite (HA) [Ca10 (PO4)6 (OH)2], is perhaps one of the most utilized biomaterial in this regard (Gao et al., 2006). The approximate composition of this synthetic material is comparable to that of bone (Silva and Lameiras, 2000; Valerio et al., 2004; Oktar et al., 2001) and teeth (Silva and Lameiras, 2000; Oktar and Altintas, 1998) (naturally occurring HA). However, it exhibits low fracture toughness due to its lack of strength and high brittleness (Silva and Lameiras, 2000; Sampaio et al., 2005; Valerio et al., 2004; Gunduz et al., 2008), thereby providing an obstacle to its application as a standalone biomedical device that must withstand high loads (Silva and Lameiras, 2000). To improve such mechanical properties of HA materials (i.e., to increase their fracture toughness), incorporation of metallic materials, ceramic oxides, whiskers or even fibers have been suggested (Salman et al., 2009; Erkmen et al., 2007). In this study, CeO2 – a rare earth oxide was used. CeO2 is generally used for stabilizing the tetragonal polycrystalline structure of zirconia (Yousefpour et al., 2011). The potential use of rare-earth elements in the development of bone tissue has been unclear up to now (Ivanchenko et al., 2009). There are also some studies with lanthanum oxide and its potential use as a reinforcement material for HA (Pazarlioglu et al., 2011; Oktar et al., 2006). Some groups are also addressing other rare-earth elements such as cerium and what potential this might have in reduction of
dental enamel demineralization (Feng and Liao, 2005) and its antibacterial abilities in preventing caries (Yingguang et al., 2007). It is also known that CeO2 is used for improving the sintering of glass ceramics and strength and thermal stability (Ivanchenko et al., 2009). Cerium has also been used as a luminophor agent in the composition of ceramic powders, which may also be beneficial in dental applications. When a dental ceramic is not fluorescent, it tends to have an appearance of reduced vitality, presenting a grayish appearance (Volpato and Fredel, 2010). The aim of this study is to improve mechanical properties of bovine derived hydroxyapatite (BHA)–CeO2 composites by using different sintering temperatures in order to assess their microstructural and mechanical properties for high loadbearing biomedical engineering applications.
2.
Materials and methods
2.1.
Materials
Naturally occurring apatite (approximate composition of synthetic apatite) was obtained from fresh bovine bones. The shaft segment of femoral bovine bones was obtained (epiphyseal components were excluded due to excessive organic residues) from an international abattoir (CarrefourSA Erenkoy, Istanbul, Turkey). CeO2 powder (99.5% purity, REO) was obtained from Alfa Aesar, USA. This displayed an average particle size range between 0.07–0.1 mm and a surface area of 12 m2/g. Sieves were purchased from Horizontal Sieve Shaker AS 400 Retsch, Haan, Germany.
2.2.
Preparation of CeO2–BHA composites
The specimens were dissected and the medulla osseo components of the samples were removed by manual abrasion. The bone samples were irrigated with copious amounts of tap water and were then deproteinized using an alkali solution (NaOH, 1%) (Goller and Oktar, 2002). Following deproteinization, BHA samples were washed thoroughly again (using copious tap water), dried and calcined at 850 1C (Goller et al., 2006; Oktar et al., 2006) to remove any prions (i.e., Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy and BSE) (Goller et al., 2004; Ozyegin et al., 2004). The samples were then crushed first in a ceramic mortar and then with a ball grinder for 4 h (Planetary Ball Mill PM 200 Retsch, Haan, Germany). The resulting BHA powder was sieved (100 mm) and was subsequently mixed with 1, 5 and 10 wt% of CeO2 content separately. Each mixture was then ball grinded for further 4 h. According to British Standard 7253 (Salman et al., 2009), the prepared powder mixtures
journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
were pressed between hardened steel dies under the pressure of 350 MPa. The cylindrical green composite compacts were sintered for 4 h at different temperatures (i.e., 1000, 1100, 1200 and 1300 1C) (Nabertherm HT 16/17, Lilienthal, Germany). For density and microhardness (HV) measurements, data was obtained using ten samples. For compression testing, four cylindrical test samples were used. For statistical analysis (and quality assurance) an independent group t-test and ANOVA were used (WINKS SDA, Version 6.0.91, TexaSoft, Houston, Texas, at 5%). This was to ensure comparison and reproducibility.
120 0 wt.% Compression Strength (MPa)
72
100 80
1 wt.% 5 wt.% 10 wt.%
60
Reference [26]
40 20 0 1000
1100
1200
1300
Temperature ( 0C)
CeO2–BHA composite characterization
Fig. 1 – Comparison of maximum compression strength results versus sintering temperature.
The mechanical properties were investigated using a universal tensile testing machine DVT (Devotrans Inc., Istanbul, Turkey). Loading rate of all compression tests was 2 mm/min. Microhardness (HV) testing was completed at 200 g load with 20 s of dwell time (Shimadzu HMV-2, Kyoto, Japan). The density of sintered samples was measured using an Archimedes method. Scanning electron microscopy (SEM JEOL 590, Tokyo, Japan), EDS analysis (JEOL 590), and X-ray diffraction tests (XRD, D8 Advance, Bruker-AXS, Germany) were performed to characterize microstructure and phases.
3.
400 300
1 wt.% 5 wt.% 10 wt.% Reference [26]
200 100
1100
1200
1300
Temperature (0C)
Table 1 shows the densities of various samples prepared using different concentrations of CeO2 along with the sintering temperature. For all samples as the sintering temperature of each concentration is increased so does the density. In all cases when the sintering temperature is increased from 1000 to 1300 1C, the density is increased by 40–45%. For each specific sintering temperature the density of each sample concentration is comparable. Fig. 1 shows the compression strength of various BHA composites (0, 1, 5 and 10 wt% CeO2) prepared at different temperatures (1000, 1100, 1200 and 1300 1C). The maximum value for compression strength was obtained for samples prepared at 1300 1C in the order 5 wt% (107 MPa), 10 wt% (106 MPa) and finally 1 wt% CeO2 (92 MPa). Although there is a little difference between 5 and 10 wt% CeO2 samples (o1%), the addition of 1 wt% CeO2 results in compression strengths that are significantly higher than samples containing no CeO2 by over 30 MPa. Previous studies focusing on the use of other materials to form BHA composites have demonstrated Table 1 – Influence of CeO2 content (1, 5 and 10 wt%) on density of CeO2–BHA composites at different temperatures.
1000 1100 1200 1300
0 wt.%
500
0 1000
Results and discussions
Temperature T (1C)
600
Microhardness (HV)
2.3.
Density ρ (g/cm3) 0 wt. %
1 wt %
5 wt %
10 wt %
1.922 2.144 2.717 2.762
1.992 2.143 2.746 2.832
2.022 2.188 2.786 2.869
2.090 2.249 2.712 2.879
Fig. 2 – Comparison of Vickers microhardness results versus sintering temperature.
similar findings. Gunduz et al. show compression strengths of 133 and 104 MPa for BHA–CIG samples (1200 1C for 10 wt% and 1300 1C for 5 wt%, respectively) (Gunduz et al., 2009). However the same study demonstrated relatively poor compression strengths of 6.9 MPa for 10 wt% CIG–BHA composites (1300 1C). Comparatively, under the same conditions, this study shows compression strengths of 106 MPa at 1300 1C for 10 wt% content (Gunduz et al., 2009). Oktar et al. have added reduced quantities of lanthanum oxide to BHA (0.25, 0.50, 1 and 2 wt%) to prepare composite structures. The best compression result was 88 MPa which was obtained at 1300 1C for 2 wt% La2O3 content (Oktar et al., 2006). Direct comparison of 1 wt% La2O3 content in BHA composites (88 MPa) shows that the CeO2 composite demonstrates slightly better compression values 93 MPa. The best results for microhardness (Fig. 2) were obtained at 1 wt% CeO2 content at 1300 and 1200 1C (582.75 and 564 HV, respectively). By comparison this is also relative to the density values at the respective temperatures (2.832 and 2.746 g/cm3), indicating that the microhardness is also affected by density, although this is not the only factor. A value of 548.25 HV was obtained for samples prepared at 1200 1C using 5 wt% CeO2 content. The microhardness values obtained in this study are superior compared to those demonstrated in a recent study utilizing inert glass (CIG) addition to HA content (Gunduz et al., 2009). The best microhardness value obtained for CIG–BHA composites in the study was 507 HV, which is appreciably lower than the
journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
highest values reported in this study. It is clearly known that CeO2 is rare earth oxide and there are limited studies in the previous literature relating to it (and other rare earth oxides) as a composite with HA. In general, when comparing microhardness and compression values of composite BHA and BHA alone, (Fig. 1 and Fig. 2) the reinforcement role of CeO2 becomes extremely evident and valuable. Fig. 3 shows electron micrographs of various BHA samples prepared. Perfect grain growth of 1300 1C sintered pure BHA samples were notable at both low and high magnifications (Fig. 3 1a and b). Samples containing 1 wt% CeO2 (Fig. 3 2a and b) content clearly show the presence of CeO2 particles,
73
which on initial observation range in size from a few to approximately 10 μm (seen here as white debris). However, under higher magnification these structures are also found to be present as smaller particles (0.1–0.5 mm). An increase in CeO2 from 1 to 5 and 10 wt% becomes clearly evident from micrographic analysis (Figs. 3 3a and b, 4a and b). The quantity of particles becomes more abundant, although at the higher concentration (10 wt%) clustering of particles is more pronounced. As with the lower concentration samples the grain growth is clearly evident for all other samples but the grain boundaries become less obvious and appear as blurred intermediate lines as the concentration of CeO2 is increased (i.e. at
Fig. 3 – Electron micrographs showing (a) low x500 and (b) high x3000 magnifications of (1) 0 (2) 1 (3) 5 and (4) 10 wt.% CeO2 as sintered (1300 1C) BSA composites.
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journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
Fig. 4 – EDS analysis for 1 wt% CeO2 composites (1300 1C) (a) at point A and (b) at point B.
5 wt%). At the maximum experimental concentration (10 wt%) the grain borders are diminished and become difficult to locate which suggests the formation of a glassy phase. From the micrographs, samples containing 5wt% CeO2 demonstrate the
best compromise between quantity and uniform distribution (reduced clustering). Referring to the compression values obtained for 5 and 10 wt% CeO2 samples (107 and 106 MPa at 1300 1C, respectively) demonstrates a small difference between
journal of the mechanical behavior of biomedical materials 35 (2014) 70 –76
the two. Previous studies demonstrate the presence of relatively high glassy phase formations at critical concentrations which result in reduced compression values when compared to composites prepared from lower concentrations (e.g. machineable fluorapatite glass–BHA at 1300 1C and 44.29 MPa (Gunduz et al., 2008), 10 wt% of inert glass–BHA at 1300 1C and 6.86 MPa (Gunduz et al., 2009)). Lower concentration samples of BHA composites (1 wt% CeO2) were assessed using Energy-dispersive X-ray spectroscopy (EDS) by selecting two points on surfaces A and B (Fig. 4). At point A, no CeO2 was detected using EDS. The standard elements from BHA component was detected (P, Ca, O). However, when performing analysis closer to a particulate cluster on the sample, Ce is detected alongside the aforementioned elements – confirming the presence of CeO2. This suggests that CeO2 found within this composite material is not sufficiently distributed and filled throughout the structure due to low concentration. Although the clustering of CeO2 becomes more pronounced in the samples as a function of weight percent, SEM images also demonstrate that the distribution of clusters vary in size. This suggests that increasing the concentration of CeO2 will also increase its distribution within the sample relatively. However, this does not insure complete uniform distribution but includes small well distributed clusters. Quantifying these clusters becomes difficult (size, number of particles, height, density etc.), especially on miniature scale and the nature of sample boundary selection (when compared to 1 wt%). Variation in cluster size and distribution (based on CeO2 content) was similar for random areas selected under the SEM. XRD patterns of selected samples are shown in Fig. 5. Different CeO2 reinforcement concentrations (1, 5 and 10 wt %) and sintering temperatures (1000 and 1300 1C) are shown. Fig. 5a and b shows the XRD patterns of HA-1 wt% CeO2 composite samples, exhibiting strong diffraction peaks belonging to the hydroxyapatite (JCPDS card number: 98005-2689) and whitlockite (JCPDS card number: 98-000-2071) stable phases. Furthermore, whitlockite phase amount decreases sharply with increasing sintering temperature. Fig. 5c and d shows the XRD patterns of HA-5 wt% CeO2 composite samples. It is also obvious that HA, whitlockite, cerium oxide (JCPDS card number: 98-006-3146) stable phases and calcium cerium phosphate (JCPDS card number: 98-03-8252) intermetallic phase peaks were observed. Also, whitlockite phase amount decrease gradually with increasing sintering temperature and increase in calcium cerium phosphate peak height after sintering process. HA10 wt% CeO2 composite samples consisting of diffraction peaks belonging to HA, whitlockite, cerium oxide and calcium cerium phosphate phases depending upon sintering temperature changing between 1000 and 1300 1C are shown in Fig. 5e and f. Although any new phase was not observed in the samples for 5 wt% CeO2–BHA. It is indicated that XRD pattern shown in Fig. 5e and f, revealed the reactions that occurred during sintering and resulted in the formation of intermetallic compounds which were totally in accordance with the phase transformations, whitlockite phase amount decreased and increased in calcium cerium phosphate phase with increasing sintering temperature as shown in Fig. 5c and d.
75
Fig. 5 – X-ray diffractograms of BHA composites doped with (a) 1 wt% of CeO2 at 1000 1C, (b) 1 wt% of CeO2 at 1300 1C, (c) 5 wt% of CeO2 at 1000 1C, (d) 5 wt% of CeO2 at 1300 1C, (e) 10 wt% of CeO2 at 1000 1C and (f) 10 wt% of CeO2 at 1300 1C. X-ray diffraction studies were applied for 1000 and 1300 1C sintered samples of 1 wt%, 5 wt% and 10 wt% of CeO2 content. For 1 wt% of CeO2 content at X-ray diffraction pattern at 1000 1C sintered sample, CeO2 and calcium cerium phosphate picks are hard to differentiate. On the other hand, due to the low content of CeO2, CeO2 and calcium cerium phosphate picks are a little bit easier to differentiate so that X-ray diffractograms are much more trustable than EDS. It can be said that the major picks for 1 wt% of CeO2 content at 1000– 1300 1C are BHA picks and with some minor whitlockite picks, especially whitlockite a biodegradable ceramic (Klein 1985) and also with some ß-tricalcium phosphate (ß-TCP) picks (Vicente et al., 1996). Regarding 5 wt% and 10 wt% CeO2 at 1000–1300 1C, the major picks were BHA and whitlockite picks. For these compositions (i.e., 5 or 10 wt%), it is notable that CeO2 picks were much more dominate when compared with 1 wt% of CeO2 content.
4.
Conclusion
The preparation and characterization of CeO2–BHA composites have demonstrated the potential of a novel biomaterial composite. Changes in microhardness and compression strength could be varied due to the concentration of CeO2
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and sintering temperature. Optimal preparation conditions have been determined for both of these properties based on the current system, demonstrating results, which are comparable to other (HA) BHA-rare earth composites previously researched for biomedical engineering applications.
Acknowledgments This study was carried out mainly with the equipment furnished (between 2007–2008) to Marmara University with the support of the Turkish Republic Government Planning Organization in the framework of the project 2003K120810 “Manufacturing and Characterization of Electro-Conductive Bioceramics”.
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