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Facilely fabricating PCL nanofibrous scaffolds with hierarchical pore structure for tissue engineering
Materials Letters 122 (2014) 62–65
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Facilely fabricating PCL nanofibrous scaffolds with hierarchical pore structure for tissue engineering Yuzhang Du a, Xiaofeng Chen b, Young Hag Koh c, Bo Lei a,n a
Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an, China School of Materials Science and Engineering, South China University of Technology, Guangzhou, China c College of Health Science, Korea University, Seoul, South Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 November 2013 Accepted 8 February 2014 Available online 16 February 2014
Highly hierarchical pore structure (macro–micro–nano) for biomimetic nanofibrous scaffolds could efficiently enhance cell infiltration and tissue formation. However, it is difficult to prepare these structures by using traditional electrostatic spinning techniques. Here, we report a facile method to fabricate polycaprolactone (PCL) nanofibrous scaffolds with multi-scale pore structure by simple phase separation. By employing water–dioxane as solvents and tris spheres as macropore template, PCL nanofibrous scaffolds with pores 300–800 mm, 1–10 mm and 100–1000 nm in diameter can be facilely produced. All scaffolds possess controlled nanofibrous morphology and porosity (90–98%). This scaffold may provide promising applications in tissue engineering and drug delivery. & 2014 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Porous materials Nanofibrous scaffolds Phase separation Hierarchical pores
1. Introduction In tissue engineering, tridimensional (3D) scaffolds play an important role because tissue formation requires the space and environment provided by scaffolds [1]. The pore structure of scaffolds usually affects the cellular migration, proliferation, and differentiation, as well as the tissue formation [2]. In addition, cell and tissue could respond according to different pore scales [3]. Therefore, the scaffolds with pore structure at different length scales from nanometer to micrometer (nm–mm) possess advantages compared to other types of scaffolds. For example, in hierarchical scaffolds, the macropores (150–500 mm) maintain the structural stability of scaffolds, support cell proliferation, extracellular matrix (ECM) deposition and tissue formation [4]. The micrometer-scale (1–50 mm) pores are also critical for nutrient diffusion and vascularization [5]. Additionally, nanoscale pore or nanofibrous structure in the scaffolds has shown important effects on controlling cell behavior such as attachment and genes expressions [6]. Therefore, recent years have seen increased interest in fabricating porous scaffolds with multi-scale pore size (from nm to mm) [7]. However, most scaffolds with hierarchical structure do not possess the nanofibrous morphology. On the other hand, most native tissues have the nanofibrous structure and possess excellent physicochemical properties and biological functions. On the side of biomimics and
materials science, it is necessary to fabricate macroporous nanofibrous scaffolds with hierarchical structure by a facile method. As a biomedical polymer, polycaprolactone (PCL) has been approved by American Food and Drug Administration (FDA). Due to its good mechanical properties and slow biodegradation, PCL has been applied in tissue engineering and drugs delivery [8]. PCL nanofibrous scaffolds were also prepared by an electrospinning method [9]. However, it is difficult to produce the macroporous nanofibrous scaffolds by the electrospinning technique. Thermally–induced phase separation technique (TIPS) is a very versatile method to fabricate polymer nanofibrous scaffolds [10,11]. Previous study showed that PCL nanofibrous scaffolds could also be fabricated by phase separation, but the scaffolds did not possess multi-scale pore structure [12]. By particle-based pore formers, macroporous polymer nanofibrous scaffolds can be facilely fabricated. Unfortunately, to the best of our knowledge, PCL nanofibrous scaffolds with hierarchical pore distributions have not been prepared yet. Therefore, in this study, the macroporous PCL nanofibrous scaffolds with multi-scale pore distributions (nm–m m–mm) are fabricated by a modified phase separation technique. The nanofiber morphology and pore structure could be controlled by polymer concentration and phase separation temperature.
2. Materials and methods
n
Corresponding author. Tel.: þ 86 29 833 95361. E-mail address: [email protected] (B. Lei).
http://dx.doi.org/10.1016/j.matlet.2014.02.031 0167-577X & 2014 Elsevier B.V. All rights reserved.
Fabrication of macroporous PCL nanofibrous scaffolds: Polycaprolactone (PCL, average Mn¼80,000), 1,4 dioxane, and tris-hydroxymethyl-aminomethane (Tris) were purchased from Sigma-Aldrich
Y. Du et al. / Materials Letters 122 (2014) 62–65
(Sigma-Aldrich, St. Louis, MO, USA). All materials were used without further treatment. Phase separation technology (PST) was used to synthesize the 3D nanofibrous structure. Tris microspheres with the range of 200–500 mm were chosen as the pore former to fabricate
63
macroporous scaffolds. Typically, a given mass of PCL was dissolved in 10 mL dioxane/H2O mixed solvents with different volume ratios (0:100, 5:95, 10:90). The samples codes and compositions are shown in Table 1S. After the formation of clear solution, the solution was
Fig. 1. Scheme display for fabricating biomimetic and hierarchical PCL nanofibrous scaffolds. (a) Scaffolds production process, (b) highly hierarchical pore distributions of nanofibrous scaffolds, and (c) photos showing the morphology of nanofibrous scaffolds with hierarchical pores.
Fig. 2. Effects of water/dioxane ratio on PCL nanofibrous scaffolds morphology: (a, d, g) PCL scaffolds with pure dioxane; (b, e, h) 5% water/95% dioxane; and (c, f, i) 10% water/90% dioxane (polymer concentration 10%, phase separation temperature 4 1C).
64
Y. Du et al. / Materials Letters 122 (2014) 62–65
immediately transferred into 70 1C, 20 1C, and 4 1C baths for freezing for 4 h. The frozen samples were freeze dried for 48 h. For making macroporous scaffolds, tris sphere template was added into plastic tubes and compacted by vacuum; then PCL solution was poured into the template, followed by freezing and drying. Characterizations of macroporous PCL nanofibrous scaffolds: The nanofibrous morphology and porous microstructure of samples were analyzed by a field emission scanning electron microscope (FE-SEM, JSM6701F, JEOL, Japan). Before observation, all the samples were coated by a layer of platinum (Pt) using a sputter coater with a pressure of 32 mTorr and a time of 150 s. The mean nanofiber length, width and pore size of hybrids were calculated by counting more than 50 species from the SEM images using image-pro Plus 6.0 software (Media Cybernetics, Inc. USA). The porosity (P) of nanofibrous matrices was calculated by evaluating the apparent density (Da) and skeleton density (Ds) according to the followed equation: P¼ (Ds Da) 100/Ds.
3. Results and discussions The hierarchical PCL nanofibrous scaffolds were synthesized by thermally-induced phase separation and pore formation method, as shown in Fig. 1. As a hydrophilic chemical, tris sphere was added into dioxane/water hybrid solution as the macropore producer. After phase separation at low temperatures ( 20 1C, 4 1C), polymer phase was self-assembled to a nanofibrous porous structure. After freeze drying, the nanofibrous PCL scaffolds with nanoscale pores were formed. The addition of tris spheres produced the macropores after final removal by water soaking. The micrometer-scale pores on walls of scaffolds were probably formed due to the slight dissolution of sphere in water-dioxane
solvent before freeze drying. The hierarchical pore structure and morphology are shown in Fig. 1c. For thermally-induced separation technique, the main factors influencing nanofibrous structure and pore structure are solvents ratios, polymer concentration and temperatures [13,14]. The effects of dioxane/water ratio, PCL concentration and temperature on nanofibrous scaffolds are shown in Figs. 2–4. The amount of water has an important effect on nanofibrous structure and morphology. Pure dioxane solvent could produce only the porous PCL structure but not the nanofibrous morphology, because no non-solvent (water) phase separation occurred (Fig. 2a, d, g). As the water phase increased, nanofibrous structure and micrometer pores began to form (Fig. 2b, e, h and c, f, i). As a result, dioxane: H2O with a ratio of 90:10 could produce a stable nanofibrous structure (Fig. 2e, f, i). It should be noted that high amount of water makes the polymer insoluble. Generally, low polymer concentration usually produced good nanofibrous structure and morphology, as well as high porosity [15]. Fig. 3 shows the effect of PCL concentration on scaffold's morphology. The PCL nanofibrous scaffolds with a content of 2% showed a long nanofiber structure (Fig. 3a, d, g). The typical micrometer-scale pores are found on the wall surface of scaffolds, as shown in Fig. 3a–c. The improved nanofibrous morphology at low PCL concentration is attributed to the high solid–liquid and liquid–liquid phase separation efficiency, which has been proved in previous reports [16]. As an important parameter, freeze temperature has a particular effect on the morphology and structure of nanofibrous scaffolds, as shown in Fig. 4. The PCL scaffolds prepared at 70 1C presented just the porous morphology but not nanofibrous structure (Fig. 4d–f). Freezing at 20 1C and 4 1C could fabricate typical nanofibrous scaffolds with micrometer-scale pore walls (Figs. 2c, f, i and 4a–c).
Fig. 3. Effects of polymer concentrations on PCL nanofibrous scaffolds morphology: (a, d, g) 2%; (b, e, h) 5%; and (c, f, i) 8% (10% water/90% dioxane, phase separation temperature 4 1C).
Y. Du et al. / Materials Letters 122 (2014) 62–65
65
Fig. 4. Effects of phase separation temperature on PCL nanofibrous scaffolds morphology. (a, b, c) 20 1C; and (d, e, f) 70 1C (10% water/90% dioxane, 10% PCL).
4. Conclusions We have demonstrated the successful fabrication of hierarchical PCL nanofibrous scaffolds with multi-scale pore structure (50– 500 nm, 1–10 mm, 300–800 mm) by phase separation technology and a soluble pore former. The nanofiber size and porosity of scaffolds could be controlled by polymer concentration and phase separation temperature. The macropore size also could be tailored by template sphere size. This biomimetic PCL nanofibrous scaffolds with hierarchical pore structure may have promising applications in tissue engineering and drugs/cell delivery. Acknowledgment We acknowledge the valuable comments of potential reviewers. This work was supported by the Scientific Research Starting Foundation from Xi’an Jiaotong University, the National Natural Science Foundation of China (51072055), and the National 973 Project of China (2011CB606204). Appendix A. Supporting materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.matlet.2014.02.031.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
Hutmacher DW. Biomaterials 2000;21:2529–43. Hollister SJ. Nat Mater 2005;4:518–24. Karageorgiou V, Kaplan D. Biomaterials 2005;26:5474–91. Zeltinger J, Sherwood JK, Graham DA, Müeller R, Griffith LG. Tissue Eng 2001;7:557–72. Chiu LL, Montgomery M, Liang Y, Liu H, Radisic M. Proc Natl Acad Sci 2012;109:E3414–23. Smith LA, Liu X, Hu J, Ma PX. Biomaterials 2009;30:2516–22. Zhong S, Zhang Y, Lim CT. Tissue Eng Part B: Rev 2011;18:77–87. Williams JM, Adewunmi A, Schek RM, Flanagan CL, Krebsbach PH, Feinberg SE, et al. Biomaterials 2005;26:4817–27. Erisken C, Kalyon DM, Wang H. Biomaterials 2008;29:4065–73. Lei B, Shin K-H, Noh D-Y, Jo I-H, Koh Y-H, Choi W-Y, et al. J Mater Chem 2012;22:14133–40. Holzwarth JM, Ma PX. Biomaterials 2011;32:9622–9. Liu S, Xiao X, Liu R, Zhong Z, Jiao J. Chem. Ind. Eng. Prog. 2011;30:1059–64. Liu X, Ma PX. Biomaterials 2009;30:4094–103. Liu X, Smith LA, Hu J, Ma PX. Biomaterials 2009;30:2252–8. Hu J, Sun X, Ma H, Xie C, Chen YE, Ma PX. Biomaterials 2010;31:7971–7. He L, Zhang Y, Zeng X, Quan D, Liao S, Zeng Y, et al. Polymer 2009;50:4128–38.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Facilely fabricating PCL nanofibrous scaffolds with hierarchical pore structure for tissue engineering Yuzhang Du a, Xiaofeng Chen b, Young Hag Koh c, Bo Lei a,n a
Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an, China School of Materials Science and Engineering, South China University of Technology, Guangzhou, China c College of Health Science, Korea University, Seoul, South Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 12 November 2013 Accepted 8 February 2014 Available online 16 February 2014
Highly hierarchical pore structure (macro–micro–nano) for biomimetic nanofibrous scaffolds could efficiently enhance cell infiltration and tissue formation. However, it is difficult to prepare these structures by using traditional electrostatic spinning techniques. Here, we report a facile method to fabricate polycaprolactone (PCL) nanofibrous scaffolds with multi-scale pore structure by simple phase separation. By employing water–dioxane as solvents and tris spheres as macropore template, PCL nanofibrous scaffolds with pores 300–800 mm, 1–10 mm and 100–1000 nm in diameter can be facilely produced. All scaffolds possess controlled nanofibrous morphology and porosity (90–98%). This scaffold may provide promising applications in tissue engineering and drug delivery. & 2014 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Porous materials Nanofibrous scaffolds Phase separation Hierarchical pores
1. Introduction In tissue engineering, tridimensional (3D) scaffolds play an important role because tissue formation requires the space and environment provided by scaffolds [1]. The pore structure of scaffolds usually affects the cellular migration, proliferation, and differentiation, as well as the tissue formation [2]. In addition, cell and tissue could respond according to different pore scales [3]. Therefore, the scaffolds with pore structure at different length scales from nanometer to micrometer (nm–mm) possess advantages compared to other types of scaffolds. For example, in hierarchical scaffolds, the macropores (150–500 mm) maintain the structural stability of scaffolds, support cell proliferation, extracellular matrix (ECM) deposition and tissue formation [4]. The micrometer-scale (1–50 mm) pores are also critical for nutrient diffusion and vascularization [5]. Additionally, nanoscale pore or nanofibrous structure in the scaffolds has shown important effects on controlling cell behavior such as attachment and genes expressions [6]. Therefore, recent years have seen increased interest in fabricating porous scaffolds with multi-scale pore size (from nm to mm) [7]. However, most scaffolds with hierarchical structure do not possess the nanofibrous morphology. On the other hand, most native tissues have the nanofibrous structure and possess excellent physicochemical properties and biological functions. On the side of biomimics and
materials science, it is necessary to fabricate macroporous nanofibrous scaffolds with hierarchical structure by a facile method. As a biomedical polymer, polycaprolactone (PCL) has been approved by American Food and Drug Administration (FDA). Due to its good mechanical properties and slow biodegradation, PCL has been applied in tissue engineering and drugs delivery [8]. PCL nanofibrous scaffolds were also prepared by an electrospinning method [9]. However, it is difficult to produce the macroporous nanofibrous scaffolds by the electrospinning technique. Thermally–induced phase separation technique (TIPS) is a very versatile method to fabricate polymer nanofibrous scaffolds [10,11]. Previous study showed that PCL nanofibrous scaffolds could also be fabricated by phase separation, but the scaffolds did not possess multi-scale pore structure [12]. By particle-based pore formers, macroporous polymer nanofibrous scaffolds can be facilely fabricated. Unfortunately, to the best of our knowledge, PCL nanofibrous scaffolds with hierarchical pore distributions have not been prepared yet. Therefore, in this study, the macroporous PCL nanofibrous scaffolds with multi-scale pore distributions (nm–m m–mm) are fabricated by a modified phase separation technique. The nanofiber morphology and pore structure could be controlled by polymer concentration and phase separation temperature.
2. Materials and methods
n
Corresponding author. Tel.: þ 86 29 833 95361. E-mail address: [email protected] (B. Lei).
http://dx.doi.org/10.1016/j.matlet.2014.02.031 0167-577X & 2014 Elsevier B.V. All rights reserved.
Fabrication of macroporous PCL nanofibrous scaffolds: Polycaprolactone (PCL, average Mn¼80,000), 1,4 dioxane, and tris-hydroxymethyl-aminomethane (Tris) were purchased from Sigma-Aldrich
Y. Du et al. / Materials Letters 122 (2014) 62–65
(Sigma-Aldrich, St. Louis, MO, USA). All materials were used without further treatment. Phase separation technology (PST) was used to synthesize the 3D nanofibrous structure. Tris microspheres with the range of 200–500 mm were chosen as the pore former to fabricate
63
macroporous scaffolds. Typically, a given mass of PCL was dissolved in 10 mL dioxane/H2O mixed solvents with different volume ratios (0:100, 5:95, 10:90). The samples codes and compositions are shown in Table 1S. After the formation of clear solution, the solution was
Fig. 1. Scheme display for fabricating biomimetic and hierarchical PCL nanofibrous scaffolds. (a) Scaffolds production process, (b) highly hierarchical pore distributions of nanofibrous scaffolds, and (c) photos showing the morphology of nanofibrous scaffolds with hierarchical pores.
Fig. 2. Effects of water/dioxane ratio on PCL nanofibrous scaffolds morphology: (a, d, g) PCL scaffolds with pure dioxane; (b, e, h) 5% water/95% dioxane; and (c, f, i) 10% water/90% dioxane (polymer concentration 10%, phase separation temperature 4 1C).
64
Y. Du et al. / Materials Letters 122 (2014) 62–65
immediately transferred into 70 1C, 20 1C, and 4 1C baths for freezing for 4 h. The frozen samples were freeze dried for 48 h. For making macroporous scaffolds, tris sphere template was added into plastic tubes and compacted by vacuum; then PCL solution was poured into the template, followed by freezing and drying. Characterizations of macroporous PCL nanofibrous scaffolds: The nanofibrous morphology and porous microstructure of samples were analyzed by a field emission scanning electron microscope (FE-SEM, JSM6701F, JEOL, Japan). Before observation, all the samples were coated by a layer of platinum (Pt) using a sputter coater with a pressure of 32 mTorr and a time of 150 s. The mean nanofiber length, width and pore size of hybrids were calculated by counting more than 50 species from the SEM images using image-pro Plus 6.0 software (Media Cybernetics, Inc. USA). The porosity (P) of nanofibrous matrices was calculated by evaluating the apparent density (Da) and skeleton density (Ds) according to the followed equation: P¼ (Ds Da) 100/Ds.
3. Results and discussions The hierarchical PCL nanofibrous scaffolds were synthesized by thermally-induced phase separation and pore formation method, as shown in Fig. 1. As a hydrophilic chemical, tris sphere was added into dioxane/water hybrid solution as the macropore producer. After phase separation at low temperatures ( 20 1C, 4 1C), polymer phase was self-assembled to a nanofibrous porous structure. After freeze drying, the nanofibrous PCL scaffolds with nanoscale pores were formed. The addition of tris spheres produced the macropores after final removal by water soaking. The micrometer-scale pores on walls of scaffolds were probably formed due to the slight dissolution of sphere in water-dioxane
solvent before freeze drying. The hierarchical pore structure and morphology are shown in Fig. 1c. For thermally-induced separation technique, the main factors influencing nanofibrous structure and pore structure are solvents ratios, polymer concentration and temperatures [13,14]. The effects of dioxane/water ratio, PCL concentration and temperature on nanofibrous scaffolds are shown in Figs. 2–4. The amount of water has an important effect on nanofibrous structure and morphology. Pure dioxane solvent could produce only the porous PCL structure but not the nanofibrous morphology, because no non-solvent (water) phase separation occurred (Fig. 2a, d, g). As the water phase increased, nanofibrous structure and micrometer pores began to form (Fig. 2b, e, h and c, f, i). As a result, dioxane: H2O with a ratio of 90:10 could produce a stable nanofibrous structure (Fig. 2e, f, i). It should be noted that high amount of water makes the polymer insoluble. Generally, low polymer concentration usually produced good nanofibrous structure and morphology, as well as high porosity [15]. Fig. 3 shows the effect of PCL concentration on scaffold's morphology. The PCL nanofibrous scaffolds with a content of 2% showed a long nanofiber structure (Fig. 3a, d, g). The typical micrometer-scale pores are found on the wall surface of scaffolds, as shown in Fig. 3a–c. The improved nanofibrous morphology at low PCL concentration is attributed to the high solid–liquid and liquid–liquid phase separation efficiency, which has been proved in previous reports [16]. As an important parameter, freeze temperature has a particular effect on the morphology and structure of nanofibrous scaffolds, as shown in Fig. 4. The PCL scaffolds prepared at 70 1C presented just the porous morphology but not nanofibrous structure (Fig. 4d–f). Freezing at 20 1C and 4 1C could fabricate typical nanofibrous scaffolds with micrometer-scale pore walls (Figs. 2c, f, i and 4a–c).
Fig. 3. Effects of polymer concentrations on PCL nanofibrous scaffolds morphology: (a, d, g) 2%; (b, e, h) 5%; and (c, f, i) 8% (10% water/90% dioxane, phase separation temperature 4 1C).
Y. Du et al. / Materials Letters 122 (2014) 62–65
65
Fig. 4. Effects of phase separation temperature on PCL nanofibrous scaffolds morphology. (a, b, c) 20 1C; and (d, e, f) 70 1C (10% water/90% dioxane, 10% PCL).
4. Conclusions We have demonstrated the successful fabrication of hierarchical PCL nanofibrous scaffolds with multi-scale pore structure (50– 500 nm, 1–10 mm, 300–800 mm) by phase separation technology and a soluble pore former. The nanofiber size and porosity of scaffolds could be controlled by polymer concentration and phase separation temperature. The macropore size also could be tailored by template sphere size. This biomimetic PCL nanofibrous scaffolds with hierarchical pore structure may have promising applications in tissue engineering and drugs/cell delivery. Acknowledgment We acknowledge the valuable comments of potential reviewers. This work was supported by the Scientific Research Starting Foundation from Xi’an Jiaotong University, the National Natural Science Foundation of China (51072055), and the National 973 Project of China (2011CB606204). Appendix A. Supporting materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.matlet.2014.02.031.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
Hutmacher DW. Biomaterials 2000;21:2529–43. Hollister SJ. Nat Mater 2005;4:518–24. Karageorgiou V, Kaplan D. Biomaterials 2005;26:5474–91. Zeltinger J, Sherwood JK, Graham DA, Müeller R, Griffith LG. Tissue Eng 2001;7:557–72. Chiu LL, Montgomery M, Liang Y, Liu H, Radisic M. Proc Natl Acad Sci 2012;109:E3414–23. Smith LA, Liu X, Hu J, Ma PX. Biomaterials 2009;30:2516–22. Zhong S, Zhang Y, Lim CT. Tissue Eng Part B: Rev 2011;18:77–87. Williams JM, Adewunmi A, Schek RM, Flanagan CL, Krebsbach PH, Feinberg SE, et al. Biomaterials 2005;26:4817–27. Erisken C, Kalyon DM, Wang H. Biomaterials 2008;29:4065–73. Lei B, Shin K-H, Noh D-Y, Jo I-H, Koh Y-H, Choi W-Y, et al. J Mater Chem 2012;22:14133–40. Holzwarth JM, Ma PX. Biomaterials 2011;32:9622–9. Liu S, Xiao X, Liu R, Zhong Z, Jiao J. Chem. Ind. Eng. Prog. 2011;30:1059–64. Liu X, Ma PX. Biomaterials 2009;30:4094–103. Liu X, Smith LA, Hu J, Ma PX. Biomaterials 2009;30:2252–8. Hu J, Sun X, Ma H, Xie C, Chen YE, Ma PX. Biomaterials 2010;31:7971–7. He L, Zhang Y, Zeng X, Quan D, Liao S, Zeng Y, et al. Polymer 2009;50:4128–38.