Control of Pore Size of the Bubble-Template Porous Carbonated Hydroxyapatite Microsphere by Adjustable Pressure Xiaokun Cheng,† Qianjun He,† Jianqiu Li,† Zhiliang Huang,*,† and Ru-an Chi‡ School of Material Science & Engineering, Wuhan Institute of Technology, Wuhan 430073, China, and School of Chemical Engineering & Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China
CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 6 2770–2775
ReceiVed December 30, 2008; ReVised Manuscript ReceiVed February 15, 2009
ABSTRACT: Flowerlike porous B-type carbonated hydroxyapatite (CHAp) microspheres have been synthesized by a novel bubbletemplate route. Their pore sizes can be well regulated in a wide range from microscale to nanoscale by smartly constructing a kind of CO2 bubble as a template and precisely adjusting the system pressure. We studied the formation of a porous structure of CHAp microspheres and the effect of the system pressure on their morphologies. Most of all, the control of the pressure over the pore sizes of bubble-template porous CHAp microspheres was investigated. The results suggested that the gathering and growth of the CO2 bubbles generated the formation of the porous structure of CHAp microspheres, and along with the increase of system pressure, the surface of the microsphere got denser and the flakes, which assembled the porous structure, became smaller. In addition, with the increased pressure, the average pore sizes of bubble-template CHAp microspheres gradually decreased from microscale to nanoscale. Furthermore, the relation between the pressure P and the pore size r was derived from the Laplace equation, the Kelvin equation, and the Clapyeron equation.
1. Introduction Carbonated hydroxyapatite (CHAp), as a traditional functional biomaterial, inherits excellent properties from hydroxyapatite (HA), which is the leading biomaterial employed today1 and can be widely applied in many fields such as biochemistry,2,3 biomedicine,4,5 environmental engineering,6,7 chemical catalysis,8,9 etc. Normally, CHAp is an acicular crystal10 and can be oriented to grow into flaky crystal along a C-axis in biological osseous tissue, which is controlled by the osteoinductive cells.11 Inspired by this template control theory, researchers obtained porous CHAp via artificial synthesis.12,13 Because of the outstanding porous structure, porous CHAp has advantages in the large surface area and the superior surface activity, which implies more potential applications.14,15 In this way, it is significant to study controlling the pore size of porous CHAp, which contributes further to their advantages in surface area and surface activity. Regularly manipulating the morphologies and the structures of inorganic crystals from microscale to nanoscale has attracted great attention.16-19 However, information about the pore size control of porous CHAp is rarely reported. Currently, porous CHAp is widely synthesized by the hard tissue burnout method and hard template casting method. However, by the former method, the porous structure may be destroyed in a sintering process and the pore sizes of products cannot be regulated.4,6,20 On the other hand, high cost, a complicated process, and low activity of products by the latter method can hardly be avoided, and it is impossible to control the pore size by the same template.21-26 Moreover, Sun et al. synthesized hollow porous CHAp spheres by a spray drying method; nevertheless, the control over the pore size was not investigated.27 * To whom correspondence should be addressed. Tel: +86-027-63210785. Fax: +86-027-87195661. E-mail:
[email protected] or
[email protected]. † School of Material Science & Engineering, Wuhan Institute of Technology. ‡ School of Chemical Engineering & Pharmacy, Wuhan Institute of Technology.
In this work, a novel CO2 bubble template was constructed to synthesize flowerlike porous CHAp microspheres. The formation of a porous structure and the effect of the pressure on the morphologies of CHAp microspheres were investigated. Furthermore, control of system pressure over the pore size of CHAp was studied, and the corresponding equation, which was derived from the Laplace, Kelvin, and Clapyeron equations, was introduced to explain this control relationship.
2. Experimental Section 2.1. Materials and Synthesis. Ca(NO3)2 · 4H2O (purity >99%, Tianjin, China), (NH4)2HPO4 (purity >98.5%, Hunan, China), HNO3 (purity ) 65-68%, Kaifeng, China), (NH2)2CO (purity >99%, Wuhan, China), and Na2EDTA (purity >99%, Guangdong, China) were used. Water used for synthesis and characterization was purified by the Milli-Q Plus water purification system (Millipore Corp., Bedford, MA). All chemicals were of analytical grade. The template-directed method28 was introduced to prepare CHAp spheres in a special autoclave (Weihai Automatic Control Reaction Kettle Co., Ltd.). The matching pressure transducer was employed to monitor the system pressure, with an accuracy of 0.5 atm. The mixed solution of Ca(NO3)2 and (NH4)2HPO4 at the molar ratio of 5:3 was titrated by HNO3 until the solution became just clear. Then, an appropriate amount of urea and Na2EDTA was dissolved in the above clear mixed solution of calcium and phosphate. Next, the homogeneous reaction solution was heated in the special autoclave at a constant temperature of 367 K, and the heating procedure lasted 5 h. In the condition of keeping other factors constant, the crystals would grow freely at a fixed pressure of 4.5 atm, which was induced by the CO2 atmosphere decomposed from urea. So, for adjusting the system pressure, the autoclave might be filled with a given mass of nitrogen before heating or some gas might be expelled from the autoclave in the heating procedure. Finally, the obtained precipitations were aged, washed, and dried. A series of experiments were carried out as follows: S1: [Ca]:[P]:[U]:[T]:[P] ) 0.06 M:0.036 M:0.3 M:2 mM:1.5 atm; S2: [Ca]:[P]:[U]:[T]:[P] ) 0.06 M:0.036 M:0.3 M:2 mM:3 atm; S3: [Ca]:[P]:[U]:[T]:[P] ) 0.06 M:0.036 M:0.3 M:2 mM:10 atm; S4: [Ca]:[P]:[U]:[T]:[P] ) 0.06 M:0.036 M:0.3 M:2 mM:26 atm; where [Ca], [P], [U], and [T] represented the initial molar concentration of calcium, phosphate, urea, and Na2EDTA, respectively. [P] was the system pressure. The pressures of S1 and S2 were adjusted via a “filling
10.1021/cg801421a CCC: $40.75 2009 American Chemical Society Published on Web 04/13/2009
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gas”, while those of the remaining two samples were adjusted by an “expelling gas”. 2.2. Model of Stability of Bubble in Liquid. Bubbles in nanoscale can exist steadily in liquid.29 The force balance concerning a spherical bubble of radius r is usually described by the Laplace equation:30
∆P ) Pin - Pout )
2δ r
(1)
where Pin and Pout are respectively defined as the pressures inside (vapor phase) and outside (liquid phase) the bubbles; δ is the surface tension. As for a stable CO2 bubble, there is a certain mass of gas inside, and it is under influence of the pressure generated from the gas. Because of the high temperature and pressure, the real CO2 gas in our study may not easily obey the ideal gas law. Thus, the real gas law (the van der Waals equation) is helpful.
(
)
a (Vm - b) ) RT Vm2
Pgas +
(2.1)
where Pgas is the pressure of CO2 gas inside the bubble; Vm is the molar volume of the CO2 gas; T is the temperature; a and b are both van der Waals constants, which, respectively, are 0.364 Pa · m6 · mol-2 and 4.27 × 10-5 m3 · mol-1 for CO2 gas.31 In this paper, with regard to the nanoscale or microscale (500 nm, ∼2 µm in diameter) stable CO2 bubble with tiny molar mass (4 × 10-17 to 1.5 × 10-16 mol), Vm is in the range from 0.34 × 10-2 m3 · mol-1 to 0.85 m3 · mol-1. Obviously, the term a/Vm2 and b can both be neglected, respectively, compared with Pgas and Vm. (The error is less than 1‰.) Thus, the real CO2 gas in this work can be approximately studied as the ideal gas, which can be described by the Clapyeron equation:
PgasV ) nRT
(2)
Figure 1. FT-IR spectra of samples. The spectrum S is the reference one of B-type CHAp, which was reported in our previous study.28
where V is the volume of the CO2 bubble; n is the amount of substance of CO2 gas in the bubble. In addition, the surface of the CO2 bubble is curved. Therefore, minor effects of the saturated vapor pressure on the surface can be described by the Kelvin equation:
ln
P*r 2δM )P* FRTr
(3)
And the relation between P*, r Pgas, and Pin can be expressed as follows:
Pin ) P*r + Pgas
(4)
where P*r and P* are the saturated vapor pressure of the curved surface of CO2 bubble and that of the liquid surface, respectively; F and M are the density and the molar mass of solution, respectively. 2.3. Characterization. Fourier transform infrared (Nicolet Impact 420 FT-IR spectrometer) and powder X-ray diffraction (Siemens XD5A XRD, Cu KR radiation at 30 kV and 20 mA, λ ) 1.54056 Å, step size 0.01°) were employed to analyze the chemical composition of CHAp. A scanning electron microscope (JSM-5510LV SEM, voltage 30 kV, samples dusted on an adhesive conductive carbon disk attached to an aluminum mount and coated with 100 Å of Au) was used to observe crystal morphology, and the average pore size was calculated by the mercury porosimetry method (Poremaster, America). The surface tension of solution was measured by the full automatic surface/interface tensiometer (Shanghai Solon Tech. A201).
3. Results and Discussion 3.1. Chemical Composition Analysis of the Products. Chemical composition analysis of CHAp has been introduced in detail in our previous study.28 As shown in Figure 1, the spectrum S1 revealed the typical bands at 1106 cm-1/1034 cm-1 (ν3 PO43-), 958 cm-1 (ν1 PO43-), and 603 cm-1/564 cm-1 (ν4 PO43-), together with those at 1457 cm-1(ν3-3 CO32-)/1414 cm-1 (ν3-4 CO32-) and 876 cm-1 (ν2 CO32-). Apparently, CO32- ions were in the B-site, which implied [PO4] was partly replaced by [CO3].28,32,33 In a similar way, compared with the spectrum S, which was the
Figure 2. XRD patterns of samples. Diffraction peaks of samples are marked by the black crosses.
reference FT-IR spectrum of B-type CHAp previously reported by us, CO32- of S2, S3, and S4 was also in the B-site. According to the XRD patterns (Figure 2), most diffraction peaks were assigned to HA (the standard JCPDS card No. 09-0432). Thus, in view of the XRD and FT-IR results, all of the samples belong to the B-type CHAp.28 Moreover, the diffraction intensity of the (0002) plane was apparently stronger, which indicates the preferred growth of CHAp along the [0001] direction.28,34 3.2. Formation of the Porous Structure of the CHAp Microsphere. As shown in Figure 3, the surface of CHAp is assembled by the flakes, most of which interconnect and form
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Figure 3. Magnified image of the flakes shown in Figure 6A.
Figure 5. Illustration of the growth process of the flakes. Formation of the flakes can be attributed to the chelated reaction of ions outside.
Figure 4. Illustration of the formation of the microsphere via coalescence of CO2 bubbles.
an open successive porous structure. Gas bubbles (specifically for CO2 bubbles), being similar to a type of template, can form open porous structure of HA microspheres 27,35 and polymeric microspheres.36 Although the synthetic method is so different, in this study, the formation of the porous structure can be similarly explicated as follows: CO2 bubbles of micrometer or nanometer size are slowly decomposed from urea with increased reaction temperature. Then, affected by the solution surface tension, CO2 bubbles are gathered into big spheres of micrometer size. (Generally, the average granularity of CHAp spheres was about 20 µm (Figure 6A-D). Under the influence of the pressure caused by the CO2 atmosphere and the surface tension, the bubbles grow up into a relatively stable state. Next, the plateaus emerge in the intersection of CO2 bubbles, and CHAp crystals start to nucleate homogeneously in the plateau center. Then, the flakes grow up via self-assembling28 along the plateau border. The process described above is shown in Figures 4 and 5. Finally, when the amount of flakes grows sufficiently, CO2 bubbles disappear and a porous structure forms. Therefore, CO2 bubbles play an important role as a novel template for the porous
structure of CHAp microspheres. In addition, clearly, the average pore size of CHAp microspheres is almost equal to the average size of the bubbles. 3.3. Effect of System Pressure on the Pore Size and the Morphology of the CHAp Microsphere. Pore sizes of CHAp microspheres obtained at different pressures were investigated. The average pore size of samples S1, S2, S3, and S4 have been calculated by the mercury porosimetry method, and the corresponding pore radius distributions were centered on 750, 600, 400, and 300 nm, respectively, so the average pore sizes were approximately 1.5 µm, 1.2 µm, 800 nm, and 600 nm, respectively. Morphologies of the resultants were characterized by SEM (Figure 6). Therefore, the regularity is declared that the average pore sizes of porous CHAp microspheres decreased gradually with the increase of system pressure, and it is significant that the average pore sizes of porous CHAp microspheres can be regulated from micrometer to nanometer size via adjusting the system pressure. The effect of the system pressures on the morphologies of CHAp microspheres was also investigated. Along with the increase of the system pressure, crystal morphologies of CHAp microspheres were very different. As for the samples at relatively low pressure (Figure 6A, 1.5 atm and Figure 6B, 3 atm), the flakes were bigger and the surfaces of the microspheres were looser. Meanwhile, as for the samples at higher pressure (Figure 6C, 10 atm and Figure 6D, 26 atm), the flakes were smaller and the surfaces of microspheres were denser. It may result from the following factor: Because of the increased pressure, the bubbles, which locate on the surface of the big microsphere, are able to become smaller primarily. Thus, more bubbles can occupy the spare room on the surface of the big microsphere, which causes distribution of the bubbles on the new surface to become compact. In this case, interfaces between the bubbles diminish, which results in the flakes becoming smaller. The converse is also valid. So, the pressure is higher (or lower), the surface of the CHAp microsphere is denser (or looser), and the “connected flakes” become smaller (or bigger). In addition, the big size bubbles determine the large interfaces
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Figure 6. SEM images of samples S1 (A), S2 (B), S3 (C), and S4 (D) at the system pressures of 1.5, 5, 10, and 26 atm, respectively. The scale bars of (A), (B), (C), and (D) all correspond to 5 µm.
and the strong compaction between the bubbles, which causes the pressure on some bubbles to decrease. Consequently, the surface morphologies of porous CHAp microspheres fabricated at lower pressure are partly irregular (Figure 6A,B). 3.4. Control Relation Between the System Pressure and the Average Pore Size of the CHAp Microsphere. According to the formation assumption of the porous structure by the bubble template, the control relationship between the pressure and the pore size is virtually the same as that between the pressure and the diameter of CO2 bubbles. According to the three eqs 1, 2, and 3 and the relation 4 above, a resultant equation can be obtained:
Pout )
nRT 2δM 2δ + P* exp 4 3 FRTr r πr 3
(
)
(5)
Compared with system pressure P, the pressure inside the solution can be neglected. So, P can be considered as Pout. Therefore, eq 5 can be simplified as follows:
P)
nRT 4δM 4δ + P* exp 1 3 FRTr r πr 6
(
)
(6)
where r is the pore size of CHAp (r ) 2r), and the term P* exp(-4δM/FRTr) is approximately considered as 0.8 atm,
which is close to the saturated vapor pressure of water at 367 K. Equation 6 is the theoretic equation which represents the control relation between the pressure P and the pore size r. No surfactant was used in the experiments; thus, the surface tension δ of the four samples should be close to each other. In addition, the effect of pressure on the surface tension could be neglected. Via a test by the surface/interface tensiometer, δ was 0.1 N/m, approximately. In order to calculate the average amount of substance of CO2 in the bubbles, a parallel experiment was carried out and the mixing ratio was as follows: S0: [Ca]:[P]:[U]:[T] ) 0.06 M:0.036 M:0.3 M:2 mM in which [Ca], [P], [U], and [T] represented the concentrations of reagents noted above. After the system reacted to a certain extent, the pressure stopped increasing and the system and pressure inside became stable. (About 1 h after the reaction started, the stable pressure was 4.5 atm.) When the system became stable, the system pressure was kept at 4.5 atm. The average pore size of S0 is about 1 µm, and its morphology is shown in Figure 7. Therefore, n could be easily taken out, which was about 1.34 × 10-16 mol. According to the obtained n and δ, the graph of eq 6 is simply drawn, which is shown in Figure 8. This equation graph revealed that with increasing pressure, the pore sizes of CHAp micro-
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(1) The results from the formation of a porous structure of CHAp microspheres suggested that the growth and gathering of the CO2 bubbles induced the growth of the CHAp flakes and formation of the porous structure. Moreover, the average pore size of CHAp microspheres was almost equal to the average diameter of the bubbles. (2) The results from the effect of the system pressure on the porous morphology of the CHAp microspheres revealed that the average pore size of porous CHAp microspheres decreased gradually with the increase of system pressure. In addition, the pressure was higher (or lower), the surface of the CHAp microsphere was denser (or looser), and the flakes became smaller (or bigger). (3) The pressure dependence of the pore size obeyed the resultant equation (eq 6). It implied a new route for controlling the pore size of porous materials. Figure 7. SEM image of sample S0 at the system pressure of 4.5 atm. The scale bar corresponds to 5 µm. Granularity of the porous CHAp microsphere was about 25 µm.
Acknowledgment. This work was supported by the National Natural Science Foundation of China (No. 50774055), the Key Project of the National Natural Science Foundation of China (No. 50834006), the Natural Science Foundation of Hubei (No. 2005ABA024), and the Key Project Foundation of the Hubei Ministry of Science and Technology (No. 2006AA101C45).
References
Figure 8. Graph of eq 6. The points corresponding to P and r of S1, S2, S3, S4, and S0 are also marked as pentagrams in this graph.
spheres decreased, which was the same relation mentioned above. Furthermore, it was clearly visible that the marks of S1, S2, S3, and S4 were close to the graph of eq 6. Consequently, the control relation between the pressure P and the pore size r was in agreement with the resulting eq 6. This equation indicated that at lower pressure (1 µm), and at higher pressure (>10 atm), the pore size was smaller and was at nanometer size (
