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Aqueous processing of hydroxyapatite
Materials Chemistry and Physics 99 (2006) 398–404
Aqueous processing of hydroxyapatite Jingxian Zhang a,∗ , Masahiko Maeda b , Noriko Kotobuki b , Motohiro Hirose b , Hajime Ohgushi b , Dongliang Jiang c , Mikio Iwasa a a b
National Institute of Advanced Industrial Science and Technology, AIST Kansai, Osaka 563-8577, Japan National Institute of Advanced Industrial Science and Technology, AIST Kansai, Hyogo 661-0974, Japan c The State Key Laboratory of High Performance Ceramics and Superfine Structure, Shanghai Institute of Ceramics, Shanghai 200050, China Received 27 November 2004; received in revised form 9 October 2005; accepted 11 November 2005
Abstract This paper describes a study of the dispersion of hydroxyapatite (OHAp) powder in aqueous media. The slurry stability is achieved by utilizing a combination of a commercially available dispersant, polyacrylic acid sodium salt (PAA-Na) and ethylenediaminetetraacetic acid, tetrasodium salt, dihydrate (EDTA·4Na·2H2 O). Adsorption, sedimentation and rheology measurements have been performed to investigate the suspensions properties. It was shown that HAp slurries could be stabilized at pH 10.50 with the PAA-Na concentration around 0.45 wt%. Rheological measurement confirmed that well dispersed slurries could be obtained with the solid content as high as 63 vol%. After sintering, the relative density of HAp sheets reached 98.8%. The as sintered HAp samples exhibited excellent properties for cell attachment and proliferation. Results showed that the aqueous processing technique is effective for preparation HAp samples for biological applications. © 2005 Elsevier B.V. All rights reserved. Keywords: Dispersant; PAA-Na; HAp; Slurry; Stability
1. Introduction There is an escalating interest in apatite, which seems to be driven mainly by the requirements for the hard tissue replacement. Hydroxyapatite (OHAp), which shows excellent biocompatibility with hard tissues and also with skin and muscles tissues, is the most appropriate ceramic material for hard-tissue replacement [1,2]. Dense, sintered HAp has many bone replacement applications and is used for repairing bone defects in dental and orthopaedic sites, immediate tooth replacement, augmentation of alveolar ridges, pulp capping material and maxillofacial reconstruction, etc. [3–6]. Presently, one of the most important applications of dense HAp is as percutaneous devices for continuous ambulatory peritoneal dialysis, monitoring of blood pressure and blood sugar, or optical observation of inner body tissue [1]. Unfortunately, due to low mechanical reliability [7], the use of HAp ceramics is very limited [2]. In recent years, efforts are made toward preparing more reliable bioceramic bod-
∗
Corresponding author. Tel.: +81 7275 19785; fax: +81 7275 19631. E-mail address: jx [email protected] (J. Zhang).
0254-0584/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2005.11.020
ies. Colloidal processing could be the most promising route for achieving this objective [8]. Colloidal approach has the potential for eliminating detrimental heterogeneities, avoiding their reintroduction during the successive processing steps and obtaining pieces with complex shapes similar to human bones [9]. Colloidal processing of ceramic powders requires control of the homogeneity, rheology and dispersion of the suspensions. Particle dispersion is often the limiting factor, affecting both rheology and homogeneity of suspensions. An addition of polymer dispersant was effective in preventing the agglomeration of ceramic powder by electrosteric stabilization and enhancing particle stability [10,11]. The dispersion of HAp powder in aqueous media has been studied in literature [12–14]. It was reported that anionic polyelectrolytes were effective for obtaining homogeneous distribution of HAp powder [15–19]. The objective of the current work is to study the effects of the dispersant, pH on the dispersing ability of HAp particles, the rheological behavior of the slurries and the tape-casting performance, focusing on the quantity of polymer dispersant. The mechanical and biological properties of as sintered HAp samples were also studied.
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sions was pored into graded tubes and the sedimentation volume was recorded; the other part of the suspensions was centrifuged and the adsorption of PAA-Na on HAp surface was determined by TG-DTA.
2.4. Rheological measurements The HAp suspensions were prepared with solids loading in the 35–60 vol% range with different amount of dispersant. The suspensions were placed in milling jars and roll-milled for 24 h before analysis. Apparent viscosity of the suspensions under different shear rate was examined under steady shear conditions by ascending and descending shear rate ramps respectively using a viscosity meter (DV-E, Brookfield Engineering Laboratories Inc., USA).
2.5. Tape casting, lamination and sintering
Fig. 1. XRD patterns of HAp200 powder.
2. Experimental 2.1. Starting materials The powder used is HAp 200 (Taihei Chemical Industrial Co., Ltd., Osaka, Japan), which is aggregate of primary elongated hexagonal grains with the diameter and the length between 0.3–0.5 m and 2–3 m, respectively. The XRD pattern is shown in Fig. 1. The bulk characteristics, which are taken from the manufacturer, indicated that the powder only contains minor amounts of compounds other than HAp, see Table 1. The dispersant was polyacrylic acid sodium salt (PAA-Na, Aldrich, MW = 30,000). Ethylenediaminetetraacetic acid tetrasodium salt dihydrate, EDTA·4Na·2H2 O (Analytical, Osaka Kishida Chemicals, Japan) was introduced to improve slurry stability. The optimal EDTA·4Na·2H2 O content is 10 wt% of that of PAA-Na. PVA2000 (Osaka Kishida Chemicals, Japan) was used as binder and ethylene glycol as plasticizer. The tape casting process was similar to that reported in literature [20,21]. Aqueous suspensions were prepared by adding HAp powders to deionized water in the presence of different amount of dispersant. The pH of suspensions was adjusted by HNO3 and NaOH (Analytical, Kanto Chemical Co. Inc., Japan).
2.2. Zeta potential test Zeta potential of the HAp particles was measured through Zetaplus (Brookhaven Instruments Corporation). HAp suspensions (0.01 vol%) were prepared in the absence and presence of PAA-Na at various pHs. The ionic strength of each slurry was adjusted with 0.001 M KCl solution. The slurries were mixed for 24 h to break agglomerates and achieve equilibrium between the powder surface and the dispersant in the suspensions.
2.3. Sedimentation and adsorption test 5 vol% HAp suspensions in the absence and presence of dispersant were made for sedimentation measurements. After mixing for 24 h, part of the suspenTable 1 Impurities in HAp-200 powders Element
Content (ppm)
Chloride Sulfate Heavy metal Fe As Mg Mn
<20 <50 <5 <10 <1 <50 <20
Initially, well-stabilized 63 vol% HAp slurries were prepared followed by addition of binder (13 wt% PVA solutions) and plasticizer. The slurries were further mixed for 2 h with a stirrer. After homogenizing, the slurries were degassed under low vacuum (∼10−5 Torr) to eliminate air bubbles. Finally, suspensions were cast onto a fixed glass plate carrier. Tapes were allowed to dry freely in open air at room temperature. Density of green sheets was measured in ethanol. After lamination and binder removal, HAp samples were sintered at 1250 ◦ C for 1 h in furnace and the density was measured in deionized water. Tests of flexural strength were performed by three point bending from specimens of size 3 mm × 4 mm × 36 mm. The calcium/phosphorous molar ratio of hydroxyapatite was determined by EDX. The phases of HAp powder and HAp samples were identified by X-ray diffractometry (XRD, Cu K␣, Rigaku, Tokyo, Japan). The step size and scan rate were fixed at 0.02 and 0.5◦ /min, respectively.
2.6. Biological test All of the dissolution tests were conducted in triplicate in simulated body fluid (SBF). The SBF was prepared by dissolving reagent-grade NaCl, KCl, K2 HPO4 ·3H2 O, MgCl2 ·H2 O, CaCl2 , and Na2 SO4 into distilled water and buffering to pH 7.4 with tris[hydroxymethyl]aminomethane [(CH2 OH3 )3 CNH3 ] and hydrochloric acid [22]. As sintered HAp samples were ground into a fine powder. After being ultrasonically washed in acetone and rinsed in deionized water, 10 mg of each granule was individually soaked into 30 mL of solution at 37 ◦ C for times varying from 1 to 60 days. After the immersion periods of 1, 3, 5, 7, 9, 11, 15, 30 and 60 days, the samples were centrifuged. The changes in the concentrations of calcium in the supernatant were measured by Shimadzu AA-6401F atomic absorption spectrophotometer (Shimadzu Corp., Tokyo, Japan). HAp specimens for flexural strength measurement were also immersed in SBF for 60 days at 37 ◦ C. HAp round disks with the diameter in the 17–19 mm range were used for cell culture test. Marrow cells were obtained from the bone shaft of femora of Fisher 344 male, 7-week-old rats. Initially, rat bone marrow cells were flushed out by a culture medium (MEM, Nacalai Tesque Inc., Kyoto, Japan) and cultured in a humidified atmosphere of 95% air with 5% CO2 at 37 ◦ C. After confluence in T75 flasks (Becton, Dickinson and Company (BD), NJ, USA), the adherent cells (MSC) from rat bone marrow were resuspended in culture medium followed by harvesting using Trypsin/EDTA. Subsequently, The cell suspension with osteogenic medium was applied to sterilized, HAp ceramic disks (18 mm in diameter and 3 mm in thick), which were placed into a 12-well plate. The culture medium was changed two or three times per week. After 2 weeks, the cell viability on HAp sample surface was checked using LIVE/DEAD Viability assay kit (Molecular Probes Inc., USA).
3. Results and discussion 3.1. Surface properties of HAp The iso-electric point (pHIEP ) of HAp particles was situated at approximately pH 7.0, thus, acrylic-type polymer is usually selected as dispersant in aqueous media [15–19]. In this paper,
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Fig. 3. Zeta potential of HAp powder in the (a) absence and (b) presence of dispersant (PAA-NA).
Fig. 2. Influence of pH on the stability of 5 vol% HAp slurries (mixed for 24 h).
point (Fig. 3), correspondingly, the sediment volume was at its lowest point. Based on the discussion above, in this paper, the HAp suspensions are stabilized around pH 10.50, similar to that reported in literature [19]. The optimal content of dispersant required was determined using 20 vol% HAp slurries at pH 10.50. Fig. 4(a) represents the viscosity, as a function of PAA-Na concentration, at three different shear rates for HAp suspensions. It was shown that minor addition of dispersant can considerably decrease the slurry viscosity. A concentration of ∼0.45 wt% dispersant helps to produce stable suspensions. When the dispersant concentration is increased beyond this point, the viscosity kept almost constant.
polyacrylic acid sodium salt (MW = 30,000) was used as dispersant. The addition of PAA-Na leads to an obvious increase of negative charge on HAp particle surface, indicating the chemical adsorption type of it. The stability of HAp slurries was characterized in term of sedimentation test, see Fig. 2. 3.2. Slurry stability As shown in Fig. 2, in the absence of dispersant, the slurry is stable in pH 10–12 range, at which the HAp powder is stabilized electrostatically due to the high surface charge. In low pH region (<5), due to the dissolution of HAp, the slurries are not stable; in the pH 5–10 range, the instability is due to the low surface charge of HAp particles (near the isoelectric point, Fig. 3). In the presence of dispersant PAA-Na, the slurry can be stabilized also in neutral region. This might be due to the adsorbed polymers, which modify the surface charge behavior of HAp particles and lead to the shift of the isoelectric point toward more acid region (pH). An instable slurry state was observed in acid pH region (pH < 7), where the surface charge of HAp particles is not so high and the PAA-Na molecules are not well expanded, similar to that observed in ZrO2 system [23]. On the contrary, at high pH (>8), the slurry is well stabilized characterized as a low sediment volume. This slurry stability can be well correlated to the electric and steric mechanism [19]. At pH 10.5, the surface charge of HAp particles was around its highest
Fig. 4. Influence of dispersant content on the stability of HAP slurries (pH 10.50): (a) viscosity of 20 vol% HAp slurries vs. dispersant content and (b) slurry properties (20 vol%).
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represents the adsorption behavior that would occur if 100% of the copolymer added was adsorbed. As evidenced in Fig. 5, the adsorption amount of PAA-Na increases with the increase in dispersant content up to 0.45 wt% and kept nearly constant thereafter, indicating the saturation adsorption of dispersant. This adsorption saturation can be well related to the viscosity measurement, see Fig. 4. At low dispersant content (<0.27 wt%), almost all the dispersant added adsorbs on the surface and the data follows the 100% line until the adsorption of PAA-Na reach approximately 0.55 mg m−2 . So the adsorption isotherms can be described as high-affinity type. This kind of adsorption behavior is commonly observed in many systems [11,24]. Fig. 5. Adsorption of PAA-Na on HAp particle surface (pH 10.5).
The viscosity change versus shear rate is further characterized in Fig. 4(b). As shown in Fig. 4(b), at low PAA-Na concentration (<0.32 wt%), the suspensions exhibit shear thinning behavior, this can be well correlated to the flocculated slurry state, which might come from the inadequate adsorption of dispersant [11]. When the dispersant content increased to 0.45 wt%, the slurry stability improved from shear thinning to near Newton behavior, indicating the achievement in slurry stability. The adsorption isotherm of PAA-Na on HAp particle surface is characterized at pH 10.5, see Fig. 5. The dash diagonal line
3.3. Flow behavior of HAp suspensions From the discussion above, it can be concluded that HAp slurries with high solid content can be obtained at pH 10.5 with the dispersant content around 0.45 wt%. Fig. 6 shows the flow behavior of HAp suspensions in the presence of PAA-Na with the solid content as 40, 50 vol% (without EDTA-Na) and 60 vol% (with EDTA-Na), respectively. Results showed that with solely PAA-Na as dispersant, it was difficult to obtain high solid content HAp suspensions (>55 vol%). After the addition of EDTA-Na s, it was possibly to prepare 60 vol% suspensions with high fluidity. The slurries showed a lower viscosity than that for 50 vol% suspensions, though the time dependent behavior is a bit significant.
Fig. 6. Properties of HAp suspensions.
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Table 2 HAp slurry formulations Materials
Function
Density
wt%
vol%
HAp PAA-Na EDTA-4Na Water PVA Ethylene glycol
Ceramic powder Dispersant Dispersant Solvent Binder Plasticizer
2.7 1 1 1 1.08 1.113
54.13 1.08 0.11 35.96 4.36 4.36
30.77 1.66 0.17 55.2 6.19 6.01
For shear thinning slurries, the relationship between shear stress and shear rate can be described by the following equation [25] τ = Kγ n
(2) Fig. 7. XRD pattern of HAp starting powder and as-sintered HAp samples.
where τ is the shear stress, r the shear rate and n is less than one. As shown in Fig. 6(c), the slip torque versus shear rate curves for 60 vol% slurries can be fitted linearly with the correlation coefficients higher than 0.99. The K and n value, which could be obtained from equation (2), are 24.17 and 0.40, respectively. Therefore, it is proposed that the 60 vol% HAp slurries exhibit power law, strong shear thinning behavior which is universal for stable ceramic slurries [25–27]. This slurry property meets the requirement for tape casting process. 3.4. Tape casting and sintering The formulation of tape casting slurries is shown in Table 2. The tape casting process was similar to that reported in literature. After drying, the green sheets exhibit a homogeneous microstructure. The relatively green density was 52.1%. The efficiency of dispersant that enables deagglomeration and the particles to flow and pack closely is the most probable explanation for the high density that has been reached. After removing the organic additives (dispersants, binder and plasticizer) and lamination, the HAp samples can be easily sintered at 1250 ◦ C and soaked for 1h in air with the relative density as 98.8%. The X-ray analyses did not show the presence of -TCP phase, which is in agreement with the literature [28], Fig. 7. A series of sharp diffraction peaks were observed, indicating that HAp grains are well crystallized. The Ca/P ratio was kept almost unchanged after sintering, see Table 3. The flexural strength of sintered HAp samples was 69.5 MPa, which is very close to the data shown in literature [7,29,30], Table 3.
Fig. 8. Continuous dissolution assay in SBF for HAp samples.
3.5. Biological properties Fig. 8 showed the calcium concentration changes in the SBF solution as a function of time. On the first day, the dissolution was dominant. After 3 days, due to the high concentration of Ca ions, the precipitation process became dominant and a decrease in calcium concentration occurred. Fourteen days after immersion, the calcium concentration reached the minimum point. This is in agreement with that shown in literature [31,32]. After 30 days, the calcium concentration increased again. However, after 60 days, the amount of material dissolution was stabilized, similar to the case shown in literature [33]. This dissolution process can be explained as a competition of dissolution of the HAp powder and the precipitation of newly formed CaP on its sur-
Table 3 Properties of HAp samples after sintering
Starting powder After sintering After immersion (60 days)
Ca/P ratio
Flexural strength (MPa)
Hardness (GPa)
Toughness (MPa m1/2 )
1.67 1.66 –
– 69.5 ± 5.8 65.7 ± 5.2
– 4.49 ± 0.12 4.41 ± 0.09
– 1.15 ± 0.14 1.21 ± 0.14
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the biocompatibility of the HAp samples. After binder removal and sintering, all the organic additives are removed and did not show any negative effect on the properties of HAp samples. 4. Conclusions The isoelectric point of HAp is at pH 7.0. Results showed that HAp slurries can be stabilized at pH 10.5 with PAA-Na content around 0.45 wt%. However, at high solid content, the stability of HAp suspensions is highly reduced with solely PAA-Na as dispersant. So EDTA-Na is introduced to improve the slurries stability. In the presence of EDTA-Na, 60 vol% HAp suspension exhibit well stabilized state which can be fitted well by shear thinning equations. The slurries are suitable for the subsequent tape casting process. After tape casting, lamination and sintering at 1250 ◦ C, HAp samples can be densified to 98.8% (RD). The samples exhibited excellent properties for cell attachment and proliferation. Results showed that it is possible to use aqueous processing route to prepare HAp samples for biological applications. References
Fig. 9. Live/Dead stain of the cultured cells after 14 days: (a) Live stain and (b) Dead stain (×19.2).
face. Initially, there was significant dissolution of the material and, with the increase of the calcium concentration in solutions, the dissolution–precipitation balance moved, and the precipitation was the main driver. After a long immersion, the dissolution and precipitation reached equilibrium and the calcium concentration was kept constant. As shown in Fig. 8, the dissolution is not so significant, which might be due to the well crystallized HAp samples after sintering [32], Fig. 7. This can also be well correlated to the mechanical properties. The flexural strength after 60 days immersion was 65.7 MPa, similar to that of assintered samples, Table 3. The HAp samples were further characterized by cell culture test, results are shown in Fig. 9. After 14 days, the viability of marrow cells was evaluated by Live/Dead staining. The assay is realized by mechanisms based on a simultaneous determination of living and dead cells with two probes, calcein-bis[(acetyloxy) methyl] ester (calcein-AM) for intracellular esterase activity and ethidium homodiner-1 (EthD-1) for plasma membrane integrity. The viable cells were dyed to green, and the nucleus of the dead cells was dyed to red. As shown in Fig. 9, most of the cells are green and only quite a few cells are red. This is in agreement with the XRD, Ca/P ratio and strength test and would confirm
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Aqueous processing of hydroxyapatite Jingxian Zhang a,∗ , Masahiko Maeda b , Noriko Kotobuki b , Motohiro Hirose b , Hajime Ohgushi b , Dongliang Jiang c , Mikio Iwasa a a b
National Institute of Advanced Industrial Science and Technology, AIST Kansai, Osaka 563-8577, Japan National Institute of Advanced Industrial Science and Technology, AIST Kansai, Hyogo 661-0974, Japan c The State Key Laboratory of High Performance Ceramics and Superfine Structure, Shanghai Institute of Ceramics, Shanghai 200050, China Received 27 November 2004; received in revised form 9 October 2005; accepted 11 November 2005
Abstract This paper describes a study of the dispersion of hydroxyapatite (OHAp) powder in aqueous media. The slurry stability is achieved by utilizing a combination of a commercially available dispersant, polyacrylic acid sodium salt (PAA-Na) and ethylenediaminetetraacetic acid, tetrasodium salt, dihydrate (EDTA·4Na·2H2 O). Adsorption, sedimentation and rheology measurements have been performed to investigate the suspensions properties. It was shown that HAp slurries could be stabilized at pH 10.50 with the PAA-Na concentration around 0.45 wt%. Rheological measurement confirmed that well dispersed slurries could be obtained with the solid content as high as 63 vol%. After sintering, the relative density of HAp sheets reached 98.8%. The as sintered HAp samples exhibited excellent properties for cell attachment and proliferation. Results showed that the aqueous processing technique is effective for preparation HAp samples for biological applications. © 2005 Elsevier B.V. All rights reserved. Keywords: Dispersant; PAA-Na; HAp; Slurry; Stability
1. Introduction There is an escalating interest in apatite, which seems to be driven mainly by the requirements for the hard tissue replacement. Hydroxyapatite (OHAp), which shows excellent biocompatibility with hard tissues and also with skin and muscles tissues, is the most appropriate ceramic material for hard-tissue replacement [1,2]. Dense, sintered HAp has many bone replacement applications and is used for repairing bone defects in dental and orthopaedic sites, immediate tooth replacement, augmentation of alveolar ridges, pulp capping material and maxillofacial reconstruction, etc. [3–6]. Presently, one of the most important applications of dense HAp is as percutaneous devices for continuous ambulatory peritoneal dialysis, monitoring of blood pressure and blood sugar, or optical observation of inner body tissue [1]. Unfortunately, due to low mechanical reliability [7], the use of HAp ceramics is very limited [2]. In recent years, efforts are made toward preparing more reliable bioceramic bod-
∗
Corresponding author. Tel.: +81 7275 19785; fax: +81 7275 19631. E-mail address: jx [email protected] (J. Zhang).
0254-0584/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2005.11.020
ies. Colloidal processing could be the most promising route for achieving this objective [8]. Colloidal approach has the potential for eliminating detrimental heterogeneities, avoiding their reintroduction during the successive processing steps and obtaining pieces with complex shapes similar to human bones [9]. Colloidal processing of ceramic powders requires control of the homogeneity, rheology and dispersion of the suspensions. Particle dispersion is often the limiting factor, affecting both rheology and homogeneity of suspensions. An addition of polymer dispersant was effective in preventing the agglomeration of ceramic powder by electrosteric stabilization and enhancing particle stability [10,11]. The dispersion of HAp powder in aqueous media has been studied in literature [12–14]. It was reported that anionic polyelectrolytes were effective for obtaining homogeneous distribution of HAp powder [15–19]. The objective of the current work is to study the effects of the dispersant, pH on the dispersing ability of HAp particles, the rheological behavior of the slurries and the tape-casting performance, focusing on the quantity of polymer dispersant. The mechanical and biological properties of as sintered HAp samples were also studied.
J. Zhang et al. / Materials Chemistry and Physics 99 (2006) 398–404
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sions was pored into graded tubes and the sedimentation volume was recorded; the other part of the suspensions was centrifuged and the adsorption of PAA-Na on HAp surface was determined by TG-DTA.
2.4. Rheological measurements The HAp suspensions were prepared with solids loading in the 35–60 vol% range with different amount of dispersant. The suspensions were placed in milling jars and roll-milled for 24 h before analysis. Apparent viscosity of the suspensions under different shear rate was examined under steady shear conditions by ascending and descending shear rate ramps respectively using a viscosity meter (DV-E, Brookfield Engineering Laboratories Inc., USA).
2.5. Tape casting, lamination and sintering
Fig. 1. XRD patterns of HAp200 powder.
2. Experimental 2.1. Starting materials The powder used is HAp 200 (Taihei Chemical Industrial Co., Ltd., Osaka, Japan), which is aggregate of primary elongated hexagonal grains with the diameter and the length between 0.3–0.5 m and 2–3 m, respectively. The XRD pattern is shown in Fig. 1. The bulk characteristics, which are taken from the manufacturer, indicated that the powder only contains minor amounts of compounds other than HAp, see Table 1. The dispersant was polyacrylic acid sodium salt (PAA-Na, Aldrich, MW = 30,000). Ethylenediaminetetraacetic acid tetrasodium salt dihydrate, EDTA·4Na·2H2 O (Analytical, Osaka Kishida Chemicals, Japan) was introduced to improve slurry stability. The optimal EDTA·4Na·2H2 O content is 10 wt% of that of PAA-Na. PVA2000 (Osaka Kishida Chemicals, Japan) was used as binder and ethylene glycol as plasticizer. The tape casting process was similar to that reported in literature [20,21]. Aqueous suspensions were prepared by adding HAp powders to deionized water in the presence of different amount of dispersant. The pH of suspensions was adjusted by HNO3 and NaOH (Analytical, Kanto Chemical Co. Inc., Japan).
2.2. Zeta potential test Zeta potential of the HAp particles was measured through Zetaplus (Brookhaven Instruments Corporation). HAp suspensions (0.01 vol%) were prepared in the absence and presence of PAA-Na at various pHs. The ionic strength of each slurry was adjusted with 0.001 M KCl solution. The slurries were mixed for 24 h to break agglomerates and achieve equilibrium between the powder surface and the dispersant in the suspensions.
2.3. Sedimentation and adsorption test 5 vol% HAp suspensions in the absence and presence of dispersant were made for sedimentation measurements. After mixing for 24 h, part of the suspenTable 1 Impurities in HAp-200 powders Element
Content (ppm)
Chloride Sulfate Heavy metal Fe As Mg Mn
<20 <50 <5 <10 <1 <50 <20
Initially, well-stabilized 63 vol% HAp slurries were prepared followed by addition of binder (13 wt% PVA solutions) and plasticizer. The slurries were further mixed for 2 h with a stirrer. After homogenizing, the slurries were degassed under low vacuum (∼10−5 Torr) to eliminate air bubbles. Finally, suspensions were cast onto a fixed glass plate carrier. Tapes were allowed to dry freely in open air at room temperature. Density of green sheets was measured in ethanol. After lamination and binder removal, HAp samples were sintered at 1250 ◦ C for 1 h in furnace and the density was measured in deionized water. Tests of flexural strength were performed by three point bending from specimens of size 3 mm × 4 mm × 36 mm. The calcium/phosphorous molar ratio of hydroxyapatite was determined by EDX. The phases of HAp powder and HAp samples were identified by X-ray diffractometry (XRD, Cu K␣, Rigaku, Tokyo, Japan). The step size and scan rate were fixed at 0.02 and 0.5◦ /min, respectively.
2.6. Biological test All of the dissolution tests were conducted in triplicate in simulated body fluid (SBF). The SBF was prepared by dissolving reagent-grade NaCl, KCl, K2 HPO4 ·3H2 O, MgCl2 ·H2 O, CaCl2 , and Na2 SO4 into distilled water and buffering to pH 7.4 with tris[hydroxymethyl]aminomethane [(CH2 OH3 )3 CNH3 ] and hydrochloric acid [22]. As sintered HAp samples were ground into a fine powder. After being ultrasonically washed in acetone and rinsed in deionized water, 10 mg of each granule was individually soaked into 30 mL of solution at 37 ◦ C for times varying from 1 to 60 days. After the immersion periods of 1, 3, 5, 7, 9, 11, 15, 30 and 60 days, the samples were centrifuged. The changes in the concentrations of calcium in the supernatant were measured by Shimadzu AA-6401F atomic absorption spectrophotometer (Shimadzu Corp., Tokyo, Japan). HAp specimens for flexural strength measurement were also immersed in SBF for 60 days at 37 ◦ C. HAp round disks with the diameter in the 17–19 mm range were used for cell culture test. Marrow cells were obtained from the bone shaft of femora of Fisher 344 male, 7-week-old rats. Initially, rat bone marrow cells were flushed out by a culture medium (MEM, Nacalai Tesque Inc., Kyoto, Japan) and cultured in a humidified atmosphere of 95% air with 5% CO2 at 37 ◦ C. After confluence in T75 flasks (Becton, Dickinson and Company (BD), NJ, USA), the adherent cells (MSC) from rat bone marrow were resuspended in culture medium followed by harvesting using Trypsin/EDTA. Subsequently, The cell suspension with osteogenic medium was applied to sterilized, HAp ceramic disks (18 mm in diameter and 3 mm in thick), which were placed into a 12-well plate. The culture medium was changed two or three times per week. After 2 weeks, the cell viability on HAp sample surface was checked using LIVE/DEAD Viability assay kit (Molecular Probes Inc., USA).
3. Results and discussion 3.1. Surface properties of HAp The iso-electric point (pHIEP ) of HAp particles was situated at approximately pH 7.0, thus, acrylic-type polymer is usually selected as dispersant in aqueous media [15–19]. In this paper,
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Fig. 3. Zeta potential of HAp powder in the (a) absence and (b) presence of dispersant (PAA-NA).
Fig. 2. Influence of pH on the stability of 5 vol% HAp slurries (mixed for 24 h).
point (Fig. 3), correspondingly, the sediment volume was at its lowest point. Based on the discussion above, in this paper, the HAp suspensions are stabilized around pH 10.50, similar to that reported in literature [19]. The optimal content of dispersant required was determined using 20 vol% HAp slurries at pH 10.50. Fig. 4(a) represents the viscosity, as a function of PAA-Na concentration, at three different shear rates for HAp suspensions. It was shown that minor addition of dispersant can considerably decrease the slurry viscosity. A concentration of ∼0.45 wt% dispersant helps to produce stable suspensions. When the dispersant concentration is increased beyond this point, the viscosity kept almost constant.
polyacrylic acid sodium salt (MW = 30,000) was used as dispersant. The addition of PAA-Na leads to an obvious increase of negative charge on HAp particle surface, indicating the chemical adsorption type of it. The stability of HAp slurries was characterized in term of sedimentation test, see Fig. 2. 3.2. Slurry stability As shown in Fig. 2, in the absence of dispersant, the slurry is stable in pH 10–12 range, at which the HAp powder is stabilized electrostatically due to the high surface charge. In low pH region (<5), due to the dissolution of HAp, the slurries are not stable; in the pH 5–10 range, the instability is due to the low surface charge of HAp particles (near the isoelectric point, Fig. 3). In the presence of dispersant PAA-Na, the slurry can be stabilized also in neutral region. This might be due to the adsorbed polymers, which modify the surface charge behavior of HAp particles and lead to the shift of the isoelectric point toward more acid region (pH). An instable slurry state was observed in acid pH region (pH < 7), where the surface charge of HAp particles is not so high and the PAA-Na molecules are not well expanded, similar to that observed in ZrO2 system [23]. On the contrary, at high pH (>8), the slurry is well stabilized characterized as a low sediment volume. This slurry stability can be well correlated to the electric and steric mechanism [19]. At pH 10.5, the surface charge of HAp particles was around its highest
Fig. 4. Influence of dispersant content on the stability of HAP slurries (pH 10.50): (a) viscosity of 20 vol% HAp slurries vs. dispersant content and (b) slurry properties (20 vol%).
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represents the adsorption behavior that would occur if 100% of the copolymer added was adsorbed. As evidenced in Fig. 5, the adsorption amount of PAA-Na increases with the increase in dispersant content up to 0.45 wt% and kept nearly constant thereafter, indicating the saturation adsorption of dispersant. This adsorption saturation can be well related to the viscosity measurement, see Fig. 4. At low dispersant content (<0.27 wt%), almost all the dispersant added adsorbs on the surface and the data follows the 100% line until the adsorption of PAA-Na reach approximately 0.55 mg m−2 . So the adsorption isotherms can be described as high-affinity type. This kind of adsorption behavior is commonly observed in many systems [11,24]. Fig. 5. Adsorption of PAA-Na on HAp particle surface (pH 10.5).
The viscosity change versus shear rate is further characterized in Fig. 4(b). As shown in Fig. 4(b), at low PAA-Na concentration (<0.32 wt%), the suspensions exhibit shear thinning behavior, this can be well correlated to the flocculated slurry state, which might come from the inadequate adsorption of dispersant [11]. When the dispersant content increased to 0.45 wt%, the slurry stability improved from shear thinning to near Newton behavior, indicating the achievement in slurry stability. The adsorption isotherm of PAA-Na on HAp particle surface is characterized at pH 10.5, see Fig. 5. The dash diagonal line
3.3. Flow behavior of HAp suspensions From the discussion above, it can be concluded that HAp slurries with high solid content can be obtained at pH 10.5 with the dispersant content around 0.45 wt%. Fig. 6 shows the flow behavior of HAp suspensions in the presence of PAA-Na with the solid content as 40, 50 vol% (without EDTA-Na) and 60 vol% (with EDTA-Na), respectively. Results showed that with solely PAA-Na as dispersant, it was difficult to obtain high solid content HAp suspensions (>55 vol%). After the addition of EDTA-Na s, it was possibly to prepare 60 vol% suspensions with high fluidity. The slurries showed a lower viscosity than that for 50 vol% suspensions, though the time dependent behavior is a bit significant.
Fig. 6. Properties of HAp suspensions.
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Table 2 HAp slurry formulations Materials
Function
Density
wt%
vol%
HAp PAA-Na EDTA-4Na Water PVA Ethylene glycol
Ceramic powder Dispersant Dispersant Solvent Binder Plasticizer
2.7 1 1 1 1.08 1.113
54.13 1.08 0.11 35.96 4.36 4.36
30.77 1.66 0.17 55.2 6.19 6.01
For shear thinning slurries, the relationship between shear stress and shear rate can be described by the following equation [25] τ = Kγ n
(2) Fig. 7. XRD pattern of HAp starting powder and as-sintered HAp samples.
where τ is the shear stress, r the shear rate and n is less than one. As shown in Fig. 6(c), the slip torque versus shear rate curves for 60 vol% slurries can be fitted linearly with the correlation coefficients higher than 0.99. The K and n value, which could be obtained from equation (2), are 24.17 and 0.40, respectively. Therefore, it is proposed that the 60 vol% HAp slurries exhibit power law, strong shear thinning behavior which is universal for stable ceramic slurries [25–27]. This slurry property meets the requirement for tape casting process. 3.4. Tape casting and sintering The formulation of tape casting slurries is shown in Table 2. The tape casting process was similar to that reported in literature. After drying, the green sheets exhibit a homogeneous microstructure. The relatively green density was 52.1%. The efficiency of dispersant that enables deagglomeration and the particles to flow and pack closely is the most probable explanation for the high density that has been reached. After removing the organic additives (dispersants, binder and plasticizer) and lamination, the HAp samples can be easily sintered at 1250 ◦ C and soaked for 1h in air with the relative density as 98.8%. The X-ray analyses did not show the presence of -TCP phase, which is in agreement with the literature [28], Fig. 7. A series of sharp diffraction peaks were observed, indicating that HAp grains are well crystallized. The Ca/P ratio was kept almost unchanged after sintering, see Table 3. The flexural strength of sintered HAp samples was 69.5 MPa, which is very close to the data shown in literature [7,29,30], Table 3.
Fig. 8. Continuous dissolution assay in SBF for HAp samples.
3.5. Biological properties Fig. 8 showed the calcium concentration changes in the SBF solution as a function of time. On the first day, the dissolution was dominant. After 3 days, due to the high concentration of Ca ions, the precipitation process became dominant and a decrease in calcium concentration occurred. Fourteen days after immersion, the calcium concentration reached the minimum point. This is in agreement with that shown in literature [31,32]. After 30 days, the calcium concentration increased again. However, after 60 days, the amount of material dissolution was stabilized, similar to the case shown in literature [33]. This dissolution process can be explained as a competition of dissolution of the HAp powder and the precipitation of newly formed CaP on its sur-
Table 3 Properties of HAp samples after sintering
Starting powder After sintering After immersion (60 days)
Ca/P ratio
Flexural strength (MPa)
Hardness (GPa)
Toughness (MPa m1/2 )
1.67 1.66 –
– 69.5 ± 5.8 65.7 ± 5.2
– 4.49 ± 0.12 4.41 ± 0.09
– 1.15 ± 0.14 1.21 ± 0.14
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the biocompatibility of the HAp samples. After binder removal and sintering, all the organic additives are removed and did not show any negative effect on the properties of HAp samples. 4. Conclusions The isoelectric point of HAp is at pH 7.0. Results showed that HAp slurries can be stabilized at pH 10.5 with PAA-Na content around 0.45 wt%. However, at high solid content, the stability of HAp suspensions is highly reduced with solely PAA-Na as dispersant. So EDTA-Na is introduced to improve the slurries stability. In the presence of EDTA-Na, 60 vol% HAp suspension exhibit well stabilized state which can be fitted well by shear thinning equations. The slurries are suitable for the subsequent tape casting process. After tape casting, lamination and sintering at 1250 ◦ C, HAp samples can be densified to 98.8% (RD). The samples exhibited excellent properties for cell attachment and proliferation. Results showed that it is possible to use aqueous processing route to prepare HAp samples for biological applications. References
Fig. 9. Live/Dead stain of the cultured cells after 14 days: (a) Live stain and (b) Dead stain (×19.2).
face. Initially, there was significant dissolution of the material and, with the increase of the calcium concentration in solutions, the dissolution–precipitation balance moved, and the precipitation was the main driver. After a long immersion, the dissolution and precipitation reached equilibrium and the calcium concentration was kept constant. As shown in Fig. 8, the dissolution is not so significant, which might be due to the well crystallized HAp samples after sintering [32], Fig. 7. This can also be well correlated to the mechanical properties. The flexural strength after 60 days immersion was 65.7 MPa, similar to that of assintered samples, Table 3. The HAp samples were further characterized by cell culture test, results are shown in Fig. 9. After 14 days, the viability of marrow cells was evaluated by Live/Dead staining. The assay is realized by mechanisms based on a simultaneous determination of living and dead cells with two probes, calcein-bis[(acetyloxy) methyl] ester (calcein-AM) for intracellular esterase activity and ethidium homodiner-1 (EthD-1) for plasma membrane integrity. The viable cells were dyed to green, and the nucleus of the dead cells was dyed to red. As shown in Fig. 9, most of the cells are green and only quite a few cells are red. This is in agreement with the XRD, Ca/P ratio and strength test and would confirm
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