Article pubs.acs.org/Macromolecules
Tunable Porous Hydrogels from Cocontinuous Polymer Blends Anne-Laure Esquirol,† Pierre Sarazin,‡,§ and Nick Virgilio*,†,§ †
CREPEC, Department of Chemical Engineering, Polytechnique Montréal, C.P. 6079 Succursale Centre-Ville, Montréal, Québec H3C 3A7, Canada ‡ Trampoline Innovations, Montréal, Québec H2G 2L3, Canada S Supporting Information *
ABSTRACT: Hydrogels embedded with networks of fully interconnected pores were prepared with microporous polylactide (PLA) molds obtained by extracting the polystyrene (PS) phase in melt-processed cocontinuous blends of PLA and PS. Quiescent annealing of the blends prior to the PS extraction allowed control over the average pore diameter from ∼1 to ∼500 μm for the PLA molds. Solutions of agar or alginate were injected within the molds and gelled in situ. Porous gels were obtained by extracting the PLA molds and X-ray microtomography was employed to characterize their microstructure. Water removal/uptake cycles were fully reversible with very fast kinetics. Freeze-drying yielded ultraporous materials without modification of the macroscopic dimensions, and rehydration yielded back porous hydrogels. It was possible to scale up the technique by using extrusion and injection molding equipment. This versatile new method allows extensive control over the gels’ porosity parameters and the use of various gel chemistries.
1. INTRODUCTION The development of methods and techniques to prepare or synthesize microstructured hydrogels has been an intensive field of research in materials science over the last 20 years1−5 because of their potential applications in fields as diverse as high performance functional membranes,6,7 materials for filtration, separation, and chromatography devices,8 materials for controlled drug release9,10 and scaffolds for tissue and biomedical engineering.5,11−13 In functional membrane applications, a porous polymer membrane can be linked to a functional hydrogel layer at the surface of the pores. The polymer imparts the membrane its mechanical properties. The functional hydrogel layer can act as a specific adsorbent for separation/purification processes, as an active layer to control and modulate selectivity and permeability (for example, pore size can be adjusted if the hydrogel layer is stimuli-sensitive), as a support for (bio)catalysts, etc. In tissue engineering of vascularised thick tissues, the porosity of the scaffold material, which is often a gel or a polymer, performs many functions: (1) pore interconnectivity allows more efficient mass transport and, as such, constitutes a microvascular-like network for oxygen, nutrients and wastes circulation; (2) it allows a uniform cell distribution within the scaffold; (3) it plays a significant role in cell survival, proliferation and migration. Furthermore, to optimize tissue regeneration, the pore size and porosity architecture need to be carefully controlled depending on the type of tissue (for example, a pore diameter of 5−15 μm for fibroblasts and 100−350 μm for bone cells).2,5 Applications in these fields then clearly require a high level of control over a number of material porosity parameters: the volume fraction of the pores, their size distribution, the average pore diameter and their interconnectivity. Preparing such porous gels has been and is still currently an experimental challenge and, consequently, © 2014 American Chemical Society
various methods have been developed to prepare porous/ microfluidic hydrogels including:3 (i) molding with microparticles followed by solvent leaching,14−19 (ii) gas foaming,20−22 (iii) cryogelation,23−25 (iv) photolithographic and micromolding (microfluidic) techniques,26−31 and (v) rapid prototyping methods.32,33 In this article, a new and versatile micromolding method for the preparation of porous gels, based on using binary cocontinuous polymer blends, is presented. A cocontinuous polymer blend is composed of two intertwined and immiscible polymer networks that are completely continuous throughout the whole volume of the blend.34−37 This type of morphology is highly sensitive on the polymers’ interfacial tension, their viscoelastic properties, their volume fractions and the processing conditions (temperature, shear rate, etc.).38 The typical microstructure length scaleoften characterized by the average diameter of the polymer domainscan range from subμm to tens of micrometers. Lower limits as low as 250 nm have been reported by using interfacial modifiers (block copolymers) that decrease the interfacial tension and partially or completely suppress coalescence.34,38−42 In complement, the upper limit can be increased to more than 1000 μm by quiescent annealing of the materials after melt processing, which leads to coarsening of the microstructure due to the interfacial tension.34,39,43 One of the polymer phases can be subsequently extracted with a selective solvent that leaves the other phase intact, yielding polymer materials comprising networks of fully interconnected pores.44−46 Finally, these microstructural features can be finely tuned and enriched by Received: December 19, 2013 Revised: April 6, 2014 Published: April 14, 2014 3068
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cyclohexane, under agitation, for 2 weeks at room temperature. To ensure complete PS extraction, the solvent was changed daily. The samples were subsequently dried in a vacuum oven at 60 °C for 2 days and the continuity of the PS phase was evaluated with eq 1: ms,ini − ms,fin mblend % continuity of PS = × × 100 ms,ini mPS in blend (1)
controlling the polymer phases volume fractions and/or by increasing the initial number of immiscible polymer phases in the blend.47−50 Molding gels in such materials, followed by the subsequent extraction of the polymer mold with a selective solvent, could represent a very versatile method to prepare porous hydrogels. To our knowledge, no such a concept has been reported so far in the literature. The objectives of this work were (1) to prepare porous hydrogels by using porous polymer molds obtained from cocontinuous polymer blends of polystyrene (PS) and poly(lactic acid) (PLA) and (2) to characterize the porous gels in terms of pore volume fraction, average pore diameter, pore interconnectivity and response to cycles of water removal and uptake within the pores. The flexibility of the approach is demonstrated by using thermoreversible (agar) and ionotropic (sodium alginate) gels.12,13,51
where ms,ini is the mass of the sample before extraction, ms,fin is the mass after extraction, mPS in blend is the mass of PS in the original blend, and mblend is the mass of the original blend. 2.4. Morphology Analysis: Polymer Blends. After melt processing in the internal mixer and quiescent annealing, polymer blend samples were microtomed using a Leica RM2165 instrument equipped with a LN21 cooling system. The PS phase was subsequently extracted with cyclohexane (see section 2.3). The samples were dried in an oven during 2 days at 60 °C, then coated with a gold−palladium layer deposited by plasma sputtering and analyzed with a JEOL JSM7600TFE field emission scanning electron microscope (SEM) operated at 2 keV and 10−6 A. Image analysis was employed to determine the specific interfacial area S between the PS and PLA phaseswhich also corresponds to the porous PLA specific surface area after PS extractionand the average pore size diameter d after the extraction of the PS phase. To do so, SEM micrographs were first converted to binary images and the interfacial perimeter P of the PS/ PLA interface was obtained with a digitizing table from Wacom and SigmaScan V.5 software. The specific interfacial area S was calculated using eq 255
2. MATERIALS AND METHODS 2.1. Materials. Polystyrene (PS 615APR, STYRON) and polylactic acid (PLA 4032D) were purveyed by Americas Styrenics and NatureWorks respectively in granular forms. The material properties are listed in Table 1.
Table 1. Homopolymer Properties homopolymers PS 615 APR (STYRON) PLA 4032D (NatureWorks)
density (g/cm3) at 20 °C
density (g/cm3) at 200 °C
Tg (°C)
1.04a
0.97b
101a
−
192 000b
a
c
d
e
e
1.24
1.11
63
Tm (°C)
169
Mw (g/mol)
S=
P A
(2)
where A is the area of the analyzed micrograph (obtained with a proper calibration). The average pore diameter was calculated with eq 356
180 000
a
Obtained from suppliers. bReference 52. cReference 39. dReference 53. eReference 54.
d=
4ϕp S
(3)
where d corresponds to the average pore diameter, ϕp is the volume fraction of the pores (here taken as 0.5 in the blend) and S is the specific interfacial area. 2.5. Injection of the Agar and Alginate Solutions in the Porous PLA Molds, In Situ Gelling, and Extraction of PLA Molds to Obtain Porous Hydrogels. Unannealed and annealed samples from blends prepared in the internal mixer were trimmed into ∼1 cm3 cubes. The PS phase was extracted with cyclohexane as described above to prepare the porous PLA molds. All molds were subsequently dried in an oven during 2 days at 60 °C, washed with DI water and dried again. Agar or alginate solutions were then injected into these molds with an injection system (Figure 1) composed of a syringe pierced in its
Commercial food grades of agar, sodium alginate, and calcium lactate (in powder forms) were purchased from La Guilde Culinaire and used as received. All solutions were prepared with deionized (DI) water: (i) 3 g/100 mL for agar, (ii) 2 g/100 mL for sodium alginate, and (iii) 1 g/100 mL for calcium lactate. All solutions were prepared by gradually adding and dissolving the powders in water while stirring during 30 min at 85 °C. Commercial food dyes from Berthelet were subsequently added to the solutions for identification purpose: red for agar and blue for sodium alginate. 2.2. Cocontinuous Polymer Blend Preparation and Quiescent Annealing Procedure. PLA granules were first dried in a vacuum oven at 60 °C for 12 h. Cocontinuous binary blends of PS and PLA (50/50 vol%) were prepared in a Plasti-Corder Digi-System internal mixer (C.W. Brabender Instrument Inc.) at 190 °C and 50 rpm for 5 min under a constant nitrogen flow. After processing, the blends were quenched in cold water to freeze-in the morphology. Quiescent annealing of blend samples was performed subsequently with a hot press at a temperature of 190 °C during 10, 30, 60, or 90 min to let the morphology coarsen. After annealing, the blends were quenched again in cold water to freeze-in the morphology and then dried. Cocontinuous PS/PLA pellets (50/50 vol%) and bars (0.95 × 1.25 × 6.3 cm3) were also prepared by melt extrusion with a co-rotating twin-screw extruder (Leistritz AG 34 mm), followed by injection molding (Sumitomo SE 50S), at 185 °C. The bars were subsequently annealed at 190 °C (10, 30, or 60 min) with a hot press and quenched to freeze-in the morphology. 2.3. Measurement of Polymer Phase Continuity. A gravimetric analysis method based on a selective solvent extraction procedure was used to determine the continuity of the PS polymer phase39 in blends prepared with the internal mixer. Typically, for all quiescent annealing times, ∼1 cm3 cubic samples were initially weighed and then placed into vials. The PS phase was subsequently selectively extracted with
Figure 1. Injection system composed of a syringe cylinder pierced in its center and of two pistons to fill the PLA mold (in gray) with the agar or alginate solutions (in blue). center, allowing purging of the air and of the excess solution, and of two pistons placed head-to-head. Porous PLA cubes were placed into the syringe in-between the two pistons, and the cylinder was filled with either the agar or the alginate solution. The pistons were then pressed manually to inject the solutions in the polymer molds at 80 °C for agar, and at room temperature for the alginate solution. The injection process is fast and the solutions are injected in micropores. As a result, the solutions are submitted to a high shear rate. The viscosities of the 3069
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pores was first absorbed with paper while trying to avoid significant shrinkage of the materials. The 2-D/3-D microstructure was characterized by X-ray microtomography (μCT) using a SkyScan 1172 microCT instrument from Brüker with the following parameters: 40 keV (source voltage), 250 μA (source current), 9.80 μm (image pixel size), 16 bits (depth), 0.4° (rotation step) and an exposure time of 147 ms. Reconstructions were then realized with the NRecon software version 1.6.6, and 2-D/3-D images were obtained with the CTan and CTvol softwares, respectively. The volume fractions of the gel phase and pores were obtained by image analysis using ImageJ software. According to Delesse’s theorem, the area fractions of the gel phase and pores on the images are equal to their respective volume fractions within the material. The specific surface S and the average pore diameter d were obtained with the equations given above (section 2.4). A minimum of 10 μCT images were used to obtain these values. Pore interconnectivity and continuity were evaluated with the 3-D microtomography reconstructions (see also video reconstruction files in the Supporting Information). 2.7. Freeze-Drying of Porous Gels. A number of porous gels were bisected and analyzed by Fourier Transform Infrared (FTIR) spectroscopy to verify for traces of solvent in the hydrated gels by using a Spectrum 65 FTIR spectrometer from PerkinElmer. Following these analyses, ∼1 cm3 porous hydrogels samples were frozen and freeze-dried for 2 days, followed by SEM and μCT analyses. Some samples were then rehydrated to verify if they would regain their original shape, and were analyzed by μCT (see preceding section) to verify for microstructural modifications.
agarose and alginate solutions were measured with a commercial rheometer (Anton-Paar MCR 501) using a double-gap Couette geometry (bob internal diameter, 24.648 mm; external diameter, 26.657 mm; cup internal diameter, 23.827 mm; external diameter, 27.595 mm) at a steady shear rate of 100 s−1. The results show that the alginate solution is more than 3 times more viscous (380 ± 5 cP) as compared to the agarose (120 ± 20 cP). After injection of the hot agar solution, the samples were placed for one night in DI water at room temperature to allow complete gelling, while the samples filled with sodium alginate were placed into a calcium lactate solution (1 g/100 mL). Following gelling, some PLA/ hydrogel materials were bisected and inspected by optical microscopy to verify the extent of solution penetration within the pores. The final preparation step consisted in the dissolution of the PLA molds with chloroform for about 2 weeks. This extraction time was chosen in order to ensure complete PLA extraction. After extraction, the excess chloroform in the porous hydrogels was absorbed with paper, the gels were rinsed in DI water five times, then stored in plastic tubes containing DI water (for agar) or a calcium lactate solution (1 g/ 100 mL) (for alginate) for subsequent analysis and characterization. Figure 2 illustrates the successive steps that were followed to prepare the porous gels, from melt-processing of the cocontinuous polymer blends to the final extraction of the PLA mold. 2.6. Morphology Analysis: Porous Hydrogels. To analyze the microstructure of cubic 1 cm3 porous hydrogels, the water within the
3. RESULTS AND DISCUSSION 3.1. Effect of Quiescent Annealing on the PS/PLA Cocontinuous Blends. The SEM micrographs in Figure 3 show the morphology of the blend right after melt-processing in the internal mixer (Figure 3a) and after quiescent annealing (Figure 3b−e). The morphology coarsens significantly and remains relatively uniform and homogeneous throughout the volume as quiescent annealing time (tanneal) increases. This coarsening phenomenon at high temperature (above the melting temperature of PLA) is driven by the PS/PLA interfacial tension and is slowed by viscosity.43 After the extraction of the PS phase with cyclohexane, a gravimetric analysis confirmed that the PS and PLA phases remained almost fully continuous (≥95%) for all annealing times (Table 2). The specific surface S and average pore diameter d results are reported in Table 2 as functions of tanneal. d increased nearly linearly until tanneal = 60 min, followed by an increasing coarsening rate. Correspondingly, S decreased significantly by prolonging tanneal. These results and trends concur with previously reported results for similar binary systems.44,45 The obtained porous PLA polymers subsequently served as molds for the preparation of the porous gels. 3.2. Injection of the Agar and Alginate Solutions in the Porous PLA Molds and In Situ Gelling. Solutions of agar (colored in red) or sodium alginate (colored in blue) were subsequently injected into the porous PLA molds with the injection system described in section 2.5 (Figure 1). After in situ gelling (temperature decrease for agar, immersion in a 1 g/ 100 mL calcium lactate solution for alginate), one sample of each PLA/gel material, for each annealing time, was bisected to verify the extent of solution penetration within the molds (Figure 4). Molds with d ≥ ∼20 μm (∼100 μm) were completely filled with the agar (sodium alginate) solution (Figure 4a,b). This can be explained by the fact that the agarose solution is less viscous than the alginate solution under the present injection conditions, which occurs at high shear rate. Closer inspections by optical microscopy of agar-filled, alginate-
Figure 2. Schematic of the successive steps followed to prepare porous hydrogels from binary cocontinuous melt-processed polymer blends. 3070
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Figure 3. SEM micrographs showing the microstructure of porous PLA materials prepared with PS/PLA blends (50/50% vol), which were subsequently used as molds: (a) tanneal = 0 min (as prepared in the internal mixer); (b) tanneal = 10 min; (c) tanneal = 30 min; (d) tanneal = 60 min; (e) tanneal = 90 min. The samples were initially annealed under quiescent conditions, cryogenically microtomed, then immersed in cyclohexane to selectively extract the PS phase. The microtomed surface is comprised within the dotted lines. The inset in part (a) is a close-up to better show the initial porosity.
Figure 4. (a) Picture of a porous PLA mold (tanneal = 10 min) filled with agar (red); (b) porous PLA mold (tanneal = 30 min) filled with alginate (blue); (c) optical microscopy picture of a porous PLA mold (tanneal = 30 min) filled with agar (red); (d) same as part c but filled with alginate (blue); (e) optical microscopy picture of an empty porous PLA mold (tanneal = 30 min); (f) picture after bisection of a porous PLA mold (tanneal = 10 min) showing alginate-filled (blue) and unfilled (white) regions within the pore network. The tiny white elongated features observed in parts (c) and (d) are artifacts caused by reflected light on the gel phase.
Table 2. Continuity of the PS Phase in Blends, Specific Surface S, and Average Pore Diameter d of Porous PLA Materials, as Functions of Quiescent Annealing Time tanneal annealing time (min) 0 10 30 60 90
PS continuity (%) 96 95 97 96 101
± ± ± ± ±
3 2 1 2 2
specific surface, S (cm−1) 5800 900 198 100 45
± ± ± ± ±
300 100 3 10 3
phases and pores throughout the whole volume of the samples, and pore interconnectivity. Two video reconstructions also clearly display these features (see Supporting Information). The pore volume fraction ϕP, the specific surface area S and the average pore diameter d of these porous gels are reported in Table 3. The agar and alginate porous gels respectively possess specific surface areas of 67 and 92 cm−1, and average pore diameters of 275 and 258 μm37.5% and 29% higher for the pores as compared to the molds. However, these values are still comparable with the PLA molds that were used (for tanneal = 60 min, S = 100 ± 10 cm−1 and d = 200 ± 20 μm, see Table 2). Furthermore, for the agar gel, the pore volume fraction ϕP remained close to 50%, while it increased to 60% for the alginate gel, which can explain the qualitative morphological differences (Figure 5c−f)the exact causes remaining to be determined. The PLA extraction procedure, possible gel syneresis, and the preparation for μCT analysis (such as emptying the pores filled with water by using paper, which causes some shrinking of the porous gels) might play some roles. It is known for example that alginate gels can display syneresis when conserved in a calcium lactate solution. It is finally interesting to note that despite their relatively high porosity, a first qualitative assessment show that these porous gels possess good mechanical properties and can be manipulated easily without damaging their structure and
average pore diameter, d (μm) 3 22 101 200 444
± ± ± ± ±
0.2 2 2 20 30
filled and unfilled PLA molds confirmed these observations (Figure 4c−e). Figure 4f shows unfilled pores, in white, at the center of molds (tanneal = 10 min, d = 22 μm, see Table 2) filled with the alginate solution. Complete filling for small pore sizes should be feasible, for example, by using a more elaborate injection system and/or by adding surfactants in the solutions to modify the wetting properties of the PLA molds. 3.3. Morphology and Properties of Porous Hydrogels. Porous hydrogels were obtained after the selective extraction of the PLA phase with chloroform. After extraction and rinsing in DI water, the porous agar and alginate hydrogels retained their original dimensions (Figure 5a,b, example for porous agar). The microstructures of the porous hydrogels were subsequently analyzed by μCT. 2-D micrographs (Figure 5, parts c and e) and 3-D reconstructions (Figure 5, parts d and f) of agar and alginate gels prepared with PLA molds from blends annealed during 60 min clearly show the cocontinuity of the gel 3071
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Figure 6. Weight ratio of porous agar gel compared to initial weight (state 1) during water removal/uptake cycles for gel prepared with 22 μm porous PLA mold (○), 101 μm porous mold (◇) and 200 μm mold (Δ). Odd numbers are gel states when pores are filled with water, while even numbers correspond to gel states with emptied pores after removal with Büchner.
Table 4. Water Removal/Uptake Gravimetric Results for Porous Agar Gels Compared to a Non-Porous Gel gel weight (g)
Figure 5. (a) 1 cm3 porous PLA polymer mold (tanneal = 60 min) before injection of the agar solution; (b) porous agar gel after the extraction of the PLA mold. The dimensions were nearly unchanged after selective PLA extraction. 2-D μCT images and 3-D reconstructions of resulting agar (c and e) and alginate (d and f) porous gels (see Supporting Information for video files).
Table 3. Characteristic Parameters of the Porous Gels Prepared with PLA Molds Annealed during 60 min porous gel
ϕP (%)
S (cm−1)
d (μm)
agar alginate agar (freeze-dried) alginate (freeze-dried)
46 ± 4 60 ± 3 ≈98.5 ≈99
67 ± 5 92 ± 4 − −
275 ± 31 258 ± 14 − −
a
PLA mold pore size (μm)
pores filled with water
empty pores
amount of water removed with Büchner (wt %)
22 101 200 nonporous
0.124 ± 0.005 0.39 ± 0.02 0.33 ± 0.01 1.37
0.058 ± 0.004 0.149 ± 0.004 0.177 ± 0.003 1.34
54 ± 2 62 ± 1 47 ± 2 2
estimated time for water uptake (s)a 18−20 2−3
