PolyHIPEs - American Chemical Society

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Biomacromolecules 2001, 2, 154-164

Emulsion-Derived Foams (PolyHIPEs) Containing Poly(E-caprolactone) as Matrixes for Tissue Engineering Wendy Busby,† Neil R. Cameron,*,† and Colin A. B. Jahoda‡ Department of Chemistry, University of Durham, South Road, Durham DH1 3LE, U.K., and Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, U.K. Received September 8, 2000; Revised Manuscript Received November 28, 2000

The preparation of PolyHIPE foams containing poly(-caprolactone) from macromonomers by free radical homo- or copolymerization is described. The macromonomers are synthesized from PCL diols and are polymerized in the continuous phase of high internal phase emulsions (HIPEs). Subsequent drying yields low-density foams with cell diameters of 5-100 µm. Foam morphology, as determined by scanning electron microscopy, depends on the type of diluent (styrene, methyl methacrylate, or toluene) added to the emulsion organic phase and on the PCL content. Increasing the latter increases the continuous phase viscosity to a point where emulsion formation is impeded. Foam swelling in toluene, 2-propanol, and water was investigated by solvent imbibition and increased with increasing solvent hydrophobicity. Furthermore, it was found generally to decrease with increasing PCL content, due to increasing cross-link density. Swelling generally increased when higher molar mass PCL macromonomer was used due to the formation of a less tightly cross-linked network. One type of foam sample was shown to support the growth of human fibroblasts over a period of 2.5 days. Introduction Arguably the greatest challenge facing modern medicine is the shortage of organs and tissue available for transplantation. This has led to the extremely rapid development of the field of tissue engineering, which employs a scaffold to act as an adhesion substrate and support for the growth of new tissue from implanted cells. Normally, the scaffold is made of biodegradable polymer that undergoes degradation as the tissue grows, resulting in nontoxic products that are eliminated by the host. Since, in addition to promoting cell adhesion and guiding tissue growth, the scaffold must also allow the supply of nutrients, growth factors, etc., to, and the removal of waste products from, the developing tissue, porous materials are usually employed.1 The porous morphology can be achieved by meshing together fibers of biodegradable polymer under heat treatment. A number of tissue-engineered products prepared using similar technology are already on the market, for example, Dermagraft and TransCyte from Advanced Tissue Sciences2 and Apligraf from Organogenesis.3 Materials that are inherently porous, i.e., foams,4 have also been investigated and can be produced in a number of ways. Healy et al.5 describe a thermally induced phase separation (TIPS) process where an emulsion of water in a dichloromethane solution of poly(lactide-glycolide) (PLGA) is frozen in liquid nitrogen and then freeze-dried to produce materials with 90-95% porosity and pore size from 15 to >200 µm. Belgian workers6 applied a similar procedure to a dioxane solution of polylactide (PLA) to yield materials * Corresponding author. E-mail: [email protected]. † Department of Chemistry, University of Durham. ‡ Department of Biological Sciences, University of Durham.

with tubular pores of ∼100 µm diameter and smaller (∼10 µm) pores within the walls of these. In an extension to this work, water was added resulting in a liquid-liquid phase separation (spinodal decomposition) process that gave smaller, interconnected pores.7 Moghe and co-workers8 examined the influence of pore size of collagen foams prepared by freezedrying on hepatocyte differentiation and found that cell spreading and infiltration into the foam were greatest as pore size increased (to ∼80 µm). Other workers9 produced poly(L-lactic acid) (PLLA) foams by freeze-drying and demonstrated some control over morphology by varying certain parameters prior to freeze-drying. Similarly, Japanese workers10 could control the structure of cross-linked gelatin foams prepared by freeze-drying, by varying the freezing temperature. The leaching of particulates from solution-cast films of biodegradable polymers is another popular method of foam production. Workers at Rice University11 cultured rat osteoblasts in 90% porous foams prepared by leaching NaCl from PLGA films.12 The same group described the production of hydroxyapatite fiber reinforced PLGA foams for bone regeneration, prepared by a similar route.13 Unfortunately, however, the most highly porous materials were not sufficiently strengthened to permit their use in load-bearing situations. Chen and co-workers14 used the same procedure to prepare a porous PLGA substrate that is used to support a cross-linked collagen layer. The resulting hybrid sponges were more hydrophilic, increasing wettability and facilitating infiltration of cells (mouse fibroblasts and bovine articular chondrocytes). Particulates other than salt have also been used to create porosity: a Canadian group15 has used glucose crystals to prepare porous PLGA materials; in a different

10.1021/bm0000889 CCC: $20.00 © 2001 American Chemical Society Published on Web 01/13/2001

Emulsion-Derived Foams Containing Poly(-caprolactone)

approach, hydrocarbon particles were leached from a variety of polymers (PLLA, PLGA, poly(methyl methacrylate) (PMMA), and poly(ethylene oxide) (PEO)) by treatment with a hydrocarbon solvent.16 Pishko and co-workers17 describe an alternative method of inducing porosity, using CO2. Emulsions of aqueous protein solution in a PLGA-solvent solution were saturated with supercritical CO2 and then the vessel was rapidly vented, causing the simultaneous formation of CO2 gas bubbles and polymer precipitation to give an interconnected foam loaded with protein. The release rate of proteins from the resulting materials was faster than that from materials prepared by salt leaching. Mooney’s group at Michigan18 has described a novel approach that is a combination of the CO2 blowing and salt-leaching techniques. PLGA loaded with DNA was compressed with NaCl and subsequently treated with CO2 as above. Leaching of the salt crystals from the resulting porous matrix yields a more highly porous and interconnected foam. Subsequent work19 has examined the release of growth factors from such matrixes. Another method for the production of foam materials involves template polymerization of high internal phase emulsions (HIPEs).20,21 These heterogeneous liquid-liquid mixtures are characterized by a droplet (internal) phase volume fraction φ of at least 0.74.22 When the nondroplet (external) phase contains monomer that is then polymerized, the resulting material is known as PolyHIPE using nomenclature devised by Unilever. PolyHIPEs can either be closed- or open-cell, depending on certain parameters.23 The latter have been studied more extensively since the open structure allows removal of the droplet phase yielding a low-density foam. Such materials have been investigated for a variety of uses, including solid-phase synthesis24 and catalyst25-27 supports, aerosol filters,28 and substrates for porous Ni electrodes.29-31 The porous structure of PolyHIPE foams is a replica of the emulsion structure at the gel point. Therefore, the foam morphology is conveniently controlled by varying such factors as φ and droplet radius, the latter being strongly influenced by the emulsion stability.23,32,33 In addition, extremely high foam surface areas (∼550 m2 g-1) can be produced by replacing some of the monomer in the nondroplet phase with an inert solvent (porogen) that is immiscible with the droplet phase.33,34 On polymerization, phase separation creates a second family of much smaller pores within the walls of the primary foam structure. It is widely recognized that the substrate structure (porosity, pore size, and surface area) can profoundly influence the behavior of cells sealed into a porous scaffold. Therefore, the ability to control these properties easily seemed to us to make PolyHIPE materials suitable candidates for evaluation as matrixes for tissue engineering. This article describes our initial experiments to prepare partially biodegradable PolyHIPE matrixes based on poly(-caprolactone). Our strategy involves free radical (co)polymerization of poly(-caprolactone) macromonomers (1 and 2) in the continuous phase of a HIPE. Recently, the use of non-biodegradable poly(styrene-divinylbenzene) PolyHIPE materials as scaffolds for tissue engineering has been described in the patent literature.35

Biomacromolecules, Vol. 2, No. 1, 2001 155

Experimental Section Materials and Instrumentation. The monomers (styrene and methyl methacrylate) were freed of inhibitor by passing through a pad of basic Al2O3; all are other species were used as supplied. Poly(-caprolactone) diol samples of Mn ) 530 and 2000 were obtained from Aldrich. Surfactants Span 60 (sorbitan monostearate), Span 80 (sorbitan monooleate), Span 85 (sorbitan trioleate), and Tween 80 (polyoxyethylene sorbitan monooleate) were obtained from Sigma. The surfactant Synperonic PE L121 is a triblock copolymer of poly(ethylene oxide) and poly(propylene oxide) (central block) and was received from ICI Surfactants. FTIR spectra (KBr disks) were recorded on a Perkin-Elmer 1600 Series FTIR spectrometer. NMR spectra were recorded on a Varian Unity-300 spectrometer at 300 MHz (1H) or 75 MHz (13C) using CDCl3 as solvent. Elemental analysis (C, H, and N) was performed using a Exeter Analytical E-440 elemental analyzer. MALDI-tof spectra of samples immobilized in a mixture of 2,5-dihydroxybenzoic acid and gentisic acid were recorded using a Kratos Kompact MALDI 4. Scanning electron microscopy was carried out at the University of Newcastle using a Hitachi S2400 electron microscope operating at 25 kV. Fractured samples were prepared for scanning electron microscopy (SEM) by mounting on aluminum stubs using a carbon paste, either to make specimens adhere better or to increase the conductivity. All samples were sputter coated with a thin layer of gold prior to viewing, to enhance conductivity. Cell suspensions were centrifuged in a Sigma/Philip Harris Scientific 3-15 centrifuge. Macromonomer Synthesis. The following general procedure was used: poly(-caprolactone) diol (Mn ) 2000, 20.0 g, ∼20 mmol of -OH groups) was dissolved in dichloromethane (200 mL) containing triethylamine (6.3 mL, 45 mmol) in a 500-mL round-bottomed flask fitted with a pressure equalizing dropping funnel containing acryloyl chloride (6.0 mL, 74 mmol) in dichloromethane (30 mL). The system was then purged with argon gas as the flask cooled to ice temperature. Subsequently, the acryloyl chloride was added dropwise with stirring to the solution of diol and triethylamine at ice temperature over ca. 1 h, then the mixture was stirred for a further 20 h during which time it was allowed to come to room temperature. The resulting solution was then filtered through Hyflo filter aid and the filtrate extracted with portions of 3% KOH solution and then 1% HCl solution until the aqueous layer was colorless in each case. The organic layer was then dried over MgSO4 and concentrated in vacuo to yield (typically) 18.9 g (95%) of product 2. IR (KBr): 2944, 2865, 1724 cm-1. 1H NMR (300 MHz, CDCl3): δ 6.40 (dd, 2H, Jtrans ) 17 Hz, Jgem ) 2 Hz), 6.10 (dd, 2H, Jtrans ) 15 Hz, Jcis ) 9 Hz), 5.80 (dd, 2H, Jcis ) 9 Hz, Jgem ) 2 Hz), 4.20 (t, 4H, J ) 5 Hz) 4.15 (t, 4H, J ) 7 Hz), 4.05 (t, 29.2H, J ) 8 Hz), 3.70 (t, 4H, J ) 5 Hz), 2.30 (t, 33.2H, J ) 8 Hz), 1.65 (m, 66.4H), 1.40 (m, 33.2H). 13C NMR (75 MHz, CDCl3): δ 173.5, 166.0, 130.0, 128.4, 72.3, 69.0, 64.1, 63.2, 62.4, 61.6, 34.0, 32.2, 28.2, 25.4, 25.2, 24.6, 24.5. Anal. Calcd for C109.6H180O38.2: C, 62.5; H, 8.5. Found: C, 62.2; H 8.8. MALDI-tof MS, m/z 2398, 2283, 2169, 2054, 1942, 1829, 1718, 1602, 1487, 1374, 1260, 1147 [(M)n + Na+].

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Network Formation. Macromonomers were either homopolymerized or copolymerized with styrene or methyl methacrylate, in toluene as solvent. The following is a representative procedure: poly(-caprolactone) macromonomer 2 (2.1 g, 1.0 mmol), methyl methacrylate (0.94 g, 9.4 mmol), AIBN (ca. 1 mg), and toluene (5 mL) were placed in a 25-mL round-bottomed flask fitted with a septum. The resulting solution was purged by Ar bubbling for 10 min and then heated at 60 °C for 20 h. After this time a soft clear gel had formed; this was removed from the flask and immersed in methanol for 10 min and then reimmersed in fresh toluene. The gel did not redissolve. PolyHIPE Preparation. PolyHIPE foams were prepared from PCL macromonomers and either styrene, methyl methacrylate, or toluene as organic phase diluent, according to a standard procedure described previously.34 A representative example is given: styrene (4.0 g, 38 mmol), PCL macromonomer (2, 1.0 g, 0.5 mmol), and Span 80 (1 g) were placed in a three-necked 100-mL round-bottomed flask and purged by Ar bubbling for 10 min. To this was added, under Ar, 45 mL of a previously purged aqueous solution containing K2S2O8 (0.1 g, 0.37 mmol) and CaCl2‚2H2O (0.5 g, 3.4 mmol), dropwise and with constant stirring at ca. 300 rpm using a D-shaped PTFE paddle powered by an overhead stirrer motor. The emulsion developed as a viscous white fluid or paste. Subsequently, the emulsion was transferred to a polyethylene container that was heated at 60 °C in an oven for 48 h. The resulting PolyHIPE was removed from the container and extracted in a Soxhlet with water and then 2-propanol (24 h each) and then dried to constant mass in vacuo. For brevity, the PolyHIPE samples prepared in this study are referred to using a code, of the form X-Y-Z where X is the percentage (by weight of total oil phase36) of PCL macromonomer in the HIPE formulation, Y indicates the macromonomer molar mass (L ) low, H ) high), and Z identifies the organic phase diluent (S ) styrene, M ) methyl methacrylate, T ) toluene). Emulsion Tests. PCL macromonomer (0.2 g), monomer (0.8 g), and surfactant (0.2 g) were mixed together and 9 mL of an aqueous solution of CaCl2‚2H2O (0.1 g, 0.7 mmol) was added dropwise with constant vigorous manual agitation. The resulting HIPE (if formed) was then heated at 60 °C, and the stability was monitored by determining the percentage of separated liquid phase produced over time. Swelling Studies. The uptake of toluene, 2-propanol, and water by each foam type was determined using a previously described procedure.37 Approximately 0.1 g of PolyHIPE was accurately weighed into a preweighed sinter stick, which was then immersed in solvent for 24 h. The wet stick plus foam was then weighed, reimmersed in solvent, and reweighed. This process was repeated until a constant mass was obtained. Each sample was analyzed in duplicate, and empty sticks were previously treated as above to determine the solvent uptake by the porous glass frit. The quantity of solvent absorbed by each foam was determined from the mass of (wet sample + stick) minus the mass of (dry stick + dry foam + solvent absorbed by stick). The values quoted (Figure 7) are the means from two runs with standard deviations

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less than 10% (higher deviations were observed for swelling ratios below 1 due to experimental error). Cell Seeding and Tissue Growth. Human fibroblasts obtained from biopsies from adult patients with informed consent were grown in culture flasks at 37 °C in a culture medium, which consisted of MEM (EAGLE) with Earle’s salts, glutamax, and NaHCO3, supplemented with fetal bovine serum (10%, 10 mL/100 mL of medium) and containing gentamicin sulfate (0.1 mL/100 mL of medium) antiobiotic. The pH of the medium was kept at ca. 7.2 by placing it in an atmosphere of CO2. Growth was monitored by optical microscopy, and the medium was changed every 2-3 days. Subsequently, excess medium was poured from the flask and 5 mL of phosphate buffer saline (PBS) containing EDTA was added. The cells were rinsed with this solution twice, then a further 2-mL portion containing 0.2 mL of a 2.5% trypsin solution was added to detach the cells from the culture flask surface. This was kept at 37 °C for 1.5 min, then 8 mL of fresh culture medium was added to quench the enzymatic reaction. The resulting solution containing the detached cells was transferred to a universal container and centrifuged at 1000 rpm for 3 min. Foam disks were placed in culture plate wells, weighed down with small pieces of 15 mm glass tubing (to prevent the disks from floating), and were presoaked in 0.5 mL of culture medium. The cells in the pellet obtained from centrifugation were resuspended in 10 mL of medium, and the resulting suspension was divided between the wells (1 mL per well). The plate was incubated at 37 °C for 2.5 days, after which the samples were divided into two batches. The foams in the first batch, for SEM analysis, were washed several times with PBS solution then treated with a fixing solution of 0.1 M NaH2PO4 buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde. The solution was poured over the samples and left for 2 h with occasional agitation. Subsequently, the samples were washed with phosphate buffer to remove glutaraldehyde, covered with buffer solution containing OsO4 (1%), and left for 1 h. Following this, the foams were washed in aqueous solutions of increasing concentration of either ethanol or acetone up to pure organic solvent and then dried in CO2 at its critical point. The resulting dry foams were coated with gold for SEM analysis. The second batch of foam samples was washed in their wells several times with PBS solution and then treated briefly with PBS/methanol (50 vol %) solution twice. The wells were then filled with 100% methanol and left for 8 min, before being transferred to Petri dishes and air-dried for 30 min. After being replaced in their wells, the samples were covered with Giemsa stain solution and left for a further 8 min. Following this, the Giemsa solution was diluted with distilled water and left a further 8 min, then the samples were rinsed several times in distilled water and allowed to air-dry overnight. They were then imaged wet with a Zeiss Stemi SV11 microscope. Results and Discussion Macromonomer Synthesis and Network Formation. The acrylate macromonomers 1 and 2 were prepared from


Figure 1. 1H NMR spectrum of 2 (see Experimental Section for reaction conditions).

Figure 2. MALDI spectrum of 1: blue trace, diol starting material; pink trace, product (see Experimental Section for reaction conditions).

commercially available diols using simple chemistry as shown in Scheme 1. Scheme 1. Macromonomer Synthesis

A large excess of acryloyl chloride ensured that the products were obtained in high yield, as pale yellow to orange viscous liquids. The presence of acrylate end groups was confirmed by characteristic resonances in the 1H NMR spectrum at 5.8, 6.1, and 6.4 ppm (Figure 1). The extent of conversion was determined unequivocally by MALDI-tof mass spectroscopy (Figure 2). Each peak of the diol starting material (blue trace) is shifted to higher molecular weight by 108 units in the diacrylate product (pink trace); this figure corresponds to the weight of two acrylate units (H2CdCH-CO-) minus two protons. Any monosubstituted product would have resulted in peaks in the product spectrum at [(M)n + 54] that, from Figure 2, are clearly not present.


Before attempting to prepare PolyHIPE materials using the macromonomers, we felt it would be wise to check their performance in the formation of networks with common vinyl monomers. Consequently, macromonomer either alone or with monomer (styrene or methyl methacrylate) in the ratio of 2:1 (by wt) were dissolved in toluene with a few crystals of AIBN initiator, and the mixtures were purged with argon then heated at 60 °C. In all cases, soft clear gels were obtained after several hours that became white and opaque on immersion in methanol and, subsequently, regained the clear gel state on reimmersion in toluene without dissolving. We took this as an indication that the macromonomers were capable of homo- or copolymerization to form networks. PolyHIPE Preparation. The experimental conditions for the preparation of PolyHIPE materials from styrene and divinylbenzene are well established34 and so, as a starting point, we decided to emulate these conditions in the present work. Our first experiments involved replacing 20 wt % of the styrene with either 1 or 2 and keeping other factors (i.e., surfactant and its concentration) constant. High internal phase emulsions are inherently unstable and can be strongly influenced by relatively small changes in emulsion composition; however, in this case it appeared that the HIPE stability was not significantly compromised by the introduction of 20% PCL and emulsions of φ ) 0.9 were obtained easily and polymerized to corresponding PolyHIPE foams. The structure of the resulting materials was investigated by SEM and is shown in Figure 3a (this is representative of foams 0.2-L/H-S). This is the classic open-cellular PolyHIPE morphology exhibited by such systems as styrene/DVB. Each material possesses cells of around 20 µm diameter, indicating that the precursor emulsions were not as stable as those prepared with high levels of 4-vinylbenzyl chloride (VBC),32 for example, which produced foams with average cell diameters of 5 µm (emulsion droplet and therefore cell diameter are inversely related to emulsion stability). To ensure that we had chosen the optimum surfactant for the formation of HIPEs of the above composition, we decided to vary its hydrophile-lipophile balance (HLB) value.38 The HLB is a measure of the relative hydrophobicity of a (normally nonionic) surfactant and is expressed as a number varying from 0 (very hydrophobic) to 20 (very hydrophilic). The HLB value can be determined39 from group contributions according to eq 1 HLB )

∑Hi - n(0.475) + 7

(1)

where Hi is the group number of hydrophilic group i and n is the number of methylene groups, each of which is assigned a value of 0.475. The values of surfactants used in this study are given in Table 1. HLB is useful for choosing a surfactant type with which to stabilize a given emulsion since oil-soluble surfactants will stabilize water-in-oil (w/o) emulsions, and vice versa. It is possible to obtain HLB values intermediate between those of pure surfactants by blending according to a simple additive formula:40 HLBmix )

∑xi HLBi

(2)

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Figure 3. SEMs of PolyHIPEs with styrene as diluent: (a) 0.2-L-S; (b) 0.3-L-S; (c) 0.4-L-S; (d) 0.3-H-S; (e) 0.4-H-S (see Experimental Section for key). Table 1. HLB Values of Surfactants Used

a

surfactant

HLB valuea

Span 80 Span 85 Tween 80 Synperonic PE L121

4.3 1.5 15 0.5

Value quoted by manufacturer.

HLBmix is the HLB value of the surfactant mixture and xi and HLBi are the weight fraction and HLB value, respectively, of surfactant i. We used this formula to prepare surfactant blends of HLB value ) 5 from Tween 80 and Span 80, and 3.6 from Spans 80 and 85 (see Table 1 for HLB values of the pure surfactants). The former blend did not result in the production of a stable HIPE, whereas the HLB ) 3.6 blend gave a stable emulsion. This and the HIPE formed with Span 80 alone were heated at 60 °C (polymerization temperature), and stability was determined by monitoring the quantity of separated liquid phase produced with time. The results shown in Figure 4 indicate that Span 80 alone produces the most stable HIPEs, and so we conclude that our chosen surfactant is near optimum for this emulsion composition. It should be stressed that the HLB value of the surfactant is not the sole criterion for determining emulsion stability, important contributions coming from the length and nature of the hydrophobic and hydrophilic moieties and from the phase inversion temperature (PIT). This has been pointed out previously by Williams41 who demonstrated that it was not possible to form stable oil-inwater (o/w) HIPEs using sorbitan monostearate (Span 60)

Figure 4. Phase separation at 60 °C of 0.2-L-S HIPEs prepared with Span 80 (HLB ) 4.3) (squares) and 0.7 Span 80/0.3 Span 85 (HLB ) 3.6) (diamonds). See Experimental Section for quantities of components used.

despite an HLB value of 4.7. However, we feel that HLB is a useful rule-of-thumb for initial surfactant choice. Following this, we attempted to incorporate increasing amounts of PCL into the foam. This is desirable since complete biodegradability is a prerequisite for in vivo use of any given matrix. Table 2 entries 2, 3, 9 and 10 indicate that up to 40 wt % of PCL could be incorporated in the PolyHIPE materials, but at levels beyond this the high organic phase viscosity inhibited efficient mixing of the emulsion phases, and nonemulsified internal phase was observed after addition of a certain volume of aqueous solution. At this point in the discussion, it is beneficial to describe the various PolyHIPE preparation parameters and how they

Emulsion-Derived Foams Containing Poly(-caprolactone) Table 2. PolyHIPE Foams Prepared with PCL foama

PCL/g

diluent/g

HLB

water/mL

φ

0.2-L-S 0.3-L-S 0.4-L-S 0.2-L-M 0.4-L-M 0.2-L-T 0.5-L-T 0.2-H-S 0.3-H-S 0.4-H-S 0.2-H-M 0.4-H-M 0.2-H-T 0.5-H-T

1.0 1.5 2.0 1.0 2.0 1.0 2.5 1.0 1.5 2.0 1.0 2.0 1.0 2.5

4.0 3.5 3.0 4.0 3.0 4.0 2.5 4.0 3.5 3.0 4.0 3.0 4.0 2.5

4.3b 4.3b 4.3b 1.0c 1.0c 4.3b 4.3b 4.3b 4.3b 4.3b 1.0c 1.0c 4.3b 4.3b

45 45 35 45 15 45 15 45 45 35 45 30 45 15

0.90 0.90 0.88 0.90 0.84 0.90 0.75 0.90 0.90 0.88 0.90 0.84 0.90 0.75

a

See Experimental Section for key. b Span 80. c 0.4 PE L121/0.6 Span

85.

can impinge on foam structure. The surfactant type, through the interfacial tension γ, controls the diameter of the HIPE droplets d (direct relationship between γ and d) and thus the diameter of the PolyHIPE cells; more stable emulsions lead to smaller cells and a narrower size distribution. However, γ also affects the size of the “windows” interconnecting adjacent cells. This is because a decrease in γ causes a thinning of the interfacial films separating emulsion droplets.42 On polymerization, shrinkage is more significant causing larger windows to appear. If an emulsion is formed with less than optimum stability, phase separation is easily determined as, following polymerization, the foam structure may be completely absent, or its volume may be less than the original HIPE with a layer of phase separated liquid lying above and/or below. Another important parameter is continuous phase viscosity, which can be increased by the addition of polymers (as is the case here). This can increase emulsion stability by providing a kinetic barrier to droplet coalescence, which can result in materials with smaller cell sizes or can even lead to the formation of a HIPE from monomers not usually amenable to this (e.g., methyl methacrylate, as described here and by Ruckenstein et al.).43 However, if the continuous phase viscosity is too high, efficient mixing of the phases is inhibited, which limits the volume of droplet phase that can be added (and thus foam porosity).44 The HIPE simply becomes too viscous to mix properly. This is easy to discern as, at a certain point during its addition, a nonemulsified layer of aqueous droplet phase is seen in addition to the viscous, opaque HIPE. Another feature that can influence HIPE morphology is mechanical strength. A weak foam, i.e., one well above its glass transition temperature (Tg), will collapse on drying, and thus the morphology as determined by SEM will be different from the true structure produced by polymerization. In all work on PolyHIPE materials, we assume that the structure of the foam is an exact copy of the HIPE morphology at the gel point; however, this is an example of a case where this does not hold. The reader might wonder how the origin of the morphology of a PolyHIPE sample can be determined, given the myriad of factors that can influence it; however, this is in


fact relatively simple. If clear liquid is seen in addition to foam following polymerization, in conjunction with a lower volume of PolyHIPE compared to emulsion, then phase separation has occurred to a certain extent. The presence of large cells (with a broad size distribution) and/or macrocavities, shown by SEM, is another strong indication. In extreme cases, the foam may have regions with a noncellular morphology that better resembles the structure of macroporous resins. The presence of a cellular structure of slightly different appearance, on the other hand, is evidence that the emulsion has remained stable but other factors, such as differences in monomer density, viscosity, or emulsion interfacial tension, have resulted in a different structure. A foam that is mechanically weak will collapse during drying, resulting in a different morphology. However, this is easy to observe and, when combined with observation of the parameters described above, it is relatively simple to determine the source of the morphology as viewed by SEM. The structures of the PolyHIPE materials with 30 and 40% PCL are shown in panels b-e of Figure 3. The foams made with 1 all possess the PolyHIPE cellular morphology, although at the highest PCL content (Figure 3c) this is less easy to discern. However, in the materials containing high molar mass PCL macromonomer (Figure 3, panel d and especially panel e), this structure is not as obvious. This is likely to result from inefficient mixing of phases (the organic phase is quite viscous due to 20 wt % of macromonomer) rather than phase separation on heating, since the latter would be less likely to produce a highly porous material (the foam in Figure 3e is obviously porous, but with a different morphology to the others). Previously33 we have found that the presence of certain chlorinated organic phase diluents can induce an apparent transition in the morphology of PolyHIPE materials to one more resembling a traditional macroporous resin. We ascribed this to an extreme increase in the diameter of the interconnects between adjacent cells, which results in a material that appears to have a noncellular morphology. However, it is clear from Figure 3e that this is not occurring here. Subsequently we investigated the incorporation of methyl methacrylate (MMA) as an alternative to styrene into the PolyHIPE foams. MMA is expected to be more acceptable than styrene as a diluent since PMMA is an accepted biocompatible biomaterial, it being used commonly as bone cement in hip replacement surgery. Replacing styrene with MMA at the same concentration and keeping the surfactant constant (Span 80) did not result in a stable HIPE. It is known that the formation of HIPEs with MMA is more difficult than with styrene as the former is appreciably more polar. Indeed, Ruckenstein et al.43 demonstrated that prepolymerization of the MMA to around 5% conversion to increase the organic phase viscosity was necessary before stable HIPEs were formed. Consequently, it is perhaps not surprising that a stable emulsion was not obtained in our case. The increased polarity of the MMA results in an organic phase that is not sufficiently “different” from the aqueous phase45 to promote HIPE formation. The way to overcome this is to use a more hydrophobic surfactant, which will tend to increase the overall hydrophobicity of the organic phase.

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Table 3. Formation and Stability of HIPEs Containing MMA at Different Surfactant HLB Valuesa PCL usedb

HLB

observation

L L L H H H

2.6c 1.8d 1.0e 2.6c 1.8d 1.0e

no HIPE formed HIPE stable for ca. 15 min at room temp HIPE stable at 60 °C for several hours -f -f HIPE stable at 60 °C for several hours

a See Experimental Section for emulsion formulations. b L ) low molar mass.; H ) high molar mass. c 0.3 Span 80/0.7 Span 85. d Span 85. e 0.4 PE L121/0.6 Span 85. f The system was not investigated.

This reduces the solubility of MMA in the aqueous phase, which is the major reason for instability of MMA HIPEs. Table 3 shows the results of varying surfactant HLB, and it can be seen that stable HIPEs were indeed obtained with a blend of Span 85 and PE L121 having an HLB value of 1.0. This HLB value was used to prepare PolyHIPEs containing 20 wt % PCL and 80 wt % MMA; an SEM of the foams prepared with 1 is shown in Figure 5a (2 resulted in a material with the same morphology). The structure resembles the open-cellular morphology of foams prepared with styrene (e.g., Figure 3a) but with notable differences: the cells appear to be composed of fused rings. The origin of this structural difference is not know but could be due to differences in the density of the MMA and styrene

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(FMMA > FS). It is believed that the formation of the interconnects occurs because of shrinkage of thin monomer/ surfactant films on conversion to polymer.46 Since MMA is more dense than styrene, this process would be expected to occur to a lesser extent in the former case so a slightly different morphology may result. However, interfacial effects cannot be ruled out since the monomer/surfactant films will be thinner in more stable emulsions, leading to a more open cellular morphology. This was observed previously by us in foams made from HIPEs containing chlorinated aromatic solvents that were implied to cause a lowering of interfacial tension.33 Conversely, less stable HIPEs will have thicker interfacial films that will result in fewer interconnects of smaller diameter. The SEM of this material at lower magnification (Figure 5b) shows the presence of very large cells that would imply that emulsion droplet coalescence was occurring to an appreciable extent. This also tends to suggest that these HIPEs are less stable than their styrene-containing counterparts. We intend to investigate the interfacial properties under compression of films corresponding to the organic phase of these HIPEs, using methodology previously employed,32,33 to elucidate this point further. Once again we attempted to increase the quantity of PCL in these foams and similarly found an upper limit of 40 wt % beyond which the organic phase viscosity was prohibitively high. Also as before, the high viscosity of the higher

Figure 5. SEMs of PolyHIPEs with methyl methacrylate as diluent: (a) 0.2-L-M at high magnification; (b) 0.2-L-M at low magnification; (c) 0.4-L-M; (d) 0.4-H-M (see Experimental Section for key).



Figure 6. SEMs of PolyHIPEs with toluene as diluent: (a) 0.2-L-T; (b) 0.5-L-T; (c) 0.2-H-T; (d) 0.5-H-T (see Experimental Section for key).

molar mass PCL limited the value of φ, to 0.84 in this case (Table 2 entry 12). The SEMs of materials prepared with 1 and 2 at this concentration are shown in panels c and d of Figure 5. Traces of a cellular structure can be seen in the foam prepared with low molar mass PCL (Figure 5c), whereas macromonomer 2 with MMA (Figure 5d) gives a material composed in part of aggregates of polymer microgel particles. This indicates that the HIPE has undergone emulsion collapse to some extent prior to network formation. It is likely that, due to the lower concentration of vinyl groups in a given mass of macromonomer, gelation with 2 is slower and occurs after significant phase separation has taken place. The final set of materials investigated were those prepared with a nonpolymerizable organic phase diluent, namely, toluene. Our aim in this case was to eliminate completely the vinyl monomer additive to give materials that were composed almost entirely of polyester. Since toluene would be expected to have similar interfacial properties to styrene, we employed Span 80 alone as the surfactant. As anticipated, this allowed the facile production of foams with 20 wt % PCL in the organic phase (Table 2, entries 6 and 13). However, these materials were mechanically very weak and tended to collapse on drying, which is not surprising as only

2 vol % of the foam in its expanded dry state is solid polymer. Consequently, we increased the PCL content to 50 wt %, which resulted in stable HIPEs but again with limited values of φ (Table 2, entries 7 and 14). Nevertheless, the resulting foams were stronger mechanically. The morphologies of these PolyHIPE materials are shown in Figure 6. It appears that in this case the foams prepared from high molar mass PCL macromonomer (Figure 6, panels c and d) have more cellular morphologies, whereas species 1 gives structures that are porous but not recognizable as cellular foams. This is probably due to collapse, to varying degrees, of these foams on drying (these were prepared without added comonomer so have less mass per given volume). The lower molar mass precursor 1 will have a lower Tg than 2, so foams from the latter would be expected to be more mechanically stable, which could explain why the foams in panels c and d of Figure 6 have a more cellular structure. It should be stressed that none of these materials displays the agglomerated particulate structure that indicates emulsion collapse, so we conclude that the HIPEs have in these cases remained stable up to the point of gelation. Swelling Studies. The swelling of the various foams in toluene, 2-propanol (IPA), and water was investigated by

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Figure 7. Swelling of foams of various composition in water, 2-propanol (IPA), and toluene, prepared from (a) low molar mass PCL and (b) high molar mass PCL.

immersing accurately weighed quantities in excess solvent and determining the equilibrium mass of solvent taken up in each case. This will provide information about the polarity of each material and how it would respond in an aqueous environment akin to that encountered in the intended end use. Swelling is expressed as mass of solvent absorbed per gram of dry polymer, and the results are shown in Figure 7. Some of the foams absorb massive amounts of solvent, up to almost 20 times their dry mass, and this is an indication of their high porosity and interconnected structure. This absorptive capacity is extremely useful in tissue-engineering applications since it allows fluid to permeate the foam easily and thus ensure the delivery of nutrients to the cells growing on the matrix. Figure 7a shows the results for foams prepared with macromonomer 1, and some trends can be discerned. For example, the swelling in toluene is greatest for the foams prepared with styrene as comonomer since these are the most hydrophobic. In addition, swelling decreases as the quantity of PCL is increased due to a combination of a higher level of cross-linking (the macromonomers are difunctional) and the lower hydrophobicity of PCL. Swelling of the 0.2-L-M foam in toluene is almost as high as that of 0.2-L-S, but the corresponding foam with a higher level of PCL (0.4-L-M) has a much lower extent of swelling. This is presumably the result of a lower foam porosity (φ ) 0.84) together with a higher level of cross-linking and, to a lesser extent, the lower affinity of MMA for toluene. The swelling in toluene of foams prepared with toluene as diluent is lower than the other foams and interestingly increases with cross-linking, despite the lower porosity in the latter case. This might reflect the fact that the 0.2-L-T foam collapsed on drying and may not retain its original level of porosity on exposure to solvent. The higher PCL content and therefore more cross-linked sample is stronger mechanically and did not collapse on drying, so has more void volume available for solvent imbibition even though it was prepared from a HIPE of lower internal phase volume. The swelling in IPA of foams prepared with 1 is lower than in toluene, but the same overall trends are observed. The lower extent of swelling simply reflects the lower overall affinity of each foam for IPA compared to toluene. However, the swelling in water is markedly different. Here, all have low swelling except 0.2L-M, which is most probably due to the higher polarity of

MMA together with the relatively low level of cross-linking and higher porosity of this foam. The swelling of foams prepared with the higher molar mass macromonomer 2 (Figure 7b) displays the same trends as with foams prepared from 1, but again some significant differences are evident. For example, the swelling of the 0.4H-M foam in toluene is around 5 times that of the 0.4-L-M sample. The likely cause of this is the different network properties induced by the different molar masses of PCL starting material. The higher molar mass species 2 will give rise to a less tightly cross-linked network than 1 with more conformational freedom and therefore a greater swelling capacity. Both foams have the same porosity so the difference is not simply due to greater void volume available to solvent in the second case. The 0.4-H-M foam also displays much greater swelling in IPA and especially water than its low molar mass PCL counterpart, presumably for the same reason. Similarly, the swelling of 0.2-H-T in toluene (Figure 7b) is around twice that of 0.2-L-T (Figure 7a). Surprisingly, this trend is not repeated with the styrene series of foams; extent of swelling is independent of molar mass of PCL macromonomer used. Since the styrene foams had higher porosities at high PCL levels (φ ) 0.88 for 0.4-L/H-S), we conclude that the greater void volume overrides the inherent differences in swelling properties of the materials resulting from differences in network structure. Presumably the lower porosity of the MMA foams at high PCL content increases the influence of the network chemical structure on its ability to swell. The 0.2-L/H-T foams have a nominal porosity of 0.9 determined from the HIPE internal phase volume, but this figure may be misleading due to the collapse of the network on drying as described above. Therefore, the network structure of these materials may also play a more important role than porosity in determining the extent of swelling. Cell Seeding and Growth. Some initial experiments were conducted with the 0.2-H-S foam to test the feasibility of using these materials as media for cell growth. Small circles of foam of approximately 12 mm diamater were cut, placed in wells of a four-well culture plate (Nunc), and presoaked in culture medium. The disks were then seeded with cells (human fibroblasts) suspended in culture medium and incubated at 37 °C for 2.5 days. Subsequently, half the foam disks were fixed and dried for SEM analysis, the other half



Figure 8. SEMs of human fibroblasts growing on 0.2-H-S PolyHIPE samples.

fibroblasts are clearly shown as dark purple species, and it is noticeable that there are a large number of these and that they have fibroblast-typical flattened spindle-shaped morphology indicative of cell proliferation and tissue growth. From these initial results we conclude that the PolyHIPE foams described in this article are suitable matrixes for the growth of cells, in this case human fibroblasts. However, it should be pointed out that we have no data as yet to indicate whether the cells are penetrating into the porous materials. Conclusions

Figure 9. Optical microscopy image of stained 0.2-H-S foam seeded with human fibroblasts. Dark purple species are stained cells, the base matrix is shown as light purple to pink.

were treated with Giemsa stain for optical microscopy evaluation. SEMs of foam samples after this process are shown in Figure 8. It is clearly seen that the cells are present on the surface of the foams and that they have flattened out and produced extracellular matrix material. The cells also present many projections and are making contact with each other. These are good indications that the PCL-containing PolyHIPE matrixes are suitable for supporting the growth of this cell type. In the higher magnification SEM (Figure 8b) the edge of a growing cell is shown and it appears that this is interacting with the matrix underneath it. This again is a strong indication of the compatibility of the foam to the cells. The stained foams were analyzed by optical microscopy, a typical image being shown in Figure 9. The

Highly porous and permeable PolyHIPE foams containing poly(-caprolactone) (PCL) have been prepared by free radical polymerization of a PCL macromonomer either alone or with a comonomer (styrene or methyl methacrylate) in precursor HIPEs. The foam morphologies were investigated by SEM and their swelling properties studied in solvents of differing polarities. The foam structure was found to depend on the nature of the diluent added to the HIPE organic phase and on the concentration of PCL macromonomer. For HIPEs containing comonomer, an upper limit of 40 wt % PCL in the organic phase was found, above which HIPE formation was inhibited by high solution viscosity. The swelling of foams was found to vary depending on solvent type in the order water < 2-propanol < toluene, reflecting the inherently hydrophobic nature of most foams. Increasing content of PCL tended to decrease swelling due to an increase in the level of cross-linking (the macromonomers are difunctional), whereas increasing the molar mass of the PCL macromonomer generally increased swelling due to the greater chain length between cross-links leading to a less tightly crosslinked network. We have also presented initial results on the growth of human fibroblasts on these porous media, which indicate that the materials are sufficiently biocompatible to support cell function and growth over a period of 2.5 days. Further studies are underway to investigate the use of the matrixes described herein as media for the growth of different

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cell types and how the foam morphology influences cell adhesion and proliferation. The results of these experiments will be reported in future articles. Acknowledgment. The authors thank the EPSRC for a studentship for W.B. References and Notes (1) Thomson, R. C.; Wake, M. C.; Yaszemski, M. J.; Mikos, A. G. AdV. Polym. Sci. 1995, 122, 245. (2) http://www.advancedtissue.com/framehome.html. (3) http://www.organogenesis.com/. (4) Baldwin, S. P.; Saltzmann, W. M. Trends Polym. Sci. (Cambridge, U.K.) 1996, 4, 177. (5) Whang, K.; Thomas, C. H.; Healy, K. E.; Nuber, G. Polymer 1995, 36, 837. (6) Schugens, C.; Maquet, V.; Grandfils, C.; Jerome, R.; Teyssie, P. Polymer 1996, 37, 1027. (7) Schugens, C.; Maquet, V.; Grandfils, C.; Jerome, R.; Teyssie, P. J. Biomed. Mater. Res. 1996, 30, 449. (8) Ranucci, C. S.; Kumar, A.; Batra, S. P.; Moghe, P. V. Biomaterials 2000, 21, 783. (9) Nam, Y. S.; Park, T. G. Biomaterials 1999, 20, 1783. (10) Kang, H. W.; Tabata, Y.; Ikada, Y. Biomaterials 1999, 20, 1339. (11) Ishaug-Riley, S. L.; Crane-Kruger, G. M.; Yaszemski, M. J.; Mikos, A. G. Biomaterials 1998, 19, 1405. (12) Mikos, A. G.; Thorsen, A. J.; Czerwonka, L. A.; Bao, Y.; Langer, R.; Winslow, D. N.; Vacanti, J. P. Polymer 1994, 35, 1068. (13) Thomson, R. C.; Yaszemski, M. J.; Powers, J. M.; Mikos, A. G. Biomaterials 1998, 19, 1935. (14) Chen, G. P.; Ushida, T.; Tateishi, T. AdV. Mater. 2000, 12, 455. (15) Holy, C. E.; Dang, S. M.; Davies, J. E.; Shoichet, M. S. Biomaterials 1999, 20, 1177. (16) Shastri, V. P.; Martin, I.; Langer, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1970. (17) Hile, D. D.; Amirpour, M. L.; Akgerman, A.; Pishko, M. V. J. Controlled Release 2000, 66, 177. (18) Shea, L. D.; Smiley, E.; Bonadio, J.; Mooney, D. J. Nature Biotechnol. 1999, 17, 551. (19) Sheridan, M. H.; Shea, L. D.; Peters, M. C.; Mooney, D. J. J. Controlled Release 2000, 64, 91. (20) Barby, D.; Haq, Z. U.S. Pat. 4,522,953, 1985.

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PolyHIPEs - American Chemical Society

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