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Production of a biomimetic apatite nanotube mesh via biotemplating a polymer nanofiber mesh
Materials Letters 64 (2010) 2655–2658
Contents lists available at ScienceDirect
Materials Letters 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 a t l e t
Production of a biomimetic apatite nanotube mesh via biotemplating a polymer nanofiber mesh Mi-Kyung Kim a,b, Jung-Ju Kim a,b, Ueon Sang Shin a,b, Hae-Won Kim a,b,c,⁎ a b c
Department of Nanobiomedical Science and WCU Research Center, Dankook University Graduate School, South Korea Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea Department of Biomaterials Science, School of Dentistry, Dankook University, South Korea
a r t i c l e
i n f o
Article history: Received 27 March 2010 Accepted 14 May 2010 Available online 27 May 2010 Keywords: Hydroxyapatite Nanotube Bone substitutes Bioceramics
a b s t r a c t Here, we report the preparation of hydroxyapatite nanotubes for specific use as bone regeneration material. The nanofiber mesh of a polymer (polycaprolactone) used as a template was mineralized within solutions via a biomimetic process. A subsequent heat-treatment (over 500 °C) completely eliminated the inner polymer, resulting in preserving the surface mineral phase in the form of nanotubes. The nanotubes had diameters of hundreds of nanometers with nonwoven mesh, replicating the initial nanofiber template. Furthermore, the nanotubes revealed a phase of poorly crystallized apatite, mimicking biological bone mineral. The developed biomimetic apatite nanotubes may be useful for bone regeneration as a new type of biomaterial. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Bone regeneration has been possible with the support of artificial materials, including bioceramics and degradable polymers [1]. The major focus of the bone substitute materials has been on the production of calcium phosphate compounds because the bone mineral phase consists of calcium phosphates, mainly hydroxyapatite [2]. Accordingly, a great deal of effort has been made to synthesize hydroxyapatite powder and its sintered products in the form of granules or porous blocks for use as bone substitutes and fillers [3]. Producing hydroxyapatite that mimics biological bone mineral has been of particular importance because this largely determines the biological activity and bone formation ability of the synthetic hydroxyapatite. When compared to the processes involving a solidstate reaction at elevated temperature, solution-mediated room temperature processes mimicking the condition of bone formation are much favored [4–6]. Indeed, it has been demonstrated that apatite crystals produced using media simulating body fluid can better mimic native bone mineral and stimulate bone cell functions [5,6]. In addition to the composition of hydroxyapatite, its exploitation into specific shapes such as nanospheres, nanocapsules, nanofibers and nanotubes is now gaining interest to enable its extended use in tissue regeneration and drug delivery systems [7–9]. Specifically, when drugs or proteins are loaded onto hydroxyapatite nanocarriers, the potential for the success of the bone filler can be greatly improved ⁎ Corresponding author. Biomaterials and Tissue Engineering Laboratory, Department of Nanobiomedical Science and WCU Research Center, Dankook University Graduate School, South Korea. Tel.: + 82 41 550 1926; fax: + 82 41 550 1926. E-mail address: [email protected] (H.-W. Kim). 0167-577X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2010.05.021
by the therapeutic actions, such as activating or blocking specific cell and tissue functions [8,9]. In this study, we attempted to produce hydroxyapatite nanotubular mesh for use as a bone substitute and drug carrier by utilizing a polymer nanofibrous template and mineralization of the surface. A solution-mediated biomimetic process was applied to obtain a composition similar to the bone mineral apatite. Elimination of the inner polymeric portion was considered to efficiently form a tubular structure made of a surface mineral phase. The preparation method and characteristics of the hydroxyapatite nanotubes are described herein. 2. Experimental The polymer nanofiber mesh to be used as a replicate for the nanotube was produced by electrospinning of poly(ε-caprolactone) (PCL; Mw = 80,000, Sigma). The electrospinning was conducted using a solution of 10% w/v PCL dissolved in dichloromethane/ethanol (4:1) at a voltage of 10 kV, a distance of 10 cm, and an injection rate of 0.5 ml/h. A total 10 ml of solution was electrospun on a metal drum to gather the PCL nanofiber mesh. Mineralization of the PCL nanofiber was based on our previous study, with slight modification [10]. First, the PCL mesh was dipped into a 2 N NaOH solution while stirring gently for 6 h, after which it was washed fully and dried. This soaking in an alkaline solution was designed to activate the hydrophobic surface by the creation of carboxyl groups that then allow the binding of calcium and phosphate ions [10]. The alkaline-treated nanofiber was then dipped into solutions of 150 mM Ca (CaCl2) and 150 mM P (Na2HPO4) alternatively to generate nuclei to accelerate further mineral growth, while
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washing with distilled water between dipping processes. After six rounds of alternative dipping, the nanofiber was incubated in a mineralization fluid (the ion concentration was 284.0 mM Na+, 10 mM K+, 3.0 mM Mg2+, 5.0 mM Ca2+, 295.6 mM Cl−, 8.4 mM 2− 2− HCO− 3 , 2.0 mM HPO4 , and 1.0 mM SO4 ) for 1, 3 and 7 days to accelerate the growth of the nanocrystalline mineral.. The mineralized sample was then dried and heat-treated at 500–800 °C for 1 h at a ramping speed of 1 °C/min. The morphology of the samples was characterized by scanning electron microscopy (SEM, Hitachi, S-3000H), after which the mineral composition was detected by energy dispersive spectroscopy (EDS, Bruker, SNE-3000M). The chemical bond structure of the nanotubes was characterized by Fourier transform infrared (FT-IR, Thermo scientific, Nicolet 380) spectroscopy. The thermal history of the mineralized sample was recorded using thermogravimetric analysis (TGA, Shimadzu, TGA50).
The surface-mineralized PCL nanofiber was subsequently heattreated to eliminate the inner polymer while preserving the outer mineral phase. Based on the TGA (not shown here) the complete removal of the organic phase above ∼420 °C was observed, with a total weight loss of ∼ 79%, suggesting the remaining mineral phase of ∼21% by weight. The SEM morphology of the nanotubular mesh produced after heat-treatment at 500 °C is shown in Fig. 2. The temperature was selected based on TGA to eliminate the polymer completely. The macroscale morphology of the mineral nanotubes heattreated at 500 °C (Fig. 2(a)) showed a nonwoven fibrous mesh that maintained the initial morphology of the mineralized PCL. The magnified image (Fig. 2(b)) showed the individual nanotubes, and some tubular structure was revealed. The morphology of samples heattreated at higher temperature (800 °C) was also observed to be similar
3. Results and discussion The morphology of the electrospun PCL nanofiber that was to be used as the template for further mineralization is shown in Fig. 1(a). A nonwoven fibrous web was produced by the electrospinning method. A series of solution-mediated reactions was then introduced to cover the nanofiber surface with calcium phosphate mineral. As shown in Fig. 1(b), when the sample was mineralized for 3 days, the nanofiber surface was almost completely covered with the mineral phase. Based on the immersion tests, which lasted for up to 7 days, a period of 3 days was appropriate to cover the surface almost completely; while the surface was not adequately covered after 1 day and excessive mineralization (clogging of the pore structure) was observed at 7 days.
Fig. 1. SEM morphology of (a) PCL nanofiber nonwoven mesh after electrospinning, and (b) its mineralized form following a series of solution-mediated reactions.
Fig. 2. (a–c) SEM morphology of the mineral nanotubular mesh following heattreatment at 500 °C (a, b) and 800 °C (c).
M.-K. Kim et al. / Materials Letters 64 (2010) 2655–2658
(Fig. 2(c)). The nanotubular morphology of the sample was better disclosed by the TEM images taken at different magnifications, as shown in Fig. 3. Long tubular fibers with an inner hollow structure were well developed, and the mineral phase was shown to line the fiber surface with relatively uniform thickness. When measured from the images, the width of inner hollow part was 722 nm (±122 nm) and the wall thickness was 185 nm (±37 nm). The minerals consisted of a number of nanocrystallites with highly elongated morphology, which is typical of bone mineral hydroxyapatite. It is believed that the diameter of the nanotubes was primarily affected by that of the initial polymer nanofiber, which is possibly modulated to be in the range of tens to
Fig. 3. TEM images of the apatite nanotubes after mineralization and heat-treatment at 500 °C, taken at different magnifications from (a) to (c), revealing a tubular structured fiber with a hollow inner part and a surface-lined with highly elongated mineral nanocrystallites.
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hundreds of nanometers and up to a few micrometers. Moreover, depending on the degree of mineralization, the shell thickness can be altered. The results of the present study indicated that mineralization over 3 to 7 days had the potential to increase the level of mineralization that proceeds in a radial direction (i.e., perpendicular to the surface). The characteristics of the mineral nanotubes were analyzed as shown in Fig. 3. The IR spectrum of the mineral nanotubes showed structural bands similar to those of the initial mineralized PCL nanofiber and hydroxyapatite used as a reference (Fig. 4(a)). In addition to the phosphate (PO4) bands that were well-characterized at 1000–1100 cm− 1 the appearance of a carbonate (CO3) band at 1400–1600 cm− 1, particularly in the mineralized samples, demonstrates that the obtained nanotubes are hydroxyapatite and partially carbonated. Energy dispersive spectroscopy analysis revealed the existence of Ca and P and a Ca/P ratio of approximately 1.69 (Fig. 4 (b)), which is similar to that of hydroxyapatite. As discussed above, in addition to control over the size and shell thickness of the nanotubes, directing the orientation of the nanotubes is also possible by fine-tuning the processes, which is generally accomplished during the electrospinning of polymers (e.g. high-speed rotating collector). In this sense, the use of an electrospun polymer network as the template for mineralization and further nanotube
Fig. 4. Analyses of the mineral nanotubes: (a) IR spectrum (mineralized PCL and commercial apatite were compared) showing PO4 and CO3 bands, and (b) EDS profile with a Ca/P ratio of ∼ 1.69.
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formation is regarded as versatile, and future work should be conducted to evaluate this modulation of nanotube morphology. Moreover, additional studies need to be conducted to evaluate the feasibility of the application of biomimetic apatite nanotubes in biomedical fields, such as in bone substituting materials and drug delivery vehicles.
Acknowledgements This work was supported by the research fund of Dankook University in 2009. References
4. Conclusions Apatite nanotubes were produced using the polymer nanofiber template and subsequent surface mineralization. An electrospun nanofibrous web of polycaprolactone was mineralized through solution-mediated reactions and subsequently heat-treated to create a mineral nanotubular mesh. Analyses of the nanotubes demonstrated the formation of a bone mineral-like apatite. This new type of biomaterial can potentially be used as a defect filler and drug carrier for bone regeneration.
[1] Kim HW, Lee HH, Knowles JC. J Biomed Mater Res A 2006;79A:643–9. [2] Palmer LC, Newcomb CJ, Kaltz SR, Spoerke ED, Stupp SI. Chem Rev 2008;108: 4754–83. [3] Hench LLJ. Am Cream Soc 1998;81:1705–28. [4] Shin KS, Jayasuriya AC, Kohn DH. J Biomed Mater Res A 2007;83:1076–86. [5] Kokubo T, Takadama H. Biomaterials 2006;27:2907–15. [6] Yu HS, Hong SJ, Kim HW. Mater Chem Phy 2009;113:873–7. [7] Lee HH, Hong SJ, Kim CH, Kim EC, Jang JH, Shin HI, et al. J Mater Sci Mater Med 2008;19:3029–34. [8] Hengst V, Oussoren C, Kissel T, Storm G. Inter J Pharm 2007;331:224–7. [9] Liu TY, Chen SY, Liu DM, Liou SC. J Control Release 2005;107:112–21. [10] Yu HS, Jang JH, Kim TI, Lee HH, Kim HW. J Biomed Mater Res A 2009;88:747–54.
Contents lists available at ScienceDirect
Materials Letters 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 a t l e t
Production of a biomimetic apatite nanotube mesh via biotemplating a polymer nanofiber mesh Mi-Kyung Kim a,b, Jung-Ju Kim a,b, Ueon Sang Shin a,b, Hae-Won Kim a,b,c,⁎ a b c
Department of Nanobiomedical Science and WCU Research Center, Dankook University Graduate School, South Korea Institute of Tissue Regeneration Engineering (ITREN), Dankook University, South Korea Department of Biomaterials Science, School of Dentistry, Dankook University, South Korea
a r t i c l e
i n f o
Article history: Received 27 March 2010 Accepted 14 May 2010 Available online 27 May 2010 Keywords: Hydroxyapatite Nanotube Bone substitutes Bioceramics
a b s t r a c t Here, we report the preparation of hydroxyapatite nanotubes for specific use as bone regeneration material. The nanofiber mesh of a polymer (polycaprolactone) used as a template was mineralized within solutions via a biomimetic process. A subsequent heat-treatment (over 500 °C) completely eliminated the inner polymer, resulting in preserving the surface mineral phase in the form of nanotubes. The nanotubes had diameters of hundreds of nanometers with nonwoven mesh, replicating the initial nanofiber template. Furthermore, the nanotubes revealed a phase of poorly crystallized apatite, mimicking biological bone mineral. The developed biomimetic apatite nanotubes may be useful for bone regeneration as a new type of biomaterial. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Bone regeneration has been possible with the support of artificial materials, including bioceramics and degradable polymers [1]. The major focus of the bone substitute materials has been on the production of calcium phosphate compounds because the bone mineral phase consists of calcium phosphates, mainly hydroxyapatite [2]. Accordingly, a great deal of effort has been made to synthesize hydroxyapatite powder and its sintered products in the form of granules or porous blocks for use as bone substitutes and fillers [3]. Producing hydroxyapatite that mimics biological bone mineral has been of particular importance because this largely determines the biological activity and bone formation ability of the synthetic hydroxyapatite. When compared to the processes involving a solidstate reaction at elevated temperature, solution-mediated room temperature processes mimicking the condition of bone formation are much favored [4–6]. Indeed, it has been demonstrated that apatite crystals produced using media simulating body fluid can better mimic native bone mineral and stimulate bone cell functions [5,6]. In addition to the composition of hydroxyapatite, its exploitation into specific shapes such as nanospheres, nanocapsules, nanofibers and nanotubes is now gaining interest to enable its extended use in tissue regeneration and drug delivery systems [7–9]. Specifically, when drugs or proteins are loaded onto hydroxyapatite nanocarriers, the potential for the success of the bone filler can be greatly improved ⁎ Corresponding author. Biomaterials and Tissue Engineering Laboratory, Department of Nanobiomedical Science and WCU Research Center, Dankook University Graduate School, South Korea. Tel.: + 82 41 550 1926; fax: + 82 41 550 1926. E-mail address: [email protected] (H.-W. Kim). 0167-577X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2010.05.021
by the therapeutic actions, such as activating or blocking specific cell and tissue functions [8,9]. In this study, we attempted to produce hydroxyapatite nanotubular mesh for use as a bone substitute and drug carrier by utilizing a polymer nanofibrous template and mineralization of the surface. A solution-mediated biomimetic process was applied to obtain a composition similar to the bone mineral apatite. Elimination of the inner polymeric portion was considered to efficiently form a tubular structure made of a surface mineral phase. The preparation method and characteristics of the hydroxyapatite nanotubes are described herein. 2. Experimental The polymer nanofiber mesh to be used as a replicate for the nanotube was produced by electrospinning of poly(ε-caprolactone) (PCL; Mw = 80,000, Sigma). The electrospinning was conducted using a solution of 10% w/v PCL dissolved in dichloromethane/ethanol (4:1) at a voltage of 10 kV, a distance of 10 cm, and an injection rate of 0.5 ml/h. A total 10 ml of solution was electrospun on a metal drum to gather the PCL nanofiber mesh. Mineralization of the PCL nanofiber was based on our previous study, with slight modification [10]. First, the PCL mesh was dipped into a 2 N NaOH solution while stirring gently for 6 h, after which it was washed fully and dried. This soaking in an alkaline solution was designed to activate the hydrophobic surface by the creation of carboxyl groups that then allow the binding of calcium and phosphate ions [10]. The alkaline-treated nanofiber was then dipped into solutions of 150 mM Ca (CaCl2) and 150 mM P (Na2HPO4) alternatively to generate nuclei to accelerate further mineral growth, while
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M.-K. Kim et al. / Materials Letters 64 (2010) 2655–2658
washing with distilled water between dipping processes. After six rounds of alternative dipping, the nanofiber was incubated in a mineralization fluid (the ion concentration was 284.0 mM Na+, 10 mM K+, 3.0 mM Mg2+, 5.0 mM Ca2+, 295.6 mM Cl−, 8.4 mM 2− 2− HCO− 3 , 2.0 mM HPO4 , and 1.0 mM SO4 ) for 1, 3 and 7 days to accelerate the growth of the nanocrystalline mineral.. The mineralized sample was then dried and heat-treated at 500–800 °C for 1 h at a ramping speed of 1 °C/min. The morphology of the samples was characterized by scanning electron microscopy (SEM, Hitachi, S-3000H), after which the mineral composition was detected by energy dispersive spectroscopy (EDS, Bruker, SNE-3000M). The chemical bond structure of the nanotubes was characterized by Fourier transform infrared (FT-IR, Thermo scientific, Nicolet 380) spectroscopy. The thermal history of the mineralized sample was recorded using thermogravimetric analysis (TGA, Shimadzu, TGA50).
The surface-mineralized PCL nanofiber was subsequently heattreated to eliminate the inner polymer while preserving the outer mineral phase. Based on the TGA (not shown here) the complete removal of the organic phase above ∼420 °C was observed, with a total weight loss of ∼ 79%, suggesting the remaining mineral phase of ∼21% by weight. The SEM morphology of the nanotubular mesh produced after heat-treatment at 500 °C is shown in Fig. 2. The temperature was selected based on TGA to eliminate the polymer completely. The macroscale morphology of the mineral nanotubes heattreated at 500 °C (Fig. 2(a)) showed a nonwoven fibrous mesh that maintained the initial morphology of the mineralized PCL. The magnified image (Fig. 2(b)) showed the individual nanotubes, and some tubular structure was revealed. The morphology of samples heattreated at higher temperature (800 °C) was also observed to be similar
3. Results and discussion The morphology of the electrospun PCL nanofiber that was to be used as the template for further mineralization is shown in Fig. 1(a). A nonwoven fibrous web was produced by the electrospinning method. A series of solution-mediated reactions was then introduced to cover the nanofiber surface with calcium phosphate mineral. As shown in Fig. 1(b), when the sample was mineralized for 3 days, the nanofiber surface was almost completely covered with the mineral phase. Based on the immersion tests, which lasted for up to 7 days, a period of 3 days was appropriate to cover the surface almost completely; while the surface was not adequately covered after 1 day and excessive mineralization (clogging of the pore structure) was observed at 7 days.
Fig. 1. SEM morphology of (a) PCL nanofiber nonwoven mesh after electrospinning, and (b) its mineralized form following a series of solution-mediated reactions.
Fig. 2. (a–c) SEM morphology of the mineral nanotubular mesh following heattreatment at 500 °C (a, b) and 800 °C (c).
M.-K. Kim et al. / Materials Letters 64 (2010) 2655–2658
(Fig. 2(c)). The nanotubular morphology of the sample was better disclosed by the TEM images taken at different magnifications, as shown in Fig. 3. Long tubular fibers with an inner hollow structure were well developed, and the mineral phase was shown to line the fiber surface with relatively uniform thickness. When measured from the images, the width of inner hollow part was 722 nm (±122 nm) and the wall thickness was 185 nm (±37 nm). The minerals consisted of a number of nanocrystallites with highly elongated morphology, which is typical of bone mineral hydroxyapatite. It is believed that the diameter of the nanotubes was primarily affected by that of the initial polymer nanofiber, which is possibly modulated to be in the range of tens to
Fig. 3. TEM images of the apatite nanotubes after mineralization and heat-treatment at 500 °C, taken at different magnifications from (a) to (c), revealing a tubular structured fiber with a hollow inner part and a surface-lined with highly elongated mineral nanocrystallites.
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hundreds of nanometers and up to a few micrometers. Moreover, depending on the degree of mineralization, the shell thickness can be altered. The results of the present study indicated that mineralization over 3 to 7 days had the potential to increase the level of mineralization that proceeds in a radial direction (i.e., perpendicular to the surface). The characteristics of the mineral nanotubes were analyzed as shown in Fig. 3. The IR spectrum of the mineral nanotubes showed structural bands similar to those of the initial mineralized PCL nanofiber and hydroxyapatite used as a reference (Fig. 4(a)). In addition to the phosphate (PO4) bands that were well-characterized at 1000–1100 cm− 1 the appearance of a carbonate (CO3) band at 1400–1600 cm− 1, particularly in the mineralized samples, demonstrates that the obtained nanotubes are hydroxyapatite and partially carbonated. Energy dispersive spectroscopy analysis revealed the existence of Ca and P and a Ca/P ratio of approximately 1.69 (Fig. 4 (b)), which is similar to that of hydroxyapatite. As discussed above, in addition to control over the size and shell thickness of the nanotubes, directing the orientation of the nanotubes is also possible by fine-tuning the processes, which is generally accomplished during the electrospinning of polymers (e.g. high-speed rotating collector). In this sense, the use of an electrospun polymer network as the template for mineralization and further nanotube
Fig. 4. Analyses of the mineral nanotubes: (a) IR spectrum (mineralized PCL and commercial apatite were compared) showing PO4 and CO3 bands, and (b) EDS profile with a Ca/P ratio of ∼ 1.69.
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formation is regarded as versatile, and future work should be conducted to evaluate this modulation of nanotube morphology. Moreover, additional studies need to be conducted to evaluate the feasibility of the application of biomimetic apatite nanotubes in biomedical fields, such as in bone substituting materials and drug delivery vehicles.
Acknowledgements This work was supported by the research fund of Dankook University in 2009. References
4. Conclusions Apatite nanotubes were produced using the polymer nanofiber template and subsequent surface mineralization. An electrospun nanofibrous web of polycaprolactone was mineralized through solution-mediated reactions and subsequently heat-treated to create a mineral nanotubular mesh. Analyses of the nanotubes demonstrated the formation of a bone mineral-like apatite. This new type of biomaterial can potentially be used as a defect filler and drug carrier for bone regeneration.
[1] Kim HW, Lee HH, Knowles JC. J Biomed Mater Res A 2006;79A:643–9. [2] Palmer LC, Newcomb CJ, Kaltz SR, Spoerke ED, Stupp SI. Chem Rev 2008;108: 4754–83. [3] Hench LLJ. Am Cream Soc 1998;81:1705–28. [4] Shin KS, Jayasuriya AC, Kohn DH. J Biomed Mater Res A 2007;83:1076–86. [5] Kokubo T, Takadama H. Biomaterials 2006;27:2907–15. [6] Yu HS, Hong SJ, Kim HW. Mater Chem Phy 2009;113:873–7. [7] Lee HH, Hong SJ, Kim CH, Kim EC, Jang JH, Shin HI, et al. J Mater Sci Mater Med 2008;19:3029–34. [8] Hengst V, Oussoren C, Kissel T, Storm G. Inter J Pharm 2007;331:224–7. [9] Liu TY, Chen SY, Liu DM, Liou SC. J Control Release 2005;107:112–21. [10] Yu HS, Jang JH, Kim TI, Lee HH, Kim HW. J Biomed Mater Res A 2009;88:747–54.