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In situ synthesis of magnesium-substituted biphasic calcium phosphate and in vitro biodegradation
Materials Research Bulletin 47 (2012) 2506–2512
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Materials Research Bulletin journal homepage: www.elsevier.com/locate/matresbu
In situ synthesis of magnesium-substituted biphasic calcium phosphate and in vitro biodegradation Tae-Wan Kim a, Hyeong-Shin Lee a, Dong-Hyun Kim a, Hyeong-Ho Jin a, Kyu-Hong Hwang b, Jong Kook Lee c, Hong-Chae Park a, Seog-Young Yoon a,* a b c
School of Materials Science and Engineering, Pusan National University, Busan 609-735, Republic of Korea School of Nano and Advanced Materials, Gyeongsang National University, Jinju, Kyungnam 660-701, Republic of Korea Department of Advanced Materials Engineering, Chosun University, Gwangju 501-759, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 January 2012 Received in revised form 13 April 2012 Accepted 8 May 2012 Available online 16 May 2012
In situ preparation of magnesium (Mg) substituted biphasic calcium phosphate (BCP) of hydroxyapatite (HAp)/b-tricalcium phosphate (b-TCP) were carried out through aqueous co-precipitation method. The concentrations of added magnesium were varied with the calcium in order to obtain constant (Ca + Mg)/P ratios of 1.602. X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy were used to characterize the structure of synthesized magnesium substituted BCP powders. The results have shown that substitution of magnesium in the calcium deficient apatites revealed the formation of biphasic mixtures of different HAp/b-TCP ratios after heating at 1000 8C. The ratios of the formation of phase mixtures were dependent on the content of magnesium. After immersing in Hanks’ balanced salt solution (HBSS) for 1 week, 1 wt% magnesium substituted BCP powders were degraded and precipitation started to be formed with small granules consisting of number of flake-like crystal onto the surface of synthesized powders. On the other hand, in the case of pure BCP powders, the formation of new precipitates was detected after immersion in HBSS for 2 weeks. On the basis of these results, magnesium substituted BCP could be able to develop a new apatite phase on the surface in contact with physiological fluids faster than BCP does. In addition, the retention time to produce the new apatite phase in implantation operation for the BCP powder could be controlled by the amount of magnesium substitution. ß 2012 Elsevier Ltd. All rights reserved.
Keywords: A. Ceramics B. Chemical synthesis C. X-ray diffraction D. Crystal structure
1. Introduction For more than 10 years, biphasic calcium phosphate (BCP) ceramics, composed of a mixture of hydroxyapatite [HAp, Ca10(PO4)6(OH)2] and b-tricalcium phosphate [b-TCP, Ca3(PO4)2], have been considered as potential substitutes for bone replacement surgical operations due to their excellent biocompatibility, ability to promote cellular functions and expressions, and osteoconductivity [1–4]. However, both materials show the difference in dissolution properties (HAp, non-resorable and bTCP, resorable) which reduce their clinical performance. Therefore, an optimum bioresorability was found when appropriately mixing both phases to provide a biphasic calcium phosphate. During implantation, it is known that bone mineral mass could be dominated by nanocrystalline multisubstituted calcium phosphate apatite [5,6]. The type and amount of ionic substitutions in the apatite phase of bone vary from the weight percent (wt%) level
* Corresponding author. Tel.: +82 51 510 2487; fax: +82 51 512 0528. E-mail address: [email protected] (S.-Y. Yoon). 0025-5408/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.materresbull.2012.05.011
[7] to the parts per million (ppm) level [8]. Among substituting ions, magnesium is known to be an important trace element in bone and teeth, despite its low concentration (generally between 0.5 and 1.5 wt% in human body). It is well known that magnesium plays a key role in bone metabolism, in particular during the early stages of osteogenesis where it stimulates osteoblast proliferation [9,10]. Due to the biological relevance of magnesium, magnesium substituted calcium phosphate compounds have attracted the interest of many scientists because magnesium incorporation is considered to be a promising way to improve the bioactivity of calcium phosphate-based biomaterials. Therefore, several studies have worked on the preparation of apatite [11–13] and calcium phosphates [14,15] implant materials containing low levels of magnesium. It has demonstrated that the incorporation of magnesium into HAp lattice sensibly affects apatite crystallization in solution and its thermal stability, promoting the formation of b-TCP and thus forming biphasic calcium phosphates. In recent, the structural analyses for the magnesium substituted HAp [16] and b-TCP [17] have been discussed with multinuclear magnetic resonance (NMR) and X-ray absorption spectroscopy (XAS), X-ray powder diffraction (XRD)
T.-W. Kim et al. / Materials Research Bulletin 47 (2012) 2506–2512
with the Rietveld refinement, respectively. However, so far not much clear emphasis has been made quantitatively on the formation of biphasic mixtures [18] and its biodegradation behavior with substituted magnesium in the BCP powder. With the aim to fabricate the magnesium substituted BCP powders, this study focused on the in situ preparation of magnesium substituted BCP powders with precise proportions of the HAp and b-TCP phase based on the input (Ca + Mg)/P molar ratio with the co-precipitation method. The success of this task would suggest the possibility of obtaining supplementally amounts of magnesium substituted BCP powders with precise ratios of two components depending on the characteristics required for the specific application by controlling the input (Ca + Mg)/P molar ratio of starting reactants. The second aim was to assess the effect of magnesium substitution on the in vitro bioactivity of BCP powder. 2. Materials and methods 2.1. Powder preparation Pure and magnesium substituted BCP powders were synthesized in a reactor at 40 8C under magnetic stirring starting from calcium nitrate tetrahydrate (Ca(NO3)24H2O, Aldrich 99.0%, MW 236.15), magnesium nitrate hexahydrate (Mg(NO3)26H2O, Aldrich 99.0%, MW 256.41) and diammonium hydrogen phosphate ((NH4)2HPO4, Aldrich 99.0%, MW 132.06). For the preparation of pure BCP powder, an appropriate amount of calcium nitrate tetrahydrate was dissolved in water by vigorously stirring at a rate of 1000 rpm. Diammonium hydrogen phosphate solution was slowly added to the calcium nitrate tetrahydrate solution to achieve predetermined Ca/P molar ratio 1.602, providing the BCP (60% HAp and 40% b-TCP) in previous our work [19]. Magnesium substituted BCP powders with different amount of magnesium (0.5, 1, and 1.5 wt%) were prepared assuming that magnesium ions would substitute for the calcium site in the HAp and b-TCP lattice in order to obtain a nominal composition in terms of (Ca + Mg)/P ratio of 1.602. The pH of mixed solution was maintained at 11 by the addition of ammonium hydroxide (NH4OH) solution. After the completion of addition, the solution was stirred constantly for 24 h by a mechanical stirrer, allowing the reaction to complete. The precipitated suspension was discharged from the reactor and allowed to settle down for 24 h for the maturation of precipitate. After 24 h, the precipitates were separated through vacuum filtration technique and were dried at 80 8C for 24 h in a drying oven (SFC-301, Dongwon Scientific System, Korea). The dried cakes were crushed to fine powders, sieved through a mesh size of 200 (>75 mm) and used for characterization. After that, the powders were calcined at 1000 8C for 24 h (heating rate was 1 8C/min) in air. 2.2. Characterization The dried powders were subjected to thermal analysis using a heating rate of 10 8C/min and peak temperature of 1000 8C in air atmosphere (SDT Q600 TG–DTA/DSC, USA) to analyze the thermal behavior during heating. An X-ray diffractometer (Rigaku D-max IIA, Japan), at 30 kV and 20 mA with a scanning speed of 18/min, was adopted to identify the phases of powders synthesized with different magnesium contents. Diffraction patterns were prepared for each control sample, and the intensity ratio was calculated based upon two unobscured peaks from each phase. The relative intensity ratio (RIR) of HAp/b-TCP was estimated using the peaks intensity of peaks (211) and (0210) of HAp and b-TCP, respectively, according to the formula RIR = Ib-TCP/(IHAp + Ib-TCP) [20]. Various functional groups present in the prepared calcium phosphates powders were identified by FTIR (Nicolet Magna IR 560, USA). Here
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1 wt% of the powder was mixed and ground with 99% of KBr. Tablets of 10 mm diameter for FTIR measurements were prepared by pressing the powder mixture at load of 5 tons for 2 min and the spectrum was taken in the range of 400–4000 cm1 with resolution 4 cm1 and 128 times scanning. Microstructural observations of the calcined samples were conducted using a scanning electron microscope (SEM; S-4800, HITACHI, Japan). 2.3. Degradation experiments Hanks’ balanced salt solution (HBSS), an extracellular solution with an ionic composition similar to human blood plasma, was used as the supporting solution for the magnesium substituted BCP powders in vitro test. The simulated solution consisted of 8.00 g NaCl, 0.35 g NaHCO3, 0.40 g KCl, 0.06 g KH2PO4, 0.10 g MgCl26H2O, 0.14 g CaCl2, 0.06 g Na2HPO42H2O, 0.06 g MgSO47H2O, 1.00 g glucose in 1000 ml distilled H2O and had an initial pH of 7.4. Pure BCP powder and magnesium substituted BCP powders were immersed in 50 mL of HBSS without organic species (pH 7.4), at 37 8C in Teflon sealed polystyrene bottle for 1, 2, and 4 weeks. The solution was not refreshed again. After immersion of samples in Hank’s solution for 1, 2, and 4 weeks, Mg2+, Ca2+ and PO43 concentrations of HBSS were measured using inductively coupled plasma atomic emission spectrometer (ICP-AES, Model ICPS-7500, Shimadzu, Japan). After immersion for 1, 2, and 4 weeks, the powders rinsed with double-distilled water to remove residual HBSS, and then immediately dried in a vacuum desiccators at 21 8C. The surface morphology, composition, and structure of the powders before and after immersion in HBSS were analyzed using a scanning electron microscope (SEM; S-4800, HITACHI, Japan).
3. Results and discussion 3.1. Synthesized powders XRD patterns for as-synthesized powders are presented in Fig. 1. All the powders provided the characteristic of crystalline HAp phase except for the difference in peak width and absolute intensity of the diffraction patterns. As can be seen in Fig. 1, the increase in the content of magnesium led to be broadening of diffraction patterns indicative of poor crystallinity of the assynthesized powders [10]. It seems that the substitution of magnesium did not appear to affect the diffraction pattern of the apatite phase of the as-synthesized powders. In fact, these results tend to agree with those reported in some previous works showing that XRD analysis of an as-synthesized apatite powder can result in diffraction patterns resembling that of HAp even though the Ca/P ratio is greater or less than the stoichiometric molar ratio of 1.67 [21]. Similar observations were made also for the apatite powders with elements substituted in trace levels [22]. FTIR spectra for the as-synthesized powders presented in Fig. 2 have indicated the vibrational modes of PO4 groups at 574, 603, and 1020–1120 cm1 and OH groups (630 and 3570 cm1) of apatite phase for all the powders [18]. FTIR patterns also tend to coincide with the results from XRD by the way that the assynthesized powders were characteristic of crystalline HAp phase. The weak absorption peak at 875 cm1 could be ascribed to the P– O–H vibration in the HPO42 group typical of non-stoichiometric HAp [23]. The presence of adsorbed water could be detected from FTIR spectra in the region around 3300–3600 cm1. Other information from FTIR spectra of as-synthesized powders is presence of carbonates groups at 1660 cm1, which are due to the adsorption of species remaining from the aqueous precipitation [24]. The presence of nitrates in the as-synthesized powders is clearly witnessed in the FTIR patterns in the region around at
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Fig. 3. TG plots of as-synthesized BCP powders with different magnesium substitutions.
Fig. 1. XRD patterns of as-synthesized BCP powders with different magnesium substitutions.
1385 cm1, resulting from the residual species in the as-synthesized powders tend to agree with the previous study [25]. TG plots illustrated in Fig. 3 indicate the weight loss along the investigated temperature range for the pure BCP powder and 0.5, 1, and 1.5 wt% magnesium substituted BCP powders. All the samples have indicated a similar behavior weight loss accounting for the removal of physically adsorbed water and residual nitrates and carbonates within temperature range between 25 and 1000 8C. Difference in weight loss among the four samples could be attributed to the structural changes associated with the different incorporation levels of magnesium and calcium ions in the biphasic mixtures. 3.2. Formation of magnesium substituted biphasic calcium phosphates
Fig. 2. FTIR spectra of as-synthesized BCP powders with different magnesium substitutions.
Calcination of the powders at 1000 8C indicates the improvement in crystallinity by the increase in the resolution of peaks when compared to the as-synthesized powders as shown in Fig. 4. At this temperature, all the obtained powders have both the b-TCP phase HAp phase confirming the formation of biphasic mixtures. These peaks assigned to the b-TCP phase are viewed by the difference in intensities corresponding to the degree of calcium deficiency of precursors. Thus, an increase in the calcium deficiency is characterized by the increased formation of b-TCP phase. However, the peaks pertaining to this b-TCP have not shown exact matches with those of JCPDS PDF #09-169, which in fact have indicated slight shift in the 2u and d-spacing values of maximum intensity plane (0 2 1 0), as represented in Table 1. These results suggest that magnesium is preferentially incorporated into the b-TCP phase, the replacement of Ca (ionic radius 0.99 A˚) by Mg (ionic radius 0.65 A˚) inducing a lattice contraction and the respective displacement toward higher 2u angles of the b-TCP reflections, as mentioned in the literature [25,26]. In addition, the HAp and b-TCP phase ratios of the synthesized BCP powders were estimated using the percentages of the different phases evaluated from the ratio of the intensity peaks of peaks (2 1 1) and (0 2 1 0) of HAp and b-TCP, indicating the most intense maxima characteristic of single phase, respectively. The amount of b-TCP phase is dependent on the magnesium substitution level as shown in Fig. 5. The pure BCP powder presents amount of b-TCP (37 wt%), and this amount increases slightly with the amount of magnesium to reach 1.0 wt%. These findings on the formation of different proportions of b-TCP with respect to the HAp phase are related with the different degrees of calcium deficiency while the added magnesium is found incorporated into the b-TCP phase
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Fig. 5. Quantitative phase estimation results: squares and circles indicate the weight amounts of HAp and b-TCP, respectively. Dotted linear lines are just guides for the eyes. The vertical line for maximum substitution is come from Ref. [17].
Fig. 4. XRD patterns of the pure BCP powder and magnesium substituted BCP powders after calcined at 1000 8C.
which is somewhat similar to those reported elsewhere [13,18]. However, at higher magnesium content of 1.5 wt%, the amount of b-TCP phase was steeply increased to approach 60 wt%, inducing the HAp/b-TCP ratio reversed to 40:60. This tendency would be due to the excess magnesium substitution into calcium, presenting the maximum magnesium substitution of nearly 15 mol % in b-TCP structure [17]. As a result, it could be explained with the following mechanism proposed to express the magnesium substitution for calcium into HAp [27]. Ca10x ðMg2 P2 O7 Þx=2 ðPO4 Þ6x ðOHÞ2x þ x=2H2 O ! ð1 xÞCa10 ðPO4 Þ6 ðOHÞ2 þ 3xðCaMg2x=9 Þ3 ðPO4 Þ2 þ xH2 O
(1)
According to Eq. (1), the higher substitution of magnesium for calcium is accompanied by an transition from HAp to b-TCP phase. Therefore, at higher content of magnesium, HAp phase could be transformed to b-TCP phase, indicating the total amount of b-TCP phase increased compared to lower magnesium content. For the reason, the increase of magnesium substitution increased the amount of b-TCP phase in BCP powder. This implied the weight
Table 1 Comparison of diffraction angle (2u) and d-spacing values of experimental results with respect to standard JCPDS values. Sample
JCPDSPDF#09-0169 (b-TCP) JCPDSPDF#09-0432 (HAp) Mg 0 wt% Mg 0.5 wt% Mg 1.0 wt% Mg 1.5 wt%
(0 2 1 0) plane
(2 1 1) plane
d-Spacing
2u
2.8800
31.02
2.8687 2.8598 2.8518 2.8598
31.15 31.25 31.40 31.25
d-Spacing
2u
2.8140 2.8038 2.8012 2.8012 2.8054
31.77 31.89 31.92 31.92 31.88
loss in plot of TG (as shown in Fig. 3) increased with increasing magnesium substitution. The morphology and particle size of the synthesized magnesium substituted BCP powders were not much varied with magnesium content as shown in Fig. 6. EDS mapping indicated that magnesium was uniformly spread out in the calcium phosphates and also more white spots can be observed which represented magnesium as increased magnesium contents. FTIR spectra for the synthesized magnesium substituted BCP powders at 1000 8C are presented in Fig. 7. The weight loss observed from the thermal analysis (as shown in Fig. 3) accounts for the loss of residual nitrates and carbonates, which are observed by the disappear and decrease in intensity of corresponding peaks after heating. From the FTIR spectra presented in Fig. 7, the overall spectra are appeared at having mainly two modes corresponding to characteristic PO43 and OH groups. Furthermore, the intensity of absorption for the peaks at 632 and 3570 cm1 assigning to the OH group decreased as well as a slightly broadening of PO43 bands with adding of magnesium content. These effects are typical for magnesium substituted calcium phosphates synthesized by wet methods and have to be associated with the increased lattice disorder due to HPO42, the latter usually increased with the magnesium content [10]. This effect could be also responsible in part for the alteration in its biological response. On the basis of the results of chemico-physical analyses the pure BCP powder, 0.5 wt% and 1 wt% magnesium substituted BCP powders were selected for in vitro evaluation. 3.3. In vitro degradation evaluation To determine the changes in the degradation behavior of the prepared three different powders as a function of soaking time, the specimens with different magnesium contents were subjected to an in vitro degradation in HBSS. Fig. 8 shows the typical features of magnesium-substituted BCP powders after immersing in HBSS for 1, 2, and 4 weeks, respectively. It was observed that the degree of degradation was strongly dependent of magnesium content in BCP powders. As can be seen in Fig. 8, the pure BCP powders and 0.5 wt% magnesium-substituted BCP powders were not degraded during immersion in HBSS for 1 week. On the other hand, at higher magnesium content of 1 wt%, BCP powders were degraded and precipitation started to be formed with individual small granules consisting of number of flake-like crystals onto the surface of synthesized powders. The EDS analysis showed the new formed precipitates had the Ca/P ratio of 1.39, indicating calcium deficient apatite phase. These results are consistent with previous
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Fig. 6. SEM micrographs and EDS magnesium mapping of magnesium substituted BCP powders after calcined at 1000 8C.
Fig. 7. FTIR spectra of the pure BCP powder and magnesium substituted BCP powders after calcined at 1000 8C.
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Fig. 8. SEM micrographs of pure BCP powder, 0.5 wt% and 1 wt% magnesium substituted BCP powders after immersion in HBSS for 1, 2, and 4 weeks.
Fig. 9. Changes of Ca2+, Mg2+, and PO43 ions concentrations in HBSS immersed with 1 wt% magnesium substituted BCP powder with 4 weeks.
experiments showing that the observation with TEM reveals the formation of nanocrystalline apatite-like calcium phosphates after immersion in SBF [28]. This enhanced reactivity, occurring in HBSS, would be resulted in reduction for the stability of the b-TCP structure due to distortion at calcium site when magnesium incorporates into BCP. On the other hand, when the immersing time in HBSS was extended to 2 weeks, both of pure BCP and 0.5 wt% magnesium substituted BCP powders were
started to show number of flake-like crystallites on the surface of specimens. The ICP-AES analysis reveals the changes of Mg2+, Ca2+, and PO43 concentrations in HBSS after immersing the pure BCP powder and 1 wt% magnesium substituted BCP powder, as shown in Fig. 9. The Mg2+ ions were slowly released with increasing the immersing time, which indicated that the Mg2+ ions were releasing from b-TCP phase in BCP powder. On the other hand, the concentration of Ca2+ and PO43 ions and in HBSS continually decreased with immersing time, suggesting that the Ca2+ and PO43 ions might be consumed by formation of a new product. This behavior could be explained that the magnesium have the ability of triggering increase of calcium and phosphor on powder surface, suggesting the enhanced Mg2+ ions concentration result in an elevated Ca2+ concentration, which is suitable for nucleation of the calcium and phosphor contained compound [29,30]. Therefore, the Ca2+ and PO43 ions in HBSS were continuously consumed, which indicated that the Ca2+ and PO43 ions were supersaturated around the magnesium substituted BCP powder and a new calcium deficient apatite phase continually grew on the sample surfaces with increase of immersing time. This mechanism was verified as that magnesium has the ability of osteoconductivity [29]. As a result, magnesium substituted BCP powder could be able to develop a new apatite phase on the surface in contact with physiological fluids faster than BCP powder does. On the basis of these results, the retention time to produce the new apatite phase in implantation operation for the BCP powder could be controlled by the amount of magnesium substitution.
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4. Conclusions Magnesium substituted biphasic mixtures of HAp/b-TCP were successfully prepared through in situ aqueous co-precipitation method. After calcination at 1000 8C, all the powders had the b-TCP phase in addition to that of HAp phase thus confirming the formation of biphasic mixtures. The amount of b-TCP phase was changed with the magnesium substitution level. When magnesium introduced into calcium phosphate structure, the substitution of magnesium led to a decrease in the unit cell volume. After immersing in HBSS for 1 week, 1 wt% magnesium substituted BCP powders were degraded and precipitation started to be formed with individual small granules consisting of number of flake-like crystals onto the surface of synthesized powders. On the other hand, when the immersing time in HBSS was extended to 2 weeks, both of pure BCP and 0.5 wt% magnesium substituted BCP powders were started to form new precipitates consisting of flake-like crystallites on the surface of specimens. This enhanced reactivity could be resulted in the reduction for the stability of the b-TCP structure due to distortion at the calcium site when magnesium incorporates into b-TCP phase in BCP powder. In addition, the existence of magnesium in BCP powder could accelerate the increase of calcium and phosphor on powder surface, which was suitable for nucleation of the calcium and phosphor contained compound. Magnesium substituted BCP powders could be able to develop a new apatite phase on the surface in contact with physiological fluids faster than BCP powder does. On the basis of these experimental results, the retention time to produce the new apatite phase in implantation operation for the BCP powder could be controlled by the amount of magnesium substitution. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011004650). Also, this work was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 20110027521).
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Contents lists available at SciVerse ScienceDirect
Materials Research Bulletin journal homepage: www.elsevier.com/locate/matresbu
In situ synthesis of magnesium-substituted biphasic calcium phosphate and in vitro biodegradation Tae-Wan Kim a, Hyeong-Shin Lee a, Dong-Hyun Kim a, Hyeong-Ho Jin a, Kyu-Hong Hwang b, Jong Kook Lee c, Hong-Chae Park a, Seog-Young Yoon a,* a b c
School of Materials Science and Engineering, Pusan National University, Busan 609-735, Republic of Korea School of Nano and Advanced Materials, Gyeongsang National University, Jinju, Kyungnam 660-701, Republic of Korea Department of Advanced Materials Engineering, Chosun University, Gwangju 501-759, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 January 2012 Received in revised form 13 April 2012 Accepted 8 May 2012 Available online 16 May 2012
In situ preparation of magnesium (Mg) substituted biphasic calcium phosphate (BCP) of hydroxyapatite (HAp)/b-tricalcium phosphate (b-TCP) were carried out through aqueous co-precipitation method. The concentrations of added magnesium were varied with the calcium in order to obtain constant (Ca + Mg)/P ratios of 1.602. X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy were used to characterize the structure of synthesized magnesium substituted BCP powders. The results have shown that substitution of magnesium in the calcium deficient apatites revealed the formation of biphasic mixtures of different HAp/b-TCP ratios after heating at 1000 8C. The ratios of the formation of phase mixtures were dependent on the content of magnesium. After immersing in Hanks’ balanced salt solution (HBSS) for 1 week, 1 wt% magnesium substituted BCP powders were degraded and precipitation started to be formed with small granules consisting of number of flake-like crystal onto the surface of synthesized powders. On the other hand, in the case of pure BCP powders, the formation of new precipitates was detected after immersion in HBSS for 2 weeks. On the basis of these results, magnesium substituted BCP could be able to develop a new apatite phase on the surface in contact with physiological fluids faster than BCP does. In addition, the retention time to produce the new apatite phase in implantation operation for the BCP powder could be controlled by the amount of magnesium substitution. ß 2012 Elsevier Ltd. All rights reserved.
Keywords: A. Ceramics B. Chemical synthesis C. X-ray diffraction D. Crystal structure
1. Introduction For more than 10 years, biphasic calcium phosphate (BCP) ceramics, composed of a mixture of hydroxyapatite [HAp, Ca10(PO4)6(OH)2] and b-tricalcium phosphate [b-TCP, Ca3(PO4)2], have been considered as potential substitutes for bone replacement surgical operations due to their excellent biocompatibility, ability to promote cellular functions and expressions, and osteoconductivity [1–4]. However, both materials show the difference in dissolution properties (HAp, non-resorable and bTCP, resorable) which reduce their clinical performance. Therefore, an optimum bioresorability was found when appropriately mixing both phases to provide a biphasic calcium phosphate. During implantation, it is known that bone mineral mass could be dominated by nanocrystalline multisubstituted calcium phosphate apatite [5,6]. The type and amount of ionic substitutions in the apatite phase of bone vary from the weight percent (wt%) level
* Corresponding author. Tel.: +82 51 510 2487; fax: +82 51 512 0528. E-mail address: [email protected] (S.-Y. Yoon). 0025-5408/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.materresbull.2012.05.011
[7] to the parts per million (ppm) level [8]. Among substituting ions, magnesium is known to be an important trace element in bone and teeth, despite its low concentration (generally between 0.5 and 1.5 wt% in human body). It is well known that magnesium plays a key role in bone metabolism, in particular during the early stages of osteogenesis where it stimulates osteoblast proliferation [9,10]. Due to the biological relevance of magnesium, magnesium substituted calcium phosphate compounds have attracted the interest of many scientists because magnesium incorporation is considered to be a promising way to improve the bioactivity of calcium phosphate-based biomaterials. Therefore, several studies have worked on the preparation of apatite [11–13] and calcium phosphates [14,15] implant materials containing low levels of magnesium. It has demonstrated that the incorporation of magnesium into HAp lattice sensibly affects apatite crystallization in solution and its thermal stability, promoting the formation of b-TCP and thus forming biphasic calcium phosphates. In recent, the structural analyses for the magnesium substituted HAp [16] and b-TCP [17] have been discussed with multinuclear magnetic resonance (NMR) and X-ray absorption spectroscopy (XAS), X-ray powder diffraction (XRD)
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with the Rietveld refinement, respectively. However, so far not much clear emphasis has been made quantitatively on the formation of biphasic mixtures [18] and its biodegradation behavior with substituted magnesium in the BCP powder. With the aim to fabricate the magnesium substituted BCP powders, this study focused on the in situ preparation of magnesium substituted BCP powders with precise proportions of the HAp and b-TCP phase based on the input (Ca + Mg)/P molar ratio with the co-precipitation method. The success of this task would suggest the possibility of obtaining supplementally amounts of magnesium substituted BCP powders with precise ratios of two components depending on the characteristics required for the specific application by controlling the input (Ca + Mg)/P molar ratio of starting reactants. The second aim was to assess the effect of magnesium substitution on the in vitro bioactivity of BCP powder. 2. Materials and methods 2.1. Powder preparation Pure and magnesium substituted BCP powders were synthesized in a reactor at 40 8C under magnetic stirring starting from calcium nitrate tetrahydrate (Ca(NO3)24H2O, Aldrich 99.0%, MW 236.15), magnesium nitrate hexahydrate (Mg(NO3)26H2O, Aldrich 99.0%, MW 256.41) and diammonium hydrogen phosphate ((NH4)2HPO4, Aldrich 99.0%, MW 132.06). For the preparation of pure BCP powder, an appropriate amount of calcium nitrate tetrahydrate was dissolved in water by vigorously stirring at a rate of 1000 rpm. Diammonium hydrogen phosphate solution was slowly added to the calcium nitrate tetrahydrate solution to achieve predetermined Ca/P molar ratio 1.602, providing the BCP (60% HAp and 40% b-TCP) in previous our work [19]. Magnesium substituted BCP powders with different amount of magnesium (0.5, 1, and 1.5 wt%) were prepared assuming that magnesium ions would substitute for the calcium site in the HAp and b-TCP lattice in order to obtain a nominal composition in terms of (Ca + Mg)/P ratio of 1.602. The pH of mixed solution was maintained at 11 by the addition of ammonium hydroxide (NH4OH) solution. After the completion of addition, the solution was stirred constantly for 24 h by a mechanical stirrer, allowing the reaction to complete. The precipitated suspension was discharged from the reactor and allowed to settle down for 24 h for the maturation of precipitate. After 24 h, the precipitates were separated through vacuum filtration technique and were dried at 80 8C for 24 h in a drying oven (SFC-301, Dongwon Scientific System, Korea). The dried cakes were crushed to fine powders, sieved through a mesh size of 200 (>75 mm) and used for characterization. After that, the powders were calcined at 1000 8C for 24 h (heating rate was 1 8C/min) in air. 2.2. Characterization The dried powders were subjected to thermal analysis using a heating rate of 10 8C/min and peak temperature of 1000 8C in air atmosphere (SDT Q600 TG–DTA/DSC, USA) to analyze the thermal behavior during heating. An X-ray diffractometer (Rigaku D-max IIA, Japan), at 30 kV and 20 mA with a scanning speed of 18/min, was adopted to identify the phases of powders synthesized with different magnesium contents. Diffraction patterns were prepared for each control sample, and the intensity ratio was calculated based upon two unobscured peaks from each phase. The relative intensity ratio (RIR) of HAp/b-TCP was estimated using the peaks intensity of peaks (211) and (0210) of HAp and b-TCP, respectively, according to the formula RIR = Ib-TCP/(IHAp + Ib-TCP) [20]. Various functional groups present in the prepared calcium phosphates powders were identified by FTIR (Nicolet Magna IR 560, USA). Here
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1 wt% of the powder was mixed and ground with 99% of KBr. Tablets of 10 mm diameter for FTIR measurements were prepared by pressing the powder mixture at load of 5 tons for 2 min and the spectrum was taken in the range of 400–4000 cm1 with resolution 4 cm1 and 128 times scanning. Microstructural observations of the calcined samples were conducted using a scanning electron microscope (SEM; S-4800, HITACHI, Japan). 2.3. Degradation experiments Hanks’ balanced salt solution (HBSS), an extracellular solution with an ionic composition similar to human blood plasma, was used as the supporting solution for the magnesium substituted BCP powders in vitro test. The simulated solution consisted of 8.00 g NaCl, 0.35 g NaHCO3, 0.40 g KCl, 0.06 g KH2PO4, 0.10 g MgCl26H2O, 0.14 g CaCl2, 0.06 g Na2HPO42H2O, 0.06 g MgSO47H2O, 1.00 g glucose in 1000 ml distilled H2O and had an initial pH of 7.4. Pure BCP powder and magnesium substituted BCP powders were immersed in 50 mL of HBSS without organic species (pH 7.4), at 37 8C in Teflon sealed polystyrene bottle for 1, 2, and 4 weeks. The solution was not refreshed again. After immersion of samples in Hank’s solution for 1, 2, and 4 weeks, Mg2+, Ca2+ and PO43 concentrations of HBSS were measured using inductively coupled plasma atomic emission spectrometer (ICP-AES, Model ICPS-7500, Shimadzu, Japan). After immersion for 1, 2, and 4 weeks, the powders rinsed with double-distilled water to remove residual HBSS, and then immediately dried in a vacuum desiccators at 21 8C. The surface morphology, composition, and structure of the powders before and after immersion in HBSS were analyzed using a scanning electron microscope (SEM; S-4800, HITACHI, Japan).
3. Results and discussion 3.1. Synthesized powders XRD patterns for as-synthesized powders are presented in Fig. 1. All the powders provided the characteristic of crystalline HAp phase except for the difference in peak width and absolute intensity of the diffraction patterns. As can be seen in Fig. 1, the increase in the content of magnesium led to be broadening of diffraction patterns indicative of poor crystallinity of the assynthesized powders [10]. It seems that the substitution of magnesium did not appear to affect the diffraction pattern of the apatite phase of the as-synthesized powders. In fact, these results tend to agree with those reported in some previous works showing that XRD analysis of an as-synthesized apatite powder can result in diffraction patterns resembling that of HAp even though the Ca/P ratio is greater or less than the stoichiometric molar ratio of 1.67 [21]. Similar observations were made also for the apatite powders with elements substituted in trace levels [22]. FTIR spectra for the as-synthesized powders presented in Fig. 2 have indicated the vibrational modes of PO4 groups at 574, 603, and 1020–1120 cm1 and OH groups (630 and 3570 cm1) of apatite phase for all the powders [18]. FTIR patterns also tend to coincide with the results from XRD by the way that the assynthesized powders were characteristic of crystalline HAp phase. The weak absorption peak at 875 cm1 could be ascribed to the P– O–H vibration in the HPO42 group typical of non-stoichiometric HAp [23]. The presence of adsorbed water could be detected from FTIR spectra in the region around 3300–3600 cm1. Other information from FTIR spectra of as-synthesized powders is presence of carbonates groups at 1660 cm1, which are due to the adsorption of species remaining from the aqueous precipitation [24]. The presence of nitrates in the as-synthesized powders is clearly witnessed in the FTIR patterns in the region around at
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Fig. 3. TG plots of as-synthesized BCP powders with different magnesium substitutions.
Fig. 1. XRD patterns of as-synthesized BCP powders with different magnesium substitutions.
1385 cm1, resulting from the residual species in the as-synthesized powders tend to agree with the previous study [25]. TG plots illustrated in Fig. 3 indicate the weight loss along the investigated temperature range for the pure BCP powder and 0.5, 1, and 1.5 wt% magnesium substituted BCP powders. All the samples have indicated a similar behavior weight loss accounting for the removal of physically adsorbed water and residual nitrates and carbonates within temperature range between 25 and 1000 8C. Difference in weight loss among the four samples could be attributed to the structural changes associated with the different incorporation levels of magnesium and calcium ions in the biphasic mixtures. 3.2. Formation of magnesium substituted biphasic calcium phosphates
Fig. 2. FTIR spectra of as-synthesized BCP powders with different magnesium substitutions.
Calcination of the powders at 1000 8C indicates the improvement in crystallinity by the increase in the resolution of peaks when compared to the as-synthesized powders as shown in Fig. 4. At this temperature, all the obtained powders have both the b-TCP phase HAp phase confirming the formation of biphasic mixtures. These peaks assigned to the b-TCP phase are viewed by the difference in intensities corresponding to the degree of calcium deficiency of precursors. Thus, an increase in the calcium deficiency is characterized by the increased formation of b-TCP phase. However, the peaks pertaining to this b-TCP have not shown exact matches with those of JCPDS PDF #09-169, which in fact have indicated slight shift in the 2u and d-spacing values of maximum intensity plane (0 2 1 0), as represented in Table 1. These results suggest that magnesium is preferentially incorporated into the b-TCP phase, the replacement of Ca (ionic radius 0.99 A˚) by Mg (ionic radius 0.65 A˚) inducing a lattice contraction and the respective displacement toward higher 2u angles of the b-TCP reflections, as mentioned in the literature [25,26]. In addition, the HAp and b-TCP phase ratios of the synthesized BCP powders were estimated using the percentages of the different phases evaluated from the ratio of the intensity peaks of peaks (2 1 1) and (0 2 1 0) of HAp and b-TCP, indicating the most intense maxima characteristic of single phase, respectively. The amount of b-TCP phase is dependent on the magnesium substitution level as shown in Fig. 5. The pure BCP powder presents amount of b-TCP (37 wt%), and this amount increases slightly with the amount of magnesium to reach 1.0 wt%. These findings on the formation of different proportions of b-TCP with respect to the HAp phase are related with the different degrees of calcium deficiency while the added magnesium is found incorporated into the b-TCP phase
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Fig. 5. Quantitative phase estimation results: squares and circles indicate the weight amounts of HAp and b-TCP, respectively. Dotted linear lines are just guides for the eyes. The vertical line for maximum substitution is come from Ref. [17].
Fig. 4. XRD patterns of the pure BCP powder and magnesium substituted BCP powders after calcined at 1000 8C.
which is somewhat similar to those reported elsewhere [13,18]. However, at higher magnesium content of 1.5 wt%, the amount of b-TCP phase was steeply increased to approach 60 wt%, inducing the HAp/b-TCP ratio reversed to 40:60. This tendency would be due to the excess magnesium substitution into calcium, presenting the maximum magnesium substitution of nearly 15 mol % in b-TCP structure [17]. As a result, it could be explained with the following mechanism proposed to express the magnesium substitution for calcium into HAp [27]. Ca10x ðMg2 P2 O7 Þx=2 ðPO4 Þ6x ðOHÞ2x þ x=2H2 O ! ð1 xÞCa10 ðPO4 Þ6 ðOHÞ2 þ 3xðCaMg2x=9 Þ3 ðPO4 Þ2 þ xH2 O
(1)
According to Eq. (1), the higher substitution of magnesium for calcium is accompanied by an transition from HAp to b-TCP phase. Therefore, at higher content of magnesium, HAp phase could be transformed to b-TCP phase, indicating the total amount of b-TCP phase increased compared to lower magnesium content. For the reason, the increase of magnesium substitution increased the amount of b-TCP phase in BCP powder. This implied the weight
Table 1 Comparison of diffraction angle (2u) and d-spacing values of experimental results with respect to standard JCPDS values. Sample
JCPDSPDF#09-0169 (b-TCP) JCPDSPDF#09-0432 (HAp) Mg 0 wt% Mg 0.5 wt% Mg 1.0 wt% Mg 1.5 wt%
(0 2 1 0) plane
(2 1 1) plane
d-Spacing
2u
2.8800
31.02
2.8687 2.8598 2.8518 2.8598
31.15 31.25 31.40 31.25
d-Spacing
2u
2.8140 2.8038 2.8012 2.8012 2.8054
31.77 31.89 31.92 31.92 31.88
loss in plot of TG (as shown in Fig. 3) increased with increasing magnesium substitution. The morphology and particle size of the synthesized magnesium substituted BCP powders were not much varied with magnesium content as shown in Fig. 6. EDS mapping indicated that magnesium was uniformly spread out in the calcium phosphates and also more white spots can be observed which represented magnesium as increased magnesium contents. FTIR spectra for the synthesized magnesium substituted BCP powders at 1000 8C are presented in Fig. 7. The weight loss observed from the thermal analysis (as shown in Fig. 3) accounts for the loss of residual nitrates and carbonates, which are observed by the disappear and decrease in intensity of corresponding peaks after heating. From the FTIR spectra presented in Fig. 7, the overall spectra are appeared at having mainly two modes corresponding to characteristic PO43 and OH groups. Furthermore, the intensity of absorption for the peaks at 632 and 3570 cm1 assigning to the OH group decreased as well as a slightly broadening of PO43 bands with adding of magnesium content. These effects are typical for magnesium substituted calcium phosphates synthesized by wet methods and have to be associated with the increased lattice disorder due to HPO42, the latter usually increased with the magnesium content [10]. This effect could be also responsible in part for the alteration in its biological response. On the basis of the results of chemico-physical analyses the pure BCP powder, 0.5 wt% and 1 wt% magnesium substituted BCP powders were selected for in vitro evaluation. 3.3. In vitro degradation evaluation To determine the changes in the degradation behavior of the prepared three different powders as a function of soaking time, the specimens with different magnesium contents were subjected to an in vitro degradation in HBSS. Fig. 8 shows the typical features of magnesium-substituted BCP powders after immersing in HBSS for 1, 2, and 4 weeks, respectively. It was observed that the degree of degradation was strongly dependent of magnesium content in BCP powders. As can be seen in Fig. 8, the pure BCP powders and 0.5 wt% magnesium-substituted BCP powders were not degraded during immersion in HBSS for 1 week. On the other hand, at higher magnesium content of 1 wt%, BCP powders were degraded and precipitation started to be formed with individual small granules consisting of number of flake-like crystals onto the surface of synthesized powders. The EDS analysis showed the new formed precipitates had the Ca/P ratio of 1.39, indicating calcium deficient apatite phase. These results are consistent with previous
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Fig. 6. SEM micrographs and EDS magnesium mapping of magnesium substituted BCP powders after calcined at 1000 8C.
Fig. 7. FTIR spectra of the pure BCP powder and magnesium substituted BCP powders after calcined at 1000 8C.
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Fig. 8. SEM micrographs of pure BCP powder, 0.5 wt% and 1 wt% magnesium substituted BCP powders after immersion in HBSS for 1, 2, and 4 weeks.
Fig. 9. Changes of Ca2+, Mg2+, and PO43 ions concentrations in HBSS immersed with 1 wt% magnesium substituted BCP powder with 4 weeks.
experiments showing that the observation with TEM reveals the formation of nanocrystalline apatite-like calcium phosphates after immersion in SBF [28]. This enhanced reactivity, occurring in HBSS, would be resulted in reduction for the stability of the b-TCP structure due to distortion at calcium site when magnesium incorporates into BCP. On the other hand, when the immersing time in HBSS was extended to 2 weeks, both of pure BCP and 0.5 wt% magnesium substituted BCP powders were
started to show number of flake-like crystallites on the surface of specimens. The ICP-AES analysis reveals the changes of Mg2+, Ca2+, and PO43 concentrations in HBSS after immersing the pure BCP powder and 1 wt% magnesium substituted BCP powder, as shown in Fig. 9. The Mg2+ ions were slowly released with increasing the immersing time, which indicated that the Mg2+ ions were releasing from b-TCP phase in BCP powder. On the other hand, the concentration of Ca2+ and PO43 ions and in HBSS continually decreased with immersing time, suggesting that the Ca2+ and PO43 ions might be consumed by formation of a new product. This behavior could be explained that the magnesium have the ability of triggering increase of calcium and phosphor on powder surface, suggesting the enhanced Mg2+ ions concentration result in an elevated Ca2+ concentration, which is suitable for nucleation of the calcium and phosphor contained compound [29,30]. Therefore, the Ca2+ and PO43 ions in HBSS were continuously consumed, which indicated that the Ca2+ and PO43 ions were supersaturated around the magnesium substituted BCP powder and a new calcium deficient apatite phase continually grew on the sample surfaces with increase of immersing time. This mechanism was verified as that magnesium has the ability of osteoconductivity [29]. As a result, magnesium substituted BCP powder could be able to develop a new apatite phase on the surface in contact with physiological fluids faster than BCP powder does. On the basis of these results, the retention time to produce the new apatite phase in implantation operation for the BCP powder could be controlled by the amount of magnesium substitution.
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4. Conclusions Magnesium substituted biphasic mixtures of HAp/b-TCP were successfully prepared through in situ aqueous co-precipitation method. After calcination at 1000 8C, all the powders had the b-TCP phase in addition to that of HAp phase thus confirming the formation of biphasic mixtures. The amount of b-TCP phase was changed with the magnesium substitution level. When magnesium introduced into calcium phosphate structure, the substitution of magnesium led to a decrease in the unit cell volume. After immersing in HBSS for 1 week, 1 wt% magnesium substituted BCP powders were degraded and precipitation started to be formed with individual small granules consisting of number of flake-like crystals onto the surface of synthesized powders. On the other hand, when the immersing time in HBSS was extended to 2 weeks, both of pure BCP and 0.5 wt% magnesium substituted BCP powders were started to form new precipitates consisting of flake-like crystallites on the surface of specimens. This enhanced reactivity could be resulted in the reduction for the stability of the b-TCP structure due to distortion at the calcium site when magnesium incorporates into b-TCP phase in BCP powder. In addition, the existence of magnesium in BCP powder could accelerate the increase of calcium and phosphor on powder surface, which was suitable for nucleation of the calcium and phosphor contained compound. Magnesium substituted BCP powders could be able to develop a new apatite phase on the surface in contact with physiological fluids faster than BCP powder does. On the basis of these experimental results, the retention time to produce the new apatite phase in implantation operation for the BCP powder could be controlled by the amount of magnesium substitution. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011004650). Also, this work was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 20110027521).
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