超临界二氧化碳

en A al Arlette Vega-Gonza´lez a, Pascale Subra-Paternault a, Ana M. Lo´pez-Periago b, by non-biodegradable and biodegradable polymers (polymethylmetacrylate/polycaprolactone). The influence of several tuted by biopolymers or blends, which induces the natural extracellular matrix that surrounds cells in the body. Hence, required characteristics include an interconnected 3D macroporous network neces- sary for cell proliferation, high microporosity essen- tial for neovascularization, and high surface area erved. * Corresponding author. Tel.: +34 93 5801853. E-mail address: conchi@icmab.es (C. Domingo). Available online at www.sciencedirect.com European Polymer Journal 44 ( EUROPEAN POLYMER 0014-3057/$ - see front matter � 2008 Elsevier Ltd. All rights res operating variables (e.g., liquid solution concentration or flow rate and nozzle design) on the polymers morphology and properties was evaluated. For all studied systems, fibers with a rough textured surface and an extremely high surface area in the order of 100–400 m2 g�1 were precipitated. Prepared materials have potential applications in tissue engineering, since they have intrinsic advantages from a biomimetic approach. � 2008 Elsevier Ltd. All rights reserved. Keywords: PMMA; L-PLA; PCL; Blends; Fibers; Supercritical CO2 1. Introduction Tissue engineering is evolving from the use of implants that repair or replace damaged parts to the use of three-dimensional (3D) scaffolds consti- formation of new functional tissues either in vitro or in vivo [1,2]. The architecture of a scaffold plays an important role in modulating tissue growth and response behavior of cultured cells. A correct archi- tecture of a scaffold should be able of mimicking the Carlos A. Garcı´a-Gonza´lez b, Concepcio´n Domingo b,* aLaboratoire d’Inge´nierie des Mate´riaux et des Hautes Pressions, C.N.R.S., Institut Galile´e, Universite´ Paris 13, 99 Avenue Jean Baptiste Cle´ment, 93430 Villetaneuse, France b Instituto de Ciencia de Materiales de Barcelona (CSIC), Campus de la UAB s/n, E-08193 Bellaterra, Spain Received 19 November 2007; received in revised form 20 December 2007; accepted 4 January 2008 Available online 12 January 2008 Abstract A supercritical carbon dioxide (SCCO2) antisolvent technique was used for the precipitation of several biopolymers into fibers organized in a three-dimensional network. The work was first focused on separately processing either a biodegrad- able (polycaprolactone or polylactic acid) or a non-biodegradable (polymethylmetacrylate) homopolymer. Second, we established that the antisolvent supercritical technique can be also used to make fibrous networks of blends constitute Supercritical CO2 antisolv networks of L-PLA, PMM for biomedic doi:10.1016/j.eurpolymj.2008.01.009 t precipitation of polymer and PMMA/PCL blends applications 2008) 1081–1094 www.elsevier.com/locate/europolj JOURNAL 1082 A. Vega-Gonza´lez et al. / European Polymer Journal 44 (2008) 1081–1094 required to promote cell adhesion [3]. Currently, laboratory designed scaffolds adopt forms ranging from monolithic microcellular structures (sponges) to networks of fibers [4–6]. Polymers used in tissue engineering applications are typically homopolymers or copolymers of lactic (L-PLA) and glycolic (PGA) acids, as well as poly (e-caprolactone) (PCL), among other biodegradable products [7–9]. Non-biodegradable materials such as polymethylmethacrylate (PMMA) are also being tested as scaffolds in order to achieve long-term mechanical stability after implantation [10]. Further- more, blends composed by biostable and biodegrad- able polymers have recently gained significant interests, since the final products can be provided with specific advantages in degradation and mechan- ical properties. Polymeric scaffolds can be obtained by numerous ways, using either conventional techniques such as solvent casting, foaming, emulsions, physical separa- tion and freeze drying [5,6,11,12], or by employing advanced processing methods such as rapid proto- typing technologies [13]. The main drawback of using liquid approaches is that they involve organic solvents and the residual molecules left in the poly- mer after processing may be harmful to the trans- planted cells. Technology based on supercritical carbon dioxide (SCCO2) has been recently estab- lished as an alternative to overcome some of the problems associated with the use of traditional organic solvents for pharmaceuticals and biomateri- als preparation [14,15]. In this respect, different approaches using SCCO2 technology for the pro- cessing of a wide range of biopolymers, with an excellent control on morphology, surface properties and purity, have been reported [11,14,16–19]. For instance, SCCO2 is widely used to produce polymer foams by pressure-induced phase separation [17–23]. This method exploits the fact that SCCO2 is an excel- lent non-solvating porogenic agent for amorphous polymers, while being a poor solvent [20]. In spite of this, when using SCCO2 as a porogenic agent, fre- quently, the obtained microcellular foams did not have a network of pores sufficiently interconnected to allow an effective cell spreading [14,17,24,25]. Similar problems are also found when using poro- genic organic liquids for the preparation of scaffolds by the temperature quenching process [7,26]. Supercritical antisolvent spray processes have also achieved considerable success in producing polymers with different morphologies [15,27–31]. It should be pointed out that most of the investiga- tions described in the supercritical antisolvent liter- ature deal with the production of fine particles, while the production of fibers has been mainly con- sidered as failed experiments. On the other hand, it has been recently established that the use of polymer fibers for tissue engineering applications has some intrinsic advantages from a biomimetic approach, since a wide variety of natural biomaterials pos- sesses fibrous structures (silk, keratin, collagen, cellulose, chitin, etc.). Moreover, the development of new and versatile techniques for the production of clean and template-free polymeric networks of fibers, as an alternative to porous sponges in tissue engineering, has reached a significant interest nowa- days. Hence, the main objective of this work is to follow a line of research that explore the possibilities of using the SCCO2 semicontinuous antisolvent (SAS) process for the preparation of intermingled fibers of homopolymers (biodegradable L-PLA or PCL and non-biodegradable PMMA) and their blends with controlled morphology. 2. Experimental 2.1. Materials Characteristics of the used polymers (L-PLA, PCL, and PMMA) are shown in Table 1. Dichloro- methane (DCM HPLC grade, Prolabo) and CO2 (99.95wt%, Air Liquide) were the used fluids. 2.2. Equipment and methods Experiments were conducted in a SAS apparatus shown schematically in Fig. 1, operated in a semi- continuous mode. The spray chamber consisted on a high pressure vessel (1, Autoclave Engineers) of 5 cm i.d. � 25 cm long. The vessel temperature was controlled by heating jackets (2). At the bottom of the vessel, a membrane filter placed on top of a stainless steel frit of 2 lm porosity allowed the col- lection of precipitated solids. The CO2 was first cooled (3) and then pumped using a reciprocating pump (4, Lewa EK3). The DCM solution of poly- mer(s) to be precipitated was compressed using a reciprocating dual-piston minipump (8, Milton Roy LDC). The pressure inside the vessel was con- trolled downstream with a micrometering valve (7). In a typical experiment, once the vessel had attained the desired temperature value (313 K), the CO2 was introduced, keeping valve (6) closed, until the selected pressure was reached (11 MPa). Then, t (g m Semicrystalline v = 50% Tg = 320 Tm = 449 PCL Powder Aldrich 14,000 A. Vega-Gonza´lez et al. / European Polymer Journal 44 (2008) 1081–1094 1083 PMMA Beads Bonar polymers 300,000 Table 1 Characteristics of employed raw polymers (v = crystallinity) Polymer Shape Supplier Molecular weigh L-PLA Pellets Biovalley 100,000 valve (6) was opened and the system was allowed to equilibrate maintaining the CO2 flow at the set value of 100 mL min�1. Next, the polymer solution was injected into the vessel, by opening valve (9), through a nozzle. Two types of nozzles were used (Fig. 1): a plain orifice (disk) and a conical spray (swirl). In addition, two different injection systems were studied. In the first one, a separate introduc- tion of the liquid solution through a nozzle and the CO2 through a 1/8 00 tubing was performed. In the second one, a coaxial nozzle was used for the introduction of both fluids concurrently. The simul- taneous mass transfer of the SCCO2 and of the liquid solvent from one phase to the other induced the supersaturation of the polymer that precipi- tated. Finally, the system was depressurized across a metering valve (7). The fluid was separated in a cyclone (10). The precipitated polymer was dried using the SCCO2 flow. 2.3. Characterization The morphology of the polymer samples was ana- lyzed by scanning electron microscopy (SEM, Leica 5440). The composition of the processed solid Fig. 1. SAS experimental set-up and schematic draws of the d Semicrystalline v = 63% Tg = 211 Tm = 339 Amorphous Tg = 393 ol�1) Structure Thermal transitions (K) samples was determined by 1H nuclear magnetic res- onance (1H NMR Bruker ARX-300 MHz spectrom- eter) in deuterated chloroform. The semicrystalline L-PLA was analyzed by X-ray diffraction (XRD) with a Rigaku Rotaflex RU200 B instrument, using Cu Ka1 radiation. The glass (Tg) and/or melting (Tm) temperatures of polymers were measured on a differential scanning calorimeter (DSC, 822e/400 Mettler Toledo). Thermograms were obtained at a heating rate of 10 K min�1 in the range 293–573 K under N2 purge. The specific surface area (Sa) and pore volume (Pv) of raw and prepared samples were determined by low-temperature N2 adsorption, using an ASAP 2000 Micromeritics INC. Previous to measurements, samples were dried under reduced pressure (<1 mPa) at 323 K for 48 h. Sa was deter- mined by the BET-method (Brunauer–Emmett– Teller) and Pv was estimated using the BJH-method (Barret–Joyner–Halenda) [32]. The average pore diameter (Dp) was calculated as 4Pv/Sa. 3. Results and discussion The SAS process was first used to precipitate the homopolymers L-PLA, PCL, and PMMA isk (hand made) and swirl (Lechler, France, SA) nozzles. separately. Further, two different compositions of PCL in the PMMA/PCL blend were processed into fibers (Table 2). This blend composition was chosen because previous studies have demonstrated that the combination of biodegradable PCL and biostable PMMA could provide materials with good mechan- ical integrity and biodegradation capabilities, being an alternative to traditional extrudable copolymers used in tissue regeneration [33]. For the SAS processing of polymers, the described main parameter influencing product mor- phology is the concentration of the liquid solution [34–36]. The uses of diluted, semidiluted and con- centrated solutions give place to different polymer 6 mL min�1 flow rate (samples PLA(1) and PLA(2), respectively, in Table 2). It should be pointed out that L-PLA polymer processed by supercritical antisolvent techniques has been gener- ally reported to precipitate as well defined indepen- dent particles [27–29,37–39], but previous results were obtained when working at much lower L-PLA concentration in the liquid solution (0.1– 1 wt%) than the one used in this work (2.8 wt%). Fig. 2 shows some SEM micrographs of the obtained samples. The observed macrostructures are a reflection of the liquid jet behavior (Fig. 2a and b). A large spreading in the axial direction was apparent in the micrographs of the L-PLA on 1084 A. Vega-Gonza´lez et al. / European Polymer Journal 44 (2008) 1081–1094 chains behavior. In the diluted regime, polymer chains behave as independent entities and the poly- mer usually precipitates as a powder. On the other hand, in the semidiluted and concentrated regions, the polymer chains are capable of entangling each other strongly to form a fibrous structure in the pre- cipitate. Since in this work the goal was to produce fibers, we worked at relatively high polymer concen- tration (semidiluted region) in the liquid solution (Table 2). Spray conditions were chosen as the important key parameters of the SAS process deter- mining polymer fibers macro and microstructure. Thus, the effects of modifying the liquid solution flow rate and the nozzle design were analyzed for each studied polymer. 3.1. L-PLA 3.1.1. Influence of liquid solution flow rate To analyze the influence of the liquid solution flow rate on polymer morphology, a 2.8 wt% L- PLA polymer solution was sprayed into SCCO2, through a plain orifice nozzle, at either 1 or Table 2 Experimental conditions for the antisolvent experiments Sample Polymer Polymer concentrati (wt%) PLA(1) L-PLA 2.8 PLA(2) L-PLA 2.8 PLA(3) L-PLA 2.8 PLA(4) L-PLA 2.8 PMMA PMMA 1 PCL PCL 0.8 Blend15(1) PMMA/15 wt% PCL 0.8 Blend15(2) PMMA/15 wt% PCL 0.2 Blend15(3) PMMA15 wt% PCL 0.8 Blend15(4) PMMA/15 wt% PCL 0.8 Blend30 PMMA30 wt% PCL 0.8 sample obtained at the low liquid flow rate of 1 mL min�1 giving place to a cobweb-like macro- structure (Fig. 2a). In contrast, at the high liquid flow rate of 6 mL min�1 viscous and inertial forces offer significant resistance to jet break-up. This led to the formation of a helical oscillation of the jet around its own axis, which can be clearly observed in Fig. 2b. These results are in accordance with pre- vious works, which have shown that, in the semidi- luted region, fibers and helical threads emerged from the nozzle, spreading in both axial and radial directions from the jet core [40]. At low liquid flow rates, the jet disintegration into the SCCO2 has been described to be characterized by axially symmetrical disturbances, while the radial direction is preferred at higher velocities [41]. A closer observation of the SEM photographs indicated that the fibers were constituted by coa- lesced polymer microparticles (Fig. 2c and d). The presence of discrete particles suggests that the tran- sition from diluted to semidiluted region occurs close to the used polymer concentration [38]. Coa- lescence of L-PLA particles seemed limited to phys- Liquid flow rate (mL min�1) Liquid/CO2 addition mode Nozzle, diameter (lm) 1 Separate Disc, 100 6 Separate Disc, 100 3 Separate Swirl, 100 3 Coaxial Swirl, 200 1 Separate Swirl, 100 0.7 Separate Swirl, 100 1 Separate Swirl, 100 1 Separate Swirl, 100 6 Separate Swirl, 100 1 Separate Disc, 100 1 Separate Swirl, 100 Fig. 2. SEM images of L-PLA precipitated using the SAS process, 2.8 wt% of polymer in DCM, injected separately through a disc nozzle at: (a, c) 1 mL min�1 (sample PLA(1)), and (b, d) 6 mL min�1 (sample PLA(2)). Fig. 3. (a) XRD, and (b) DSC or raw L-PLA and PLA(1) sample. A. Vega-Gonza´lez et al. / European Polymer Journal 44 (2008) 1081–1094 1085 ical interaction. Indeed, the semicrystalline structure of the L-PLA makes agglomeration from plasticiza- tion difficult [34]. At 1 mL min�1 of liquid solution flow rate, particles with a size of 2–3 lm were pre- cipitated along with 0.5 lm particles (Fig. 2c). The bimodal size distribution may suggest the formation of particles in the jet and in the solution. At 6 mL min�1 of liquid solution flow rate, particles with a size smaller than 1 lm were produced (Fig. 2d). In this case, a high flow rate resulted on enhanced turbulence and convective mass transfer [29,42]. Therefore, smaller liquid droplet sizes were formed resulting in considerably smaller particles than in the run with the low liquid solution flow rate. Realistic models of fluids mixing have shown the complexity of the theoretical work [40–44]. Those, will be the subject of a further work and, therefore, they are not deeply analyzed in this investigation, where a more qualitative assessment of the effect of a whole set of experimental conditions in precip- itate characteristics was performed. Fibers of L-PLA obtained at 1 mL min�1 flow rate (sample PLA(1)) were further characterized regarding the microstructure. X-ray diffraction pattern of raw L-PLA displayed the characteristic narrow diffraction lines at 16.5� and 19�, corre- sponding to the a crystalline form (Fig. 3a). Despite the very fast precipitation rate, XRD analysis showed that the PLA(1) sample was still semicrys- talline (Fig. 3a) by displaying the more intense dif- fraction peak at 16.5�. Moreover, peak broadening indicated small crystal size in the precipitated fibers. DSC analysis (Fig. 3b) did not evidence significant changes with respect to the raw material regarding the melting point or melting enthalpy (compare values in Tables 1 and 3). In addition, the values of crystallinity estimated from the DSC data for both raw and processed L-PLA were similar Table 3 Melting point, crystallinity (calculated from the melting enthalpy) and surface area of some representative prepared samples Sample Tm (K) v (%) Sa (m 2 g�1) PLA(1) 452 K (L-PLA) 48 60 ± 8 PMMA – – 150 ± 15 Blend15(2) 337 K (PCL) – 400 ± 25 Blend30 338 K (PCL) – 240 ± 30 DCM 1086 A. Vega-Gonza´lez et al. / European Polymer Journal 44 (2008) 1081–1094 Fig. 4. SEM images of precipitated L-PLA, 2.8 wt% of polymer in (sample PLA(3)), and (c, d) coaxially (sample PLA(4)). , injected at 3 mL min�1 through a swirl nozzle: (a, b) separately (about 50%). BET analysis revealed a specific sur- face area value of �60 m2 g�1 for PLA(1) sample (Table 3), which comprised both meso and macro- pores. 3.1.2. Influence of the injection mode and nozzle design In order to examine the effect of the injection mode in polymer morphology, the 2.8 wt% L-PLA liquid solution was introduced into the precipitation chamber through the swirl nozzle, at a flow rate of 3 mL min�1, either separately or co-currently with the SCCO2 (samples PLA(3) and PLA(4), respec- tively, in Table 2). The hydrodynamics of the differ- ent used nozzles have been already described previously [45]. SEM photographs of the sample obtained using the separate injection mode (Fig. 4a and b) showed coalesced L-PLA particles with a similar macrostructure to that of the system precipitated using the disk nozzle and a solution flow rate of 1 mL min�1 (Fig. 2a and c). According to that, no significant effect of the nozzle design (disc or swirl) was observed. However, for the sys- tem where the CO2 was injected co-currently in the annular region over the liquid solution, smooth fibers were formed (Fig. 4c and d) with different morphology than those obtained using the separate injection mode (Fig. 4a and b). The co-currently injection mode enhanced the mixing of the system [29,44], and L-PLA apparently precipitated into fibers before individual droplets could be formed in the liquid jet. The SEM images of sample PLA(4) showed some 0.5 lm beads deposited in fibers (Fig. 4d). 3.2. PMMA Under the working experimental conditions of polymer concentration, pressure and temperature (Table 2), PMMA polymer always precipitated as DCM A. Vega-Gonza´lez et al. / European Polymer Journal 44 (2008) 1081–1094 1087 Fig. 5. SEM images of precipitated PMMA, 1 wt% of polymer in to different magnifications. , injected at 1 mL min�1 through a swirl nozzle. (a–c) correspond a fibrous network with a similar appearance to that shown in the SEM images of Fig. 5. The observed macrostructure can be described as an open airy net- work (Fig. 5a) composed of very small subfibers of less than 0.5 lm diameter (Fig. 5b). Once more, the formation of fine particles appears to indicate that the transition from diluted to semidiluted region occurs close to the working polymer concentration. For PMMA the agglomeration and flocculation was extended in comparison with L-PLA due to the PMMA amorphous nature and SCCO2 plasticiza- tion effect. A value of 150 m2 g�1 of specific