Tailoring the Porosity and Morphology of Gelatin ... - ACS Publications

Nov 12, 2005 - di Biologia Cellulare e dello Sviluppo, University of Rome “La Sapienza”, ... Polymerization of the continuous phase gave rise to a...
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Langmuir 2005, 21, 12333-12341

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Tailoring the Porosity and Morphology of Gelatin-Methacrylate PolyHIPE Scaffolds for Tissue Engineering Applications Andrea Barbetta,*,† Mariella Dentini,*,† Elisabetta M. Zannoni,† and Maria E. De Stefano‡ Department of Chemistry, University of Rome “La Sapienza”, Rome, Italy, and Dipartimento di Biologia Cellulare e dello Sviluppo, University of Rome “La Sapienza”, Rome, Italy Received July 26, 2005. In Final Form: October 3, 2005 Gelatin is a natural protein with many desirable properties for application as a biomaterial, including scaffolding for tissue engineering. In this work gelatin A with a molecular weight in the range 50-100 kg mol-1 was modified with methacrylic anhydride and processed into a concentrated oil-in-water emulsion. Polymerization of the continuous phase gave rise to a polyHIPE, a porous material possessing a highly interconnected, trabecular morphology. In the paper, we focused on the goal of obtaining matrixes characterized by suitable sizes of both voids and interconnects, to allow an in depth colonization from transplanted cells. In this respect, we investigated the role of the volume percentage of the dispersed phase and the effect of additives. It was established that high pore volumes (g90%) are to be preferred, because they allow the production of solid foams characterized by average void and interconnect diameters of approximately 20 and 10 µm, respectively. These values are still inadequate for the intended application of these scaffolds but represent a good starting point for further improvements. These were achieved through the use of additives, namely sodium chloride and dimethyl sulfoxide, which partially destabilized the precursor emulsion and allowed a solid foam to be obtained with void and interconnect diameters in the range of 30-150 µm and 10-50 µm, respectively.

Introduction PolyHIPEs are a class of porous and permeable materials prepared by curing the continuous phase of a high internal phase emulsion (i.e., in which the volume fraction of the droplet phase is g0.74). The ensuing porous matrixes are characterized by a trabecular morphology: cavities (hereinafter defined as voids) of approximately spherical shapes are interconnected by a plurality of window holes (hereinafter defined as interconnects). Of vital importance to the use of polyHIPEs in advanced materials applications is the ability to control morphology (surface porosity, void/ interconnect size) and physical properties (surface area). In this respect many investigations have been carried out. For instance, it is possible to tune the solid foam morphology by either processing condition1 and/or by introducing additives or proper comonomers2-4 in the emulsion formulation. High surface area polyHIPE materials can be obtained by replacing some of the monomer in the HIPE continuous phase with a non-polymerizable organic solvent. The combination of both a thermodynamically compatible solvent with the growing polymer network and a suitable surfactant mixture made it possible to obtain surface area in excess of 700 m2 g-1. 4-6 * To whom correspondence should be addressed. Tel: +39-0649913633. Fax: +39-06-4457112. E-mail: andrea.barbetta@ uniroma1.it (A.B.); [email protected] (M.D.). † Department of Chemistry. ‡ Dipartimento di Biologia Cellulare e dello Sviluppo. (1) Akay, G. Flow induced phase inversion in powder structuring by polymers. In Polymer Powder Technology; Ins, M., Rosenzweig, N., Eds.; Wiley: New York, 1995; Chapter 20, pp 541-87. (2) Akay, G.; Price, V. J.; Downes, S. European Patent No. 1,183,328, 2002. (3) Barbetta, A.; Cameron, N. R.; Cooper, S. J. Chem. Commun. 2000, 221-222. (4) Cameron, N. R.; Barbetta, A. J. Mater. Chem. 2000, 10, 24662472. (5) Barbetta, A.; Cameron, N. R. Macromolecules 2004, 37, 32023213.

In an attempt to widen the possible applications, the traditional composition (based mostly on the styrenedivinylbenzene couple) of water-in-oil (W/O) HIPEs was expanded to include water soluble monomers and crosslinkers. In a series of patents and articles, methodologies for the productions of either monolithic and beads-like polyHIPEs from acrylic acid/N,N′-methylene-bis-acrylamide,7 acrylamide, N-isopropylacrylamide, and HEMA8 are described. As far as the biomedical applications of polyHIPEs are concerned, an aspect which needs a substantial improvement is the chemical composition of the matrixes. Both the academic and patent literature describe the use of styrene-divinylbenzene in the preparation of polyHIPEs as cell growth supports and microreactor. For instance, both Akay et al1,9 and Busby et al10 based their polyHIPE matrixes on styrene-divinylbenzene or a blend of either polycaprolactone or polylactic and styrene. However, these synthetic polymers are very hydrophobic. The wettability of a polymer scaffold is considered very important for homogeneous and sufficient cell seeding in three dimensions.11 In the case of synthetic hydrophobic scaffolds, it is difficult to deliver a cell suspension in a manner that (6) Barbetta, A.; Cameron, N. R. Macromolecules 2004, 37, 31883202. (7) Benson, J. R. Am. Lab. 2003, 35, 44-52. Li, N.-H.; Benson, J. B.; Kitagawa, N. U.S. Patent No. 5,760,097, 1998. Kitagawa, N. WO 99/ 00187, 1999. Li, N.-H.; Benson, J. B.; Kitagawa, N. U.S. Patent No. 5,863,957. Kitagawa, N. U.S. Patent No. 6,048,908, 2000. (8) Zhang, H.; Cooper, A. I. Chem. Mater. 2002, 14, 4017-4020. (9) Akay, G.; Birch, M. A.; Bokhari, M. A. Biomaterials 2004, 25, 3991-4000. (10) Busby, W.; Cameron, N. R.; Jahoda, C. A. B. Biomacromolecules 2001, 2, 154-164. Busby, W.; Cameron, N. R.; Jahoda, C. A. B. Polym. Int. 2002, 51, 871-881. (11) Freed, L. E.; Marquis, J. C.; Nohria, A.; Emmanual, J.; Mikos, A. C.; Langer, R. J. Biomed. Mater. Res. 1993, 27, 11-23. Wald, H. L.; Sarakinos, G.; Lyman, M. D.; Mikos, A. G.; Vacanti, J. P.; Langer, R. Biomaterials 1993, 14, 270-278.

10.1021/la0520233 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/12/2005

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uniformly distributes transplanted cells throughout the porous scaffolds. Another aspect concerns the biocompatibility of such hydrophobic scaffolds. Generally, our living system recognizes the biomaterials foreign bodies from their surface. Therefore, a biomaterial that has a surface quite different from that of the living structures may be very poor in interfacial biocompatibility. One way to change the surface of a man-made material into a biocompatible one is to cover the material surface with biological components. As the major constituents of the human body in contact with biomaterials include water, hydroxyapatite, and collagen in amounts of approximately 60, 7, and 4% of body weight, respectively, it seems likely that the living system will not be able to recognize an implanted material as a foreign body if the surface is fully covered with water, collagen, or hydroxyapatite. This rationale induced Akay et al.1,9 and Hayman et al.12 to coat a styrene-divinylbenzene polyHIPE with a layer of hydroxyapatite and poly-D-lysine, respectively, to improve either the hydrophilicity and the surface biocompatibility towards the seeded cells. This strategy was relatively successful but it tends to hide the core problems inherent with such an approach: (1) the use of toxic monomers and (2) their polymerization in a W/O emulsion. These two issues are intimately related to each other. The polymerization of the hydrophobic monomers takes place in the organic phase of the emulsion. As a consequence, it may be difficult to purify the scaffolds exhaustively from unreacted monomers, additives, and surfactant that may remain trapped in the cross-linked monolithic matrix. Residues of organic contaminants which can remain in these polymers after processing may damage the transplanted cells and nearby tissue. Additionally, many biologically active factors (e.g., growth factors) are inactivated by exposure to organic solvents. Thus, incorporated growth factors may be inactivated during the emulsion formation and polymerization. Finally, styrene and divinylbenzene are scarcely or at all biodegradable. Aware of these problems, we have adopted a completely new synthetic route for the production of polyHIPE scaffolds suitable for the culture of cells.13,14 Our approach makes use of biopolymers as the constituents of the solid foams. This approach presents numerous advantages with respect to conventional polyHIPEs obtained from synthetic monomers: (1) The solid foams are highly hydrophilic. (2) They possess many chemical groups amenable to functionalization which can be exploited for the introduction of, for instance, bioactive species. (3) The solid foams are derived from O/W emulsions. This means that the dispersed organic, droplet phase is separated from the external, polymerizable phase. This represents a guarantee that the organic contaminants will be exhaustively removed. (4) The scaffold pore architecture is highly interconnected, a feature that is essential for three-dimensional colonization in tissue engineering applications. The denaturated type collagen, gelatin, has been used in medicine as a plasma expander, wound dressing, adhesive and adsorbent pad for surgical use. Although collagen, also known to have wide biomedical applications, expresses antigenicity in physiological condition, gelatin (12) Hayman, M. W.; Smith, K. H.; Cameron, N. R.; Przyborski, S. A. Biochem. Biophys. Res. Commun. 2004, 314, 483-488. Hayman, M. W.; Smith, K. H.; Cameron, N. R.; Przyborski, S. A. Biochem. Biophys. Res. Commun. 2005, 62, 231-240. (13) Barbetta, A.; Dentini, M. Italian Patent, A000307, 2003. (14) Barbetta, A.; Dentini, M.; De Vecchis, M. S.; Filippini, P.; Formisano, G.; Caiazza, S. Adv. Funct. Mater. 2005, 15, 118-124.

Barbetta et al.

is known to have no such antigenicity.15 Recently, gelatin has shown to exhibit activation to macrophages16 and high hemostatic effect.17 Finally, gelatin is practically more convenient than collagen because a concentrated collagen solution is extremely difficult to prepare, and furthermore, gelatin is by far more economical than collagen.18 The aim of this article is to supply a detailed experimental and theoretical framework as a guidance for the preparation of polyHIPEs made from gelatin and to show how to tailor the porosity and morphology of such solid foams (size of voids and interconnects) in order to render them suitable as scaffolds for tissue engineering applications. Experimental Section Materials. Gelatin A3 (extracted from porcine skin and with a Bloom number equal to 300) was supplied by Sigma-Aldrich. The surfactant Triton X-405 (70% w/v solution in water), methacrylic anhidride (MA), 2,2′-azoisobutyronitrile (AIBN), sodium chloride, dimethyl sulfoxide (DMSO) and toluene were purchased from Aldrich and used without further purification. Gelatin A3 Purification. Prior to use gelatin was purified according to the following procedure: twenty grams of A3 commercial gelatin was suspended in 100 mL of water (20% w/v), and the solution was kept under stirring for 30 min. The mixture was heated at 60 °C for 10 min, then cooled to room temperature, and left to stand overnight. The gel obtained was dialyzed (at 4 °C) against repeated changes of distilled water for several days to eliminate salts (dialysis water finally reaches the nominal conductivity of distilled water). The gel was put in 5 L of distilled water, stirred, and heated at 60 °C to disaggregate the physical network. The final solution was filtered on sintered glass and then freeze-dried. Gelatin Vinylation. Gelatin methacrylate (GMA) was prepared by reaction of the gelatin primary amines groups with methacrylic anhydride. After dissolution of gelatin (15% w/v) in phosphate buffer (pH ) 8) at 45 °C, an excess of methacrylic anhydride was added while vigorously stirring. After 2 h of reaction, the reaction mixture was brought to room temperature and let to stand still overnight. The gel was dialyzed against acetone for 3 days and water at 4 °C until the nominal conductivity of water was reached. The DS as determined through the method of Habeeb was 92%.19 Preparation of PolyHIPEs. The procedure for the production of methacrylate gelatin based PolyHIPEs is as follows: the calculated amount of the vinylated biopolymer (20 or 25% w/v) was dissolved in 2.5 mL of water together with the surfactant Triton X-405 (net 7 or 8.5% w/v). This solution was placed in a three necked round-bottom flask partially submerged in a water bath thermostated at 50 °C in order to keep the gelatin solution in liquid form. The flask was equipped with a dropping funnel consisting of a condenser connected to the thermostat and provided with a valve. This assured that the dispersed phase, 1% w/v of AIBN in toluene, was maintained at the same temperature of the gelatin solution. The shear necessary for the dispersion of the organic phase into the continuous aqueous solution was provided by a D-shaped paddle driven by a mechanical stirrer set at 350 rpm. After completing the addition of toluene, the emulsion was kept under stirring for further 20 min, transferred into a cylindrical plastic container and placed into an oven at 60 °C for 1 day. At the end of this time the solid foam was soaked into DMSO, which was changed regularly (typically three times a day) for one weak. This procedure was (15) Chvapil, M. J. Biomater. Mater. Res. 1977, 11, 721-741. Sela, M.; Arnon, R. Biochem. J. 1960, 75, 91-102. (16) Anderson, J. M.; Miller, K. M. Biomaterials 1984, 5, 5-10. Tabata, Y.; Ikada, Y. J. Pharm. Pharmacol. 1987, 39, 698-704. (17) Chvapil, M. J. Biomater. Mater. Res. 1982, 16, 245-263. (18) Choi, Y. S.; Hong, S. R.; Lee, Y. M.; Song, K. W.; Park, M. H.; Nam, Y. S. Biomaterials 1999, 20, 409-417. Choi, Y. S.; Hong, S. R.; Lee, Y. M.; Song, K. W.; Park, M. H.; Nam, Y. S. J. Biomed. Mater. Res. 1999, 48, 631-639. Choi, Y. S.; Hong, S. R.; Lee, Y. M.; Song, K. W.; Park, M. H.; Nam, Y. S. J. Mater. Sci.: Mater. Med. 2001, 12, 67-73. (19) Habeeb, A. F. Anal. Biochem. 1966, 14, 328-336.

Tissue Engineering Applications of PolyHIPE aimed to displace toluene thoroughly. The solid foam was then Soxhlet extracted with water for 1 day and finally freeze-dried. PolyHIPE Coding System. The polyHIPE materials are classified by a code which is dependent on the polymer concentration, pore volume, nature, and amount of additives present in either the aqueous or organic phases. The codes for the basic gelatin-methacrylate systems have the general form GMAaPVbScDdTe, where a is GMA concentration in the aqueous phase, PV is the nominal pore volume, as determined by the organic phase content, as a percentage, S indicates the presence of NaCl whose concentration is expressed in moles/l (c) with respect to the volume of aqueous phase, D indicates the presence of dimethyl sulfoxide (DMSO) whose concentration (d) is expressed in % v/v with respect to the volume of the dispersed phase and T stands for Triton X-405 dissolved in the aqueous phase at a concentration e % w/v. Thus GMA20PV92S0.01D1%T8.5 would represent a polyHIPE material of 20% w/v of GMA in the aqueous phase, 92% pore volume, containing NaCl 0.01 M and DMSO 1% v/v and prepared by using Triton X-405 at a concentration of 8.5% w/v. Where the surfactant concentration is not specified it is assumed to be 7% w/v. Characterization. Foams morphologies were investigated with a LEO 1450VP scanning electron microscope (SEM). The inner area of fractured segments were mounted onto circular carbon adhesive pads attached to cylindrical aluminum stubs and were gold coated using a sputter coater (SEM COATING UNIT 953, Agar Scientific). Morphometry of void and interconnect diameters were conducted on micrographs obtained by light microscopy (Nikon 104 equipped with a JVC TK-1070 E video camera). Specimens were treated with a 5% w/v of glutaraldehyde in PBS buffer in order to strengthen their structure and were repeatedly washed with water to remove excess glutaraldehyde and then freeze-dried. Afterward, they were embedded with a resins (Lowcryl K4M, Polyscience). Sections 1 µm thick, were cut with an ultramicrotome (UltracutE, Reichert Jung), collected on a glass slide, stained with a 0.1% w/v of an aqueous solution of Toluidine Blue and 0.1% w/v Borax, and cover slipped by using Eukit balsam. The measurement of voids and interconnects was carried out on micrograph taken with the light microscope (LM) at a magnification of 200 and 400× using Scion Image (ScionCorporation) 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 size of each porous matrix. Density and Porosity. The density and porosity of the polyHIPEs were measured by liquid displacement.20 Hexane was used as the displacement liquid since it is a nonsolvent for gelatin, 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 evacuation-repressurization cycle to force the hexane through the pores. The total volume of the hexane and the hexaneimpregnated 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), 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 Discussion Derivatization of Gelatin. Functionalization of gelatin with methacrylic moieties is readily accomplished through the reaction with methacrylic anhydride in basic (20) Zhang, R. Y.; Ma, P. X. J. J. Biomed. Mater. Sci. 1999, 44, 446455.

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Figure 1. Scanning electron (1) and light (2) micrographs (magnification 400 ×, scale bar 20 µm) of GMA PolyHIPE solid foams characterized by an increasing nominal pore volume (PV): (a) 75%; (b) 85%; (c) 90%; (d) 92%; (e) 95%. Cp ) 20% w/v for the solid foams shown in (a), (b) and (c) and 25% w/v for the solid foams shown in (d) and (e). Cs ) 7% w/v.

aqueous media.21 The degree of functionalization was 92% (see the Experimental Section). Effect of the Pore Volume. As a first attempt to vary the porosity and morphology of the gelatin-MA (GMA) polyHIPEs, we investigated the influence of the volume percentage of the internal phase (PV) and of GMA concentration in the aqueous phase (Cp). Surfactant concentration (Cs ) 7% w/v) was kept constant in all the range of PVs and Cp explored. Cp was set at 20% w/v in the range of PV e 90% while for PV > 90% (i.e., 92 and 95%) it was increased to 25% w/v. In Figure 1, SEM and LM micrographs of polyHIPE solid foams are displayed according to increasing PV. The internal phase volume fraction in a HIPE, φ, is larger than the critical value of the most compact arrangement of uniform, undeformed spherical drops φc ) 0.74.22 As the volumetric fraction of (21) Wang, L. F.; Shen, S. C. Carbohydr. Polym. 2003, 52, 389-396. Van den Bulcke, A. I.; Bogdanov, B.; De Rooze, N.; Schacht, E. H.; Cornelissen, M.; Berghmans, H. Biomacromolecules 2000, 1, 31-38. (22) Ostwald, W. Kolloid Z. 1910, 6, 103; 1910, 7, 63.

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dispersed phase increases beyond the maximum compaction of spherical drops, film formation starts at the faces of the deformed drops. In the limiting case, the drops assume polyhedrical shapes separated by thin films consisting of the surfactant and the GMA solution making up the continuous phase. The plateau borders at the edges act as reservoirs of surfactant and GMA solution, which depletes as the dispersed phase fraction approaches one. Micrographs reported in Figure 1 are coherent with such a theoretical picture: as PV increases voids adopt a more pronounced polyhedrical shape. At the same time the skeletal framework of the solid foams 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 continuous phase around the droplets of the dispersed phase. Mechanical properties (i.e., compression moduli) are adversely affected by too high a thinning of the solid foam walls as predicted on theoretical grounds.23 One of the most attractive features a scaffold must possess is a high degree of interconnection among voids, a factor that is critical for maintaining cell penetration and tissue formation throughout the depth of the scaffold. A high degree of interconnection in polyHIPEs is assured by a high PV (g90%). In an attempt to stabilize high PV (92 and 95%) solid foams, at least partially, a Cp of 25% w/v was employed. Even with this relatively high Cp, GMA25PV95 resulted comparatively weaker than PV < 95% solid foams. A feeling of this can be drawn from the comparison between SEM and LM micrographs of GMA25PV95 (Figure 1, e1 and e2). SEM shows a solid foam with a well-defined morphology, on the contrary the corresponding LM micrograph shows a distorted void pattern. This reflects the inherent mechanical weakness of such a solid foam characterized by a very thin skeletal framework that under the sectioning procedure (see the Experimental Section) suffered some degree of damage. Attempts to use even higher Cp (i.e., 30% w/v) were unsuccessful: the emulsion did not form, probably because the viscosity of the continuous phase was so high that it prevented the efficient dispersion of the droplet phase. A quantitative analysis of the morphology of the scaffolds reported in Figure 1 can be carried out in term of average dimensions of voids and interconnects as well as their distributions. In this respect, LM micrographs are particularly valuable because they represent a remarkable simplification of the complex morphology of polyHIPEs which results from projection in 2D. The average void and interconnect diameters of GMA25PV95, 〈D〉 and 〈d〉 , were estimated from the SEM micrograph which for the reason stated above were considered more reliable (even though less precise) than the corresponding LM micrograph. 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, one of the most important of which is the interfacial tension. A relatively low interfacial tension value will favor the dispersion of the internal phase because the work required to break droplets into smaller ones is relatively low. This translates into the formation of voids of smaller dimension. The three-dimensional (23) Gibson, L. J.; Ashby, M. F. Cellular Solids: Structure and Properties, 2nd ed.; Cambridge University Press: Cambridge, U.K., 1997.

Barbetta et al. Table 1. Morphological Characteristics of Gelatin-Methacrylate PolyHIPEs Obtained by Varying the Volume Percentage of the Dispersed Phase and the Concentration of Polymer in the Aqueous Phase samples

〈d〉 Ff 〈D〉a (µm) P(D)b (µm)c P(d)d 〈d〉/〈D〉e (mg/mL)

GMA20PV75 7.0 GMA20PV85 9.1 GMA20PV90 13.8 GMA25PV92 14.0 GMA25PV95* 23

2.3 5.0 5.5 6.8 9

4.3 4.9 6.8 7.5 13

1.5 2.4 2.5 2.8 4

0.61 0.54 0.49 0.54 0.57

59 ( 1 53 ( 1 38 ( 2 34 ( 2 26 ( 2

g 81 ( 2 89 ( 2 92 ( 2 95 ( 3 98 ( 3

a Average void diameter. b Void diameter polydispersities. c Average interconnect diameter. d Interconnect diameter polydispersities. e Degree of interconnection. f Solid foam densities. g Solid foam porosities.

packing of such an array of droplets will be more compact, the W/O interfacial area will be higher and as a consequence the thickness of the layer of continuous phase surrounding droplets of the dispersed phase will be thinner. On polymerization of the continuous phase, this will lead to the formation of larger interconnects, relative to void dimension (i.e., large 〈d〉/〈D〉).24 It can be observed (Table 1) that the average void diameter and void polydispersity (P(D)) increase with PV. These results agree with observation made in other systems.14 The increase in 〈D〉 and P(D) for PV < 90% solid foams could be explained by considering the process of emulsion formation. We add the dispersed phase to the continuous phase dropwise. At the beginning, small droplets can be formed. As we keep increasing the amount of the dispersed phase, there is less continuous phase available to form the interfacial film; therefore, bigger droplets are formed. 25 This interpretation is not sufficient to explain the details of the morphological features of the solid foams shown in Figure 1 as evidenced by the behavior of void and interconnect diameter distributions (hereinafter indicated with the acronyms VDD and IDD) (Figure 2) as well as 〈d〉/〈D〉 (Table 1) as a function of PV. As it can be seen (Table 1), 〈d〉/〈D〉 decreases upon an increase in PV for PV e 90% solid foams, while for PV > 90% the trend is reversed. VDD and IDD exhibit a clear trend as a function of PV: for PV e 90% solid foams both kind of distributions on the whole shift toward the large diameter side and become progressively broader and right skewed as evidenced by the polydispersity indexes (Table 1). In particular, the void fraction number in the range 0-10 µm undergoes a progressive decrease with an increase of PV while in the range 10-25 µm undergoes a proportional increase. Similar observation can be made for IDD (Figure 2, a2, b2, and c2). A more thorough full explanation of the data presented above can be drawn by making reference on what has been established on a firm basis about the structure of high internal phase emulsion (HIPE). Phase behavior studies of gel emulsions-forming systems, i.e., water/polyoxyethylene nonionic surfactant/oil systems, indicate that at equilibrium they separate into two isotropic liquid phases.26 In O/W gel emulsion, the equilibrium phases are an oil phase and an oil-swollen aqueous micellar solution (or O/W microemulsion).27 Since the surfactant aggregates formed are microemulsion droplets, the critical concen(24) Cameron, N. R.; Sherrington, D. C.; Albiston, L.; Gregory, D. P. Colloid Polym. Sci. 1996, 254, 592-595. (25) Pons, R.; Solans, C.; Stebe`, M. J.; Erra, P.; Ravey, J. C. Prog. Colloid Polym. Sci. 1992, 89, 110-113. (26) Auvray, L.; Cotton, J. P.; Ober, R.; Taupin, C. J. Phys. 1984, 45, 913-928. (27) Kunieda, H.; Evans, D. F.; Solans, C.; Yoshida, M. Colloids Surf. 1990, 47, 35-43.

Tissue Engineering Applications of PolyHIPE

Figure 2. Number-distributions of voids (1) and interconnects (2) size of GMA polyHIPE solid foams characterized by an increasing nominal pore volume (PV): (a) 75%; (b) 85%; (c) 90%; (d) 92%. The area of a histogram bar is proportional to the number fraction of either voids or interconnects within a size range.

tration of surfactant required for their formation is referred to as the critical microemulsion concentration (cµc). After reaching this point, the surfactant chemical potential stays essentially constant, because all the surfactant added is consumed by the micelles. The interfacial tension at the cµc is the lowest possible under given experimental conditions. In the nonequilibrium state, the number of micelles decreases with increasing oil content (i.e., PV) and finally no micelles are present in the continuous medium at very high oil content. This picture of the emulsion structure is useful in the interpretation of the morphologies and of quantitative data obtained by analysis of LM micrographs. As shown by SEM (Figure 1a), GMA20PV75 presents a strut-type morphology which only resembles that characteristic of polyHIPEs. On examination of the correspondent LM micrograph it can be seen that the origin of this somewhat peculiar morphology is due to extremely large interconnects in relation to void size as it is also evidenced by a very large value of 〈d〉/〈D〉 (Table 1). A loss of cellular structure in polyHIPEs were observed when either a very high surfactant level was used in the preparation of poly(styrene-divinylbenzene) polyHIPEs28 or in the presence of relevant amounts of an interfacially active porogen in the continuous phase of a divinybenzene based HIPE.5 In the present case for PV e 90%, the emulsion composition of both the aqueous and organic phases have been kept constant so differences in morphologies must have a physical origin. As stated previously, in O/W emulsions, surfactant molecules are (28) Williams, J. M.; Gray, A. J.; Wilkerson, M. H. Langmuir 1990, 6, 437-444.

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partitioned between interface and the O/W microemulsion phase present in the bulk of the continuous phase. At low PV (i.e., 75%), the surfactant/oil ratio is relatively high. This favors the creation of the largest interfacial area compatibly with such a PV and surfactant content. As the PV is increased, the surfactant/oil ratio decreases. This means the surfactant available for the creation of new interfacial area becomes less and less. It may be that this re-distribution of surfactant is accompanied also by an increase in interfacial tension. Such a phenomenon has been observed quantitatively in W/O HIPEs by Pons et al.29 This would imply that the surfactant concentration in the bulk of the continuous phase may be above surfactant cµc already at PV ) 75% or may pass across cµc at the given experimental conditions (Cp and temperature of emulsion preparation) at 75% e PV e 85% with the consequence that the interface may not be saturated with surfactant molecules. The combined set of data referring to 〈D〉, 〈d〉 and 〈d〉/ 〈D〉 (Table 1) supports the theoretical picture outlined above in the case of PV e 90% solid foams, and it is coherent with the shift of the whole IDD toward large diameter side. If factors other than surfactant/oil ratio and/or interfacial tension interfacial increase were playing a role upon the increase in PV, only tailing of VDD would have been observed. Another physical variable that has a pronounced effect on final foam morphology is emulsion viscosity. The viscosity, defined as the ratio of shear stress to shear viscosity, depends on several factors: the viscosity of the continuous and the dispersed phase, the volume fraction of the dispersed phase, the average particle size and particle size distribution, shear rate, the nature of the emulsifier, and temperature.30 Composition and preparation conditions were kept constant for emulsions with PV e 90, as a consequence emulsions viscosities depend on a first approximation on the volume fraction of the dispersed phase. Emulsion viscosity, η, is related to the volume fraction of the dispersed phase through the relationship

η ) ηcon(1 + φ + bφ2 + cφ3 + .....)

(1)

where ηcon is the viscosity of the continuous phase and φ is the volume fraction of the droplet phase.31 Although this equation was originally derived for colloid suspensions, it may be applicable as a qualitative guide for identifying processing parameters in emulsion systems that contains emulsifiers. In this equation, η is shown to be directly related to φ . The smaller voids in the VVD are retained from the small organic phase droplets formed in the early stages of HIPE synthesis (low φ), when the high shear stress of mixing the low viscosity HIPE breaks up larger droplets. The larger voids are formed in the late stage of HIPE synthesis (high φ) when mixing the more viscous HIPE does not generate sufficient shear stress to break up larger droplets. The solid foam structure thus reflects the surfactant/oil ratio/interfacial tension and viscosity of the HIPEs. The relatively high surfactant/oil ratio and/or low interfacial tension coupled to a relatively low emulsion viscosity experienced at low value of PV (75%) favors the comminution of the added internal phase and the creation of a relatively high interfacial area in (29) Pons, R.; Ravey, J. C.; Sauvage, S.; Stebe, M. J.; Erra, P.; Solans, C. Colloids Surf. A 1993, 76, 171-177. (30) Barnes, H. A. Colloids Surf. A 1994, 91, 89-95. (31) Sherman, P. Rheological Properties of Emulsions. In Encyclopedia of Emulsion Technology; Becher, P., Ed.; Marcel Dekker: New York, 1983; Vol. 1.

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accordance with a high 〈d〉/〈D〉 value (Table 1). As it is evidenced by Figure 2(a1) the shape of VDD of GMA20PV75 is approximately symmetric with respect to the maximum and most of both voids and interconnects diameters are comprised within a narrow range (5-10 µm and 2-8 µm, respectively) and as a result polydispersities are relatively low (Table 1). GMA20PV85 presents a more well defined morphology than GMA20PV75 although the degree of interconnection is still high (Table 1). Its LM micrograph (Figure 1b2) shows the presence of relatively large voids surrounded by a number of smaller ones and this may reflect the enhanced viscosity the emulsion starts experiencing above a certain φ. The heterogeneity in void diameter in GMA20PV85 solid foam is manifested in a value of void polydispersity (Table 1) much larger than GMA20PV75 which further supports the hypothesis of the influence of viscosity enhancement in determining the width of VDD. If VDD increase was driven only by the decrease of surfactant/oil ratio and an increase of interfacial tension this would have been translated into a shift of the whole distribution toward larger void sizes. On the contrary, we also observe a broadening of the distribution. The trend consisting of the increase in both void and interconnect sizes with PV is fulfilled by GMA20PV90 also. Void and interconnect polydispersities remain approximately constant, whereas there is a substantial decrease in the degree of interconnection with respect to GMA20PV85 (Table 1). GMA25PV92 solid foam morphology is characterized by a very similar value of the average void diameter, a modest increase of the interconnect average diameter, and an increase in the degree of interconnection with respect to GMA20PV90. As the concentration of the surfactant has been kept constant, the increase of PV should lead to larger voids in accordance with that observed with the solid foams with PV < 90%. The coupled effect of lower level of available surfactant and increased emulsion viscosity as predicted by eq 1 should translate into both an increase in void and interconnect polydispersities as observed (Table 1) and an increase in 〈D〉. On the contrary, 〈D〉 is very similar to that of GMA20PV90. The larger polydispersity arguably reflects the increase in emulsion viscosity due to both the increase in PV and in Cp. Interestingly, when the GMA20PV90 VDD is compared with those of lower PVs, the trend consisting in the shift of the whole distributions toward larger void diameter is not fulfilled. Especially in the diameter range 0-10 µm, an increase in the fraction number is observed with respect to GMA20PV90 VDD. The increase in 〈d〉/〈D〉 with respect to that of GMA20PV90 may be attributed to two possible effects: the increase in PV and/or a decrease in interfacial tension. The increase in PV is accompanied by a thinning of the continuous phase surrounding the droplets of the dispersed phase. On polymerization, this will produce larger interconnects.24 The combined evidence of the constancy of the average void diameter with respect to that of GMA20PV90 and a higher degree of interconnection together with the detailed comparison between GMA20PV90 and GMA25PV92 VDD seem to support the hypothesis of a reduction of the interfacial tension. Data presented are more coherent with such an hypothesis. The lowering of the interfacial tension must follow a variation in emulsion composition. The only difference between GMA25PV92 emulsion and those characterized by lower PVs is the concentration of GMA (25% instead of 20% w/v). Thus GMA itself seems to exert an interfacial activity which effectively counterbalances the postulated decrease in γ. This is not surprising, considering that

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gelatin is used as a stabilizer in oil-in-water emulsions.32 Upon a further increase in PV (95%) the predominant effect seems to be the increase in interfacial tension as witnessed by the remarkable increase in 〈D〉 (Table 1). This is the consequence of an amount of surfactant in the bulk of the continuous phase that is far from saturating the O/W interface at such high PV. The degree of interconnection is predominantly determined this time by the thinning of the continuous phase. Data relative to foams densities (Table 1) coherently follow an inverse relationship with PV. Porosity data are constantly higher than nominal pore volumes as determined by the volume of the dispersed phase. The origin of this disagreement is due to the fact that the solid foams were freeze-dried from the swollen state, which may cause an expansion of the void volume. Experimental pore volumes (Table 1) increase monotonically with nominal pore volumes and this indicates that the solid foam did not suffer any shrinkage during the dehydration procedure. For this reason, we can conclude that the morphological features as evidenced by both SEM and LM are representative of the emulsion structure prior of gel point attainment. Effect of Additives. From an application point of view, the polyHIPE solid foams presented in the previous section are still inadequate as scaffolds for tissue engineering. The minimum void size required depends on the cell type to be cultured but as a rough guide the average void and interconnect diameters should be as much as three times and at least equal to cell dimension (∼20 µm), respectively. We are then faced with the problem of increasing drastically both void and interconnect dimensions. The discussion about the influence of PV and Cp on porosity and morphology of polyHIPEs offers some hints in this respect. First, high PVs (i.e., 90 and 92%) are to be preferred because larger voids and interconnects are associated with them (Table 1). Second, a Cp of 20% w/v in the water phase is to be preferred to that of 25% w/v because the experimental evidence collected points to an interfacial activity of GMA which contributes in lowering the emulsion interfacial tension and leads to the creation of smaller voids. A possible route to follow in order to increase substantially the voids dimension is to cause a controlled destabilization of the precursor emulsion. In general, the stability of emulsion thin film is determined by the interactions between surfactantsurfactant, surfactant-solvent, and surfactant-dispersed bulk phase components.33 Emulsion partial destabilization can be induced through the use of additives (either inorganic or organic). The effect of additives on nonionic surfactants is to change their real HLB (hydrophilic lipophilic balance) at the O/W interface as manifested by the variation in the PIT (phase inversion temperature) of the emulsion.34 Nonionic surfactans owe their solubility in water to the hydration of poly(ethylene oxide) chains, so solubility increases as the chain length increases. As the temperature is increased, hydrogen bonds break35 and the solubility of the surfactant in water diminishes. Most important, the size and shape of the surfactant aggregates (32) Jones, R. T. Process Biochem. 1971, 6, 19-22. Livshitz, V. A.; Dzykowski, B. G.; Pirogov, N. O. Colloid Surf. A 1993, 72, 313-320. Olijve, J.; Mori, F.; Toda, Y. J. Colloid Interface Sci. 2001, 243, 476482. Mu¨ller, H. J.; Hermel, H. Colloid Polym. Sci. 1994, 272, 433-439. (33) Friberg, S.; Larsson, K. In Advances in Liquid Crystals; Brown, G. H., Ed.; Academic Press: London, 1975; Vol. 2, pp 173-197. (34) Shinoda, K.; Friberg, S. Emulsions and Solubilization; John Wiley & Sons: New York, 1986. (35) Corti, M.; Minero, C.; Degiorgio, V. J. Phys. Chem. 1984, 88, 309-317. Rosen, M. J. Surfactants and Interfacial Phenomena, 2nd ed.; Wiley: New York, 1989.

Tissue Engineering Applications of PolyHIPE

change strongly with temperature, as has been documented both in studies of micellar solutions and in establishing the phase equilibrium of the entire amphiphile concentration range. For sufficiently long polyethoxylene chains and low temperatures, the micelles are spherical, but with increasing temperature they grow into rod micelles. At higher temperatures, there is another isotropic solution phase forming in many system, the L3 (lamellar liquid-crystal) phase, which is built up of closely planar zero-mean-curvature surfactant bilayers.36 Ultimately, at a certain temperature, known as the cloud point, the surfactant solution separates into two miscible phases: a surfactant-rich phase and a surfactant-lean phase. The effect of temperature on nonionic surfactants can be mimicked by salts whose effect is to cause a vertical shift of the phase diagram (T vs oil/water ratio) in agreement with the linear relationship between PIT and salt concentration.34 Many researcher have investigated the effect of inorganic additives on the cloud point of nonionic surfactants.37 It has long been known that simple salts, e.g., NaCl and CaCl2, lower the cloud point of nonionic surfactants, with sodium chloride causing more depression than calcium chloride.38 Al-Ghandi et al39 have studied the influence of different additives on the cloud point of surfactants belonging to the family of Triton. In the present case, it must be borne in mind that Triton X-405 is blended with a large amount of GMA whose influence on its cloud point is not easily predictable. As a consequence, literature data39 can serve only as a rough guide. For this reason, in an attempt to induce partial emulsion destabilization, the effect of different concentrations of NaCl were tested on the GMA20PV90 emulsion system. In Figure 3, SEMs of GMA20PV90 solid foams prepared by employing three different NaCl concentrations (0.01, 0.1, and 1 M) are shown. The two lower concentrations of NaCl allowed to obtain solid foams possessing well-defined morphologies, characteristic of polyHIPEs. On the contrary, a concentration of NaCl 1 M has a very destabilizing effect: voids as large as 200-400 µm appear and on the whole the morphology is very irregular and inhomogeneous. For instance, interconnect sizes are extremely polydispersed. It is useful at this point to compare void and interconnect sizes of GMA20PV90S0.01-0.1 with those of GMA20PV90 in order to verify whether the addition of NaCl resulted in an increase of either void and/or interconnect average diameters. Qualitatively, it is evident that on increasing the NaCl concentration from 0.01 to 0.1 M void size decreases. This is shown on a quantitative basis in Figure 4 which summarizes results regarding average void and interconnect diameters and allows a comparison among all GMA20PV90 and GMA20PV90S0.01-0.1 solid foams. Interestingly, a nonlinear trend is observed: at a NaCl concentration of 0.01 M an as large as double increase in void and interconnect average diameters is observed with respect to GMA20PV90. Upon a further increase of NaCl to 0.1 M, the void average diameter drops to a value similar (36) Anderson, D.; Wennerstro¨m, H.; Olsson, U. J. Phys. Chem. 1989, 93, 4243-4253. (37) Hinze, W. L.; Pramauro, E. Crit. Rev. Anal. Chem. 1993, 24, 133-177. Schott, H. J. Colloid Interface Sci. 1973, 43, 150-155. Schott, H.; Royce, A. H.; Han, S. K. J. Colloid Interface Sci. 1984, 98, 196-201. Deguchi, K.; Meguro, K. J. Colloid Interface Sci. 1975, 50, 223-227. Sadaghiani, A. S.; Khan, A. J. Colloid Interface Sci. 1991, 144, 191200. (38) Doscher, T. M.; Myers, G. E.; Atkins, D. C. J. Colloid Interface Sci. 1951, 6, 223-235. (39) Al-Ghandi, A. M.; Nasr-El-Din, H. A. Colloids Surf. A 1997, 125, 5-18.

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Figure 3. Effect of the addition of different amounts of NaCl on the morphologies of GMA polyHIPE solid foams. Nominal pore volume ) 90%, Cp ) 20% w/v, Cs ) 7% w/v. (a) 0.01 M; (b) 0.1 M; (c) 1 M.

to that characterizing GMA20PV90. The degree of interconnection of the solid foams reported in Figure 4 follows the order 0 > 0.1 > 0.01 M with respect to salt content (0.49, 0.38, and 0.42, respectively) which indicates that interfacial tension varies in an inverse fashion. This implies that at a NaCl concentration of 0.01 M the surfactant interfacial film is less densely packed with surfactant molecules than in the absence or with a NaCl content of 0.1 M. The nonlinear trend observed with respect to 〈d〉/〈D〉 seems to indicate the superimposition of two simultaneous phenomena, presumably the increased partitioning of Triton X-405 in favor of the organic phase and a reorganization of the interfacial surfactant film structure. Only the determination of surfactant phase diagram at the given experimental conditions can disclose

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Figure 4. Voids and interconnects average diameters of polyHIPE solid foams as a function of NaCl concentration in the continuous phase. Pore volume ) 90%; Cp ) 20% w/v; Cs ) 7% w/v.

the structural changes the interface undergoes upon the addition of NaCl. Even though encouraging, the improvements in term of increase in void and interconnect dimensions recorded as a result of the addition of NaCl 0.01 M are still insufficient for our purposes. It is a well-established experimental fact that the interfacial film can be disrupted by adding a cosolvent to the emulsion.40 For instance, certain materials such as acetone or methanol are often soluble in both the oil and the water phases. If added to an emulsion, such materials may tend to dilute the interfacial layer and cause some of the surfactant to migrate away from the interface thus destabilizing the emulsion and allowing in the extreme case droplets of the dispersed phase to coalesce. In an attempt to further destabilize in a controlled manner the emulsions such an approach was implemented. A small amount (1% v/v) of DMSO was added to the organic phase. The continuous phase was represented by an aqueous solution of NaCl 0.01 M containing GMA with a Cp ) 20% w/v. PV was set at 90 and 92% and Cs ) 7% w/v. As it can be seen in Figure 5 (A1,2 and B1,2) and Table 2, the outcome was dramatic. Void and interconnect average diameters increased as much as 6-7 and 2-4 times, respectively, compared to the correspondent polyHIPEs obtained without the addition of any additives. Solid foams morphologies are quite homogeneous and this rules out the occurrence of coalescence to any significant extent. The occurrence 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 it has already been documented.41 The droplets size distributions of a stable emulsion and of one undergoing coalescence phenomena differ by the presence in the latter case of a long tail extending toward large diameter side while the position of the maximum remain approximately unchanged.42 Such features are absent in both void distributions referring to GMA20PV90s0.01D and GMA20PV92s0.01D (Figure 6, A1a and B1a). Comparison of the degrees of interconnection between solid foams prepared by employing additives and the correspondent ones without (Tables 1 and 2) seems to indicate that in the former ones the interfacial tension in the precursor emulsion was significantly lower. For this reason we can assume that surfactant depletion in the interfacial film as well as the probable increase in surfactant cµc in the (40) Lissant, K. J. Emulsion and Emulsion Technology; Marcel Dekker: New York, 1974; Vol. 6. (41) Tai, H.; Sergienko, A.; Silverstein, M. S. Polym. Eng. Sci. 2001, 41, 1540-1552. Krajnc, P.; Stefanec, D.; Brown, J. F.; Cameron, N. R. J. Polym. Sci. Part A 2005, 43, 296-303. (42) Aronson, M. P.; Petko, M. F. J. Colloid Interface Sci. 1993, 159, 134-149.

Figure 5. Effect of the presence of additives in HIPE formulation (NaCl 0.01 M and DMSO 1% v/v) on GMA polyHIPE solid foams as evidenced by scanning electron (1) and light microscopy (2) (magnification 200 ×, scale bar 40 µm). Pore volumes: (A) and (B) 90%; (C) and (D) 92%. Cp ) 20% w/v. Cs ) (a) 7% w/v and (b) 8.5% w/v.

bulk of the aqueous phase as a consequence of the presence of DMSO are mainly responsible for the remarkable increase in void size. Both void and interconnect distributions are rather broad in comparison with solid foams obtained without additives as evidenced by the relative polydispersities (Table 2). Another phenomenon that we can be safely assumed is taking place in the emulsion prior to gelation of the aqueous phase is Ostwald ripening. This phenomenon is driven by the difference in the chemical potential of the material within the drops. This difference arises from the difference in the radius of curvature of the drops.43 The addition of an additive soluble in both phases enhances the rate of ripening because the solubility of the dispersed phase in the continuous one is increased. Some authors12 have ascribed the increase in void size on addition of small quantities of a cosolvent to the emulsion exclusively to Ostwald ripening. This seems an oversimplification of a more complex phenomenon. Ostwald ripening alone cannot explain the differences in the morphologies of the solid foams prepared from emulsions whose compositions differs by the presence or not of additives, such as the variation in the degree of interconnection. To better elucidate this point, we have carried out the following experiment: the composition of the emulsions were maintained constant with the exception of Cs which was increased from 7% to 8.5% w/v and void and interconnect diameter distributions were determined as for the previous (43) Taylor, P. Adv. Colloid Interface Sci. 1998, 75, 107-163.

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Table 2. Morphological Characteristics of Gelatin-Methacrylate PolyHIPEs Obtained by Using Additives and by Varying the Volume Percentage of the Dispersed Phase and the Concentration of Surfactant

e

samples

〈D〉a (µm)

P(D)b

〈d〉c (µm)

P(d)d

〈d〉/〈D〉e

Ff (mg/mL)

g

GMA20S0.01MPV90D1% GMA20S0.01MPV92D1% GMA20S0.01MPV90D1%T8.5 GMA20S0.01MPV92D1%T8.5

61.4 83.9 19.3 28.2

17.9 32.3 7.3 13.0

12.2 27.6 8.7 14.1

4.6 12.7 3.5 5.4

0.20 0.33 0.45 0.50

37 ( 2 32 ( 2 40 ( 2 28 ( 2

93 ( 2 96 ( 2 91 ( 2 95 ( 2

a Average void diameter. b Void diameter polydispersities. c Average interconnect diameter. Degree of interconnection. f Solid foam densities. g Solid foam porosities.

d

Interconnect diameter polydispersities.

without the use of any additives (Table 1) and similar considerations can be applied. Conclusions

Figure 6. Effect of the presence of additives in HIPE formulation (NaCl 0.01 M and DMSO 1% v/v) on the numberdistributions of voids (1) and interconnects (2) size of polyHIPE solid foams. Pore volumes: (A) and (C) 90%; (B) and (D) 92%. Surfactant concentrations Cs: (a) 7% w/v and (b) 8.5% w/v. The area of a histogram bar is proportional to the number-fraction of either voids or interconnects within a size range.

samples. If the increase in void size has to be accounted to Ostwald ripening solely, then the increase in Cs should have a negligible influence on void diameter. Experimentally, a drastic decrease in void size as well as a relevant increase in the degree of interconnection are observed (Table 2). These quantities are inferior in value to those of GMA20PV90 and GMA25PV92 (Table 1). This seems to indicate that the amount of surfactant interfacially active is lower although a higher content of surfactant was used. Polydispersities are also remarkably lower than those referring to GMA20S0.01PV90D1% and GMA20S0.01PV92D1%, and this again points to a lower interfacial tension that makes it easier to break larger drops of the dispersed phase. Thus, although Ostwald ripening might play a role, we believe that the effect of additives on the interfacial surfactant excess is the predominant factor in determining the overall morphology of the ensuing polyHIPEs. Porosities and densities of the solid foams reported in Table 2 are very similar to the corresponding ones obtained

This work presents a systematic study of the ability to fabricate gelatin-methacrylate polyHIPE scaffolds with a wide range of morphological features. Specifically, by varying PV it was noted that the average void diameter increased with PV reaching the maximum value at PV 95%. In the range of PV explored, all solid foams exhibited an open cellular morphology characterized by an excellent degree of interconnectivity. From an application point of view in the tissue-engineering field, even the scaffolds characterized by the highest PVs (92 and 95%) possessed an insufficient average dimensions of both voids and interconnects to allow their viable colonization by cells in 3D. For this reason, in the attempt to increase considerably void and interconnect sizes, we have explored the effect of additives, namely NaCl and DMSO, to the emulsion phases. The outcome was dramatic: we succeeded in obtaining percentage increases up to 500% and 330% in void and interconnect dimensions, respectively, as compared to foams characterized by the same composition and PV but without the employment of additives. These results are promising in view of the exploitation of our scaffolds in the tissue engineering field as a result of the combination of their expected biocompatibility and attractive morphological characteristics. Further developments under study are focusing on the following issue: to avoid the introduction of foreign chemical functionalities (e.g., methacrylate groups) which may jeopardize the biocompatible nature of the starting biopolymer, it may be more convenient to use a more “natural” cross-linking procedure which exploits the presence of functional groups already present in the biopolymer structure. Two such possibilities currently under study are represented by the use of the microbial transglutaminase which catalyses the formation of isopeptide bonds between the γ-carbonyl group of a glutamine residue and the -amino group of a lysine residue44 and the autocrosslinking reaction between either hydroxyls or amines and carboxylic groups promoted by the system EDC (1-ethyl3-(3-dimethylaminopropyl)carbodiimide) and NHS (nhydroxysiccinimmide).45 Results from these studies will be the topic of future publications. Acknowledgment. The authors thank the University of Rome (Ateneo funds) and Consorzio Interuniversitario Biotecnologie (CIB) for funding this research and Professor Camillo La Mesa for valuable discussions. LA0520233 (44) Crescenzi, V.; Francescangeli, A.; Taglienti, A. Biomacromolecules 2002, 3, 1384-1391. (45) Lee, J. M. J. Mater. Sci.: Mater. Med. 1996, 7, 531-541. Rich, D. H.; Singh, J. The carbodiimide method. In The Peptides; Academic Press Inc.: New York, 1979; pp 241-261. Pieper, J. S.; Oosterhof, A.; Dijkstra, P. J.; Veerkamp, J. H.; van Kuppevelt, T. Biomaterials 1999, 20, 847-858.