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Polycaprolactone scaffolds or anisotropic particles: The initial solution temperature dependence in a gelatin particle-leaching method
Polymer 54 (2013) 277e283
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Polymer journal homepage: www.elsevier.com/locate/polymer
Polycaprolactone scaffolds or anisotropic particles: The initial solution temperature dependence in a gelatin particle-leaching method Meicong Wang, Lie Ma*, Dan Li, Changyou Gao* MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2012 Received in revised form 1 October 2012 Accepted 1 November 2012 Available online 7 November 2012
A porogen-leaching method was applied to intend fabrication of polycaprolactone (PCL) scaffolds. Following with a routine solution infiltration, freeze-drying and porogen-leaching process, the porous scaffolds were normally prepared at an initial solution temperature of 25 C. However, the PCL anisotropic particles with the smooth and fuzzy surfaces toward the gelatin porogen and the solution, respectively, were unexpectedly obtained when the initial solution temperature was maintained at 37 C. The freezing temperature was a governing factor for formation of the different PCL products too, while the coarsening time and the PCL concentration within 10e20% had no substantial influence. The PCL anisotropic particles are highly crystallized than the PCL raw materials. To clarify the intrinsic mechanisms, the temperature, cloud point, crystalline ability, and particle size in the solution were quantified. It is demonstrated that the sponges are formed by the traditional liquideliquid demixing for the 25 C solution, whereas the anisotropic particles are obtained by the solideliquid demixing for the 37 C solution and under the assistance of gelatin particles as nucleation sites. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Polycaprolactone (PCL) Anisotropic particles Porous scaffold
1. Introduction As a crucial factor in tissue engineering and regenerative medicine, the scaffold functions as a physical template to support cell adhesion, proliferation and differentiation, and provide channels for the exchange of nutrients and wastes [1e8]. The materials used for fabricating the 3-D scaffolds can be both nature-originated and synthetic. Polycaprolactone (PCL) is a kind of semi-crystalline and biodegradable polymers obtained through petrochemical process, and has been widely used for bone and cartilage tissue engineering [9e13], vascular reconstruction [14,15], and bioartificial liver and so on [16,17]. Its crystallization property enriches its versatility in making many functional materials and devices [18e21]. Recently, many methods such as solvent-casting/particleleaching [22,23], thermally induced phase separation (TIPS)/ freeze-drying [24e27], electrospinning [28,29], and supercritical fluid-gassing [30,31] have been applied for preparation of the 3-D scaffolds. Among which the particle-leaching method is frequently adopted because of the controlled pore size and distribution, which is mainly determined by the size of the porogens, and the
* Corresponding authors. Tel./fax: þ86 571 87951108. E-mail addresses: [email protected] (L. Ma), [email protected] (C. Gao). 0032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymer.2012.11.001
controlled morphology on the pore walls, which is a key factor to determine the cell and tissue response in vitro and in vivo [2,32,33]. So far many kinds of particles such as NaCl [34], sugar [35], gelatin [24], and paraffin [36] have been used in the preparation of porous scaffolds. For example, Wan’s group used the NaCl particles to fabricate a chitosan-g-PCL scaffold with a gradient pore size. The pore size and porosity increase gradually along with the longitudinal direction and can be controlled by selecting particles with different sizes [37]. Chen’s group used ice particulate templates to prepare funnel-like collagen sponges for cartilage tissue engineering [38]. Our group used gelatin particles to fabricate poly(Llactic acid) scaffold [39] poly(lactide-co-glycolide) sponge for cartilage tissue engineering [40]. As demonstrated previously, introduction of gelatin can simultaneously improve the positive interaction between the materials and cells without any potential side effects. Moreover, the annealing at high temperature and humidity is optionally used to bind the gelatin particles, so that the replica pores can have good interconnectivity. When the scaffold is fabricated by the particle-leaching method, TIPS process is generally accompanied, resulting in tiny pores on the walls of macro-pores and thereby a hierarchical structure of the scaffold. The TIPS process is mainly determined by four aspects, i.e. polymer, solvent with high boiling point and low molecular weight, cooling process and diluent-removing process [41]. Many factors such as material composition, solvent composition, polymer
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concentration, frozen temperature, quenching route and coarsening time are used to adjust the TIPS process to obtain the scaffolds with different microstructures [38,42e45]. Therefore, the combination effects of the porogen-leaching and the TIPS will decide the scaffold microstructures. For example, Ma’s group reported the fabrication of scaffolds with macro-pores by using sugar particles as porogen, and the pore walls of the scaffold can be adjusted from nanofibers, partial nanofibers to smooth surface [35,46]. It is widely proved that the nanoscale structure of the scaffold has a great impact on the cell responses such as adhesion [3,47], proliferation [48], differentiation [49e51] and gene expression [52]. Therefore, it is very important to fabricate the scaffold with different topographical features by adjusting the fabrication parameters. In this study, it is found for the first time that the anisotropic PCL particles or sponges are formed during the scaffold fabrication by the method of gelatin particle-leaching. The microstructure is mainly governed by the initial temperature of the PCL/tetrahydrofuran solution, which is a factor often neglected in traditional TIPS. Attention is then paid to the influencing factors and the intrinsic mechanisms, which are finally clarified based on the experimental results and theoretical analysis. This finding is not only important for developing a scaffold of new microstructures, but also attractive for basic science and materials chemistry since the anisotropic particles have very unique properties yet are difficult to fabricate in big batch [53]. 2. Experimental section 2.1. Materials Polycaprolactone (PCL, Mn ¼ 80 kDa) was purchased from SigmaeAldrich. Gelatin was obtained from Chinese Medicine Company and sieved to collect the particles with a size of 280e 450 mm before use. Camphene (with a purity of 97%) was purchased from Aladding Company Ltd., China. All other reagents were of analytical grade and used as received.
2.4. X-ray diffraction The PCL particles were characterized by X-ray diffraction (X’Pert PRO, the Netherlands) at a glancing angle from 3 to 60 , and with the supplied voltage and current of 40 kV and 40 mA, respectively. Samples were exposed at a scan rate of 0.0167 /10 s. 2.5. Differential scanning calorimetry The differential scanning calorimetry (DSC) analysis was carried out using a PerkineElmer Pyris 1 instrument. The PCL/THF solutions were heated to 70 C to eliminate the thermal history, and then decreased to 60 C at a rate of 10 C/min. 2.6. Dynamic light scattering The variance of mean diameter of 10% PCL/THF solution was monitored by using a Brookhaven BI-200SM dynamic light scattering instrument. The PCL/THF solution was heated to 70 C and then decreased to 5 C by an ethanol circulating cooling system. Data were collected at the predetermined temperature. 3. Results and discussion The porogen-leaching method has been very frequently employed to prepare the spongy scaffolds with good control over the macroscopic shape and microstructure as well as other physicochemical properties [35,39,46]. Particularly, the gelatin particles/ spheres are demonstrated as one of the most promising porogens to prepare the porous scaffolds with very good interconnectivity between pores and cytoviability due to the simultaneous entrapment of gelatin molecules on the pore walls [40,54]. Therefore, in this study the gelatin particles template was infiltrated with the PCL/THF solution of different concentrations and initial solution temperatures to prepare the PCL scaffold after freeze-drying and particle-leaching in water. 3.1. Influence of initial temperature and coarsening time
2.2. Preparation of PCL scaffolds The gelatin template was fabricated via a method reported previously [54]. Briefly, the gelatin particles within a size of 280e 450 mm were added into a cylindrical glass vial with a diameter of 12 mm, followed by a slight press on the top, and then treated with saturated water vapor at 70 C for 4 h. After freeze-dried, the gelatin template was immersed into PCL/tetrahydrofuran (THF) solution and maintained under a low pressure of 0.07e0.08 MPa to evolve the trapped air bubbles. When the pressure was released, the polymer solution was spontaneously infiltrated into the cavities between the gelatin particles. The PCL/THF solution-filled gelatin template was kept at a certain temperature (noted as the initial temperature) for 3 h, coarsened in a 20 C refrigerator for a certain period of time, and then freeze-dried. The gelatin template was leached by incubation in 300 ml Millipore water at 37 C for 3 d. Finally, the PCL scaffold was obtained after freeze-drying. 2.3. Morphological characterization The morphology of the PCL porous scaffold or PCL particles was characterized by scanning electron microscopy (SEM, SIRION-100, the Netherlands). To view the cross-sectional morphology, the PCL scaffold was frozen in liquid nitrogen for 5 min and then cut by a razor blade before gold-coating. The PCL particles were also observed under a polarized optical microscope (Olympus BX51, Japan).
It is known that the freeze-drying can simultaneously induce the so-called phase separation, resulting in smaller pores on the walls of big pores which are the replica of the gelatin particles (280e450 mm) [55]. These small pores can improve the connectivity between the big pores and enhance the exchange of nutrients, metabolic products and oxygen etc., and thus are important for the overall performance of the scaffolds. Among the various factors influencing the formation and size of the smaller pores during freeze-drying, the initial temperature and polymer concentration take the major roles since they can change the quenching rate and the location in the binodal/spinodal phase separation curve, and thereby influence significantly the phase separation behaviors [56]. For this context, the systems of infiltrated PCL/THF solution and gelatin template were firstly maintained at 25 C and 37 C to reach the equilibrate state, and then moved to a refrigerator at 20 C, allowing the coarsening of the solution for 3, 8 and 20 h, respectively (Fig. 1). When the initial solution temperature was 25 C, PCL scaffolds with a porous structure were obtained regardless of the coarsening time (Fig. 1aec). However, the size of the small pores was gradually increased along with the prolongation of the coarsening time, accompanying with the thickening of the walls of big pores. It was unexpected, however, that no scaffold was obtained when the initial solution temperature was maintained at 37 C. Instead, only particles were formed regardless of the coarsening time (Fig. 1def). The average size of the PCL particles increased
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Fig. 1. SEM images to show the influence of the initial temperature of 10% PCL/THF solution and the coarsening time on the morphology of the products. The initial temperature was set at (aec) 25 C and (def) 37 C, and the coarsening time was set for (a,d) 3 h, (b,e) 8 h, and (c,f) 20 h at 20 C, respectively.
along with the coarsening time from 45 10 mm (3 h) to 65 20 mm (8 h), and further to 90 20 mm (20 h). Detail examination revealed that the particles showed a discoid structure with an anisotropic morphology, i.e. one smooth face and one rough face (For more detail, see Fig. 4a). This phenomenon has been hardly observed in previous reports, and thus is deserved further studies. 3.2. Influence of polymer concentration To reveal the universality of the anisotropic particles formation, several other factors were varied during the scaffold fabrication. Fig. 2 compares the influence of polymer concentrations at 15% and
20%. Again, only porous scaffolds were obtained when the initial solution temperature was set at 25 C, accompanying with the gradual denser of the pore walls and the size decrease of the smaller pores at a higher PCL concentration (Fig. 2a,b). By contrast, only discoid anisotropic particles were formed at an initial solution temperature of 37 C regardless of the increase of PCL concentration (Fig. 2c,d). At the 3 h coarsening time, the size of the anisotropic particles increased from 45 10 mm to 110 10 mm along with the increase of PCL concentration from 10% to 20%. This result suggests that the initial solution temperature is at least one of the governing factors for the anisotropic particle formation in this system.
Fig. 2. SEM images to show the influence of the concentration of PCL/THF solutions on the morphology of PCL scaffolds. The solution concentrations were (a,c) 15%, and (b,d) 20% with an initial temperature of (a,b) 25 C and (c,d) 37 C, respectively. All the scaffolds were fabricated at a coarsening temperature of 20 C for 3 h.
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Fig. 3. SEM images to show the influence of the coarsening temperature on the morphology of PCL scaffolds. The coarsening temperature was set at 198 C, and the initial temperature of 10% PCL/THF was set at (a) 25 C and (b) 37 C, respectively. (c,d) are higher magnification of (a,b), respectively.
3.3. Influence of coarsening temperature Now that the initial temperature has a big impact, alteration of the coarsening temperature will similarly change the phase separation process, which may in turn bring influence on the morphology of the obtained structures [43]. Fig. 3 shows that at a freezing temperature of 198 C in liquid nitrogen only porous scaffolds were obtained regardless of the initial solution
temperature. Some tiny particles with a size of 5e8 mm appeared on the pore walls of the scaffold prepared with 10% PCL and an initial solution temperature of 25 C (Fig. 3c). 3.4. PCL anisotropic particles So far it has been clarified that the initial solution temperature and the coarsening temperature take the major role in governing
Fig. 4. SEM images (a,b) and the polarized optical microscopy photo (c) of the PCL anisotropic particles. (d) XRD patterns of gelatin, PCL raw material and PCL anisotropic particles.
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acid) (PDLLA), poly(L-lactic acid) (PLLA), and poly(lactide-coglycolide) (PLGA) in 87/13 dioxane/water after quenching at 15 C. The bead size is very small (<10 mm) and independent on the polymers used [27]. The authors explained their phenomenon by the nucleation and growth mechanism. Herein the experimental conditions are very different in terms of the types of polymers, polymer concentration, and solvent quality and solidification temperature. Most importantly, keeping all the other factors constant, variation of the initial solution temperature resulted in the very different PCL sponge or anisotropic particles, which to the best of our knowledge have not been observed previously. In order to unveil the mechanism the solution temperature was in situ monitored upon putting the system into a refrigerator at 20 C. Fig. 5a shows that at both initial temperatures (25 and 37 C) the solution temperature decreased rapidly to <0 C within 50 min, and then showed a general decrease trend with a very slow rate. Basically, the temperatures of the 37 C solution and the 25 C solution were maintained below 5 C during the whole monitoring process, respectively. It has to be pointed out that after coarsening of the 37 C solution for 70 min the solution temperature increased gradually from 6.4 C to 5.2 C after 95 min, and then decreased to 6.4 C again until 150 min. The whole process lasted for 80 min. Comparatively, there was only a slight temperature increase between the coarsening time of 55 min and 59 min and lasted for a very short period of time for the 25 C solution. The cloud points of the PCL/THF solution with different concentrations were also measured and shown in Fig. 5b. It increases along with the increase of PCL concentration. The cloud point of the 10% PCL/THF solution is around 5 C. To better understand the heat exchange during the cooling process, DSC measurements were conducted. Fig. 5c shows that crystallization of PCL occurs at 20e30 C for all the PCL solutions of different concentrations. The other strongest crystallization took place at 18 w 50 C depending on the polymer concentration. Finally, Fig. 5d shows that there was no particulate-like substance inside
the formation of scaffolds or anisotropic particles. Fig. 4a shows clearly that the anisotropic particles have one smooth face and one rough face. FTIR spectroscopy confirms that the anisotropic particles are composed of pure PCL without noticeable gelatin (data not shown). Observation in their cross-section reveals that the particles have a solid rather than a hollow structure (Fig. 4b). Polarized optical microscopy (Fig. 4c) shows that the PCL anisotropic particles have a crystalline and spherulite structure, which was identified by XRD characterization too (Fig. 4d). Compared with the gelatin particles, both the PCL anisotropic particles and the PCL raw materials show two characteristic peaks at 21.5 and 23.5 . However, a much higher crystallization degree (78%) was found for the PCL anisotropic particles according to DSC measurement, which is much higher than that of the PCL raw material (46%). 3.5. Formation mechanism of the anisotropic particles The above results show that the porous scaffolds were normally obtained at an initial solution temperature of 25 C (Fig. 1aec and Fig. 2a,b) following with a routine freeze-drying and porogenleaching process. However, the PCL anisotropic particles were unexpectedly obtained if the initial solution temperature was maintained at 37 C (Fig. 1def and Fig. 2c,d). The temperature of the freezing solution was a governing factor too (Fig. 3), while the coarsening time and PCL concentration within 10e20% had no significant influence. Apparently, the different microstructures of the as-prepared PCL products should be exclusively attributed to the difference of phase separation mechanisms during the coarsening process. Normally, a TIPS process may include liquideliquid demixing, solideliquid demixing, polymer crystallization, gelation and vitrification of the polymer solution, crystallization of the solvent and so on. Some of these processes may occur simultaneously, making the system become more complex [31,57]. Nam et al. observed the formation of beady sponges from very dilute solutions (1%) of poly(D,L-lactic
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Fig. 5. (a) Temperature of the 10% PCL/THF solutions with an initial temperature of 25 C and 37 C as a function of incubation time in a 20 C refrigerator, respectively. (b) Cloud point of the PCL/THF solution with different concentration. (c) DSC curves of PCL/THF solutions with different concentration recorded from 60 C to 60 C at a cooling rate of 10 C/ min. (d) The mean diameter of particles determined in the 10% PCL/THF solution along with the decrease of the temperature.
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o l 5C itia 2 In ure= t era p id tem iqu L id- ing qu ix Li dem
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Fig. 6. Scheme of the forming mechanisms of PCL scaffold and PCL anisotropic particles.
the PCL solution above 37 C. Along with the temperature decrease, particles appeared and their size was progressively enlarged. For example, at 25 C the particles with a size of 90 nm were detected, which reached about 10 mm at 0 C. However, if the solution was maintained undisturbed at 25 C very tiny particles with a size of 2 nm were measured. This discrepancy should be attributed to the hysteresis effect of the dynamic cooling process. As shown in Fig. 6c, the maximum crystallization temperature of 10% PCL solution (23 C) is slightly lower than 25 C. Taking all the results into consideration, it is likely that the PCL solution with a lower initial temperature (25 C) experiences a liquideliquid demixing process, since it passes very quickly through the first crystallization point at 23 C (Fig. 5c), and eventually stays far below the cloud point of the solution (Fig. 5a,b), which might locate below the spinodal curve. At this case, the solution shall separate into both the bicontinuous polymer-rich phase and the polymer-lean phase, which form the pore wall and the small pore after freeze-drying (Fig. 6 upper), respectively. By contrast, the PCL solution with an initial solution temperature of 37 C experiences a solideliquid demixing process (Fig. 6 lower), since it has longer time to pass through the crystallization point at 23 C and thereby a bigger chance to form larger mount of nuclei (Fig. 5d) during the cooling process. The eventual solution temperature is slightly below the cloud point, which locates in the metastable region between binary and spinodal curves. All these situations will accelerate the further nucleation and growth of the PCL particles, which can be demonstrated by the abnormal temperature increase during the coarsening process (Fig. 5a) and the steady increase of the particle size along with the prolongation of the coarsening time (Fig. 1). When the freezing temperature is critical low such as 198 C, the PCL solution will pass through the temperature of nucleation and growth of particles with an extremely fast rate, leading to the frozen of the whole system. Therefore, only sponge is obtained after freeze-drying. Formation of the tiny beads on the scaffold at an initial solution temperature of 25 C (Fig. 3c) is likely attributed to the initially formed particles in the solution, since in practice the operation always requires a couple of minutes and the temperature cannot be controlled very precisely either.
The PCL anisotropic particles are formed due to the existence of the gelatin particles. Indeed, the smooth face is always toward the particle side, whereas the rough surface to the solution. This would mean that the nucleation of the PCL particles should start from the gelatin particles, which is a normal phenomenon for the crystal formation and growth [58]. In the coarsening process, the further growth of the particles can only take place on the free side (namely the unprotected side), leading to the final fuzzy surface as shown in Fig. 4a. 4. Conclusions The polycaprolactone (PCL) sponges and anisotropic particles were obtained by a particle-leaching method at different initial solution temperature, respectively. It was found for the first time that the initial temperature of the PCL/THF solution plays a key role in determining the microstructures of the PCL products. Compared to the normal sponges obtained with the 25 C PCL solution, the PCL anisotropic particles with the smooth face toward gelatin particles and the fuzzy face toward the solution were formed with the 37 C PCL solution at a coarsening temperature of 20 C. When the freezing temperature was critically low, for example, 198 C, only sponges were obtained regardless of the initial solution temperature. The coarsening time and PCL concentration within 10e20% had no significant influence. The PCL anisotropic particles are highly crystallized than that of the PCL raw materials. Taking into account the facts of cooling rate and the final temperature of the solutions, cloud point, DSC results and particle size during the cooling process, the formation mechanisms of the sponges and anisotropic particles are proposed. It happens that the liquideliquid demixing takes place for the 25 C solution, resulting in the sponges. However, the solideliquid demixing dominates the phase separation process for the 37 C solution, leading to the formation of the anisotropic particles under the assistance of gelatin particles as nucleation sites. Acknowledgment Financial support by the Natural Science Foundation of China (20934003), the National Basic Research Program of China
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Polymer journal homepage: www.elsevier.com/locate/polymer
Polycaprolactone scaffolds or anisotropic particles: The initial solution temperature dependence in a gelatin particle-leaching method Meicong Wang, Lie Ma*, Dan Li, Changyou Gao* MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 July 2012 Received in revised form 1 October 2012 Accepted 1 November 2012 Available online 7 November 2012
A porogen-leaching method was applied to intend fabrication of polycaprolactone (PCL) scaffolds. Following with a routine solution infiltration, freeze-drying and porogen-leaching process, the porous scaffolds were normally prepared at an initial solution temperature of 25 C. However, the PCL anisotropic particles with the smooth and fuzzy surfaces toward the gelatin porogen and the solution, respectively, were unexpectedly obtained when the initial solution temperature was maintained at 37 C. The freezing temperature was a governing factor for formation of the different PCL products too, while the coarsening time and the PCL concentration within 10e20% had no substantial influence. The PCL anisotropic particles are highly crystallized than the PCL raw materials. To clarify the intrinsic mechanisms, the temperature, cloud point, crystalline ability, and particle size in the solution were quantified. It is demonstrated that the sponges are formed by the traditional liquideliquid demixing for the 25 C solution, whereas the anisotropic particles are obtained by the solideliquid demixing for the 37 C solution and under the assistance of gelatin particles as nucleation sites. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Polycaprolactone (PCL) Anisotropic particles Porous scaffold
1. Introduction As a crucial factor in tissue engineering and regenerative medicine, the scaffold functions as a physical template to support cell adhesion, proliferation and differentiation, and provide channels for the exchange of nutrients and wastes [1e8]. The materials used for fabricating the 3-D scaffolds can be both nature-originated and synthetic. Polycaprolactone (PCL) is a kind of semi-crystalline and biodegradable polymers obtained through petrochemical process, and has been widely used for bone and cartilage tissue engineering [9e13], vascular reconstruction [14,15], and bioartificial liver and so on [16,17]. Its crystallization property enriches its versatility in making many functional materials and devices [18e21]. Recently, many methods such as solvent-casting/particleleaching [22,23], thermally induced phase separation (TIPS)/ freeze-drying [24e27], electrospinning [28,29], and supercritical fluid-gassing [30,31] have been applied for preparation of the 3-D scaffolds. Among which the particle-leaching method is frequently adopted because of the controlled pore size and distribution, which is mainly determined by the size of the porogens, and the
* Corresponding authors. Tel./fax: þ86 571 87951108. E-mail addresses: [email protected] (L. Ma), [email protected] (C. Gao). 0032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymer.2012.11.001
controlled morphology on the pore walls, which is a key factor to determine the cell and tissue response in vitro and in vivo [2,32,33]. So far many kinds of particles such as NaCl [34], sugar [35], gelatin [24], and paraffin [36] have been used in the preparation of porous scaffolds. For example, Wan’s group used the NaCl particles to fabricate a chitosan-g-PCL scaffold with a gradient pore size. The pore size and porosity increase gradually along with the longitudinal direction and can be controlled by selecting particles with different sizes [37]. Chen’s group used ice particulate templates to prepare funnel-like collagen sponges for cartilage tissue engineering [38]. Our group used gelatin particles to fabricate poly(Llactic acid) scaffold [39] poly(lactide-co-glycolide) sponge for cartilage tissue engineering [40]. As demonstrated previously, introduction of gelatin can simultaneously improve the positive interaction between the materials and cells without any potential side effects. Moreover, the annealing at high temperature and humidity is optionally used to bind the gelatin particles, so that the replica pores can have good interconnectivity. When the scaffold is fabricated by the particle-leaching method, TIPS process is generally accompanied, resulting in tiny pores on the walls of macro-pores and thereby a hierarchical structure of the scaffold. The TIPS process is mainly determined by four aspects, i.e. polymer, solvent with high boiling point and low molecular weight, cooling process and diluent-removing process [41]. Many factors such as material composition, solvent composition, polymer
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concentration, frozen temperature, quenching route and coarsening time are used to adjust the TIPS process to obtain the scaffolds with different microstructures [38,42e45]. Therefore, the combination effects of the porogen-leaching and the TIPS will decide the scaffold microstructures. For example, Ma’s group reported the fabrication of scaffolds with macro-pores by using sugar particles as porogen, and the pore walls of the scaffold can be adjusted from nanofibers, partial nanofibers to smooth surface [35,46]. It is widely proved that the nanoscale structure of the scaffold has a great impact on the cell responses such as adhesion [3,47], proliferation [48], differentiation [49e51] and gene expression [52]. Therefore, it is very important to fabricate the scaffold with different topographical features by adjusting the fabrication parameters. In this study, it is found for the first time that the anisotropic PCL particles or sponges are formed during the scaffold fabrication by the method of gelatin particle-leaching. The microstructure is mainly governed by the initial temperature of the PCL/tetrahydrofuran solution, which is a factor often neglected in traditional TIPS. Attention is then paid to the influencing factors and the intrinsic mechanisms, which are finally clarified based on the experimental results and theoretical analysis. This finding is not only important for developing a scaffold of new microstructures, but also attractive for basic science and materials chemistry since the anisotropic particles have very unique properties yet are difficult to fabricate in big batch [53]. 2. Experimental section 2.1. Materials Polycaprolactone (PCL, Mn ¼ 80 kDa) was purchased from SigmaeAldrich. Gelatin was obtained from Chinese Medicine Company and sieved to collect the particles with a size of 280e 450 mm before use. Camphene (with a purity of 97%) was purchased from Aladding Company Ltd., China. All other reagents were of analytical grade and used as received.
2.4. X-ray diffraction The PCL particles were characterized by X-ray diffraction (X’Pert PRO, the Netherlands) at a glancing angle from 3 to 60 , and with the supplied voltage and current of 40 kV and 40 mA, respectively. Samples were exposed at a scan rate of 0.0167 /10 s. 2.5. Differential scanning calorimetry The differential scanning calorimetry (DSC) analysis was carried out using a PerkineElmer Pyris 1 instrument. The PCL/THF solutions were heated to 70 C to eliminate the thermal history, and then decreased to 60 C at a rate of 10 C/min. 2.6. Dynamic light scattering The variance of mean diameter of 10% PCL/THF solution was monitored by using a Brookhaven BI-200SM dynamic light scattering instrument. The PCL/THF solution was heated to 70 C and then decreased to 5 C by an ethanol circulating cooling system. Data were collected at the predetermined temperature. 3. Results and discussion The porogen-leaching method has been very frequently employed to prepare the spongy scaffolds with good control over the macroscopic shape and microstructure as well as other physicochemical properties [35,39,46]. Particularly, the gelatin particles/ spheres are demonstrated as one of the most promising porogens to prepare the porous scaffolds with very good interconnectivity between pores and cytoviability due to the simultaneous entrapment of gelatin molecules on the pore walls [40,54]. Therefore, in this study the gelatin particles template was infiltrated with the PCL/THF solution of different concentrations and initial solution temperatures to prepare the PCL scaffold after freeze-drying and particle-leaching in water. 3.1. Influence of initial temperature and coarsening time
2.2. Preparation of PCL scaffolds The gelatin template was fabricated via a method reported previously [54]. Briefly, the gelatin particles within a size of 280e 450 mm were added into a cylindrical glass vial with a diameter of 12 mm, followed by a slight press on the top, and then treated with saturated water vapor at 70 C for 4 h. After freeze-dried, the gelatin template was immersed into PCL/tetrahydrofuran (THF) solution and maintained under a low pressure of 0.07e0.08 MPa to evolve the trapped air bubbles. When the pressure was released, the polymer solution was spontaneously infiltrated into the cavities between the gelatin particles. The PCL/THF solution-filled gelatin template was kept at a certain temperature (noted as the initial temperature) for 3 h, coarsened in a 20 C refrigerator for a certain period of time, and then freeze-dried. The gelatin template was leached by incubation in 300 ml Millipore water at 37 C for 3 d. Finally, the PCL scaffold was obtained after freeze-drying. 2.3. Morphological characterization The morphology of the PCL porous scaffold or PCL particles was characterized by scanning electron microscopy (SEM, SIRION-100, the Netherlands). To view the cross-sectional morphology, the PCL scaffold was frozen in liquid nitrogen for 5 min and then cut by a razor blade before gold-coating. The PCL particles were also observed under a polarized optical microscope (Olympus BX51, Japan).
It is known that the freeze-drying can simultaneously induce the so-called phase separation, resulting in smaller pores on the walls of big pores which are the replica of the gelatin particles (280e450 mm) [55]. These small pores can improve the connectivity between the big pores and enhance the exchange of nutrients, metabolic products and oxygen etc., and thus are important for the overall performance of the scaffolds. Among the various factors influencing the formation and size of the smaller pores during freeze-drying, the initial temperature and polymer concentration take the major roles since they can change the quenching rate and the location in the binodal/spinodal phase separation curve, and thereby influence significantly the phase separation behaviors [56]. For this context, the systems of infiltrated PCL/THF solution and gelatin template were firstly maintained at 25 C and 37 C to reach the equilibrate state, and then moved to a refrigerator at 20 C, allowing the coarsening of the solution for 3, 8 and 20 h, respectively (Fig. 1). When the initial solution temperature was 25 C, PCL scaffolds with a porous structure were obtained regardless of the coarsening time (Fig. 1aec). However, the size of the small pores was gradually increased along with the prolongation of the coarsening time, accompanying with the thickening of the walls of big pores. It was unexpected, however, that no scaffold was obtained when the initial solution temperature was maintained at 37 C. Instead, only particles were formed regardless of the coarsening time (Fig. 1def). The average size of the PCL particles increased
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Fig. 1. SEM images to show the influence of the initial temperature of 10% PCL/THF solution and the coarsening time on the morphology of the products. The initial temperature was set at (aec) 25 C and (def) 37 C, and the coarsening time was set for (a,d) 3 h, (b,e) 8 h, and (c,f) 20 h at 20 C, respectively.
along with the coarsening time from 45 10 mm (3 h) to 65 20 mm (8 h), and further to 90 20 mm (20 h). Detail examination revealed that the particles showed a discoid structure with an anisotropic morphology, i.e. one smooth face and one rough face (For more detail, see Fig. 4a). This phenomenon has been hardly observed in previous reports, and thus is deserved further studies. 3.2. Influence of polymer concentration To reveal the universality of the anisotropic particles formation, several other factors were varied during the scaffold fabrication. Fig. 2 compares the influence of polymer concentrations at 15% and
20%. Again, only porous scaffolds were obtained when the initial solution temperature was set at 25 C, accompanying with the gradual denser of the pore walls and the size decrease of the smaller pores at a higher PCL concentration (Fig. 2a,b). By contrast, only discoid anisotropic particles were formed at an initial solution temperature of 37 C regardless of the increase of PCL concentration (Fig. 2c,d). At the 3 h coarsening time, the size of the anisotropic particles increased from 45 10 mm to 110 10 mm along with the increase of PCL concentration from 10% to 20%. This result suggests that the initial solution temperature is at least one of the governing factors for the anisotropic particle formation in this system.
Fig. 2. SEM images to show the influence of the concentration of PCL/THF solutions on the morphology of PCL scaffolds. The solution concentrations were (a,c) 15%, and (b,d) 20% with an initial temperature of (a,b) 25 C and (c,d) 37 C, respectively. All the scaffolds were fabricated at a coarsening temperature of 20 C for 3 h.
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Fig. 3. SEM images to show the influence of the coarsening temperature on the morphology of PCL scaffolds. The coarsening temperature was set at 198 C, and the initial temperature of 10% PCL/THF was set at (a) 25 C and (b) 37 C, respectively. (c,d) are higher magnification of (a,b), respectively.
3.3. Influence of coarsening temperature Now that the initial temperature has a big impact, alteration of the coarsening temperature will similarly change the phase separation process, which may in turn bring influence on the morphology of the obtained structures [43]. Fig. 3 shows that at a freezing temperature of 198 C in liquid nitrogen only porous scaffolds were obtained regardless of the initial solution
temperature. Some tiny particles with a size of 5e8 mm appeared on the pore walls of the scaffold prepared with 10% PCL and an initial solution temperature of 25 C (Fig. 3c). 3.4. PCL anisotropic particles So far it has been clarified that the initial solution temperature and the coarsening temperature take the major role in governing
Fig. 4. SEM images (a,b) and the polarized optical microscopy photo (c) of the PCL anisotropic particles. (d) XRD patterns of gelatin, PCL raw material and PCL anisotropic particles.
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acid) (PDLLA), poly(L-lactic acid) (PLLA), and poly(lactide-coglycolide) (PLGA) in 87/13 dioxane/water after quenching at 15 C. The bead size is very small (<10 mm) and independent on the polymers used [27]. The authors explained their phenomenon by the nucleation and growth mechanism. Herein the experimental conditions are very different in terms of the types of polymers, polymer concentration, and solvent quality and solidification temperature. Most importantly, keeping all the other factors constant, variation of the initial solution temperature resulted in the very different PCL sponge or anisotropic particles, which to the best of our knowledge have not been observed previously. In order to unveil the mechanism the solution temperature was in situ monitored upon putting the system into a refrigerator at 20 C. Fig. 5a shows that at both initial temperatures (25 and 37 C) the solution temperature decreased rapidly to <0 C within 50 min, and then showed a general decrease trend with a very slow rate. Basically, the temperatures of the 37 C solution and the 25 C solution were maintained below 5 C during the whole monitoring process, respectively. It has to be pointed out that after coarsening of the 37 C solution for 70 min the solution temperature increased gradually from 6.4 C to 5.2 C after 95 min, and then decreased to 6.4 C again until 150 min. The whole process lasted for 80 min. Comparatively, there was only a slight temperature increase between the coarsening time of 55 min and 59 min and lasted for a very short period of time for the 25 C solution. The cloud points of the PCL/THF solution with different concentrations were also measured and shown in Fig. 5b. It increases along with the increase of PCL concentration. The cloud point of the 10% PCL/THF solution is around 5 C. To better understand the heat exchange during the cooling process, DSC measurements were conducted. Fig. 5c shows that crystallization of PCL occurs at 20e30 C for all the PCL solutions of different concentrations. The other strongest crystallization took place at 18 w 50 C depending on the polymer concentration. Finally, Fig. 5d shows that there was no particulate-like substance inside
the formation of scaffolds or anisotropic particles. Fig. 4a shows clearly that the anisotropic particles have one smooth face and one rough face. FTIR spectroscopy confirms that the anisotropic particles are composed of pure PCL without noticeable gelatin (data not shown). Observation in their cross-section reveals that the particles have a solid rather than a hollow structure (Fig. 4b). Polarized optical microscopy (Fig. 4c) shows that the PCL anisotropic particles have a crystalline and spherulite structure, which was identified by XRD characterization too (Fig. 4d). Compared with the gelatin particles, both the PCL anisotropic particles and the PCL raw materials show two characteristic peaks at 21.5 and 23.5 . However, a much higher crystallization degree (78%) was found for the PCL anisotropic particles according to DSC measurement, which is much higher than that of the PCL raw material (46%). 3.5. Formation mechanism of the anisotropic particles The above results show that the porous scaffolds were normally obtained at an initial solution temperature of 25 C (Fig. 1aec and Fig. 2a,b) following with a routine freeze-drying and porogenleaching process. However, the PCL anisotropic particles were unexpectedly obtained if the initial solution temperature was maintained at 37 C (Fig. 1def and Fig. 2c,d). The temperature of the freezing solution was a governing factor too (Fig. 3), while the coarsening time and PCL concentration within 10e20% had no significant influence. Apparently, the different microstructures of the as-prepared PCL products should be exclusively attributed to the difference of phase separation mechanisms during the coarsening process. Normally, a TIPS process may include liquideliquid demixing, solideliquid demixing, polymer crystallization, gelation and vitrification of the polymer solution, crystallization of the solvent and so on. Some of these processes may occur simultaneously, making the system become more complex [31,57]. Nam et al. observed the formation of beady sponges from very dilute solutions (1%) of poly(D,L-lactic
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Fig. 5. (a) Temperature of the 10% PCL/THF solutions with an initial temperature of 25 C and 37 C as a function of incubation time in a 20 C refrigerator, respectively. (b) Cloud point of the PCL/THF solution with different concentration. (c) DSC curves of PCL/THF solutions with different concentration recorded from 60 C to 60 C at a cooling rate of 10 C/ min. (d) The mean diameter of particles determined in the 10% PCL/THF solution along with the decrease of the temperature.
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o l 5C itia 2 In ure= t era p id tem iqu L id- ing qu ix Li dem
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Fig. 6. Scheme of the forming mechanisms of PCL scaffold and PCL anisotropic particles.
the PCL solution above 37 C. Along with the temperature decrease, particles appeared and their size was progressively enlarged. For example, at 25 C the particles with a size of 90 nm were detected, which reached about 10 mm at 0 C. However, if the solution was maintained undisturbed at 25 C very tiny particles with a size of 2 nm were measured. This discrepancy should be attributed to the hysteresis effect of the dynamic cooling process. As shown in Fig. 6c, the maximum crystallization temperature of 10% PCL solution (23 C) is slightly lower than 25 C. Taking all the results into consideration, it is likely that the PCL solution with a lower initial temperature (25 C) experiences a liquideliquid demixing process, since it passes very quickly through the first crystallization point at 23 C (Fig. 5c), and eventually stays far below the cloud point of the solution (Fig. 5a,b), which might locate below the spinodal curve. At this case, the solution shall separate into both the bicontinuous polymer-rich phase and the polymer-lean phase, which form the pore wall and the small pore after freeze-drying (Fig. 6 upper), respectively. By contrast, the PCL solution with an initial solution temperature of 37 C experiences a solideliquid demixing process (Fig. 6 lower), since it has longer time to pass through the crystallization point at 23 C and thereby a bigger chance to form larger mount of nuclei (Fig. 5d) during the cooling process. The eventual solution temperature is slightly below the cloud point, which locates in the metastable region between binary and spinodal curves. All these situations will accelerate the further nucleation and growth of the PCL particles, which can be demonstrated by the abnormal temperature increase during the coarsening process (Fig. 5a) and the steady increase of the particle size along with the prolongation of the coarsening time (Fig. 1). When the freezing temperature is critical low such as 198 C, the PCL solution will pass through the temperature of nucleation and growth of particles with an extremely fast rate, leading to the frozen of the whole system. Therefore, only sponge is obtained after freeze-drying. Formation of the tiny beads on the scaffold at an initial solution temperature of 25 C (Fig. 3c) is likely attributed to the initially formed particles in the solution, since in practice the operation always requires a couple of minutes and the temperature cannot be controlled very precisely either.
The PCL anisotropic particles are formed due to the existence of the gelatin particles. Indeed, the smooth face is always toward the particle side, whereas the rough surface to the solution. This would mean that the nucleation of the PCL particles should start from the gelatin particles, which is a normal phenomenon for the crystal formation and growth [58]. In the coarsening process, the further growth of the particles can only take place on the free side (namely the unprotected side), leading to the final fuzzy surface as shown in Fig. 4a. 4. Conclusions The polycaprolactone (PCL) sponges and anisotropic particles were obtained by a particle-leaching method at different initial solution temperature, respectively. It was found for the first time that the initial temperature of the PCL/THF solution plays a key role in determining the microstructures of the PCL products. Compared to the normal sponges obtained with the 25 C PCL solution, the PCL anisotropic particles with the smooth face toward gelatin particles and the fuzzy face toward the solution were formed with the 37 C PCL solution at a coarsening temperature of 20 C. When the freezing temperature was critically low, for example, 198 C, only sponges were obtained regardless of the initial solution temperature. The coarsening time and PCL concentration within 10e20% had no significant influence. The PCL anisotropic particles are highly crystallized than that of the PCL raw materials. Taking into account the facts of cooling rate and the final temperature of the solutions, cloud point, DSC results and particle size during the cooling process, the formation mechanisms of the sponges and anisotropic particles are proposed. It happens that the liquideliquid demixing takes place for the 25 C solution, resulting in the sponges. However, the solideliquid demixing dominates the phase separation process for the 37 C solution, leading to the formation of the anisotropic particles under the assistance of gelatin particles as nucleation sites. Acknowledgment Financial support by the Natural Science Foundation of China (20934003), the National Basic Research Program of China
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