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Micropatterned TiO2 effects on calcium phosphate mineralization
Materials Science and Engineering C 29 (2009) 2355–2359
Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Micropatterned TiO2 effects on calcium phosphate mineralization Lili Jiang a, Xiong Lu a,b,⁎, Yang Leng b, Shuxin Qu a, Bo Feng a, Jie Weng a a b
Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, China Department of Mechanical Engineering, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 17 February 2009 Received in revised form 22 April 2009 Accepted 11 June 2009 Available online 21 June 2009 Keywords: TiO2 Calcium phosphate Microgrooves Biomineralization
a b s t r a c t The effects of implant surface topography and chemistry on biomineralization have been a research focus because of their potential importance in orthopedic and bone replacement applications. While a vast amount of research is focusing on chemical modified surfaces and rough surfaces, little attention has been paid to the well-defined micropatterned surface effects on calcium phosphate mineralization process due to the difficulties in preparing microfabricated biomaterial surfaces. This work focuses on the effects of microgrooved TiO2 surfaces on the calcium phosphate mineralization process. Firstly, we developed a new process that can prepare microgrooved TiO2 coatings on glass substrates using soft-lithography and sol–gel technology. Then microgrooved TiO2 surfaces were used to induce Ca–P mineralization under biomimetic conditions. The results revealed that topography dominated the growth and distribution of mineralization at the initial days and then the effects of topography become weak with the extended immersion days. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Biomineralization is a process of forming minerals in organisms, where minerals nucleate and grow from a supersaturated aqueous solution under the biomimetic conditions [1,2]. Biomineralization widely exists in nature and several important bioceramics such as calcium phosphates (Ca–P), which are the major component of bone and teeth and have good osteoconductivity, can be obtained by this process [3,4]. Inspired by the biological process of mineralization, various researchers employed mineralization process to obtain Ca–P in order to improve biocompatibility and bone inductivity of human bone implants [5–7]. The authors' group also obtained Ca–P coatings on nitric-acid-treated titanium surfaces in simulated body fluid under physiological conditions [8]. However, the mechanism of the biomineralization is far from being fully understood. It is generally accepted that the material surface chemistry plays an important role on the mineralization process [9,10]. Surface topography, which is another important surface property, might also be a critical factor that affects the Ca–P mineralization process [11]. Recently, researchers indicated that Ca–P mineralization ability depended upon the topography of the substrate surface to a certain extent [12,13]. Researchers also proved that the Ca–P mineralization on the rough substrate surfaces was easier than that on the plane one and porous titanium surfaces exhibit excellent ability of inducing Ca–P
⁎ Corresponding author. Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, China. Tel.: +86 28 87634023; fax: +86 28 87601371. E-mail address: [email protected] (X. Lu). 0928-4931/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2009.06.005
mineralization [14]. The author's previous study already demonstrated that Ca–P deposition on the microhole-patterned titanium surfaces was better than that on the plane surfaces and the Ca–P preferred to grow on the inner wall of the microholes [15]. In the present study, we proposed a new method to prepare well-designed micropatterned TiO2 surfaces in order to further explain the surface topography effects on Ca–P mineralization process accurately. Titanium dioxide attracted our interest with its good biocompatibility and soft-lithography technology was used in this paper to obtain microgrooved TiO2 surfaces. 2. Materials and methods 2.1. The TiO2 microgrooves Microgrooved TiO2 surfaces were fabricated onto a glass substrate using soft-lithography technology. Soft-lithography is a well known technique that uses an elastomeric silicone rubber, mostly polydimethylsiloxane (PDMS), as a template for structuring materials on the micro scale technology [16,17]. The process was schematically shown step by step in Fig. 1. In the first step, a PDMS microstamp was fabricated by replicating the microgrooves of a micromachined silicon substrate obtained by photolithography and wet etching. Firstly, the Sylgard 184 (Dow Corning) and the initiator with the mass ratio of 10:1 was coated on the microgrooved silicon wafer; then the sample was placed in a vacuum oven at 80 °C for 2 h to exclude bubbles and cure; finally the cured polymer was peeled off from the silicon wafer and the microgrooved PDMS stamp was obtained. In the second step, the TiO2 sol–gel precursor solution was prepared using a mixture of tetrabutyltitanate/ethanol/hydrochloric acid/distilled
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L. Jiang et al. / Materials Science and Engineering C 29 (2009) 2355–2359 Table 2 The contact angles of probe liquid on microgrooved and plane TiO2 surfaces. Surface
Contact angle
Microgrooves (water) Microgrooves (glycerol) Plane (water) Plane (glycerol)
76.6° ± 0.9° 73.6° ± 6.5° 25.2° ± 0.9° 27.3° ± 5.9°
microgrooves was hung and immersed in the solution in a polystyrene tube at 36.5 °C for 4 and 7 days. After soaking in the SCPS for different times, the substrates were taken out and washed with distilled water. 2.3. TiO2 surface characterization
Fig. 1. The process of the fabrication of the TiO2 microgrooves. (a) TiO2 sol–gel was coated on the glass substrate. (b) PDMS microstamp was embossed onto TiO2 sol–gel. (c) Microgrooved TiO2 was obtained by heat treatments after removing the PDMS microstamp.
water/1,5-pentanediol with a molar ratio of 1/30/0.2/2/2. Firstly, tetrabutyltitanate was dissolved in two-thirds ethanol and stirred vigorously for 30 min at room temperature, which was named as solution I; water, hydrochloric acid and 1,5-pentanediol were dissolved in ethanol, which was named as solution II. Then solution II was slowly dropped into solution I under vigorous stirring. The mixed solution was kept overnight at room temperature to obtain the TiO2 sol. The asprepared TiO2 sol was coated on the glass substrate and the PDMS stamp was embossed onto the TiO2 sol. TiO2 sol was condensed and solidified in a vacuum oven at 25 °C for 12 h. After drying, the PDMS stamp was removed and the gel was sintered at 400 °C for 2 h and finally the microgrooved TiO2 was obtained. The plane TiO2 specimen was also prepared using sol–gel technique as control samples. 2.2. Ca–P mineralization A simple supersaturated calcium phosphate solution (SCPS) with high calcium and phosphate ion concentrations was used for biomineralization study. SCPS was prepared by dissolving NaCl (7.714 g), CaCl2 (1.387 g), Na2HPO4·2H2O (0.89 g) and 1 M HCl 50 ml in 1 L of deionized water. Tris-hydroxymethyl aminomethane (TRIS) was used to adjust the pH value to 6.0. SCPS has been successfully applied to investigate the Ca–P formation by previous study [8,18]. In SCPS, Ca2+ and HPO2− 4 concentrations were five times that of human blood plasma and Mg2+ and HCO− 3 which were generally regarded as Ca–P crystallization inhibitors were removed. Thus, the formation of Ca–P mineralization in SCPS could be faster than that in SBF. The ion concentrations of SCPS and human blood plasma are listed in Table 1. The as-prepared TiO2 with or without
Table 1 Ion concentrations of human blood plasma and SCPS. Ion
Concentration (mM/L) Blood plasma
SCPS
Na+ K+ Mg2+ Ca2+ Cl− HCO− 3 HPO2− 4 2− SO4
142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5
142.0 – – 12.5 217.0 – 5.0 –
Scanning electron microscopy (SEM; QUANTA-200, FEI, The Netherlands) was used to analyze the morphology of the TiO2 microgrooves before and after the mineralization. The crystal structure and phase composition of as-prepared TiO2 surfaces and the precipitated Ca–P were examined using thin film X-ray diffraction (TF-XRD; X'pert proMPD, PANalytical, The Netherlands). The XRD measurements were performed on a stage using a Cu–Kα (wavelength = 1.5056 ) X-ray source with step rate of 0.02° /s. The depth of the as-prepared TiO2 microgrooves was measured by a surface profilometer (Ambios XP-2, Ambios, USA). The surface energies of the microgrooved and plane TiO2 surfaces were obtained from Owens–Wendt (OW) method through the following equation [19]: qffiffiffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffi ð1 + cosθÞγL = 2ð γSd γLd + γSP γLP Þ
ð1Þ
where θ is the equilibrium contact angle; γL is the surface tension of testing liquid and γdL and γpL is the dispersion and polar components; the surface energy of a solid (γS) is equal to a sum of the dispersion (γdS ) and polar (γpS ) components (γS = γdS + γpS ). The two probe liquids used in the present study are deionized water (γ = 72.8 mJ/m2, γp = 51.0 mJ/m2, γd = 21.8 mJ/m2) and glycerol (γ = 63.4 mJ/m2, γp = 26.4 mJ/m2, γd = 37.0 mJ/m2). The contact angle measurements were carried out using the sessile-drop technique (DSA100, Kruss GmbH, Germany). The liquid droplets were laid onto the microgrooved and plane surfaces by a micro syringe. Around 8 samples were tested for every type of microgrooved and plane surfaces and only one drop was laid on every single sample. The average contact angles were listed in Table 2 and were substituted into Eq. (1) to calculate the corresponding surface energies which were listed in Table 3. 3. Results 3.1. TiO2 microgrooves Microgrooved TiO2 was successfully produced on glass substrates by soft-lithography technology as shown in the SEM micrograph (Fig. 2a). Surface profiler measurement shows the periodic width of the microgrooves is ∼10 µm and the depth of the microgrooved TiO2 is ∼0.3 µm (Fig. 2b). The TF-XRD spectrum clearly revealed that crystalline phase of microgrooved TiO2 surface after sintered at 400 °C for 2 h is
Table 3 The surface energies calculated from OW method.
Microgrooved surfaces Plane surfaces
γSd (mJ/m2)
γSp (mJ/m2)
γS (mJ/m2)
9.5 13.5
18.2 53.4
27.7 66.9
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microgrooves in the SCPS for 4 days. However, nearly no Ca–P nucleus was found on plane TiO2 surfaces (Fig. 4c). The Ca and P elements shown in EDX spectrum (Fig. 4d) probably come from the Ca–P nucleus in the cracks of the plane TiO2 surfaces, which were formed during the sintering process. This result revealed that the Ca–P nucleated more easily on the microgrooved TiO2 surface than on the plane one. After 7 days of soaking, SEM observation found that large crystals precipitated on the microgrooved surfaces and the effects of microgrooves on Ca–P growth were weak (Fig. 5). The TF-XRD spectrum revealed that the crystalline phase was dicalcium phosphate dehydrate (DCPD, CaHPO4·2H2O) (Fig. 6). The peaks at 2θ = 10.7°, 21.4° and 31.7° correspond to the (020), (121̄) and (211̄) crystal planes of DCPD, which are the characteristic peaks of well-crystallized DCPD as listed in JCPDS card (72-0713). It has been demonstrated that DCPD is the kinetically favorable phase in a supersaturated Ca–P solution with high Ca2+ and HPO2− 4 concentrations at pH larger than 5 [21,22]. Therefore, it is not surprising that DCPD was obtained in the present study considering the ion concentration and the pH value of SCPS. Combining the results of 4 days and 7 days of mineralization, it was found that the surface microgeometry can affect the Ca–P nucleation at the initial days while they have weak influence on Ca–P growth with the extension of immersion days. 4. Discussion
Fig. 2. (a) SEM micrograph of the microgrooved surface. (b) The dimension of the microogrooved surface.
anatase (Fig. 3). The peaks at 2θ = 25.2°, 47.9° correspond to the (101), (200) crystal planes of TiO2 anatase, which are the characteristic peaks of well-crystallized anatase as listed in JCPDS card (71-1168). The TFXRD spectrum also shows a dome peak ranging from 20° to 40°, which is attributed to the amorphous glass substrate. Surface energy measurement indicated that the surface energies of microgrooved surfaces were smaller than those of the plane TiO2 surfaces (Table 3), which could not be simply explained by the conventional view of liquid in contact with rough surfaces, such as the Wenzel and Cassie models. That probably could be ascribed to the size effects of micropatterns. Neither the Wenzel nor Cassie model takes into account the local geometry and therefore is not expected to be valid when the drop size is comparable to a characteristic dimension of the surface roughness [20]. In the present study, the length of the microgroove is in millimeter level and the width is in micrometer level, which is comparable with the drop size in the contact angle measurement. The detailed investigation of microgroove geometry effect on the TiO2 surface energy will be our next step research focus.
Comparison of Ca–P mineralization on the plane TiO2 surfaces and microgrooved TiO2 surfaces revealed that the deposition of Ca–P on the ridges of the microgrooves was much easier than on the plane ones. This result clearly indicates that the nucleation of Ca–P prefers to happen at the ridges of TiO2 microgrooves and the topography dominates the nucleation and distribution of Ca–P at the initial days. This phenomenon might be partially explained by the surface energy difference between the ridges and plane area. As is known to all, the surface energy of substrate is a critical factor affecting Ca–P nucleation and Ca–P formation on TiO2 surfaces in supersaturated aqueous solution is a heterogeneous nucleation process [23]. In classical nucleation theory, the critical free energy for heterogeneous nucleation depends on the supersaturation of the solution (S), the temperature (T), the surface energies of the substrate (σS) and the nucleus (σ), and the interfacial energy of the substrate/nucleus (σi) [24]: ⁎
⁎
ΔGheter = ΔGhom
2
3
Δσ 16πν σ σ + σi −σs = • 2σ 2σ 3k2 T 2 ðlnSÞ2
where k is the Boltzmann constant; Δσ is the net interfacial energy; and ΔGhom⁎ is the critical free energy for the homogenous nucleation.
3.2. Ca–P mineralization The morphologies of the Ca–P deposited on TiO2 surface under biomimetic environments were observed under SEM. SEM micrograph (Fig. 4a) and EDX spectrum (Fig. 4b) revealed that Ca–P nucleated on the ridges of the microgrooves by immersing the TiO2
ð2Þ
Fig. 3. TF-XRD spectrum of TiO2 surface prepared by sol–gel method.
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L. Jiang et al. / Materials Science and Engineering C 29 (2009) 2355–2359
Fig. 4. (a) The SEM micrograph of calcium phosphate mineralization on microgrooved TiO2 surface for 4 days. (b) EDX of (a). (c) Calcium phosphate mineralization on plane TiO2 surface for 4 days. (d) EDX of (c).
Reduction of the critical free energy, resulting in a lower energy barrier to heterogeneous nucleation, can be achieved by increasing the supersaturation and reducing the net interfacial energy (Δσ). We proposed that the dangling bonds on the ridges of the TiO2 microgrooves generated in the preparation of microgrooves and heat
treatments probably are the critical factors for Ca–P mineralization. In this study, the atoms on the ridges of the microgrooves might have more unsaturated bonds than those on the plane surfaces, as schematically demonstrated in Fig. 7. Although the surface energies of microgrooved surfaces are smaller than those of the plane TiO2 surfaces, the dangling bonds on the ridges of the TiO2 microgrooves result in an increase of the surface energy (σS) at the ridge area [25]. Thus ΔGheter⁎ on the ridges of
Fig. 5. The SEM micrograph of large calcium phosphate crystals on microgrooved TiO2 surface after being immersed in SCPS for 7 days.
Fig. 6. TF-XRD spectrum indicated that the calcium phosphate on the microgrooved TiO2 after immersed in SCPS for 7 days was DCPD.
L. Jiang et al. / Materials Science and Engineering C 29 (2009) 2355–2359
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plays an important role at the initial stage of biomineralization and its effects become weak with the extended days. Acknowledgements This project was financially supported by the National Natural Science Foundation of China (No. 30700172), NSFC/RGC Joint Research Funding (N_HKUST601/08, 30831160509), Specialized Research Fund for the Doctoral Program of Higher Education for Young Teacher (20070613019) and National Key Project of Scientific and Technical Supporting Programs Fund from MSTC (2006BAI16B01). Fig. 7. The sketch shows the unsaturated bonds of the ridges of the microgrooves and those on plane bottoms of the microgrooves.
the microgrooves is reduced and therefore Ca–P nucleation happens in this area. Note that Jarn et al. reported that both the TiO2 surface topography and surface energy controlled CaP precipitation kinetics [12]. They found that the surface with high surface energy but insufficient roughness has no CaP precipitation. On the other hand, they also found that high roughness enhances the CaP mineralization. In the present study, the effects of surface topography on biomineralization were investigated on well-defined TiO2 microgrooves, which further confirmed that surface topography affects the mineralization process, especially at the initial days.
5. Conclusions This work investigated biomimetic Ca–P mineralization on microgrooved TiO2 surfaces and plane TiO2 surfaces, respectively. The softlithography and sol–gel technology were employed to produce micropatterned TiO2 surfaces. The phase of the TiO2 after heat treatments is anatase and the phase of the Ca–P obtained from biomineralization process is DCPD. The results indicate that Ca–P mineralization prefers to occur on the ridges of microgrooves at the initial days. With the extension of the immersion days, the effects of microgrooves on mineralization become weak. The investigation confirms that the surface topography
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Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Micropatterned TiO2 effects on calcium phosphate mineralization Lili Jiang a, Xiong Lu a,b,⁎, Yang Leng b, Shuxin Qu a, Bo Feng a, Jie Weng a a b
Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, China Department of Mechanical Engineering, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 17 February 2009 Received in revised form 22 April 2009 Accepted 11 June 2009 Available online 21 June 2009 Keywords: TiO2 Calcium phosphate Microgrooves Biomineralization
a b s t r a c t The effects of implant surface topography and chemistry on biomineralization have been a research focus because of their potential importance in orthopedic and bone replacement applications. While a vast amount of research is focusing on chemical modified surfaces and rough surfaces, little attention has been paid to the well-defined micropatterned surface effects on calcium phosphate mineralization process due to the difficulties in preparing microfabricated biomaterial surfaces. This work focuses on the effects of microgrooved TiO2 surfaces on the calcium phosphate mineralization process. Firstly, we developed a new process that can prepare microgrooved TiO2 coatings on glass substrates using soft-lithography and sol–gel technology. Then microgrooved TiO2 surfaces were used to induce Ca–P mineralization under biomimetic conditions. The results revealed that topography dominated the growth and distribution of mineralization at the initial days and then the effects of topography become weak with the extended immersion days. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Biomineralization is a process of forming minerals in organisms, where minerals nucleate and grow from a supersaturated aqueous solution under the biomimetic conditions [1,2]. Biomineralization widely exists in nature and several important bioceramics such as calcium phosphates (Ca–P), which are the major component of bone and teeth and have good osteoconductivity, can be obtained by this process [3,4]. Inspired by the biological process of mineralization, various researchers employed mineralization process to obtain Ca–P in order to improve biocompatibility and bone inductivity of human bone implants [5–7]. The authors' group also obtained Ca–P coatings on nitric-acid-treated titanium surfaces in simulated body fluid under physiological conditions [8]. However, the mechanism of the biomineralization is far from being fully understood. It is generally accepted that the material surface chemistry plays an important role on the mineralization process [9,10]. Surface topography, which is another important surface property, might also be a critical factor that affects the Ca–P mineralization process [11]. Recently, researchers indicated that Ca–P mineralization ability depended upon the topography of the substrate surface to a certain extent [12,13]. Researchers also proved that the Ca–P mineralization on the rough substrate surfaces was easier than that on the plane one and porous titanium surfaces exhibit excellent ability of inducing Ca–P
⁎ Corresponding author. Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, China. Tel.: +86 28 87634023; fax: +86 28 87601371. E-mail address: [email protected] (X. Lu). 0928-4931/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2009.06.005
mineralization [14]. The author's previous study already demonstrated that Ca–P deposition on the microhole-patterned titanium surfaces was better than that on the plane surfaces and the Ca–P preferred to grow on the inner wall of the microholes [15]. In the present study, we proposed a new method to prepare well-designed micropatterned TiO2 surfaces in order to further explain the surface topography effects on Ca–P mineralization process accurately. Titanium dioxide attracted our interest with its good biocompatibility and soft-lithography technology was used in this paper to obtain microgrooved TiO2 surfaces. 2. Materials and methods 2.1. The TiO2 microgrooves Microgrooved TiO2 surfaces were fabricated onto a glass substrate using soft-lithography technology. Soft-lithography is a well known technique that uses an elastomeric silicone rubber, mostly polydimethylsiloxane (PDMS), as a template for structuring materials on the micro scale technology [16,17]. The process was schematically shown step by step in Fig. 1. In the first step, a PDMS microstamp was fabricated by replicating the microgrooves of a micromachined silicon substrate obtained by photolithography and wet etching. Firstly, the Sylgard 184 (Dow Corning) and the initiator with the mass ratio of 10:1 was coated on the microgrooved silicon wafer; then the sample was placed in a vacuum oven at 80 °C for 2 h to exclude bubbles and cure; finally the cured polymer was peeled off from the silicon wafer and the microgrooved PDMS stamp was obtained. In the second step, the TiO2 sol–gel precursor solution was prepared using a mixture of tetrabutyltitanate/ethanol/hydrochloric acid/distilled
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L. Jiang et al. / Materials Science and Engineering C 29 (2009) 2355–2359 Table 2 The contact angles of probe liquid on microgrooved and plane TiO2 surfaces. Surface
Contact angle
Microgrooves (water) Microgrooves (glycerol) Plane (water) Plane (glycerol)
76.6° ± 0.9° 73.6° ± 6.5° 25.2° ± 0.9° 27.3° ± 5.9°
microgrooves was hung and immersed in the solution in a polystyrene tube at 36.5 °C for 4 and 7 days. After soaking in the SCPS for different times, the substrates were taken out and washed with distilled water. 2.3. TiO2 surface characterization
Fig. 1. The process of the fabrication of the TiO2 microgrooves. (a) TiO2 sol–gel was coated on the glass substrate. (b) PDMS microstamp was embossed onto TiO2 sol–gel. (c) Microgrooved TiO2 was obtained by heat treatments after removing the PDMS microstamp.
water/1,5-pentanediol with a molar ratio of 1/30/0.2/2/2. Firstly, tetrabutyltitanate was dissolved in two-thirds ethanol and stirred vigorously for 30 min at room temperature, which was named as solution I; water, hydrochloric acid and 1,5-pentanediol were dissolved in ethanol, which was named as solution II. Then solution II was slowly dropped into solution I under vigorous stirring. The mixed solution was kept overnight at room temperature to obtain the TiO2 sol. The asprepared TiO2 sol was coated on the glass substrate and the PDMS stamp was embossed onto the TiO2 sol. TiO2 sol was condensed and solidified in a vacuum oven at 25 °C for 12 h. After drying, the PDMS stamp was removed and the gel was sintered at 400 °C for 2 h and finally the microgrooved TiO2 was obtained. The plane TiO2 specimen was also prepared using sol–gel technique as control samples. 2.2. Ca–P mineralization A simple supersaturated calcium phosphate solution (SCPS) with high calcium and phosphate ion concentrations was used for biomineralization study. SCPS was prepared by dissolving NaCl (7.714 g), CaCl2 (1.387 g), Na2HPO4·2H2O (0.89 g) and 1 M HCl 50 ml in 1 L of deionized water. Tris-hydroxymethyl aminomethane (TRIS) was used to adjust the pH value to 6.0. SCPS has been successfully applied to investigate the Ca–P formation by previous study [8,18]. In SCPS, Ca2+ and HPO2− 4 concentrations were five times that of human blood plasma and Mg2+ and HCO− 3 which were generally regarded as Ca–P crystallization inhibitors were removed. Thus, the formation of Ca–P mineralization in SCPS could be faster than that in SBF. The ion concentrations of SCPS and human blood plasma are listed in Table 1. The as-prepared TiO2 with or without
Table 1 Ion concentrations of human blood plasma and SCPS. Ion
Concentration (mM/L) Blood plasma
SCPS
Na+ K+ Mg2+ Ca2+ Cl− HCO− 3 HPO2− 4 2− SO4
142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5
142.0 – – 12.5 217.0 – 5.0 –
Scanning electron microscopy (SEM; QUANTA-200, FEI, The Netherlands) was used to analyze the morphology of the TiO2 microgrooves before and after the mineralization. The crystal structure and phase composition of as-prepared TiO2 surfaces and the precipitated Ca–P were examined using thin film X-ray diffraction (TF-XRD; X'pert proMPD, PANalytical, The Netherlands). The XRD measurements were performed on a stage using a Cu–Kα (wavelength = 1.5056 ) X-ray source with step rate of 0.02° /s. The depth of the as-prepared TiO2 microgrooves was measured by a surface profilometer (Ambios XP-2, Ambios, USA). The surface energies of the microgrooved and plane TiO2 surfaces were obtained from Owens–Wendt (OW) method through the following equation [19]: qffiffiffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffi ð1 + cosθÞγL = 2ð γSd γLd + γSP γLP Þ
ð1Þ
where θ is the equilibrium contact angle; γL is the surface tension of testing liquid and γdL and γpL is the dispersion and polar components; the surface energy of a solid (γS) is equal to a sum of the dispersion (γdS ) and polar (γpS ) components (γS = γdS + γpS ). The two probe liquids used in the present study are deionized water (γ = 72.8 mJ/m2, γp = 51.0 mJ/m2, γd = 21.8 mJ/m2) and glycerol (γ = 63.4 mJ/m2, γp = 26.4 mJ/m2, γd = 37.0 mJ/m2). The contact angle measurements were carried out using the sessile-drop technique (DSA100, Kruss GmbH, Germany). The liquid droplets were laid onto the microgrooved and plane surfaces by a micro syringe. Around 8 samples were tested for every type of microgrooved and plane surfaces and only one drop was laid on every single sample. The average contact angles were listed in Table 2 and were substituted into Eq. (1) to calculate the corresponding surface energies which were listed in Table 3. 3. Results 3.1. TiO2 microgrooves Microgrooved TiO2 was successfully produced on glass substrates by soft-lithography technology as shown in the SEM micrograph (Fig. 2a). Surface profiler measurement shows the periodic width of the microgrooves is ∼10 µm and the depth of the microgrooved TiO2 is ∼0.3 µm (Fig. 2b). The TF-XRD spectrum clearly revealed that crystalline phase of microgrooved TiO2 surface after sintered at 400 °C for 2 h is
Table 3 The surface energies calculated from OW method.
Microgrooved surfaces Plane surfaces
γSd (mJ/m2)
γSp (mJ/m2)
γS (mJ/m2)
9.5 13.5
18.2 53.4
27.7 66.9
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microgrooves in the SCPS for 4 days. However, nearly no Ca–P nucleus was found on plane TiO2 surfaces (Fig. 4c). The Ca and P elements shown in EDX spectrum (Fig. 4d) probably come from the Ca–P nucleus in the cracks of the plane TiO2 surfaces, which were formed during the sintering process. This result revealed that the Ca–P nucleated more easily on the microgrooved TiO2 surface than on the plane one. After 7 days of soaking, SEM observation found that large crystals precipitated on the microgrooved surfaces and the effects of microgrooves on Ca–P growth were weak (Fig. 5). The TF-XRD spectrum revealed that the crystalline phase was dicalcium phosphate dehydrate (DCPD, CaHPO4·2H2O) (Fig. 6). The peaks at 2θ = 10.7°, 21.4° and 31.7° correspond to the (020), (121̄) and (211̄) crystal planes of DCPD, which are the characteristic peaks of well-crystallized DCPD as listed in JCPDS card (72-0713). It has been demonstrated that DCPD is the kinetically favorable phase in a supersaturated Ca–P solution with high Ca2+ and HPO2− 4 concentrations at pH larger than 5 [21,22]. Therefore, it is not surprising that DCPD was obtained in the present study considering the ion concentration and the pH value of SCPS. Combining the results of 4 days and 7 days of mineralization, it was found that the surface microgeometry can affect the Ca–P nucleation at the initial days while they have weak influence on Ca–P growth with the extension of immersion days. 4. Discussion
Fig. 2. (a) SEM micrograph of the microgrooved surface. (b) The dimension of the microogrooved surface.
anatase (Fig. 3). The peaks at 2θ = 25.2°, 47.9° correspond to the (101), (200) crystal planes of TiO2 anatase, which are the characteristic peaks of well-crystallized anatase as listed in JCPDS card (71-1168). The TFXRD spectrum also shows a dome peak ranging from 20° to 40°, which is attributed to the amorphous glass substrate. Surface energy measurement indicated that the surface energies of microgrooved surfaces were smaller than those of the plane TiO2 surfaces (Table 3), which could not be simply explained by the conventional view of liquid in contact with rough surfaces, such as the Wenzel and Cassie models. That probably could be ascribed to the size effects of micropatterns. Neither the Wenzel nor Cassie model takes into account the local geometry and therefore is not expected to be valid when the drop size is comparable to a characteristic dimension of the surface roughness [20]. In the present study, the length of the microgroove is in millimeter level and the width is in micrometer level, which is comparable with the drop size in the contact angle measurement. The detailed investigation of microgroove geometry effect on the TiO2 surface energy will be our next step research focus.
Comparison of Ca–P mineralization on the plane TiO2 surfaces and microgrooved TiO2 surfaces revealed that the deposition of Ca–P on the ridges of the microgrooves was much easier than on the plane ones. This result clearly indicates that the nucleation of Ca–P prefers to happen at the ridges of TiO2 microgrooves and the topography dominates the nucleation and distribution of Ca–P at the initial days. This phenomenon might be partially explained by the surface energy difference between the ridges and plane area. As is known to all, the surface energy of substrate is a critical factor affecting Ca–P nucleation and Ca–P formation on TiO2 surfaces in supersaturated aqueous solution is a heterogeneous nucleation process [23]. In classical nucleation theory, the critical free energy for heterogeneous nucleation depends on the supersaturation of the solution (S), the temperature (T), the surface energies of the substrate (σS) and the nucleus (σ), and the interfacial energy of the substrate/nucleus (σi) [24]: ⁎
⁎
ΔGheter = ΔGhom
2
3
Δσ 16πν σ σ + σi −σs = • 2σ 2σ 3k2 T 2 ðlnSÞ2
where k is the Boltzmann constant; Δσ is the net interfacial energy; and ΔGhom⁎ is the critical free energy for the homogenous nucleation.
3.2. Ca–P mineralization The morphologies of the Ca–P deposited on TiO2 surface under biomimetic environments were observed under SEM. SEM micrograph (Fig. 4a) and EDX spectrum (Fig. 4b) revealed that Ca–P nucleated on the ridges of the microgrooves by immersing the TiO2
ð2Þ
Fig. 3. TF-XRD spectrum of TiO2 surface prepared by sol–gel method.
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Fig. 4. (a) The SEM micrograph of calcium phosphate mineralization on microgrooved TiO2 surface for 4 days. (b) EDX of (a). (c) Calcium phosphate mineralization on plane TiO2 surface for 4 days. (d) EDX of (c).
Reduction of the critical free energy, resulting in a lower energy barrier to heterogeneous nucleation, can be achieved by increasing the supersaturation and reducing the net interfacial energy (Δσ). We proposed that the dangling bonds on the ridges of the TiO2 microgrooves generated in the preparation of microgrooves and heat
treatments probably are the critical factors for Ca–P mineralization. In this study, the atoms on the ridges of the microgrooves might have more unsaturated bonds than those on the plane surfaces, as schematically demonstrated in Fig. 7. Although the surface energies of microgrooved surfaces are smaller than those of the plane TiO2 surfaces, the dangling bonds on the ridges of the TiO2 microgrooves result in an increase of the surface energy (σS) at the ridge area [25]. Thus ΔGheter⁎ on the ridges of
Fig. 5. The SEM micrograph of large calcium phosphate crystals on microgrooved TiO2 surface after being immersed in SCPS for 7 days.
Fig. 6. TF-XRD spectrum indicated that the calcium phosphate on the microgrooved TiO2 after immersed in SCPS for 7 days was DCPD.
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plays an important role at the initial stage of biomineralization and its effects become weak with the extended days. Acknowledgements This project was financially supported by the National Natural Science Foundation of China (No. 30700172), NSFC/RGC Joint Research Funding (N_HKUST601/08, 30831160509), Specialized Research Fund for the Doctoral Program of Higher Education for Young Teacher (20070613019) and National Key Project of Scientific and Technical Supporting Programs Fund from MSTC (2006BAI16B01). Fig. 7. The sketch shows the unsaturated bonds of the ridges of the microgrooves and those on plane bottoms of the microgrooves.
the microgrooves is reduced and therefore Ca–P nucleation happens in this area. Note that Jarn et al. reported that both the TiO2 surface topography and surface energy controlled CaP precipitation kinetics [12]. They found that the surface with high surface energy but insufficient roughness has no CaP precipitation. On the other hand, they also found that high roughness enhances the CaP mineralization. In the present study, the effects of surface topography on biomineralization were investigated on well-defined TiO2 microgrooves, which further confirmed that surface topography affects the mineralization process, especially at the initial days.
5. Conclusions This work investigated biomimetic Ca–P mineralization on microgrooved TiO2 surfaces and plane TiO2 surfaces, respectively. The softlithography and sol–gel technology were employed to produce micropatterned TiO2 surfaces. The phase of the TiO2 after heat treatments is anatase and the phase of the Ca–P obtained from biomineralization process is DCPD. The results indicate that Ca–P mineralization prefers to occur on the ridges of microgrooves at the initial days. With the extension of the immersion days, the effects of microgrooves on mineralization become weak. The investigation confirms that the surface topography
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