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Porous Biomaterials Obtained Using Supercritical CO2-Water Emulsions Cleofe Palocci, Andrea Barbetta,* Angelo La Grotta, and Mariella Dentini Department of Chemistry, UniVersity of Rome, “La Sapienza”, Piazzale A. Moro, 5 00185 Rome, Italy ReceiVed April 2, 2007. In Final Form: May 18, 2007 Highly porous, hydrophilic porous matrices were fabricated by using a high internal phase supercritical-CO2 (scCO2) emulsion templating technique. The novel aspect of the work resides in the combination of a natural biopolymer (dextran) as the building component of the matrices and of an environmentally benign solvent (supercritical-CO2) as the pore-generating phase. The synthetic route to the porous biomaterials involved the preliminary functionalization of the dextran chains with methacrylic moieties, formation of a scCO2-in-water concentrated emulsion, and curing of the external phase of the emulsion by radical polymerization. As the emulsion stabilizer a perfluoropolyether surfactant was chosen. The matrices obtained exhibit highly interconnected, trabecular morphologies. The porous biomaterial morphologies were qualitatively characterized by scanning electron microscopy (SEM) and the evaluation of void and interconnect sizes was carried out on the micrographs taken with the light microscope. To tailor the morphologies of the porous structures, the influence of the volume fraction of the internal phase and of the surfactant/ internal phase ratio was investigated. It was established that the variation of the volume fraction of the internal phase exerted only a limited influence on void and interconnect sizes. On the contrary the increase of surfactant concentration alters dramatically the distribution of void size, a large proportion of the void space enclosed within the matrix being attributable to voids with a diameter exceeding 100 µm. The free toxic solvent process of fabrication of the porous structures, the high water content, the expected biocompatibility, and the mechanical properties that resemble natural tissues make these porous hydrogels potentially useful for tissue engineering applications.
Introduction The level of interest in supercritical fluid (SFC) technology can be gauged from the growing number of participating academic and industrial research groups worldwide. Supercritical carbon dioxide (scCO2) has been considered as the solvent of choice for many applications of industrial interest because of its intriguing attributes; e.g., it is environmentally benign, nonhazardous, and very inexpensive. CO2 has a critical temperature near room temperature, a modest critical pressure, and a higher density than most supercritical fluids, which means that, at temperature slightly above room temperature, it is possible to obtain liquidlike densities and liquid-like solvent characteristics. By contrast the economics of using dense CO2 on an industrial scale are usually complex and must be assessed on a case-by-case basis.1 On this basis, significant efforts have been devoted to the use of alternatives to conventional organic and halogenated solvents in manufacturing and processing. The use of scCO2, as a process solvent, could give significant contributions to global environmental pollution, and it represents a versatile solvent for the synthesis and processing of a range of materials. According to this view, one of the fields of research that has taken advantage of the use of scCO2 is the synthesis of porous polymeric materials. Typically, a polymeric porous monolithic material characterized by a mesopore pore size distribution (2-50 nm) is obtained by blending an inert solvent with the monomeric phase. Polymeric matrices characterized by a pore size distribution in the range of 1-100 µm can be obtained by an emulsion templating technique. However, such an approach in the case of oil-inwater (O/W) emulsion is very solvent intensive due to the large volume of oil phase and to the need to remove effectively the * Corresponding author. Telephone: +39-6-49913633. E-mail: andrea
[email protected]. (1) (a) Hautal, W. H. Chemosphere 2001, 43, 123-135. (b) Cruz, F. J.; Szwajcer, D. E. Acta Microbiol. Pol. 2003, 52, 35-43. (c) Williams, J. R.; Clifford, A. A.; Al-Saidi, S. H. Mol. Biotechnol. 2002, 22, 263-86.
organic phase after reaction. To overcame this problems, a few groups have investigated the production of porous polymeric materials using scCO2 as a solvent2 and foaming agent.3 The removal of the template phase is simple since the CO2 reverts to gaseous phase upon depressurization. As part of our ongoing research program aimed at producing scaffolds that meet, both from a chemical composition and processing point of view, the demands placed by tissue engineering, we have exploited the high internal phase emulsion (HIPE) templating technique in the preparation of porous biomaterials. The procedure we developed consisted of dispersing toluene in an aqueous solution of a biopolymer (dextran, pullulan, gelatin, and recently hyaluronic acid and chondroitin sulfate) in the presence of an appropriate surfactant.4 This technique has wide appeal because it allows the synthesis of porous materials with a highly porous and interconnected morphology. The final goal was to develop templates that mimic the cellular microenvironment. However, as stated above, a significant disadvantage of such an approach is that concentrated O/W emulsions are very solvent intensive. The internal oil phase constitutes between 75 and 95% of the total reaction volume, and it may be difficult to remove completely this solvent from the material at the end of reaction. Since the limits on the tolerated amount of residual toxic contaminants that remain in the materials used in biomedical applications are strict, we were induced to seek nontoxic solvent (2) (a) Cooper, A. I. AdV. Mater. 2003, 15, 1049-1059. (b) Hebb, A. K.; Senoo, K; Bhat, R.; Cooper, A. I. Chem. Mater. 2003, 15, 2061-2069. (c) Butler, R.; Davies, C. M.; Cooper, A. I. AdV. Mater. 2001, 13, 1459-1063. (d) Butler, R.; Hopkinson, I.; Cooper, A. I. J. Am. Chem. Soc. 2003, 125, 14473-14481. (3) Howdle, S. M.; Watson, M. S.; Whitaker, M. J.; Popov, V. K.; Davies, M. C.; Mandel, F. S.; Wang, J. D.; Shakesheff, K. M. Chem. Commun. (Cambridge) 2001, 109-110. (4) (a) Barbetta, A.; Dentini, M.; De Vecchis, M. S.; Filippini, P.; Formisano, G.; Caiazza, S. AdV. Funct. Mater. 2005, 15, 118-124. (b) Barbetta, A.; Dentini, M.; Zannoni, E. M.; De Stefano, M. E. Langmuir 2005, 15, 118-124. (c) Barbetta, A.; Dentini, M.; Massimi, M.; Conti Devirgiliis, L. Biomacromolecules 2006, 7, 3059-3068.
10.1021/la700947g CCC: $37.00 © 2007 American Chemical Society Published on Web 06/23/2007
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alternatives. As far as we know only very limited work5 has been carried out on the preparation of porous biomaterials using biopolymers in combination with scCO2 as the pore-generating templating phase. In this work we describe the preparation of dextran-based porous structures by using scCO2 as the internal phase and outline the experimental and theoretical criteria for tailoring their porosity and morphology (sizes of voids and interconnects). Materials and Methods Materials. Dextran T40 with Mw ) 40 kg mol-1 was obtained by Amersham Biosciences (Sweden). Dimethyl sulfoxide (DMSO), glycidyl methacrylate (GMA), 4-(N,N-dimethylammino)pyridine (DMAP, 99%), and potassium peroxydisulfate were purchased from Sigma-Aldrich. Ammonium salt of perfluoropolyether (PFPE, Mw ) 550 g/mol) carboxylic acid was a gift from Solvay Solexis and was used as received. Synthesis of Methacrylated Dextran. The preparation of methacrylated dextran (DMA) was carried out using the synthetic procedure described previously.6 Dextran (40 g) was dissolved in DMSO (360 mL) in a stoppered round-bottom flask. After dissolution of DMAP (8.0 g), a calculated amount of GMA (10.5 g, equivalent to a theoretical degree of substitution of 30%) was added. The solution was stirred at room temperature for 48 h, after which the solution was stopped by adding an equimolar amount of concentrated HCl to neutralize the DMAP. The reaction mixture was transferred into a dialysis tube and extensively dialyzed against demineralized water at 4 °C until the nominal conductivity of water was reached. The solution of DMA was lyophilized, and the white fluffy product was stored at 4 °C before use. Quantitative determination of the degree of vinylic substitution (DS) was obtained by measuring the ratio between the average value of the integrals of the double-bond protons (δ ) 5.8-6.3 ppm) and the integrals of the anomeric protons signals (δ ) 4.8-5.6 ppm) in the 1H NMR spectra. The DS of DMA was 25%. Preparation of Dextran-Based Porous Strucutures. Highpressure reactions were carried out in a stainless steel reactor (20 cm3) stirred with a magnetic stir bar. The reactor was charged with a volume of an aqueous solution of DMA, PFPE, and K2S2O8 (1% (w/v) based on the volume of water) correspondent to the desired volume fraction of the external phase. Stirring and temperature were set at 600 rpm and 20 ( 0.5 °C, respectively. After the complete dissolution of the components was reached, the reactor was closed and stirring was continued for a further 30 min. The reactor was then charged with CO2 at a constant flow of 1 mL/min to obtain a final pressure of 10.0 ( 0.1 MPa. Stirring was continued for 30 min and then ceased before heating the reactor to the required reaction temperature (60 °C) for 20 h. After cooling to 20 °C, the CO2 was vented slowly and the solid foam was removed from the reactor. Samples were Soxhlet-extracted with water for 48 h and finally freeze-dried. The solid foams were designed with a two-numbers code: the first one indicates the nominal pore volume (volume fraction of the dispersed phase × 100); the second one indicates the concentration of the surfactant in the aqueous phase expressed as percent (w/v). For instance, the code 90-3.5 stands for a solid foam with a nominal pore volume of 90% (v/v) and synthesized using 3.5% (v/w) of PEFE. Characterization. Foam morphologies were investigated with a LEO 1450VP scanning electron microscope (SEM). The inner area of the fractured segments was mounted onto circular carbon adhesive pads attached to aluminum stubs and were gold-coated using a sputter coater (SEM coating unit 953, Agar Scientific). Morphometry of void and interconnect diameters was conducted on micrographs of specimen sections obtained by light microscopy (AXIOSCOP 2 (5) Partap, S.; Rehman, I.; Jones, J. R.; Darr, J. A. AdV. Mater. 2006, 18, 501-504. (6) van Dijk-Wolthuis, W. N. E.; Franssen, O.; Talsma, H.; van Steenbergen, M. J.; Kettens-van den Bosch, J. J.; Hennink, W. E. Macromolecules 1995, 28, 6317-6322.
Palocci et al. (plus) ZEISS). Specimens were fixed in 5% (w/v) of glutaraldehyde in phosphate-buffered saline (PBS) at room temperature. Samples were then repeatedly rinsed with water to remove the excess glutaraldehyde and then freeze-dried. Afterward, they were repeatedly soaked in 1,2-propylene oxide in order to fully dehydrate them. After most of the propylene oxide was evaporated, the specimen fragments were soaked in a 1:1 solution of propylene oxide and epoxy embedding resin (Fluka) for about 2 h. The samples were removed from this solution and soaked in pure resin for 3 h. Specimens were placed in plastic pyramidal molds and polymerized for 3 days at 60 °C. Sections 1 µm thick were cut with an ultramicrotome (UltracutE, Reichert Jung), collected on a glass slide, stained with 0.1% (w/v) of an aqueous solution of methylene blue, and coverslipped by using Eukit balsam. The measurement of voids and interconnects (several hundreds were taken into account) was carried out on the micrographs taken with the light microscope (LM) at a magnification of 200× and 400× using Scion Image (Scion Corp.) as a software tool. Raw data were used to calculate number distributions of both voids and interconnects as well as their relative averages. These were then taken as the void and interconnect sizes of each porous matrix. Density and Porosity. The density and porosity of the porous matrices were measured by liquid displacement.7 Hexane was used as the displacement liquid since it is a nonsolvent for the DMA solid foams, is able to easily permeate through the matrix, and does not cause swelling or shrinkage. A sample of weight W was immersed in a known volume (V1) of hexane in a graduated cylinder. The sample was left in the hexane covered for approximately 5 min. In this time, the contents in the cylinder underwent an evacuationrepressurization cycle to force the hexane through the pores. The total volume of the hexane and the hexane-impregnated matrix is V2. The volume difference (V2 - V1) is the volume of the polymeric matrix. The hexane-impregnated matrix was then removed from the cylinder, and the residual hexane volume was recorded as V3. The quantity V1 - V3, the volume of hexane within the scaffold, was determined as the void volume of the scaffold. The total volume of the scaffold was V ) (V2 - V1) + (V1 - V3) The density (F) of the solid foam is expressed as F ) W/(V2 - V3) and the porosity of the foam () was obtained by ) (V1 - V3)/(V2 - V3)
Results and Discussions Effect of the Volume Fraction of scCO2. As a model biopolymer for the preparation of concentrated C/W emulsions, dextran was chosen, an anhydroglucose polymer consisting mainly of R-(1-6)-glucosidic linkages with occasional branches at C(3).4a This highly flexible structure confers to dextran a high solubility in water. Furthermore, to make sure that the viscosity of the continuous phase is maintained at a level that allows the homogeneous dispersion of the internal phase, a dextran product characterized by a relatively low molecular weight (40 kg/mol) was used. To lock-in the structure of the external phase, once the concentrated emulsion is formed, the dextran chains were previously functionalized with methacrylic moieties (Figure 1). This derivation was accomplished through the reaction of the polysaccharide hydroxyls with glycidyl methacrylate catalyzed by 4-(dimetylamino)pyridine in DMSO. The degree of substitution as determined by 1H NMR was 25%. The subsequent step was the choice of a suitable surfactant to stabilize the C/W emulsions. As is well-known, many conventional hydrocarbon surfactants used to form O/W emulsions (7) Zhang, R. Y.; Ma, P. X. J. J. Biomed. Mater. Sci. 1999, 44, 446-455.
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Figure 1. (I) Reaction scheme for the synthesis of metacrylated dextran. (II) CO2/W HIPE formation and curing. A schematic representation of the polymeric network making up polyHIPE walls is shown in a.
Figure 2. Photograph of a low-density C/W emulsion templated methacrylated-dextran-based solid foam prepared according the procedure reported in Materials and Methods (see the text). The solid foam obtained conforms closely to the cylindrical interior of the reaction vessel.
exhibit low solubility in CO2 and are unable to generate W/C or C/W emulsions. By contrast and according to Johnston’s studies8 surfactants containing fluoroalkyl or fluoroether tails show good solubility. We used a PFPE surfactant characterized by a low molecular weight (550 g mol-1) since it was demonstrated that this surfactant exhibits significant water solubility and has the ability to form C/W rather than W/C emulsions. An aqueous solution of the DMA (20 or 25% (w/v)) and PFPE constituted the external phase of the emulsion. A radical initiator present in the aqueous phase (K2S2O8, 1% (w/v)) was another essential component of the formulation as it allows one to trigger the radical polymerization among the vinylic functionalities (Figure 1). The distinctive aspect of this work is that scCO2 was used as the internal phase. The solidified matrices conformed closely to the interior of the reaction vessel (Figure 2), suggesting that most of the scCO2 volume added was emulsified and templated and no significant shrinkage was observed upon CO2 venting. Morphologies of emulsion-templated polymeric materials depend on several chemical composition and processing parameters of the parent emulsions: concentration of the polymer dissolved in the continuous phase, type, and concentration of the surfactant used to stabilize the emulsion, the volume fraction of the dispersed phase (φ), and the method of emulsion preparation. In the case of C/W emulsion an additional parameter is represented by the density of CO2, which in turn is determined by the physical variables temperature and pressure. To aid finding a correlation (8) Lee, C. T., Jr.; Petros, P. A.; Johnston, K. P. Langmuir 1999, 15, 67816791.
between the chemical composition and physical variables of the parent emulsion and the morphologies of the ensuing porous structures, all the variables quoted above, with the exception of φ and surfactant concentration (Cs), were kept constant. Polymer concentration (Cp) was set at 20 or 25% (w/v). As specified in Materials and Methods, the pressure of scCO2 was set at 10 atm. In the first instance, Cs was kept constant at a value of 3.5% (w/v) with respect to the volume of the dispersed phase and φ was varied in the range between 0.75 and 0.95. In Figure 3 SEM and LM micrographs are displayed according to increasing φ. The qualitative inspection of both SEM and LM micrographs of the porous structures characterized by a nominal φ in the range between 0.75 and 0.92 (Figure 3a-c) shows that there is a significant increase in both the void and interconnect sizes when φ changes from 0.75 to 0.90. Above 0.90 such an increase is relatively less pronounced. A quantitative analysis of the morphology of the matrices reported in Figure 3 can be carried out in terms of the average dimensions of voids (〈D〉) and interconnects (〈d〉) as well as their distributions. In this respect, LM micrographs are particularly valuable because they represent a remarkable simplification of the complex morphology of the solid foams which result from projection in 2D. The results of such measurements are reported in Table 1 as well as the ratio 〈d〉/〈D〉, which effectively represents the degree of interconnection among voids. These data provide useful information on the structure of the emulsion prior to gelation. This in turn depends on the physical parameters of the emulsion, such as φ, the scCO2/surfactant ratio, and the interfacial tension. When the volume fraction (φ) of the dispersed phase in a monodisperse emulsion equals 0.74, the droplets can fit in a hexagonal close-packing arrangement without being deformed.9 When the concentration of the disperse phase exceeds this value, each droplet is deformed, and thin flat films of continuous phase are formed at each point where droplets touch. Each film is under a “compressive pressure” which is counteracted by a “disjoining pressure” that is developed within the thin liquid film. The disjoining pressure10 is a force normal to the droplet surfaces, which keeps them apart from each other. The magnitude of this pressure (π) is very much affected by the presence of surfactant molecules adsorbed at the C/W interface and can be calculated using the expression
π ) πa + πe + πsr
(1)
(9) Ostwald, W. Z. Chem. Ind. Kolloide 1910, 6, 103; 1910, 7, 63. (10) (a) Derjaguin, B. V.; Obukhov, E. V. Acta Physicochim. URSS 1936, 5, 1. (b) Derjaguin, B. V. Theory of Stability of Colloids and Thin Liquid Films; Plenum/Consultants Bureau: New York, 1989.
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Figure 3. Scanning electron (1) and light (2) micrographs (magnification, 400×; scale bar, 20 µm) of methacrylated-dextran solid foams characterized by an increasing nominal volume fraction (φ): (a) 75; (b) 90; (c) 92; (d) 95%. Cp ) 25% (w/v); Cs ) 3.5% (w/v).
where πa, πe, and πrs are the contribution to the local disjoining pressure of the van der Waals attraction forces, the double-layer repulsion forces, and the short-range attraction or repulsion forces, respectively. Micrographs reported in Figure 3 are coherent with such a theoretical picture: as φ increases, voids adopt a more pronounced polyhedrical shape. At the same time the skeletal framework of the porous structures becomes progressively thinner. This is the consequence of the distribution of the
continuous phase over a progressively larger interfacial area, which causes a concomitant thinning of the film of the continuous phase around the droplets of the dispersed phase. When the continuous phase is a polymerizing medium, phase separation of the polymeric network is accompanied by the appearance of interconnecting holes at the points of maximum vicinity among neighboring droplets due to volume contraction, which occurs on conversion of polymer to a network.11
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Table 1. Average Void (〈D〉) and Interconnect (〈d〉) Diameters and Corresponding Polydispersities (σD, σd), Density (G), and Porosity (E) of Methacrylated-Dextran Solid Foams 〈D〉 〈d〉 sample (µm) (µm) 〈d〉/〈D〉 σD〈D〉 σd〈d〉 F (mg/mL) 75-3.5 90-3.5 92-3.5 95-3.5 75-5.0 90-5.0
6.0 13.4 14.5 25.7 6.3 15.2
2.1 6.7 8.5 10.2 3.1 4.4
0.35 0.50 0.59 0.40 0.49 0.29
0.7 0.7 0.6 0.9 0.6 1.2
0.6 0.6 0.4 0.8 0.5 1.1
104 ( 1 33 ( 2 27 ( 3 38 ( 2 74 ( 2 68 ( 2
80 ( 2 94 ( 3 96 ( 4 91 ( 2 83 ( 2 84 ( 2
At a φ ) 0.75 (that is just above the close packing limit) the droplets of the dispersed phase are essentially spherical in shape; therefore, the areas of maximum approach among neighboring droplets will be very limited, and as a result the window holes among voids in the ensuing porous structures are very small (Figure 3(a1)). Conversely, when φ ) 0.92, droplets are considerably more compressed and deformed. In such circumstances the area of maximum approach among neighboring droplets is constituted by planar thin films which following polymerization bring about the formation of large interconnecting holes (Figure 3(c1)). The 〈d〉/〈D〉 data of Table 1 support the qualitative observation of a progressive thinning of the skeletal framework of the matrices. Also the behavior of the corresponding void (VDD) and interconnect diameter (IDD) distributions gives useful information about the characteristics of the parent emulsions (Figure 4(a1a3)). As can be observed, VDD and IDD tend to shift toward the increasing diameter side with the increase of φ and the tail on the same side tends to span over a broader diameter range. The position of the maximum of the distributions shifts in the same direction as well. The process of forming concentrated or high internal phase emulsions is a complex one, and the mechanism is not well understood. Theoretical treatments of emulsification are generally limited to the rupture of isolated droplets as first derived by Taylor.12 When an isolated, spherical droplet of radius R0 and relatively low viscosity ηi is sheared in a fluid of viscosity ηe, the droplet will deform into an ellipsoid or elongated cylinder only when the shear stress ηeγ˘ surpasses the interfacial stress σ/R0, where γ˘ is the shear rate and σ is the interfacial tension. When the capillary number (Ca ) ηeγ˘ R0/σ, the ratio of the two competing stresses) exceeds a critical value, Cacrit, the elongated droplet will rupture to give smaller droplets of average radius R:
σ R ∝ Cacrit ηeγ˘
(2)
Cacrit depends on the viscosity ratio (ηi/ηe) and the type of flow. Taylor’s analysis assumes a Newtonian continuous or external phase such that ηe is independent of γ˘ , a condition that holds for dilute emulsions with low concentration of surfactant. Nevertheless, this equation has proven to be effective in describing the average drop radius for some systems with a shear thinning continuous phase or a high internal phase volume fraction (φ), if eq 2 is modified by replacing ηe with an effective external phase viscosity, ηeff. Here, ηeff is a function of γ˘ (i.e., ηeff ∝ γ˘ -x) and represents the viscosity of either a concentrated surfactant solution or the highly viscous, shear-thinning concentrated emulsion itself. Thus, when φ e 0.74, the droplets of the dispersed (11) Cameron, N. R.; Sherrington, D. C.; Albiston, L.; Gregory, D. P. Colloid Polym. Sci. 1996, 274, 592-95. (12) (a) Taylor, G. I. Proc. R. Soc. London, Ser. A 1932, 138, 41. (b) Taylor, G. I. Proc. R. Soc. London, Ser. A 1934, 146, 501.
phase are spherical in shape, and when subjected to an external stress, they can flow past one another without being deformed and the rheological behavior of the emulsion is Newtonian. When the droplets are concentrated above the random close-packing limit, they cannot move freely and are trapped by their neighbors and take the shape of polyhedron. Furthermore, the C/W interfacial area increases. As a consequence ηeff increases with φ because part of the stress imposed will be dissipated as the work of droplets deformation. Thus, during the process of addition of the dispersed phase as φ increases the constant shear stress supplied will encounter increasing difficulties in incorporating the dispersed phase and homogenizing into a collection of droplets characterized by narrow size distribution. As a result, high φ porous structures tend to be characterized by a large polydispersity with respect to void size. If factors other than viscosity were operating such as an increase of the interfacial tension, or a certain degree of emulsion coalescence, this would have translated in characteristic morphological features in the ensuing solid foams. For instance, an increase of the interfacial tension accompanying the increase in φ would have implied a correspondent decrease in 〈d〉/〈D〉. On the contrary, experimentally, a reversed trend is observed. Also coalescence is accompanied by a decrease in 〈d〉/〈D〉 because the formation of larger droplets would decrease the interfacial C/W area. Density and porosity data increase monotonically with φ (Table 1), ruling out the occurring of coalescence. The two behaviors outlined above regarding void diameter distributions translate in an increase in the values of 〈D〉 and 〈d〉, as it is evidenced by data reported in Table 1. On the other hand the values of the polydispersity indexes normalized with respect to 〈D〉 and 〈d〉 (σD/〈D〉 and σd/〈d〉) are constant for φ ) 0.75 ÷ 0.92 solid foams. This indicates that the degree of tailing in both VDD and IDD is correlated with the shift of the entire distributions. On the contrary, both σD/〈D〉 and σd/〈d〉, referring to the solid foam characterized by a nominal φ ) 0.95, are significantly higher than the other samples of the series. Furthermore, while 〈d〉/〈D〉 increases pronouncedly (Table 1) for φ < 0.95 solid foams, that relative to the φ ) 0.95 one is markedly lower. These data combined with those relative to density and porosity data (Table 1) indicate that the parent emulsion of 9535 underwent coalescence to a certain degree. Coalescence is the irreversible process by which two or more droplets of the dispersed phase merge together forming a larger one. For an emulsion to be stable, the compressive pressure has to be balanced by the disjoining pressure developed within the thin liquid film, as a result of repulsive forces between the approaching droplet surfaces. As φ is increased further and further, a stage is reached where the disjoining pressure can no longer balance the compressive force, and the rupture of the interfacial film occurs. Film rupture could lead to an increase in the mean droplet diameter of the emulsion (coalescence). This effect reduces the compressive pressure to a value below the maximum disjoining pressure, and the interfacial liquid film becomes thicker. This is what is observed in practice as evidenced by the 〈d〉/〈D〉 value (Table 1). Effect of Surfactant Concentration. As is evident from the data presented so far, the variation of φ offers a limited tool to change over a wide range the morphological features of the matrices (see the 〈D〉 and 〈d〉 data of Table 1). On the contrary, the possibility of tailoring the size of voids and interconnects according to those required by applications represents a very important issue. For instance, the porous structures presented in the previous section are inadequate as scaffolds for tissue engineering. Void and interconnect dimensions are significantly
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Figure 4. Void pore diameter (a) and interconnect diameter distributions (b) of methacrylated-dextran solid foams characterized by an increasing pore volume: (1) 75; (2) 90; (3) 92; (4) 95%. Cp ) 25% (w/v); Cs ) 3.5% (w/v).
undersized with respect to those considered optimal for cell culture (100-200 and 40 µm, respectively). Therefore we were faced with the problem of increasing drastically both void and interconnect dimensions. In emulsion science an effect which has been known for a long time and well-understood from a theoretical point of view is depletion attraction.13 This attractive force arises when small particles are present within the continuous phase of the emulsion, for example, surfactant aggregates. Indeed, surfactants form small (nanometer-sized), approximately spherical aggregates called micelles when present at a concentration above the critical micellar concentration (cmc). As the surfaces of two neighboring droplets (13) Jenkins, P.; Snowden, M. AdV. Colloid Interface Sci. 1999, 68, 57-96.
are drawn close together as a consequence of the increase in φ, the gap between the two surfaces eventually becomes less than the diameter of the micelle and only solvent is present in the interstitial space. The non-adsorbed species effectively form an exclusion radius around each droplet. This is known as the depletion layer with a characteristic thickness dependent on the size of the adsorbing species. The concentrations of non-adsorbed entities increase outside of the gap and create an osmotic pressure gradient between the interstitial space and the solution at the plateau borders. As a consequence the droplets of the dispersed phase are pushed into contact. The interface in such circumstances may become very weak and not able to withstand the pressures of droplet contact and as a consequence is more prone to coalescence phenomena.
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Figure 5. Scanning electron (1) and light (2) micrographs (magnification, 400×; scale bar, 20 µm) of methacrylated-dextran solid foams characterized by an increasing nominal volume fraction (φ): (a) 75; (b) 90%. Cp ) 25% (w/v); Cs ) 5% (w/v).
The tendency of fluorosurfactants to form large aggregates of anisotropic shape in water solution is well-documented.14 Another important characteristic of fluorocarbon surfactants is their tendency to form stiff bilayer aggregates and structures with curvature lower than that of hydrocarbon surfactants.15 Rossi et al.16 have shown that the ammonium salt of perfluoroether carboxilic acid (M ) 680) in water form multilamellar vesicles very polydispersed in size (radii ranging from 10 to 120 nm). We speculated that depletion attraction in a concentrated emulsion may be exploited to induce a partial, controlled coalescence among neighboring droplets, thus leading to larger droplets (and eventually larger interconnects). With the aim being to confirm our hypothesis, the concentration of the PFE surfactant dissolved in the aqueous phase was increased from 3.5 to 5% (w/v and φ was set at 0.75, and 0.90. In Figure 5 the SEM and LM micrographs of 75-5.0 and 90-5.0 are shown. While the morphology of 75-5.0 underwent only minor changes with respect to 75-3.5, as evidenced by very similar values of 〈D〉 and 〈d〉 (the only significant difference is represented by a decrease of the polydispersed index and an increase in 〈d〉/〈D〉 which are a consequence of the increase in the surfactant/scCO2 ratio), that of 90-5.0 changed dramatically. Already qualitatively, it is evident from SEM micrographs that the morphology became much more heterogeneous with respect to void and interconnect sizes as compared to 90-3.5, and the LM micrograph (Figure 5(b2) shows that the void distribution is characterized by large voids surrounded by much smaller ones. As a result the σD/〈D〉 and σd/〈d〉 values are markedly higher than that referring to 903.5 and with respect to all the other porous structures listed in Table 1. Only a slight increase in 〈D〉 and 〈d〉 is recorded as a consequence of the increase in Cs, while 〈d〉/〈D〉 decreases. The 〈D〉 value of 90-5.0 is comparable to that of 90-3.5 because the (14) Kissa, E.; Fluorinated Surfactants; Science Series, No. 50; Marcel Dekker: New York, 1994, and references therein. (15) (a) Hoffmann, H.; Wu¨rtz, J. J. Mol. Liq. 1997, 72, 191. (b) Wang, K.; Karlsson, G. J. Phys. Chem. B 1999, 103, 9237. (16) Rossi, S.; Larlsson, G.; Ristori, S.; Martini, G.; Edwards, K. Langmuir 2001, 17, 2340-2345.
increase of the surfactant/scCO2 ratio, besides inducing the depletion attraction according to the mechanism outlined above, favors the dispersion and breaking of large droplets into finer ones. Thus, at the end of emulsion preparation, the population of droplets will be characterized by smaller dimension and a narrower distribution than the 90-3.5 emulsion. Afterward, coalescence phenomena induced by depletion attraction will alter considerably such a situation until the continuous phase reaches the gel point. The comparison of void and interconnect size distribution between 90 and 3.5 and 90 and 5.0 reveals that the position of the maximum VDD and IDD is negligibly changed (Figure 6). All these features indicate that the parent emulsion of 90-5.0 underwent coalescence to some extent. The occurring of coalescence is easily recognized because it causes the appearance of isolated very large voids characterized by dimension 1 or even 2 orders of magnitude larger than the surrounding ones, as has been documented by other authors.17 The droplets size distribution of a stable emulsion and of one undergoing coalescence phenomena differ by the presence in the latter case of a long tail extending toward the large diameter side, while the position of the maximum remains approximately unchanged.18 The experimental evidence reported strongly supports that the mechanism underlying the morphology of the solid foams of Figure 5 is due to depletion attraction. The occurrence of coalescence in emulsion containing a higher content of PFPE is a phenomenon which is clearly correlated with φ. This is because the distance at which the vesicles are excluded from the interstices between droplets is evidently dependent on φ. Thus, in the 755.0 emulsion the thickness of the film of continuous phase may be comparable to or lower than the diameter of the vesicles. This picture is coherent with the absence of any significant features characteristic of coalescence (Figure 5(a2)) in the morphology of 75-5.0. On the contrary, when φ ) 0.90, the thickness of the (17) (a) Tai, H.; Sergienko, A.; Silverstein, M. S. Polym. Eng. Sci. 2001, 41, 1540-1552. (b) Krajnc, P.; Stefanec, D.; Brown, J. F.; Cameron, N. R. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 296-303. (18) Aronson, M. P.; Petko, M. F. J. Colloid Interface Sci. 1993, 159, 134149.
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Figure 6. Void pore diameter (a) and interconnect diameter distributions (b) of methacrylated-dextran solid foams characterized by an increasing pore volume: (1) 75; (2) 90%. Cp ) 25% (w/v); Cs ) 5% (w/v).
in Figure 7 the void volume and area interconnect distributions of 90-3.5 and 90-5.0 are reported. It is evident that only in the latter type of porous structure there is a significant contribution to matrix void volume from voids exceeding of 100 µm in diameter (57%). The analogous comparison carried out with respect to interconnect size shows that 37% of interconnects have a diameter g 20 µm. Another positive feature of the 90-5.0 porous structure is that the lower pore volume (see porosity data of Table 1) implies thicker walls, as is evident from Figure 5(b2). Since in cellular solids there is strict correlation between the thickness of the foam’s walls and its mechanical properties,19 the 90-5.0 porous structure is expected to exhibit better mechanical properties (e.g., compressive moduli) than 90-3.5. Overall the 90-5.0 porous structure may be a good candidate as a scaffold for tissue engineering applications.
Conclusion
Figure 7. Volume distribution of voids (a) and area distribution of interconnects (b) of 90-35 (red line) and 90-50 (black line).
film of the continuous phase at the region of maximum approach among neighboring droplets is lower than the vesicles dimension. This induces depletion attraction and to a certain extent coalescence as is evidenced in Figure 5(b2). Attempts to prepare emulsions characterized by higher φ and containing 5% (w/v) of surfactant failed. The porous structures obtained were characterized by a collapsed morphology, indicating that the parent emulsion underwent extensive phase separation. An attractive feature of the approach described above is that it is possible to increase significantly the size of voids in a very simple way, that is, by adjusting the concentration of surfactant to a value that induces a controlled coalescence in the parent emulsion. In this manner, a relevant proportion of the scaffold void volume is due to large voids. To better illustrate this point,
This paper presents one of the very few attempts to synthesize porous biomaterials through the employment of a natural polymer such as dextran in combination with a pore-generating procedure based on scCO2/W emulsion templating. Such an approach allowed the synthesis of matrixes characterized by an open highly interconnected porous architecture, a feature which is very important for tissue and cells in-growth and 3D colonization. Moreover, this methodology does not involve any volatile organic or toxic solvents either in the synthesis or in the purification steps. It was shown that the morphology of the porous structure can be tailored only to a limited extent by varying the volume fraction of the dispersed phase and in any case the size of the voids and interconnects are not adequate for the viable colonization by seeded cells. In an effort to increase both void and interconnect sizes drastically, a partial destabilization of the parent emulsion was induced by exploiting depletion attraction, an effect which was shown to be related to surfactant concentration. At a Cs ) 5% (w/v) the parent emulsion characterized by a relatively high (19) Gibson, L. J.; Ashby, M. F. Cellular Solids: Structure and Properties, 2nd ed.; Cambridge University Press: Cambridge, U.K., 1997.
Biomaterials Using Supercritical CO2-H2O Emulsions
φ (0.90) underwent partial coalescence, enabling the formation of a high percentage of large voids in the ensuing porous structure. The suitability of such biomaterials as supports for cell cultures will be tested in the near future. Future work will be addressed to improve further the morphology of the matrices (especially the size of the interconnects) and to extend this approach to other biologically important biopolymers such as gelatin, hyaluronic acid, and chondroitin sulfate. In this context, it is worth reporting that recently hydrocarbon alternatives to perfluroether surfactants have been developed.20 These surfactants are block copolymers of poly(vinyl acetate) and poly(ethylene glycol). The biocompatible and potentially biodegradable nature of such components make these surfactants especially suited in the preparation of porous biomaterials intended for biomedical applications.
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Another potentially positive feature of the block copolymers developed by Cooper et al.20 resides in their polymeric nature. It is well-known that polymeric surfactants tend to lower the interfacial tension of, for instance, O/W emulsion, to a lesser extent than low molecular weight surfactants,21 thus enabling the formation of porous materials characterized by larger voids and interconnects. This would represent an important step forward to the synthesis of scaffolds characterized by adequate dimensions of voids and interconnects by using the C/W emulsion templating technique. Acknowledgment. The authors thank the Univerity of Rome (Ateneo funds) and Consorzio Interuniversitario Biotechnologie (CIB) for funding this research and Dr. Daniela Ferro for invaluable help with the scanning electron microscope. LA700947G
(20) (a) Lee, J.-Y.; Tan, B.; Cooper, A. I. Macromolecules 2007, 40, 19551961. (b) Tan, B.; Lee, J.-Y.; Cooper, A. I. Macromolecules 2007, 40, 19451954. (c) Tan, B.; Cooper, A. I. J. Am. Chem. Soc. 2005, 127, 8938-8939.
(21) Rotureau, E.; Marie, E.; Dellacherie, E.; Durand, A. Colloids Surf., A 2007, 301, 229-238.
