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Fabrication of polycaprolactone nanofibrous scaffolds by facile phase separation approach
Materials Science and Engineering C 44 (2014) 201–208
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Fabrication of polycaprolactone nanofibrous scaffolds by facile phase separation approach Shuqiong Liu a,b, Zhihang He a, Guojie Xu a, Xiufeng Xiao a,⁎ a b
College of Chemistry and Chemical Engineering, Fujian Normal University, Fujian, Fuzhou 350007, China Ecology and Resource Engineering, Wuyi University, Fujian, Nanping, Wuyishan 354300, China
a r t i c l e
i n f o
Article history: Received 25 February 2014 Received in revised form 2 July 2014 Accepted 1 August 2014 Available online 10 August 2014 Keywords: Polycaprolactone Nanofibrous Phase separation Spherulite
a b s t r a c t Three-dimensional polycaprolactone (PCL) scaffolds with spherulite and nanofibrous structures were fabricated for the first time by thermally induced phase separation from a ternary PCL/dioxane/water system. Moreover, the effects of polymer concentration, aging temperature and the ratio of dioxane to water on the morphology of nanofibrous scaffolds were investigated. The result revealed that gelation, aging temperature, and ratio of solvents significantly influenced the formation of the unique spherulite and nanofibrous structures. The apatiteformation ability test showed relatively rapid growth of carbonate hydroxyapatite in the nanofibrous PCL scaffold with macropore compared to the other two scaffolds with smooth structure and nanofibrous structure without macropore, respectively, indicating good apatite-formation ability of the macroporous and nanofibrous PCL scaffolds. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering has attracted remarkable attention because it exhibits an excellent potential for the regeneration of damaged tissue, circumventing the limitations of autologous tissue repair. Tissue engineering strategy requires the use of biodegradable scaffolds providing structural support and acting as reservoir for bioactive molecules. Recently, nanofibrous scaffolds have attracted significant interest in the field of tissue engineering [1,2], mainly due to their imitation of extracellular matrix (ECM) collagen. Collagen fibers, the major ECM component in tissues, have diameters ranging from 50 to 500 nm. According to the literature, the synthetic biodegradable nanofibrous scaffolds act as promising candidates for improving cell adhesion, proliferation, migration, and differentiation in various cell types [3]. Moreover, an increase in protein adsorption in the nanofibrous scaffolds leads to an increase in cell attachment, essential for proliferation, migration, and differentiation [4]. Among the synthetic biodegradable polymers, polycaprolactone (PCL) is biodegradable aliphatic polyester which has been approved by the Food and Drug Administration for certain human clinical applications, such as surgical sutures and some implantable devices [5]. Moreover, it is an ideal scaffold material because of its biocompatibility, nontoxicity for organism, gradual resorption after implantation, and good mechanical properties. PCL-based scaffolds act as promising candidates for tissue engineering
⁎ Corresponding author. Tel.: +86 591 83465190; fax: +86 591 87560183. E-mail address: [email protected] (X. Xiao).
http://dx.doi.org/10.1016/j.msec.2014.08.012 0928-4931/© 2014 Elsevier B.V. All rights reserved.
to carry cells or growth factors and act as templates for tissue regeneration [6–9]. Many literatures demonstrated that the micro-nano or nano structure surface of the scaffold enhanced the adhesion, proliferation, and phenotype of cell better than the scaffold with smooth surface [10–12]. Moreover, nanofibrous scaffold affects phenotype of many types of cells such as nerve cell, hepatocyte, and fibroblast [13,14]. Ruckh et al. demonstrated the fabrication of nanofibrous PCL scaffolds by electrospinning technique [15]. The results indicated that 3D synthetic biodegradable nanofibrous scaffolds acted as excellent framework for enhanced cell adhesion, viability, and increased levels of alkaline phosphatase activity compared to the smooth PCL substrates. The investigations revealed that calcium phosphate mineralization was substantially accelerated on nanofibrous scaffolds compared to the smooth PCL. Till date, three basic techniques have been used to fabricate scaffolds with nanofibrous structure, namely electrospinning, self-assembly, and thermally induced phase separation (TIPS) [16,17]. The electrospinning techniques produce polymer fibers with diameters ranging from nanometers to micrometers scale; however, the process involves a significant challenge in creating three-dimensional (3D) scaffolds with well-defined pore architecture and complex geometries [2,18]. Although, molecular self-assembly is a fairly new technique for designing nanoscale scaffolds, it demonstrates limited ability to control the pore size and structure, essential for cell incorporation, migration, and proliferation. Moreover, an improvement in the mechanical strength of self-assembled scaffolds is required before employing them for tissue engineering applications [2]. TIPS has been effectively used to fabricate nanofibrous scaffolds
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because it involves simple equipment, easy operation, and controllable 3D pore arrangement similar to natural ECM structure. In this study, 3D PCL scaffold with spherulite and nanofibrous structure was fabricated by TIPS from a ternary PCL/dioxane/water system. The effects of gelation process, gelation temperature (Tgel), and ratio of dioxane to water on the morphology of designed nanofibrous scaffolds were investigated, and the structure–activity relationship of nanofibrous PCL scaffold was examined. 2. Materials and methods 2.1. Materials PCL with an inherent viscosity of 1.68 dL g−1 was purchased from Jiangsu Youli Technologies Ltd. All other reagents and solvents with analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. 2.2. Fabrication of nanofibrous PCL and macroporous and nanofibrous PCL scaffolds The nanofibrous PCL scaffolds were fabricated by liquid–liquid phase separation from a PCL/dioxane/water ternary system. A given mass of PCL (5%, 10%, and 15%) was dissolved in dioxane/water mixed solvents with different mass ratios (90/10, 88/12, and 85/15), respectively, at 40 °C. By cooling the transparent polymer solution in a rate of 2 °C/30 min, the cloud point was determined as the temperature when the solution became turbid. The gelation temperature was determined as the solution could no longer flow when the sample was held at a temperature. 5 ml of PCL transparent solution was poured into a beaker mold (3.5 cm in diameter and 5 cm in height). Subsequently, it was quenched to a designed temperature (−8, −4, 0, 4, 8, and 12 °C) and aged for 2 h, followed by quenching to −40 °C for another 2 h to completely freeze the samples. During this period, the solution would come in the form of solid state. The scaffold (3.5 cm in diameter and 1 cm in thickness) was obtained after lyophilization under 0.940 mbar at − 54 °C for three days (FD-1A-50 freeze dryer, Beijing Boyikang Experimental Instrument Co., Ltd.). To study the effect of aging on the morphology of the sample, in the gel status, 10% (w/w) PCL/dioxane/water solution was directly quenched to − 40 °C and maintained for 2 h followed by lyophilization under 0.940 mbar at −54 °C for three days. Moreover, macroporous nanofibrous PCL scaffolds were also fabricated for comparison, by a technique combining the unique phaseseparation method with sugar leaching process, from PCL/dioxane/ water ternary system. Mixture solution (50%) was obtained by dissolving a certain amount of sugar (diameter in 150–500 μm) in clear solution of PCL (10%, w/w) at room temperature with stirring. The resulting mixture solution was quenched to −4 °C and maintained for 2 h, followed by quenching to − 40 °C for another 2 h to completely freeze the samples. Subsequently, the samples were immersed in cold ultrapure water (4 °C) for three days for leaching out the sugar by changing the water three times per day. The sample so obtained was freeze-dried under 0.940 mbar at −54 °C for three days. 2.3. Characterization of nanofibrous PCL scaffolds The morphology of PCL scaffolds prepared under different conditions was observed by scanning electron microscopy (SEM) (FE-SEM, JSM-7500 F, JEOL Ltd.) at 5 KV. The samples were fractured after liquid nitrogen treatment and coated with gold for 150 s using a sputter coater (AUTO FINE COATER, JFC-1600, JEOL Ltd.). The precipitated apatite layer of nanofibrous PCL scaffolds after immersion in the SBF were analyzed using a Philips X'pert MPD X diffractometer using Cu Ka generated at 40 KV and 40 mA. The samples were scanned from 10° to 90° with a step size of 0.02° and a count rate of 3.0°/min.
FTIR spectra were measured to confirm the formation of nanofibrous PCL scaffolds after immersion in the SBF. A nicolet Avatar 360 spectrometer, with KBr pastille, was used for FTIR characterization. The analysis range was from wave number 4000 to 400 cm−1. 2.4. Porosity test The porosity of the scaffolds was determined by a simple method [19]. To determine the porosity, different samples were dried at 40 °C for a day. After weighing (Ws), each sample was placed into a pycnometer filled with ethanol, with the weight of pycnometer and ethanol taken together as W1. Subsequently, the pycnometer was placed into a vacuum container to extract the air out of the sample, thus pushing ethanol into the space originally occupied by air bubble. During the vacuum process, the fluid level in the pycnometer fell down. Therefore, the pycnometer was taken out and filled up, and the entire weight (W2) was taken. Subsequently, the sample was taken out and the surface ethanol was dripped back into the pycnometer to obtain the weight of the remaining ethanol and the pycnometer (W3). The porosity can be calculated from the following equations [20], where ρ is the density of ethanol: Scaffold volume : V s ¼ ðW 1 −W 2 þ W s Þ=ρ
Pore volume : V p ¼ ðW 2 −W 3 −W s Þ=ρ
Porosity : ε ¼ V p = V p þ V s ¼ ðW 2 −W 3 −W s Þ=ðW 1 −W 3 Þ
2.5. Apatite-formation ability Bioactivity is a critical factor in facilitating the chemical fixation of biomaterials to bone tissue, and ultimately the in vivo success of the bone grafting material [21,22]. The nanofibrous PCL scaffold and macroporous and nanofibrous scaffold were immersed in a 1.5 simulated body fluid (1.5SBF) with ionic concentration nearly equal to human blood plasma. 1.5SBF was prepared as described in the literature [23], the pH of the 1.5SBF is 7.4 and ionic concentrations of 1.5SBF and human plasma was listed at Table 1. The scaffolds were immersed in SBF for 14 days. Finally, the samples were rinsed thoroughly with distilled water and dried at 40 °C. 3. Results 3.1. The cloud point and gelation point of the PCL Fig. 1 shows the dependence of the cloud points and the gelation points on the polymer concentrations and the solvent composition with different mass ratios. The cloud points and gelation points increased as the increase of the polymer concentration and the water content in mixed solvents. The dependence on water content was greater than those of the polymer concentration. Table 1 Ion concentrations of SBF and human plasma (mmoL/L).
Human plasma 1.5SBF
Na+
K+
Mg2+
Ca2+
Cl−
HCO2− 3
HPO2− 4
SO2− 4
142.0 213.00
5.0 7.50
1.5 3.80
2.5 2.30
103.0 6.30
27.0 223.00
1.0 1.50
0.5 0.75
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3.2. Effects of aging in the gel status on the morphologies of the PCL scaffold Gelation of the PCL/dioxane/water ternary system played a crucial role in creating the unique nanofibrous structure. Fig. 2 illustrates the morphologies of the PCL scaffolds prepared with and without aging in the gel status at −8 °C. When the ternary PCL/dioxane/water system was directly quenched to − 40 °C, an open microporous structure is formed with a solid pore wall and an average pore size of 20–50 μm (Fig. 2b). However, the scaffold prepared with an aging time of 2 h in the gel status at −8 °C displayed the formation of matrix with specifically connected microspheres, before quenching to − 40 °C. Fig. 2a exhibits that the diameters of microspheres are in the range of 60–70 μm; however, some spherical pore sizes with diameters of 5–25 μm are observed in-between the microspheres. Moreover, the wall of the microspheres is nanofibrous instead of being solid. 3.3. Effects of gelation temperature on the morphologies of the PCL scaffolds
Fig. 1. Dependence of cloud point and gelation point on PCL concentration in dioxane/ water (mass ratio 90/10) (a); Dependence of cloud point and gelation point on water content in mixed solvent, PCL concentration was 10% (b).
Fig. 3 displays the morphologic evolution of PCL scaffolds as a function of Tgel. These scaffolds were fabricated from a PCL solution (10 wt.%) in 90/10 (w/w) dioxane/water at different Tgel (−4, 0, 4, 8, and 12 °C) with aging for 2 h in the gel status. Fig. 3 displays the SEM images under low and high magnification, demonstrating evidently that different morphologies were obtained at different Tgel. Microspheres formed the matrices, and the walls of the micropores in the microspheres were composed of nanofibrous structures at low Tgel (Tgel ≤ 4 °C) (Fig. 3a–f). However, for higher Tgel (Tgel ≥ 8 °C), an open microporous structure with solid pore wall was obtained instead of the microspheres matrices and nanofibrous structure (Fig. 3i–j). Therefore, Tgel not only affects the nanofibrous matrices, but also the form of the specific microspheres. Microspheres structure fades away with increasing Tgel. At a Tgel of 4 °C, the boundary between the microspheres becomes blurred and faintly visible spherical structure is observed. Noticeably, the spherical structure made of tufted sheets and solid mesh disappeared especially at the Tgel of 8 °C. While the Tgel lower than 0 °C, the average fiber diameter of the fibrous matrices did not obviously change with the change in Tgel. But the Tgel higher than 0 °C, the average fiber diameter of the fibrous matrices obviously increase twice of the original (Fig. 3d,f), and gathered into a bundle while the Tgel at 8 °C (Fig. 3h).
Fig. 2. SEM of PCL scaffolds prepared from different conditions (a): 10% PCL gelation at −8 °C, 2 h; (b): 10% PCL freezing at −40 °C, 2 h directly, The mass ratio of dioxane/water is 90:10.
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Fig. 3. SEM of PCL scaffolds prepared from 10% PCL in dioxane/water (mass ratio 90/10) at different aging temperatures for 2 h. (a, b): − 4 °C; (c, d): 0 °C; (e, f): 4 °C; (g, h): 8 °C; (i, j): 12 °C.
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Fig. 4. SEM of PCL scaffolds prepared from 10% PCL in different mass ratio of dioxane/water (a, b): 88/12; (c, d): 85/15. Tgel is −8 °C for 2 h.
3.4. Effects of the volume ratio of dioxane/water on the morphologies of the nanofibrous PCL scaffolds
content was increased to 15%, microspheres disappeared generating a porous network structure.
The volume ratio of dioxane/water played a significant role in creating the unique nanofibrous structure. Fig. 4 shows entanglement and merging of the nanofibrous structure leading to the disappearance of the microsphere matrices and nanofibrous assembly with increasing amount of water in the solvent mixture. In particular, when the water
3.5. Effects of PCL concentration on the morphologies of the nanofibrous PCL scaffold As can be seen from Fig. 5, the polymer concentration has no significant effect on the formation of nanofibrous structure, only a little
Fig. 5. SEM of PCL scaffolds prepared from different PCL concentration in dioxane/water (mass ratio 90/10). (a, b): 5%; (c, d): 15%. Tgel is −8 °C for 2 h.
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Table 2 The porosity of the PCL scaffolds with different condition. Concentration (%)
Aging temperature (°C)
Porosity (%)
10
−8 −4 0 4 8 12 −4
93.77 90.55 90.98 90.33 90.67 90.57 94.76
10 (with porogen)
± ± ± ± ± ± ±
0. 80 0.21 0.49 0.58 0.28 0.43 0.57
influence on the fiber diameter. Microspheres were interconnected when the polymer concentration was 5% or 15%. 3.6. Effects of the porogen on the morphologies of the nanofibrous PCL scaffold Nanofibrous matrices with particular macropores were generated from PCL with sugar as porogen by a phase separation technique. These matrices have low density and high porosity (Table 2). SEM micrographs demonstrate the interconnected open pore structure and nanofibrous pore walls (Fig. 6). Basically, three size scales are involved in these matrices, i.e., the macropore size, interfiber distance, and fiber diameter. The size of the macropores, determined by the used porogen particle size, was a few hundred micrometers. Sugar particles generated macropore structures for the synthetic polymer matrices. The interfiber distance was determined by the phase separation process, similar to that of nanofibrous PCL matrices prepared without sugar porogen. The fiber diameter was approximately in the range of 50–500 nm, identical to that prepared without sugar porogen; however, the fiber spacing and diameter became more nonuniform. 3.7. Porosity As listed in Table 2, the scaffolds exhibit a high porosity of about 90%. The porosity of scaffolds did not show significant change with different Tgel. The nanofibrous PCL scaffolds prepared using sugar as porogen had a higher porosity due to the leaching effect of porogen. 3.8. Apatite-formation ability Fig. 7a–e shows the surface morphology of the PCL scaffolds prepared under different conditions after immersing the scaffolds in SBF for 14 days. Apparently, soaking in SBF for 14 days leads to the growth of sporadic mineral on the convex parts of the network structured scaffolds; however, sediments are not observed on the smooth parts (Fig. 7a). Identical results were obtained for Tgel = 8 °C, the sediments grow on the areatus of the scaffolds and not on the smooth parts (Fig. 7b). Fig. 7c and d shows the obvious formation of coating on the surface of the nanofibrous PCL scaffolds without and with macropores.
Numerous leaves are observed on the surface of spherical particles at high magnification (Fig. 7e), and the morphology is almost similar to that of the deposited apatite on a substrate through biomimetic deposition in SBF. To identify the coating on the scaffolds, X-ray diffraction and Fourier transform infrared spectroscopy were performed and the results are shown in Fig. 8. Fig. 8a shows the appearance of the weak diffraction peaks at 26 and 32°, indicating the formation of apatite with low crystallinity. Fig. 8b shows the peaks at 3416 and 1636 cm−1 attributed to the characteristic absorption of H2O, the absorption bands at 1043, 602, 556, and 462 cm−1 are assigned to PO34 −, the peaks at 876 and 1400–1460 cm−1 are the characteristics of CO2− 3 , an important indication of carbonate group entering into the apatite [24]. Therefore, the coatings obtained after immersing the scaffolds in SBF consisted of carbonated hydroxyapatite with low crystallinity, indicating the excellent apatite-formation ability of the nanofibrous PCL scaffolds.
4. Discussions In this study, the PCL scaffold showed a spherical structure, which might be caused by the growth of PCL spherulites during the phase separation of homogeneous PCL solution. The crystal stacks grow radially outward from the center during the crystallization, resulting in spherulite shapes. In the polymer melt, multiple crystal stacks often grow outward simultaneously from the crystal nucleus. The more the nuclei, the more the spherulites growing simultaneously, resulting in smaller size spherulites. When two or more spherulites interact, the molecular chains grow on the edges of spherulites, which is why the junction of spherulites tends to be flat [25]. The PCL spherulites are thought to undergo an outward radial growth after the crystals are generated via phase separation at crystal nuclei. However, only the radial growth of crystal plates is not sufficient for the formation of spherulites; two other mandatory conditions are required: (i) high viscosity and (ii) a certain amount of impurities. In this case, the solvent and non-solvent act as impurities. When large impurities are present, they cannot be removed to either side of phases because of the high viscosity of the system, and therefore, these impurities tend to remain in the middle of the phases resulting in the forked growth of the crystals. Like a growing tree, crystals grow in all directions through numerous branching while maintaining a spherical shape. As shown in Figs. 2 and 3, the PCL scaffold show some round pore structures on the PCL microspheres, formed by the aggregated solvent molecules due to the repulsion of impurities during freeze drying. The nanofibers in the spherical matrix are formed by the growth of crystal plates. Gelation of the polymer solution system played a crucial role in creating the unique nanofibrous structure. First, when the polymer solution was cooled to − 40 °C for phase separation, the scaffold with a highly porous structure rather than a nanofibrous structure was obtained (Fig. 2b). This is because at such a low temperature (− 40 °C), the homogeneous polymer solution directly entered into spinodal
Fig. 6. SEM of PCL scaffolds prepared from 10% PCL in dioxane/water (mass ratio 90/10) with sugar porogen. The mass ratio of PCL solution/sugar is 1:1. Tgel is −8 °C for 2 h.
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Fig. 7. SEM of different topography of scaffold after immersed in SBF 14 days. (a, b and c: Tgel = 12 °C, 8 °C and −4 °C for 2 h without porogen; d, e: Tgel = −8 °C for 2 h with porogen).
phase separation to form a two-phase consecutive structure, leading to the highly porous structure after freeze drying. In contrast, when the polymer solution was cooled to a higher temperature (≥−8 °C) for phase separation, the PCL crystal nuclei were formed first, and the molecular chain began to grow in the form of a crystal plate with the
Fig. 8. XRD and FTIR of nanofibrous PCL scaffold after immersed in SBF for 14 days.
spherulite growth pattern, thus showing a primary phase separation mechanism for polymer crystallization. As shown in Fig. 3, the size of spherulites gradually reduced with increasing temperature until they finally disappear. This result may be explained by the fact that the growth of spherulites is mainly manifested as the growth of crystal plates in the longitudinal and lateral directions; in general, the greater the degree of supercooling, the larger the longitudinal speed constant G, and the narrower the fiber width. The degree of supercooling increases with decreasing aging temperature (Tgel), resulting in the formation of larger spherulites [25]. Another possible reason is that a higher temperature is more favorable for the growth of crystal nucleus; thus, under the same conditions, the higher the number of crystal nuclei, the higher the number of spherulites grown and the smaller the size of spherulites. The micrographs also show that the boundary of growing spherulites becomes obscured at a higher temperature, probably because of the fact that, although the growing spherulites are small, the crystal plates constantly grow in the aging process. Therefore, the overlapping and aggregation of crystal plates occur, i.e., the crystal plates of spherulites will overlap and intertwine with each other, resulting in aggregation. Finally, the spherulites (nanofiber structure) gradually disappear, resulting in a highly porous structure. Nanofibrous entanglement and mergence occurred accompanied with microsphere matrices and nanofibrous structure fade away with increasing amount of water content in the system. This is because high non-solvent volume fraction deteriorated the dissolvability of polymer, a weaker polymer–diluent interaction and lowered viscosity
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in the cosolvent system might induce the formation of polymer poor phase with greater droplet domains [26,27]. In addition, the gradual addition of water would increase the gelation point [28], which is the same as decreasing the degree of supercooling of the system. Therefore, the size of spherulite decreases with increasing solvent to non-solvent ratio while the fibers are gradually broadened, finally leading to the disappearance of both spherulites and nanofibrous matrix. However, the concentration of polymer has an insignificant effect on the growth of spherulite within the experimental range, except that more crystal nuclei were generated in the initial stage of phase separation with increasing concentration, resulting in smaller spherulites and overlapped growth of crystal plates. By varying sugar sphere size and content, one can easily control the macropore size and macropore structure of the nanofibrous PCL scaffold. Not only macroporous and nanofibrous scaffold have a high porosity but also nanofibrous PCL scaffold without macropores have a high porosity. Numerous pores between nanofibrous network structures is the main reason for its high porosity. Meanwhile, the scaffold without nanofibers also has a high porosity because of its highly connected network structure. The porosity of different structure scaffold was similar, but the nanofibrous scaffold proved beneficial for cell attachment, migration and proliferation due to its specific nano-effects. To be effectively used for bone tissue regeneration, it is beneficial that a scaffold promotes bone-like apatite formation when in contact with physiological fluid. To evaluate the bioactivity of the novel macroporous and nanofibrous PCL scaffolds developed in this study, we have investigated their ability to form bone-like apatite in a SBF. Apatite particles were deposited uniformly throughout the macroporous and nanofibrous PCL scaffolds. Noting very uniform growth of apatite layers throughout the macroporous and nanofibrous PCL scaffolds but not on the scaffolds without the nanofeatures [29,30], it is likely that the physical and surface characteristics of the scaffold (macropores, interpore openings, and nanotopography) affect the formation kinetics and morphology of apatite layers [30]. 5. Conclusions In this study, nanofibrous PCL scaffolds with 3D macro/microporous structure were fabricated by TIPS technique combined with sugar leaching process, from a ternary PCL/dioxane/water system. macroporous and nanofibrous PCL scaffold were obtained by controlling the Tgel, ratio of dioxane to water, and content of porogen. The spherulite-like morphology of the scaffold was disrupted with increasing Tgel or water content in the mixed solvent system. Moreover, coalescence of nanofibers occurred, followed by the disappearance of spherulite-like structure at Tgel greater than 8 °C and freezing at −40 °C directly without gelation, or when the proportion of water exceeded 12%. Apatite-formation ability test revealed that the macroporous and nanofibrous PCL scaffolds exhibited good ability to trigger the formation of apatite. Acknowledgments This work was supported by National Nature Science Foundation of China under grant 30970887, the Science Research Foundation of Ministry of Health under grant WKJ 2008-2-037, Fujian Province Nature Science Foundation under grants 2012J01194 and 2011J06019, and Foundation for Young Teachers of Wuyi University under grant xq201102.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Fabrication of polycaprolactone nanofibrous scaffolds by facile phase separation approach Shuqiong Liu a,b, Zhihang He a, Guojie Xu a, Xiufeng Xiao a,⁎ a b
College of Chemistry and Chemical Engineering, Fujian Normal University, Fujian, Fuzhou 350007, China Ecology and Resource Engineering, Wuyi University, Fujian, Nanping, Wuyishan 354300, China
a r t i c l e
i n f o
Article history: Received 25 February 2014 Received in revised form 2 July 2014 Accepted 1 August 2014 Available online 10 August 2014 Keywords: Polycaprolactone Nanofibrous Phase separation Spherulite
a b s t r a c t Three-dimensional polycaprolactone (PCL) scaffolds with spherulite and nanofibrous structures were fabricated for the first time by thermally induced phase separation from a ternary PCL/dioxane/water system. Moreover, the effects of polymer concentration, aging temperature and the ratio of dioxane to water on the morphology of nanofibrous scaffolds were investigated. The result revealed that gelation, aging temperature, and ratio of solvents significantly influenced the formation of the unique spherulite and nanofibrous structures. The apatiteformation ability test showed relatively rapid growth of carbonate hydroxyapatite in the nanofibrous PCL scaffold with macropore compared to the other two scaffolds with smooth structure and nanofibrous structure without macropore, respectively, indicating good apatite-formation ability of the macroporous and nanofibrous PCL scaffolds. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering has attracted remarkable attention because it exhibits an excellent potential for the regeneration of damaged tissue, circumventing the limitations of autologous tissue repair. Tissue engineering strategy requires the use of biodegradable scaffolds providing structural support and acting as reservoir for bioactive molecules. Recently, nanofibrous scaffolds have attracted significant interest in the field of tissue engineering [1,2], mainly due to their imitation of extracellular matrix (ECM) collagen. Collagen fibers, the major ECM component in tissues, have diameters ranging from 50 to 500 nm. According to the literature, the synthetic biodegradable nanofibrous scaffolds act as promising candidates for improving cell adhesion, proliferation, migration, and differentiation in various cell types [3]. Moreover, an increase in protein adsorption in the nanofibrous scaffolds leads to an increase in cell attachment, essential for proliferation, migration, and differentiation [4]. Among the synthetic biodegradable polymers, polycaprolactone (PCL) is biodegradable aliphatic polyester which has been approved by the Food and Drug Administration for certain human clinical applications, such as surgical sutures and some implantable devices [5]. Moreover, it is an ideal scaffold material because of its biocompatibility, nontoxicity for organism, gradual resorption after implantation, and good mechanical properties. PCL-based scaffolds act as promising candidates for tissue engineering
⁎ Corresponding author. Tel.: +86 591 83465190; fax: +86 591 87560183. E-mail address: [email protected] (X. Xiao).
http://dx.doi.org/10.1016/j.msec.2014.08.012 0928-4931/© 2014 Elsevier B.V. All rights reserved.
to carry cells or growth factors and act as templates for tissue regeneration [6–9]. Many literatures demonstrated that the micro-nano or nano structure surface of the scaffold enhanced the adhesion, proliferation, and phenotype of cell better than the scaffold with smooth surface [10–12]. Moreover, nanofibrous scaffold affects phenotype of many types of cells such as nerve cell, hepatocyte, and fibroblast [13,14]. Ruckh et al. demonstrated the fabrication of nanofibrous PCL scaffolds by electrospinning technique [15]. The results indicated that 3D synthetic biodegradable nanofibrous scaffolds acted as excellent framework for enhanced cell adhesion, viability, and increased levels of alkaline phosphatase activity compared to the smooth PCL substrates. The investigations revealed that calcium phosphate mineralization was substantially accelerated on nanofibrous scaffolds compared to the smooth PCL. Till date, three basic techniques have been used to fabricate scaffolds with nanofibrous structure, namely electrospinning, self-assembly, and thermally induced phase separation (TIPS) [16,17]. The electrospinning techniques produce polymer fibers with diameters ranging from nanometers to micrometers scale; however, the process involves a significant challenge in creating three-dimensional (3D) scaffolds with well-defined pore architecture and complex geometries [2,18]. Although, molecular self-assembly is a fairly new technique for designing nanoscale scaffolds, it demonstrates limited ability to control the pore size and structure, essential for cell incorporation, migration, and proliferation. Moreover, an improvement in the mechanical strength of self-assembled scaffolds is required before employing them for tissue engineering applications [2]. TIPS has been effectively used to fabricate nanofibrous scaffolds
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because it involves simple equipment, easy operation, and controllable 3D pore arrangement similar to natural ECM structure. In this study, 3D PCL scaffold with spherulite and nanofibrous structure was fabricated by TIPS from a ternary PCL/dioxane/water system. The effects of gelation process, gelation temperature (Tgel), and ratio of dioxane to water on the morphology of designed nanofibrous scaffolds were investigated, and the structure–activity relationship of nanofibrous PCL scaffold was examined. 2. Materials and methods 2.1. Materials PCL with an inherent viscosity of 1.68 dL g−1 was purchased from Jiangsu Youli Technologies Ltd. All other reagents and solvents with analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. 2.2. Fabrication of nanofibrous PCL and macroporous and nanofibrous PCL scaffolds The nanofibrous PCL scaffolds were fabricated by liquid–liquid phase separation from a PCL/dioxane/water ternary system. A given mass of PCL (5%, 10%, and 15%) was dissolved in dioxane/water mixed solvents with different mass ratios (90/10, 88/12, and 85/15), respectively, at 40 °C. By cooling the transparent polymer solution in a rate of 2 °C/30 min, the cloud point was determined as the temperature when the solution became turbid. The gelation temperature was determined as the solution could no longer flow when the sample was held at a temperature. 5 ml of PCL transparent solution was poured into a beaker mold (3.5 cm in diameter and 5 cm in height). Subsequently, it was quenched to a designed temperature (−8, −4, 0, 4, 8, and 12 °C) and aged for 2 h, followed by quenching to −40 °C for another 2 h to completely freeze the samples. During this period, the solution would come in the form of solid state. The scaffold (3.5 cm in diameter and 1 cm in thickness) was obtained after lyophilization under 0.940 mbar at − 54 °C for three days (FD-1A-50 freeze dryer, Beijing Boyikang Experimental Instrument Co., Ltd.). To study the effect of aging on the morphology of the sample, in the gel status, 10% (w/w) PCL/dioxane/water solution was directly quenched to − 40 °C and maintained for 2 h followed by lyophilization under 0.940 mbar at −54 °C for three days. Moreover, macroporous nanofibrous PCL scaffolds were also fabricated for comparison, by a technique combining the unique phaseseparation method with sugar leaching process, from PCL/dioxane/ water ternary system. Mixture solution (50%) was obtained by dissolving a certain amount of sugar (diameter in 150–500 μm) in clear solution of PCL (10%, w/w) at room temperature with stirring. The resulting mixture solution was quenched to −4 °C and maintained for 2 h, followed by quenching to − 40 °C for another 2 h to completely freeze the samples. Subsequently, the samples were immersed in cold ultrapure water (4 °C) for three days for leaching out the sugar by changing the water three times per day. The sample so obtained was freeze-dried under 0.940 mbar at −54 °C for three days. 2.3. Characterization of nanofibrous PCL scaffolds The morphology of PCL scaffolds prepared under different conditions was observed by scanning electron microscopy (SEM) (FE-SEM, JSM-7500 F, JEOL Ltd.) at 5 KV. The samples were fractured after liquid nitrogen treatment and coated with gold for 150 s using a sputter coater (AUTO FINE COATER, JFC-1600, JEOL Ltd.). The precipitated apatite layer of nanofibrous PCL scaffolds after immersion in the SBF were analyzed using a Philips X'pert MPD X diffractometer using Cu Ka generated at 40 KV and 40 mA. The samples were scanned from 10° to 90° with a step size of 0.02° and a count rate of 3.0°/min.
FTIR spectra were measured to confirm the formation of nanofibrous PCL scaffolds after immersion in the SBF. A nicolet Avatar 360 spectrometer, with KBr pastille, was used for FTIR characterization. The analysis range was from wave number 4000 to 400 cm−1. 2.4. Porosity test The porosity of the scaffolds was determined by a simple method [19]. To determine the porosity, different samples were dried at 40 °C for a day. After weighing (Ws), each sample was placed into a pycnometer filled with ethanol, with the weight of pycnometer and ethanol taken together as W1. Subsequently, the pycnometer was placed into a vacuum container to extract the air out of the sample, thus pushing ethanol into the space originally occupied by air bubble. During the vacuum process, the fluid level in the pycnometer fell down. Therefore, the pycnometer was taken out and filled up, and the entire weight (W2) was taken. Subsequently, the sample was taken out and the surface ethanol was dripped back into the pycnometer to obtain the weight of the remaining ethanol and the pycnometer (W3). The porosity can be calculated from the following equations [20], where ρ is the density of ethanol: Scaffold volume : V s ¼ ðW 1 −W 2 þ W s Þ=ρ
Pore volume : V p ¼ ðW 2 −W 3 −W s Þ=ρ
Porosity : ε ¼ V p = V p þ V s ¼ ðW 2 −W 3 −W s Þ=ðW 1 −W 3 Þ
2.5. Apatite-formation ability Bioactivity is a critical factor in facilitating the chemical fixation of biomaterials to bone tissue, and ultimately the in vivo success of the bone grafting material [21,22]. The nanofibrous PCL scaffold and macroporous and nanofibrous scaffold were immersed in a 1.5 simulated body fluid (1.5SBF) with ionic concentration nearly equal to human blood plasma. 1.5SBF was prepared as described in the literature [23], the pH of the 1.5SBF is 7.4 and ionic concentrations of 1.5SBF and human plasma was listed at Table 1. The scaffolds were immersed in SBF for 14 days. Finally, the samples were rinsed thoroughly with distilled water and dried at 40 °C. 3. Results 3.1. The cloud point and gelation point of the PCL Fig. 1 shows the dependence of the cloud points and the gelation points on the polymer concentrations and the solvent composition with different mass ratios. The cloud points and gelation points increased as the increase of the polymer concentration and the water content in mixed solvents. The dependence on water content was greater than those of the polymer concentration. Table 1 Ion concentrations of SBF and human plasma (mmoL/L).
Human plasma 1.5SBF
Na+
K+
Mg2+
Ca2+
Cl−
HCO2− 3
HPO2− 4
SO2− 4
142.0 213.00
5.0 7.50
1.5 3.80
2.5 2.30
103.0 6.30
27.0 223.00
1.0 1.50
0.5 0.75
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3.2. Effects of aging in the gel status on the morphologies of the PCL scaffold Gelation of the PCL/dioxane/water ternary system played a crucial role in creating the unique nanofibrous structure. Fig. 2 illustrates the morphologies of the PCL scaffolds prepared with and without aging in the gel status at −8 °C. When the ternary PCL/dioxane/water system was directly quenched to − 40 °C, an open microporous structure is formed with a solid pore wall and an average pore size of 20–50 μm (Fig. 2b). However, the scaffold prepared with an aging time of 2 h in the gel status at −8 °C displayed the formation of matrix with specifically connected microspheres, before quenching to − 40 °C. Fig. 2a exhibits that the diameters of microspheres are in the range of 60–70 μm; however, some spherical pore sizes with diameters of 5–25 μm are observed in-between the microspheres. Moreover, the wall of the microspheres is nanofibrous instead of being solid. 3.3. Effects of gelation temperature on the morphologies of the PCL scaffolds
Fig. 1. Dependence of cloud point and gelation point on PCL concentration in dioxane/ water (mass ratio 90/10) (a); Dependence of cloud point and gelation point on water content in mixed solvent, PCL concentration was 10% (b).
Fig. 3 displays the morphologic evolution of PCL scaffolds as a function of Tgel. These scaffolds were fabricated from a PCL solution (10 wt.%) in 90/10 (w/w) dioxane/water at different Tgel (−4, 0, 4, 8, and 12 °C) with aging for 2 h in the gel status. Fig. 3 displays the SEM images under low and high magnification, demonstrating evidently that different morphologies were obtained at different Tgel. Microspheres formed the matrices, and the walls of the micropores in the microspheres were composed of nanofibrous structures at low Tgel (Tgel ≤ 4 °C) (Fig. 3a–f). However, for higher Tgel (Tgel ≥ 8 °C), an open microporous structure with solid pore wall was obtained instead of the microspheres matrices and nanofibrous structure (Fig. 3i–j). Therefore, Tgel not only affects the nanofibrous matrices, but also the form of the specific microspheres. Microspheres structure fades away with increasing Tgel. At a Tgel of 4 °C, the boundary between the microspheres becomes blurred and faintly visible spherical structure is observed. Noticeably, the spherical structure made of tufted sheets and solid mesh disappeared especially at the Tgel of 8 °C. While the Tgel lower than 0 °C, the average fiber diameter of the fibrous matrices did not obviously change with the change in Tgel. But the Tgel higher than 0 °C, the average fiber diameter of the fibrous matrices obviously increase twice of the original (Fig. 3d,f), and gathered into a bundle while the Tgel at 8 °C (Fig. 3h).
Fig. 2. SEM of PCL scaffolds prepared from different conditions (a): 10% PCL gelation at −8 °C, 2 h; (b): 10% PCL freezing at −40 °C, 2 h directly, The mass ratio of dioxane/water is 90:10.
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Fig. 3. SEM of PCL scaffolds prepared from 10% PCL in dioxane/water (mass ratio 90/10) at different aging temperatures for 2 h. (a, b): − 4 °C; (c, d): 0 °C; (e, f): 4 °C; (g, h): 8 °C; (i, j): 12 °C.
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Fig. 4. SEM of PCL scaffolds prepared from 10% PCL in different mass ratio of dioxane/water (a, b): 88/12; (c, d): 85/15. Tgel is −8 °C for 2 h.
3.4. Effects of the volume ratio of dioxane/water on the morphologies of the nanofibrous PCL scaffolds
content was increased to 15%, microspheres disappeared generating a porous network structure.
The volume ratio of dioxane/water played a significant role in creating the unique nanofibrous structure. Fig. 4 shows entanglement and merging of the nanofibrous structure leading to the disappearance of the microsphere matrices and nanofibrous assembly with increasing amount of water in the solvent mixture. In particular, when the water
3.5. Effects of PCL concentration on the morphologies of the nanofibrous PCL scaffold As can be seen from Fig. 5, the polymer concentration has no significant effect on the formation of nanofibrous structure, only a little
Fig. 5. SEM of PCL scaffolds prepared from different PCL concentration in dioxane/water (mass ratio 90/10). (a, b): 5%; (c, d): 15%. Tgel is −8 °C for 2 h.
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Table 2 The porosity of the PCL scaffolds with different condition. Concentration (%)
Aging temperature (°C)
Porosity (%)
10
−8 −4 0 4 8 12 −4
93.77 90.55 90.98 90.33 90.67 90.57 94.76
10 (with porogen)
± ± ± ± ± ± ±
0. 80 0.21 0.49 0.58 0.28 0.43 0.57
influence on the fiber diameter. Microspheres were interconnected when the polymer concentration was 5% or 15%. 3.6. Effects of the porogen on the morphologies of the nanofibrous PCL scaffold Nanofibrous matrices with particular macropores were generated from PCL with sugar as porogen by a phase separation technique. These matrices have low density and high porosity (Table 2). SEM micrographs demonstrate the interconnected open pore structure and nanofibrous pore walls (Fig. 6). Basically, three size scales are involved in these matrices, i.e., the macropore size, interfiber distance, and fiber diameter. The size of the macropores, determined by the used porogen particle size, was a few hundred micrometers. Sugar particles generated macropore structures for the synthetic polymer matrices. The interfiber distance was determined by the phase separation process, similar to that of nanofibrous PCL matrices prepared without sugar porogen. The fiber diameter was approximately in the range of 50–500 nm, identical to that prepared without sugar porogen; however, the fiber spacing and diameter became more nonuniform. 3.7. Porosity As listed in Table 2, the scaffolds exhibit a high porosity of about 90%. The porosity of scaffolds did not show significant change with different Tgel. The nanofibrous PCL scaffolds prepared using sugar as porogen had a higher porosity due to the leaching effect of porogen. 3.8. Apatite-formation ability Fig. 7a–e shows the surface morphology of the PCL scaffolds prepared under different conditions after immersing the scaffolds in SBF for 14 days. Apparently, soaking in SBF for 14 days leads to the growth of sporadic mineral on the convex parts of the network structured scaffolds; however, sediments are not observed on the smooth parts (Fig. 7a). Identical results were obtained for Tgel = 8 °C, the sediments grow on the areatus of the scaffolds and not on the smooth parts (Fig. 7b). Fig. 7c and d shows the obvious formation of coating on the surface of the nanofibrous PCL scaffolds without and with macropores.
Numerous leaves are observed on the surface of spherical particles at high magnification (Fig. 7e), and the morphology is almost similar to that of the deposited apatite on a substrate through biomimetic deposition in SBF. To identify the coating on the scaffolds, X-ray diffraction and Fourier transform infrared spectroscopy were performed and the results are shown in Fig. 8. Fig. 8a shows the appearance of the weak diffraction peaks at 26 and 32°, indicating the formation of apatite with low crystallinity. Fig. 8b shows the peaks at 3416 and 1636 cm−1 attributed to the characteristic absorption of H2O, the absorption bands at 1043, 602, 556, and 462 cm−1 are assigned to PO34 −, the peaks at 876 and 1400–1460 cm−1 are the characteristics of CO2− 3 , an important indication of carbonate group entering into the apatite [24]. Therefore, the coatings obtained after immersing the scaffolds in SBF consisted of carbonated hydroxyapatite with low crystallinity, indicating the excellent apatite-formation ability of the nanofibrous PCL scaffolds.
4. Discussions In this study, the PCL scaffold showed a spherical structure, which might be caused by the growth of PCL spherulites during the phase separation of homogeneous PCL solution. The crystal stacks grow radially outward from the center during the crystallization, resulting in spherulite shapes. In the polymer melt, multiple crystal stacks often grow outward simultaneously from the crystal nucleus. The more the nuclei, the more the spherulites growing simultaneously, resulting in smaller size spherulites. When two or more spherulites interact, the molecular chains grow on the edges of spherulites, which is why the junction of spherulites tends to be flat [25]. The PCL spherulites are thought to undergo an outward radial growth after the crystals are generated via phase separation at crystal nuclei. However, only the radial growth of crystal plates is not sufficient for the formation of spherulites; two other mandatory conditions are required: (i) high viscosity and (ii) a certain amount of impurities. In this case, the solvent and non-solvent act as impurities. When large impurities are present, they cannot be removed to either side of phases because of the high viscosity of the system, and therefore, these impurities tend to remain in the middle of the phases resulting in the forked growth of the crystals. Like a growing tree, crystals grow in all directions through numerous branching while maintaining a spherical shape. As shown in Figs. 2 and 3, the PCL scaffold show some round pore structures on the PCL microspheres, formed by the aggregated solvent molecules due to the repulsion of impurities during freeze drying. The nanofibers in the spherical matrix are formed by the growth of crystal plates. Gelation of the polymer solution system played a crucial role in creating the unique nanofibrous structure. First, when the polymer solution was cooled to − 40 °C for phase separation, the scaffold with a highly porous structure rather than a nanofibrous structure was obtained (Fig. 2b). This is because at such a low temperature (− 40 °C), the homogeneous polymer solution directly entered into spinodal
Fig. 6. SEM of PCL scaffolds prepared from 10% PCL in dioxane/water (mass ratio 90/10) with sugar porogen. The mass ratio of PCL solution/sugar is 1:1. Tgel is −8 °C for 2 h.
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Fig. 7. SEM of different topography of scaffold after immersed in SBF 14 days. (a, b and c: Tgel = 12 °C, 8 °C and −4 °C for 2 h without porogen; d, e: Tgel = −8 °C for 2 h with porogen).
phase separation to form a two-phase consecutive structure, leading to the highly porous structure after freeze drying. In contrast, when the polymer solution was cooled to a higher temperature (≥−8 °C) for phase separation, the PCL crystal nuclei were formed first, and the molecular chain began to grow in the form of a crystal plate with the
Fig. 8. XRD and FTIR of nanofibrous PCL scaffold after immersed in SBF for 14 days.
spherulite growth pattern, thus showing a primary phase separation mechanism for polymer crystallization. As shown in Fig. 3, the size of spherulites gradually reduced with increasing temperature until they finally disappear. This result may be explained by the fact that the growth of spherulites is mainly manifested as the growth of crystal plates in the longitudinal and lateral directions; in general, the greater the degree of supercooling, the larger the longitudinal speed constant G, and the narrower the fiber width. The degree of supercooling increases with decreasing aging temperature (Tgel), resulting in the formation of larger spherulites [25]. Another possible reason is that a higher temperature is more favorable for the growth of crystal nucleus; thus, under the same conditions, the higher the number of crystal nuclei, the higher the number of spherulites grown and the smaller the size of spherulites. The micrographs also show that the boundary of growing spherulites becomes obscured at a higher temperature, probably because of the fact that, although the growing spherulites are small, the crystal plates constantly grow in the aging process. Therefore, the overlapping and aggregation of crystal plates occur, i.e., the crystal plates of spherulites will overlap and intertwine with each other, resulting in aggregation. Finally, the spherulites (nanofiber structure) gradually disappear, resulting in a highly porous structure. Nanofibrous entanglement and mergence occurred accompanied with microsphere matrices and nanofibrous structure fade away with increasing amount of water content in the system. This is because high non-solvent volume fraction deteriorated the dissolvability of polymer, a weaker polymer–diluent interaction and lowered viscosity
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in the cosolvent system might induce the formation of polymer poor phase with greater droplet domains [26,27]. In addition, the gradual addition of water would increase the gelation point [28], which is the same as decreasing the degree of supercooling of the system. Therefore, the size of spherulite decreases with increasing solvent to non-solvent ratio while the fibers are gradually broadened, finally leading to the disappearance of both spherulites and nanofibrous matrix. However, the concentration of polymer has an insignificant effect on the growth of spherulite within the experimental range, except that more crystal nuclei were generated in the initial stage of phase separation with increasing concentration, resulting in smaller spherulites and overlapped growth of crystal plates. By varying sugar sphere size and content, one can easily control the macropore size and macropore structure of the nanofibrous PCL scaffold. Not only macroporous and nanofibrous scaffold have a high porosity but also nanofibrous PCL scaffold without macropores have a high porosity. Numerous pores between nanofibrous network structures is the main reason for its high porosity. Meanwhile, the scaffold without nanofibers also has a high porosity because of its highly connected network structure. The porosity of different structure scaffold was similar, but the nanofibrous scaffold proved beneficial for cell attachment, migration and proliferation due to its specific nano-effects. To be effectively used for bone tissue regeneration, it is beneficial that a scaffold promotes bone-like apatite formation when in contact with physiological fluid. To evaluate the bioactivity of the novel macroporous and nanofibrous PCL scaffolds developed in this study, we have investigated their ability to form bone-like apatite in a SBF. Apatite particles were deposited uniformly throughout the macroporous and nanofibrous PCL scaffolds. Noting very uniform growth of apatite layers throughout the macroporous and nanofibrous PCL scaffolds but not on the scaffolds without the nanofeatures [29,30], it is likely that the physical and surface characteristics of the scaffold (macropores, interpore openings, and nanotopography) affect the formation kinetics and morphology of apatite layers [30]. 5. Conclusions In this study, nanofibrous PCL scaffolds with 3D macro/microporous structure were fabricated by TIPS technique combined with sugar leaching process, from a ternary PCL/dioxane/water system. macroporous and nanofibrous PCL scaffold were obtained by controlling the Tgel, ratio of dioxane to water, and content of porogen. The spherulite-like morphology of the scaffold was disrupted with increasing Tgel or water content in the mixed solvent system. Moreover, coalescence of nanofibers occurred, followed by the disappearance of spherulite-like structure at Tgel greater than 8 °C and freezing at −40 °C directly without gelation, or when the proportion of water exceeded 12%. Apatite-formation ability test revealed that the macroporous and nanofibrous PCL scaffolds exhibited good ability to trigger the formation of apatite. Acknowledgments This work was supported by National Nature Science Foundation of China under grant 30970887, the Science Research Foundation of Ministry of Health under grant WKJ 2008-2-037, Fujian Province Nature Science Foundation under grants 2012J01194 and 2011J06019, and Foundation for Young Teachers of Wuyi University under grant xq201102.
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