Porous Alginate Hydrogels: Synthetic Methods for Tailoring thePorous Texture

Andrea Barbetta,* Elena Barigelli, and Mariella Dentini*

Department of Chemistry, University of Rome “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy

Received May 6, 2009; Revised Manuscript Received June 18, 2009

Alginate is a versatile, renewable biopolymer that has found numerous applications in diverse areas such asadsorbent materials of water pollutants and scaffolds for tissue engineering. In such kinds of applications themost convenient physical form of alginate-based materials is as porous matrices. The pore scale dimension hasto be carefully engineered to meet the requirements posed by the specific application. The aim of this paper is todescribe two synthetic methodologies that allow the preparation of alginate porous materials characterized bypores lying in well separated dimension ranges. One process is based on emulsion templating, which consists ofdispersing an organic phase into an aqueous solution of alginate in the presence of a suitable emulsion stabilizerand locking in the structure of the continuous phase by chemical cross-linking. This approach required thepreliminary degradation of alginate to reduce its molecular weight and, hence, the viscosity of the external phaseof the concentrated emulsion. Porous matrices were characterized by pores and interconnects of about 10-20and 2-5 µm, respectively, and a surface area of 230 m2/g. The second process consisted of replacing the organic,internal phase with a gas, namely, CO2, generated in situ the aqueous solution of alginate. The chemical reactionfor CO2 generation, nature of the surfactant, and cross-linking method were carefully selected to give highlyporous, stable matrices with pores and interconnects of the order of 300 and 80 µm, respectively.

Introduction

Among polysaccharides, alginate, a block copolymer of 1,4-�-D-mannuronate (M) and R-L-glucuronate (G) residues, is oneof the most versatile from an application point of view. Twomajor fields of applications of alginate-based materials are asadsorbents for the elimination of heavy metals and organicpollutants from contaminated environments1-8 and as devicesfor tissue engineering and drug delivery.9-20 In the former typeof application, alginate is one of the most extensively investi-gated biopolymers as it is inexpensive, nontoxic, and efficient.Insolubility in the majority of the organic solvents and thepresence of numerous and different surface functionalities suchas hydroxyl and carboxyl groups and excellent selectively forcertain metals are the key features that make of alginate as avalid alternative to conventional methods of metal recovery.20,21

As a biomaterial, alginate has a number of advantageous featuresincluding biocompatibility and nonimmunogenicity, which arelikely related to its hydrophilicity.22 Due to its nontoxicity,unique tissue compatibility, and biodegradability, alginate hasbeen studied extensively in tissue engineering, including theregeneration of skin,23 cartilage,24,25 bone,26 liver,27 and cardiactissue.28 A critical advantage is its gentle gelling behavior, whichallows encapsulation of various substances with minimaltrauma.29 A chemically modified alginate has been usedclinically as a drug delivery vehicle for proteins that promoteregeneration of mineralized tissue30 and as a carrier fortransplanted cells.31 Furthermore, in recent years geneticengineering allowed to produce tailor-made alginates withdefinite relative amount of glucuronic and mannuronic residuesarranged in the desired type of sequence.32-36 This opened up

the possibility to induce specific biological responses once thecorrelation between structure and biological activity is disclosed.

From a practical point of view, in both kind of applicationsthe more convenient physical form of the devices is as a poroussupport with the respective pore sizes lying in a different scalerange. A suitable adsorbent for polar molecules or metal ionsshould present a high concentration of adsorption sites, highsurface area, and an interconnected pore morphology for goodcirculation of the solution. To have a high surface area material,a significant fraction of mesopores (2-50 nm) should be present.

In the case of scaffolds for tissue engineering applications itis generally accepted that pore and interconnects sizes shouldbe at least 100 and 40 µm, respectively, to favor cell colonizationof the entire scaffold volume, metabolic waste removal, etc.

Two synthetic methodologies that allow satisfying themorphological requirements posed by the aforementioned typeof applications is high internal phase emulsion (HIPE) and foamtemplating. A considerable number of publications dealt withthe preparation and characterization of HIPE templated porousmaterials exhibiting high surface areas and with applicationsranging from scavengers, catalysis support and photonic to quotea few.37-43

Unfortunately, so far, HIPE templating did not allow access-ing to scaffolds characterized by pores and interconnects withaverage diameters >100 and 40 µm, respectively.44-50 As aconsequence, there is the need of a complementary fabricationtechnique which permits the synthesis of materials characterizedby larger pores and interconnects. It is well-known that gasblown plastics (foams) are often characterized by pores (eitherclosed or open) of the order of 100s of µm. For instance, inpolyurethane synthesis, the blowing gas (CO2) is developed insitu as a byproduct during the condensation reaction betweenan di-isocyanide and a dicarboxylic reagent. We envisaged thata similar approach could be adapted to our purposes. In otherwords, the liquid phase that represents the discontinuous phase

* To whom correspondence should be addressed. Tel.: +39-06-49913630(A.B.); +39-06-49913630 (M.D.). Fax: +39-06-4457112 (A.D.); +39-06-4457112 (M.D.). E-mail: andrea.barbetta@uniroma1.it (A.D.); mariella.dentini@uniroma1.it (M.D.).

Biomacromolecules 2009, 10, 2328–23372328

10.1021/bm900517q CCC: $40.75 2009 American Chemical SocietyPublished on Web 07/10/2009

in HIPEs could be replaced by an inert gas generated withinthe aqueous solution of alginate. Such a gas development shouldbe gradual to mimic the dropwise addition of the internal phaseduring HIPE making.

The present article aims at showing how alginate porousmaterials characterized by very different scale dimension ofpores and interconnects can be obtained by employing either aclassical HIPE approach and a new, innovative high internalphase foam (HIPF) approach.

Experimental Section

Materials. Two types of alginates were used in this work: Laminariahyperborea (Stipe LF 10/60 S12727, with a fraction of guluronic acid,FG ) 0.65), which was kindly provided by Prof. Skjak-Bræk of theUniversity of Trondheim, Norway, and a commercial alginate providedby Fluka, FG ) 0.40. Triton X-405 (solution 70% w/v, Aldrich),ethylendiaminetetraacetic acid disodium salt dihydrate (ETDA; Fluka),calcium chloride dihydrate (Fluka), calcium carbonate (Carlo ErbaAnalitycals), D-glucone-δ-lactone (DGL) (Fluka), 2-morpholinoethane-sulfonic acid monohydrate (MES; Fluka), N-hydroxysuccinimide (NHS;Fluka), 1-ethyl-3,3-[3-(dimethylamino)propyl]carbodiimide hydrochlo-ride (EDC; Fluka), toluene (Carlo Erba), dimethylsuphoxide (CarloErba), n-hexane (Carlo Erba), sodium chloride (Fluka), citric acid,tartaric acid, pluronic F-108, tyloxapol (all Adrich), and polyquaternium10JR400 (Amerchol) were used as received.

Mechanical Degradation of Alginate. Alginate degradation wascarried out with high energy ball milling. About 80 g of alginate (Fluka)was placed inside a jar that was allocated in a ball mill ZOZ GmbH-D5910 Kreuztal (a picture of the machine is reported in the SupportingInformation, Figure S1). The apparatus was provided with a coolingsystem to keep the jar at room temperature and was connected to avacuum pump to exclude oxygen during the degradation process. Ironballs (diameter 5 mm, ZOZ GmbH-D57482 Wenden Grinding Media)were used in a ratio of 10/1 g/g with respect to the weight of alginate.The angular velocity was set at 1000 rpm. At predetermined times,grinding was stopped and aliquots of alginate were taken out of the jarand subjected to molecular weight determination. Prior to use, thedegraded alginate samples were dissolved in water and filtered with aseries of descending pore size filters (from 3 to 0.45 µm) andsuccessively dialyzed against water. Finally, water was removed byfreeze-drying.

PolyHIPE Synthesis. A total of 0.5 g of degraded alginate wasdissolved in 2.2 mL of MES buffer (pH ) 4.5) in the presence of TritonX-405 (6.0% w/v). The solution was poured into a thermostated (18°C), three necked, round-bottom flask provided with a dropping funnel.Toluene (the dispersed phase, 6.6, 12.5, or 19.8 mL) was addeddropwise under stirring (300 rpm). At the end of the addition, stirringwas prolonged for another 15 min to allow better homogenization ofthe HIPE. Afterward, 0.3 mL of a solution of EDC (EDC/alginate-COONa ) 2.5 mol/mol) and NHS (NHS/EDC ) 0.2 mol/mol) wereadded to the HIPE and stirring was maintained for 2 min. The HIPEwas transferred into a polyethylene bottle that was placed inside anoven set at 40 °C for 24 h. After this period, the polyHIPE was soakedin DMSO, which was changed regularly (typically three times a day)for 1 week. This procedure was aimed at displacing toluene thoroughly.The polyHIPE was then Soxhlet extracted with water for 1 day andfinally freeze-dried.

Solid Foam Synthesis and Foam Stability Evaluation. Alginatefrom Laminaria hyperborea was used. A solution of 5% w/v (0.125 gin 2.5 mL) of alginate, pluronic F-108 (4.0 or 6.0% w/v), and sodiumbicarbonate (2% w/v) was prepared and poured into the reactor (ascheme of the experimental apparatus is reported in Figure S2 of theSupporting Information). An equivalent amount, with respect to themoles of NaHCO3, of acid (tartaric, citric, and gluconic) was addedwhile keeping the alginate solution under stirring (500 rpm). Stirringwas continued for 15 min to allow CO2 to fully develop. Afterward,

the foam was frozen in liquid nitrogen and freeze-dried. The resultingsolid foam was soaked in a solution 2.0 M of CaCl2 to induce theformation of the calcium-based physical gel for 24 h and then dialyzedagainst a solution of CaCl2 0.1 M. Finally, the solid foam was freeze-dried. Chemical cross-linking of the Ca2+-based solid foam was carriedout with EDC/NHS (EDC/alginate-COONa ) 2.5 mol/mol, HNS/EDC ) 0.2 mol/mol) in MES buffer (pH ) 4.5) under mild stirring for24 h. The solid foams were dialyzed against water and then freeze-dried.

The selection of the surfactant more effective for the stabilizationof the CO2-in-water foam was carried out by measuring the decline ofthe foam volume inside a graduated cylinder as a function of time.Foams were prepared by placing 5 mL of solution (15% w/v ofdegraded alginate, NaHCO3, organic acid, and surfactant) in a glass-stoppered 25 mL graduated cylinder. The cylinder was shaken 10 timesby hand through an arc of 180°. The initial volume of foam producedwas recorded and additional measurements were made every 2 min.The inspection of the size distribution of the foam bubbles wasmonitored by light microscopy observations (Leica Z16 APO).

Characterization. Elemental Analysis. Elemental analysis wascarried out by emission optical spectroscopy inductively coupled plasma(ICP-OES) using a VARIAN VISTA MPX CCD Simultaneous ICP-OES equipped with an ultrasound nebulizer. Sample solutions with aconcentration of alginate of 2.8 × 10-7 M in G and M residues wereobtained by dilution of a stock solution with a concentration of 2.8 ×10-2 M. Solution pH was adjusted to 3.5 with HCl.

Particle Size Determination. For the determination of particles size,a laser diffraction particle analyzer Beckam Coulter LS 13 320 wasused. Alginate powders from the original sample and from the degradedproduct (40 h of milling) were suspended in n-hexane and analyzed.Average particle size and particle size distributions provided are themean values from three independent measurements.

Viscosity and Gel Permeation (GPC) Measurements. The intrinsicviscosity [η] was determined using the viscosity measuring unitAVS379 (Schott-Gerate, Hofheim, Germany), connected to a Visco-doser AVS20 piston buret (for automatic dilutions), to make automatedmeasurements of the flow-through times in a capillary viscometer(Ubbelholde viscometer, Φ ) 0.53 mm, for dilution sequences). Theviscometer was immersed in a precision water bath (transparentthermostat CT 1150, Schott Gerate, Hofheim, Germany) to maintainthe temperature at 25 ( 0.1 °C. The solutions were prepared bydissolving the lyophilized Alg and Algdeg (starting alginate and thevarious alginate samples at different degrading times, respectively) withmagnetic stirring for at least 24 h at room temperature, followed byfiltration through a Millipore filter of 0.45 µm. Solutions had relativeviscosities from about 1.2 to 2.0 to ensure good accuracy and linearityof extrapolation to zero concentration. The intrinsic viscosity, [η], wasobtained by double extrapolation to zero concentration of Huggins’and Kraemer’s equations, respectively

where ηrel and ηsp are the (dimensionless) relative and specificviscosities, k′ and k′′ are the Huggins’ and Kraemer’s coefficients,respectively, and C is the solution concentration.

Alg and Algdeg were also subjected to GPC analysis. The GPC usedis a modular system (Lab flow 4000) with a refractometer (ShimadzuRID-10A). The samples were run using two TSK-GEL GM-PW (30× 7.5, 17 µm) columns and an aqueous solution of NaCl 0.01 N as themobile phase. The flow was set at 0.8 mL/min and the temperature at25 °C. Calibration of the GPC was achieved using pullulan standards.

NMR Experiments. The samples (Alg and Algdeg ∼ 2 mg) weredissolved in 700 µL of phosphate buffered (pD ) 7) D2O solution,0.01 M NaCl. 1H NMR experiments were performed on a BrukerAVANCE AQS 600 spectrometer operating at 600.13 and 150.92 MHz,

ηsp

C) [η] + k′[η]2C

(ln ηrel)

C) [η] + k′′[η]2C

Porous Alginate Hydrogels Biomacromolecules, Vol. 10, No. 8, 2009 2329

respectively, with a Bruker inverse multinuclear z-gradient probehead.Because at room temperature the 1H spectra appear broad and poorlyresolved, spectra were recorded at 343 K; at this temperature, in fact,all resonances appear sufficiently resolved to be assigned. Whennecessary, to avoid some signal overlapping, the spectra were also runat 333 K. In all 1H spectra, a soft presaturation of the HOD residualsignal was performed.

Chemical shifts of 1H spectra are reported in ppm with respect to2,2-dimethyl-2 silapentane-5-sulfonate sodium salt (DSS) used as aninternal standard.

The deconvolution of the 1H NMR spectra was performed using theSHAPE2000 (version 2.1) software package. Applying the deconvo-lution program, the areas Ii of peaks resonating in the 4.4-5.1 ppmspectral region are obtained along with errors ∆Ii. It is worth notingthat the obtained errors are always well within 4-5% of the nominalIi values. Therefore, the error on the area of each resonance is small,which is satisfactory. Because it is not possible to obtain errors on thecalculated molar fractions, on the doublet and triplet frequencies, andon the average block lengths, straight from the deconvolution program,the propagation error theory has been applied. However, as is well-known, this theory may overestimate the errors. As a consequence,errors reported in Table 3 are overestimated.

In our notation, M ) mannuronic acid unit and G ) guluronic acidunit.

Electron Microscopy. Scanning electron microscopy (SEM) imagesof both polyHIPEs and solid foams were obtained by using a LEO1450 VP electron microscope. Samples were mounted on aluminumstubs using a carbon tape and sputter coated with a gold layer.

Surface Area/Pore Size Distribution. After the samples werevacuum-dried at 473 K overnight, nitrogen adsorption/desorptionisotherms were determined at 77.3 K using a Micromeritics ASAP 2010apparatus. The specific surface areas were calculated by the Brunauer-Emmet-Teller (BET) equation utilizing eight points from the adsorptionisotherm collected over the relative pressure range 0.05-0.20 (P/P0).Pore size distributions were obtained from the analysis of the adsorptionbranch of the isotherm using the Barrett-Joyner-Halenda (BJH)method.51 N2 sorption measurements were carried out on both thefreeze-dried samples or previously treated with a solution of CaCl2 (1.0M). The latter type of sample was dehydrated with supercritical CO2

(scCO2). The Ca2+-polyHIPE was first dehydrated by immersion in aseries of successive ethanol-water baths of increasing alcohol con-centration (10, 30, 50, 70, 90, and 100%) for 15 min each. Finally, itwas dried under supercritical CO2 conditions (74 bar, 31.5 °C) in aPolaron 3100 apparatus.

Results and Discussion

Emulsion Templated Porous Materials. A major problemin the preparation of concentrated emulsions from polymericaqueous solutions is their viscosity. Experience teaches47,48 thattoo high a viscosity of the continuous phase can hinder thedispersion of the internal phase, which precludes the possibilityof incorporating it completely within the emulsion and obtaininga homogeneous polyHIPE. Commercial alginate is usually highmolecular weight. This joined to its intrinsic stiffness andpolyelectrolytic nature makes it impossible to employ it directlyin the production of a HIPE (which by definition is characterizedby a water to oil ratio of at least 0.75). Therefore, a necessarystep preceding its employment is represented by its degradationto reduce substantially its molecular weight. To this end, wehave employed mechanical degradation by high energy ballmilling. To our knowledge this is the first time that thistechnique has been used in the degradation of a complexbiopolymer such as alginate; therefore, it is of interest to givea full description of its effectiveness outlining advantages anddisadvantages.

Degradation of Alginate by Ball Milling. In ball milling,hard balls are placed inside a cylindrical container (jar) alongwith the bulk polymer. The cylinder is turned horizontally alongits long axis to cause the balls to repeatedly tumble over oneanother, thereby fragmenting in the first instance the sampleparticles into smaller ones and then causing the breakage ofthe polymer chemical bonds.

One of the main advantages of this technique is that it operatesin the solid phase, thus allowing large quantities of polymer tobe processed. Furthermore, it guarantees the complete recoveryof the employed material. In comparison, hydrolytic degradationwould necessitate dissolving the same amount of alginate in avery large amount of solvent (water, in our case), making thedegradation process less practical.

A problem associated with the ball milling technology is theerosion from the milling material and container walls duringthe milling process that inevitably contaminate the product witha very fine powder. For this reason, the degradation productneeds purification by filtration and extensive dialysis. Duringmilling, relevant energies are involved and internal frictionscause heating of the sample, therefore, radical species may begenerated, which may induce side reactions with O2 or moistureor induce the formation of branched species through thecombination of macroradicals. To avoid such side reactions,degradation was carried out under vacuum and the jar wasrefrigerated.

The first parameter to be monitored during degradation wasthe molecular weight. At prefixed times, the milling processwas stopped and a sample of alginate was taken out of the jar.The molecular weight was determined by viscometry and GPC(Figure S2 of the Supporting Information). In Figure 1a, Mj w,Mj n, and Mj η are plotted as a function of the degradation time.Numerical values are reported in Table SI of the SupportingInformation. It can be noted that the degradation is more efficientwithin the first 5 h. For more extended periods of time,molecular weights tend toward asymptotic values. In Figure 1band in Table SI it can be seen that the polydipsersity index, I,is approximately constant during the first 4 h of milling. Thismay reflect the random nature of chain scission, which in the

Figure 1. Behavior of the viscosity (Mη), number (Mn), and weight(Mw) average molecular weights (A) and polydipsersity index (B) ofalginate sample as a function of the degradation time.

2330 Biomacromolecules, Vol. 10, No. 8, 2009 Barbetta et al.

solid phase may involve different chains to a different extent.As the degradation proceeds, all chains undergo to a similarnumber of chain cuts, with the consequence that I at longerdegradation times decreases. It may be wondered the reasonthe molecular weight tends for long degradation times to anasymptotic value. The efficiency of degradation depends on therheology of the sample powder, which in turn is dependent onparticles size. Therefore, the particle size distributions of startingalginate (Alg0h) and degraded alginate after 40 h of milling(Alg40h) were determined by laser diffraction. Such distributionsare displayed in Figure 2. Both distributions are single peakedand from their comparison it is evident that that relative toAlg40h is shifted remarkably toward the low diameter side andis narrower with respect to Alg0h one. In Table 1, thecorresponding average particle diameters and standard deviations(SD) are reported. The data of Table 1 support in morequantitative terms the qualitative analysis of Figure 2. Thestandard deviations of Alg40h and Alg0h are 34 and 61 µm,respectively. Furthermore 75% of Alg0h is constituted byparticles of dimension in the range between 160-310 µm, while54% of Alg40h contains particle of dimension in the rangebetween 14-48 µm. It has been recognized52 that as the aver-age particle dimension decreases the powder acquires a morepseudoplastic behavior with the consequence that the efficiencyof energy transfer from the milling apparatus to particlesfragmentation and chain degradation decreases progressively.

Another aspect of concern in the mechanical degradation ofcomplex biomacromolecules such as alginate is whether de-graded alginate retains its starting primary structure. It istherefore useful to make a quantitative comparison between thesequences of G, M, and MG blocks in both Alg0h and Alg40h.To this end, 1H NMR spectra have been recorded for both Alg0hand Alg40h and analyzed (Figure S3a,b of the SupportingInformation). The deconvolution of the anomeric region (4-5ppm) allowed the estimation of the relative percentages of thedifferent kind of diads and triads.53 From Table 2 it can be seenthat no major differences characterize the primary structures ofAlg0h and Alg40h. The most notable differences regard therelative amount of M1G diads and MG5M triads. The total

content of M and G residues are ∼62 and 38% for Alg0h and66 and 34% for Alg40h, These results indicate that thedegradation takes place predominantly at the glycosidic bond(with some preference for the glycosidic bond between amannuronic and glucuronic residues) and does not involve theglucopyranose ring.

To evaluate the magnitude of contamination caused by themilling process on the sample of alginate, the quantitativedetermination of the metallic elements present in both the jarand milling balls (Fe, Cr, Ni) was carried out on Alg0h, Alg20h,and Alg40h by ICP-OES. Results are displayed in Table 3. Itis evident that the content of metallic elements increasedexponentially with the time of degradation. In fact, most of thecontamination occurs in the last 20 h of milling when thedegradation process becomes progressively less efficient. As aconsequence, before using degraded alginate, it was necessaryto purify it extensively. Filtration and dialysis (see ExperimentalSection) proved successful.

Preparation of Emulsion Templated Porous Materials.The methodology for the preparation of HIPE templated porousmaterials is well established. Biopolymers of a different nature,that is, polysaccharides and proteins,44-48 have been successfullyincluded in polyHIPE formulation. A general requirementunderlying the employment of biopolymers as the main com-ponents of the external phase of a HIPE is the control overthe continuous phase viscosity. The molecular weight of thebiopolymer must be controlled to keep the viscosity of thecontinuous phase within values that allow the incorporation oflarge volume fractions of the internal phase (φ g 0.75). In thisrespect, the intrinsic stiffness of the biopolymer plays animportant role. A flexible polymer such as dextran even of highmolecular weight is immediately employable. On the contrary,a stiff polymer such as alginate must be subjected to aconsiderable reduction of its molecular weight to reduce theviscosity of its concentrated solutions to processable values.

The various degraded alginate samples (Table SI) have beensubjected to emulsification tests. The concentration of alginatein the continuous phase chosen was 15 or 20% w/v and φ

(defined as the ratio of the organic phase volume to totalemulsion volume) was set at 0.75, 0.85, or 0.90. Only Alg40hgave rise to macroscopically homogeneous HIPEs, the lessdegraded alginate samples (Algxh with x < 40 h) did not al-low the full incorporation of the entire volume of the dispersed

Figure 2. Particle size volume distributions of starting alginate (Alg0h)and degraded alginate after 40 h of milling (Alg40h).

Table 1. Average Particle Dimensions and Standard Deviations(SD) and Normalized SD (CV) of Alg0h and Alg40h

sampleaverage

dimensions (µm)SDa

(µm)CVb

(%)d10

c

(%; µm)d50

c

(%; µm)d90

c

(%; µm)

Alg0h 229 60.9 27 155 223 312Alg40h 43 34 78 93 33 94

a Standard deviation. b CV ) (SD × 100)/average dimensions. c % ofparticles within prefixed dimension ranges (dx).

Table 2. NMR Peak Assignements (ppm), Intensities, and WeightPercentages of Diads and Triads in Alg0h and Alg40h

Alg0h Alg40h

diads or triadsa ppm intensity weight % intensity weight %

MG5G 4.42 3256 4.8 3060 2.9GG5G 4.44 6282 9.3 12624 12.0M1M 4.66 21365 31.8 33491 31.9M1G 4.69 13430 20.0 14942 14.2MG5M 4.70 2580 3.8 8422 8.0GG5M 4.72 2342 3.8 2447 2.3G1G-MG1M 5.05 18007 26.8 29981 28.6a M: mannuronis residues; G: glucuronic residues.

Table 3. Results of ICP-OES on Alginate Alg0h and itsDegradation Products Alg20 and Alg40h

sample Nia Cra Fea

Alg0h 0.2 ndb ndb

Alg20h 8.6 2.8 23.9Alg40h 364.7 116.3 1010.5

a Element content is expressed as ppm in 1.0 g of sample. b Belowdetectable limit.

Porous Alginate Hydrogels Biomacromolecules, Vol. 10, No. 8, 2009 2331

phase. Thus, in the following of this section it is implicitlyassumed that the alginate material used is Alg40h.

The simplest way to modulate the morphology of polyHIPEsis by varying φ. If the volume of the internal phase is keptconstant, the increase of φ will cause the thinning of the filmof aqueous phase surrounding the droplets of the dispersedphase. On polymerization,54 this will bring about materialscharacterized, on the average, by larger interconnecting holes.In flow through applications this translates into a higherpermeability.

In Figure 3a-d, the SEM micrographs illustrate this con-ceptual approach to morphology tailoring. For instance, φ )0.75 and a polymer concentration, Cp, of 15% w/v (Figure 3a)voids and interconnecting holes are in the range of 3-15 and1-8 µm. Increasing φ to 0.90 (Cp ) 15% w/v; Figure 4b) causesa considerable increase in both void and interconnect dimen-sions, 10-40 and 2-20 µm, respectively. In general, theviscosity of HIPEs (ηHIPE) is directly proportional to the viscosityof the external phase (ηe) and on φ. When the external phasecontains a polymer, the relationship between ηe and polymerconcentration (Cp) is obvious. Also, the effect of φ on ηHIPE isintuitive: when the droplets are concentrated above the randomclose-packing limit (φ g 0.74), they cannot move freely andare trapped by their neighbors and take the shape of apolyhedron. As a consequence, ηHIPE increases with φ, becausepart of the stress imposed will be dissipated as the work of

droplets deformation. The combined effect of φ and Cp is wellillustrated by the morphology of the polyHIPEs shown in Figure3c,d. When φ ) 0.85 and Cp ) 20% w/v (Figure 3c), largevoids of the order of 20-70 µm are interdispersed into a morehomogeneous porous structure consisting of much smaller voids.This effect is even more evident in the polyHIPE characterizedby φ ) 0.90 and Cp ) 20% w/v, where voids as large as 50-100µm appear. This is clearly the outcome of the combined effectsof the increase of φ and Cp on the HIPE viscosity. During HIPEformation, as the amount of the added dispersed phase increases,the emulsion becomes more and more viscous and the shearstress provided is not effective enough to break the droplets ofthe final portion of the added internal phase into smaller ones.Another aspect of the matrices presented that needs to beevaluated is the textural morphology of the polyHIPE walls. Inapplications such as removal of pollutants and as supports inbiocatalysis it would be desirable to have high surface materials.Valentin et al.55,56 have shown that the drying procedure ofionically cross-linked alginate gels is critical to preserve theporous texture of the gel. In this respect, extraction of solventwith scCO2 proved to be the most effective procedure. Thistechnique is commonly processed with inorganic solids toachieve very high specific surface areas.57 On the contrary,freeze-drying caused the alginate chains to collapse over oneanother causing the disappearance of macro (>50 nm) andmesopores (5-50 nm). Both approaches were implemented with

Figure 3. Scanning electron micrographs of Alg40h-based polyHIPEs obtained by varying the volume fraction of the dispersed phase (φ) andthe concentration of polymer in the aqueous phase (Cp): (a) φ ) 0.75, Cp ) 15% w/v; (b) φ ) 0.90, Cp ) 15% w/v; (c) φ ) 0.85, Cp ) 20% w/v;(d) φ ) 0.90, Cp ) 20% w/v. Surfactant concentration (Triton X-405): 6% w/v.

2332 Biomacromolecules, Vol. 10, No. 8, 2009 Barbetta et al.

the alginate-based polyHIPEs. Treatment with scCO2 must bepreceded by exchange of water with a series of graded ethanolsolutions. During such a process, the polyHIPE underwent to aremarkable shrinking. We hypothesized that this phenomenamight be due to a level of chemical cross-linking not highenough to avoid the pore collapse phenomenon consequent tothe exposure of highly hydrophilic gels to a nonsolvent. Toconfer a higher rigidity to the polyHIPEs, the ability of alginateto undergo physical cross-linking in the presence of divalentions (Ca2+, Ba2+, Cd2+, Sr2+, etc.) was exploited. A polyHIPE(φ ) 0.75, Cp ) 15% w/v) was treated before subjecting themto exchange with ethanol with a concentrated solution of CaCl2

(1.0 M). We hoped in this way to increase the cross-linkingdensity (chemical and physical) and obtain a more rigid material.It was indeed observed that when the Ca2+-polyHIPE wereexposed to ethanol the degree of shrinkage was much less.

The influence of the drying procedure (freeze-drying vstreatment with scCO2) on the morphology of the polyHIPEs isillustrated in Figure 4a,b. The difference between the twoprocedures of drying is clear when we observe the details ofthe polyHIPE walls. In the case of freeze-dried sample the wallsappear completely smooth (Figure 4a). On the contrary, in thecase of scCO2 dried sample (termed aerogel-polyHIPE), wallsexhibit a porous texture.

Further information about the textural properties of thepolyHIPE-based alginate was obtained by nitrogen adsorption-desorption hysotherms. The nitrogen adsorption-desorptionhysotherms of N2 at 77 K of the freeze-dried and aerogel-polyHIPEs are presented in Figure 4c. All isotherms of Figure4c are of type IV at the borderline with type II in the IUPACclassification. From their qualitative comparison it evident thatN2 adsorption from the aerogel-polyHIPE is much higher thanthe freeze-dried polyHIPE and the hysteresis loop much broader.Specific surface areas were 237 and 10 m2/g, respectively. Thepore size distributions show (Figure 4d) that in the case offreeze-dried polyHIPE no mesopores are present at all as it is

clearly observed in the relative SEM micrograph (Figure 4a).Evidently, the surface area measured comes mainly from themacrovoids. The aerogel-polyHIPE exhibited a pore sizedistribution characterized by a significant presence of mesopores.Nevertheless, the porosity is mainly in the macropore region.

Gas Templated Solid Foams. While the emulsion templatedalginate porous materials presented in the previous section maybe useful in areas such as purification of wastewater and enzymeimmobilization due to their excellent permeability and goodmechanical stability, they are inadequate for applications suchscaffolds for tissue engineering. In this area of application, afundamental prerequisite a scaffold must possess is a poroustexture composed of voids and interconnects of at least 100 and40 µm, respectively. It has already been established that in thecase of HIPE templated porous materials the upper limits ofvoid and interconnect sizes do not overcome the values reportedabove.45,47

For this reason we searched for a related technique of porousmaterials production that would bring in this respect significantimprovements. We wondered if the replacement of the internalliquid phase of the HIPE with a gas would represent the keyfactor to achieve a substantial increase of voids and interconnectssizes. Such an approach would be beneficial also under otheraspects: the synthetic procedure and purification would besimplified because it does not involve extraction of the organicinternal phase with another organic solvent followed withextensive washing with water; the avoidance of any organicsolvents as the internal phase represents an advantage as farbiocompatibility of the ensuing biomaterials is concerned.

On the other end, a drawback of foam templating isrepresented by the inferior kinetic stability of foams with respectto HIPEs. For this reason, the choice of surfactant and the gasgenerating system is critical and must therefore be carefullyselected.

Figure 4. SEM micrographs of EDC cross-linked aerogel (A) and of EDC and calcium cross-linked xerogel alginate polyHIPE (B). Nitrogenadsorption-desorption isotherms (C) and BHJ pore size distributions of alginate aerogel (black line) and xerogel polyHIPEs (red line).

Porous Alginate Hydrogels Biomacromolecules, Vol. 10, No. 8, 2009 2333

Choice of the Gas Generating System. There are essentiallytwo inert gas that can be exploited for foam formation: N2 andCO2. N2 can be generated in situ the alginate solution by meanof the oxidative/reductive reaction:

CO2 can be generated by virtue of the acid/base reaction:

where R can be D-glucono-δ-lactone, citric acid, and tartaricacid (see Supporting Information, Figure S4, for chemicalformulas).

It is well-known58,59 that alginates undergo gelation in acidicenvironment (pH < 3); as a consequence, it was necessary tomake a screening of the final equilibrium pH for each reaction((a) and (b)). Results are summarized in Table 4. As expected,the equilibrium pH of reaction (a) is rather low and is thusinapplicable for our purpose. On the contrary, all reactions basedon the development of CO2 (b) give rise to a pH > 3 and arethus exploitable. Another aspect to be evaluated regards theintensity of CO2 development. In Table 4, such a qualitative

evaluation is given. As it can be seen, the reactions involvingTA and, to a lesser extent, CA are the most promising.

Another component of paramount importance is the surfactantwhose function is to stabilize the foam for a time long enoughto allow cross-linking to lock-in the structure of the externalphase before destabilization process take place to any significantextent.

Foams are systems kinetically less stable than emulsions. Dueto the relatively large density difference between the gas phaseand the liquid, the bubbles rise in the gravity field to the top ofthe liquid. There they form a more or less close-packed,honeycomb-like, bubble structure from which the liquid drains,forming foams that become dryer and dryer. The thin filmsformed can collapse for some reason, causing the coalescenceof bubbles. The lifetime of a foam can be extended significantlythrough one of the following: (1) a high viscosity of the liquidphase, which retards drainage of the liquid from between thebubble interface, as well as providing a cushion effect to adsorbshocks resulting from random or induced motion; (2) a highsurface viscosity, which also retards liquid loss from betweeninterfaces and dampens film deformation prior to bubblecollapse; (3) surface effects such as the Gibbs and Marangonieffects,60 which act to “heal” areas of film thinning due lo liquidloss; and (4) electrostatic and steric repulsion between adjacentinterfaces due to the adsorption of ionic and nonionic surfactants,polymers, and so on. Most of the guidelines 1-4 can be fulfilledby using a combination of a high molecular weight alginateand a surfactant. Alginate from Laminaria hyperborea (Mj η )2.3 × 105) was used.

Foaming properties of a given surfactant are necessarilycharacterized by at least two parameters: one specifying the easeof foam production under specified conditions and one specify-ing the persistence of this foam under quiescent conditions.Shaking the surfactant solution in a cylinder and observing for20 min the decline in foam volume is a simple proceduresatisfying these conditions. A certain number of surfactants,either low and macromolecular weight (Figure S5 of theSupporting Information), were tested and their performanceswere evaluated. Results are summarized in Table 5. As it can

Table 4. pH of the Aqueous Solutions Containing the IndicatedReactants for the In Situ Development of Gas and RelatedObservations

solutiona pH experimental observations

SA 2% 0.6SA + SN 1.1 intense development of N2

BC 2% 8.7BC + CA 4.6 fair development of CO2

BC + TA 3.4 intense development of CO2

BC + DGL 7.2 modest development of CO2

a SA, sulfamic acid; SN, sodium nitrite; BC, sodium bicarbonate; CA,citric acid; TA, tartaric acid; DGL, D-glucono-δ-lactone.

Table 5. Remarks on the Kinetics of Foams Decay as a Function of Surfactant Type and its Concentrationa

surfactant % w/w foaming 2 min 10 min 30 min 45 min

PF 4 intense no variation no variation coalescence phenomenaobserved

phase separation

6 intense no variation no variation coalescence phenomenaobserved

phase separation

8 intense no variation no variation coalescence phenomena andincipient phase separationobserved

complete phaseseparation

PQ-SDS 0.1-0.01 scarce <2 min some phaseseparation observed

0.4-0.04 scarce <2 min some phaseseparation observed

0.8-0.08 modest no variation phase separationSDS 4 intense no variation phase separation

6 intense no variation coalescence phenomena andincipient phase separationobserved

complete phaseseparation

8 intense no variation phase separationTY 4 modest <2 min some phase

separation observed6 intense <2 min some phase

separation observed10 intense no variation phase separation

TR 5 scarce <2 min some phaseseparation observed

10 scarce no variation coalescence phenomena andincipient phase separationobserved

complete phaseseparation

12 scarce no variation phase separation

a PF, pluronic F-108; PQ-SDS, polyquaternium 10 JR400-sodium dodecylsulphate; SDS, sodium dodecylsulphate; TY, tyloxapol; TR, triton X-405.

H2NSO3H + NaNO2 f N2 + NaHSO3 + H2O (a)

RCOOH + NaHCO3 f RCOONa + H2O + CO2

(b)

2334 Biomacromolecules, Vol. 10, No. 8, 2009 Barbetta et al.

be seen, the polymeric surfactant Pluronic F-108 proved to bethe most effective foam stabilizer. For the generating gas phaseit was chosen the system TA/NaHCO3 (molar ratio ) 2/1),which as reported in Table 4 gave rise to the most intense CO2

development.The alginate chains autocross-linking carried out with the

system EDC/NHS proved not to be satisfactory, probablybecause these reagents themselves adversely affected the stabil-ity of the foam. The most effective procedure consisted offreezing the foam in liquid nitrogen, freeze-drying it, and thensoaking the solid foam obtained in a solution of CaCl2 (molarratio CO2Na/Ca2+, R ) 1) to lock-in the structure of the solid

foam by physical cross-linking. In Figure 5 the SEM micro-graphs of two solid foams synthesized according to foamcompositions reported in Table 6 are shown. It can be seen thatthe concentration of the surfactant allows to modulate to a largeextent the morphological characteristics of the solid foams. Byincreasing [PF-108] from 4 to 6% w/v, a considerable increaseof void and interconnect dimensions is obtained (Figure 5B andTable 6). Probably the use of higher surfactant concentration ismore effective in trapping the gas developed within the foam,thus, allowing to obtain a more expanded porous structure.

Physical cross-linking does not guarantee long-term durabilityof the scaffold in an aqueous environment. This drawback canbe easily overcome by a post chemical cross-linking of thecalcium/alginate-based scaffold with the system EDC/NHS. InFigure 6 it can be seen the different fate the two differentlycross-linked scaffold met during the time when soaked in water.After four weeks, the calcium-based network was almostcompletely dissolved. On the contrary, the chemically stabilizednetwork retained completely its shape. After chemical cross-linking, it can be chosen whether to remove calcium from thematrices or not. In the first case, it is just sufficient to dialyzethe scaffolds against a solution of EDTA for a few days andthen freeze-dry the scaffolds again.

Figure 5. SEM micrographs of alginate-based foams. Foaming system: sodium bicarbonate (3% w/v) and tartaric acid (2.7% w/v); surfactantused: pluronic F106; concentration surfactant: (A) 4% w/v, (B) 6% w/v. Foams were first physically cross-linked with CaCl2 (R ) 1) and then withEDC/NHS in acetone.

Table 6. Composition (% w/v) of the Aqueous Phase of theFoams and of the CO2 Generating System (NaCO3/Tartaric Acid)

[Alg]a

(% w/v)[PF-108]b

(% w/v)[NaHCO3]c

(% w/v)[TA]d

(% w/v)<D>e

(µm)<d>e

(µm)

5 4 3 2.7 180 305 6 3 2.7 260 80

a Concentration of alginate from Laminaria hyperborea with respect tothe volume of aqueous phase. b Concentration of pluronic F/108 withrespect to the volume of the aqueous phase. c Weight % of sodiumbicarbonate with respect to the volume of the aqueous phase. d Weight% of tartaric acid with respect to the volume of the aqueous phase.e Estimation of the average void <D> and interconnect diameters <d>.

Figure 6. Photographs of alginate solid foams obtained by physically calcium-induced gelation (a-c) and further stabilized by chemical cross-linking with EDC/NHS (d-f) after 1 day (a,d), 2 weeks (b,e), and 4 weeks (c,f) of dialysis against water.

Porous Alginate Hydrogels Biomacromolecules, Vol. 10, No. 8, 2009 2335

Conclusions

Tailoring the porous structure of alginate-based materialsis vital for meeting end-usage material characteristics. Asan example, considerable research efforts have been devotedin the preparation of alginate matrices for the removal ofpollutants (especially heavy metals) from wastewater and fortissue engineering applications. Such kinds of applicationsrequire porous matrices with interconnected pores of dimen-sion lying in very different size ranges. In this paper wedescribed two methods particularly suited for obtaining twoclasses of porous alginate-based materials with pore andinterconnects spanning over two well-separated ranges ofdimensions. The first one, which allows access to a porousmorphology characterized by pores and interconnects of theorder of 10 and a few µm, respectively, was obtained by aHIPE templating approach. Alcohol exchange and CO2

supercritical drying have proved to be efficient methods topreserve the volume and microscopic texture of the poly-HIPEs. The second consisted of replacing the liquid internalphase with a gas generated in situ the external aqueous phase,namely, CO2. The CO2 generating system and experimentalconditions were optimized to obtain materials with a well-defined morphology and with pores and interconnects in therange of 100-300 and 30-80 µm, respectively.

Acknowledgment. The authors thank Dr. Daniela Ferro forperforming SEM analysis, Dr. Franco Padella of ENEA-UTSMAT-CR Casaccia for the help given with the ball millingfacility, and “La Sapienza” University of Rome for financialsupport (Ateneo Funds).

Supporting Information Available. A photograph of theball milling apparatus, GPC chromatograms of startingalginate, numerical values of Mj w, Mj n, Mj η, and I of startingalginate and degraded alginate samples, 1H NMR spectra ofstarting alginate and degraded alginate after 40 h of milling,chemical reactions used for the generation in situ of CO2,chemical formulas of the surfactants used and their physicalparameters, and scheme of the apparatus used for foamsynthesis. This material is available free of charge via theInternet at http://pubs.acs.org.

References and Notes

(1) Escudero, R. R.; Robitzer, M.; Di Renzo, F.; Quignard, F. Carbohydr.Polym. 2009, 75, 52–57.

(2) Sone, H.; Fugetsu, B.; Tanaka, S. J. Hazard. Mater. 2009, 162, 423–429.

(3) Valentin, R.; Molvinger, K.; Viton, C.; Domard, A.; Quignard, F.Biomacromolecules 2005, 6, 2785–2792.

(4) Banerjee, A.; Nayakb, D.; Lahiri, S. Biochem. Eng. J. 2007, 33, 260–262.

(5) Peretz, S.; Cinteza, O. Colloids Surf., A 2008, 319, 165–172.(6) Park, H. G.; Kim, T. W.; Chae, M. Y.; Yoo, I.-K. Process Biochem.

2007, 42, 1371–1377.(7) Seki, H.; Suzuki, A. Ind. Eng. Chem. Res. 1996, 35 (4), 1378–1382.(8) Bajpai, J.; Shrivastava, R.; Bajpai, A. K. J. Appl. Polym. Sci. 2007,

103, 2581–2590.(9) Glicklis, R.; Shapiro, L.; Agraria, R.; Merchuk, J. C.; Cohen, S.

Biotechnol. Bioeng. 2000, 67, 344–353.(10) Gerecht-Nir, S.; Cohen, S.; Ziskind, A.; Itskovitz-Eldor, J. Biotechnol.

Bioeng. 2004, 88, 313–320.(11) Augst, A. D.; Kong, H. J.; Mooney, D. J. Macromol. Biosci. 2006, 6,

623–633.(12) Kuo, C. K.; Ma, P. X. J. Biomed. Mater. Res. 2008, 84A, 899–907.(13) Li, Z.; Gunn, J.; Chen, M.-H.; Cooper, A.; Zhang, M. J. Biomed. Mater.

Res. 2008, 86A, 552–559.(14) Dittrichw, R.; Tomandl, G.; Despang, F.; Bernhardt, A.; Hanke, Th.;

Pompe, W.; Gelinsky, M. J. Am. Ceram. Soc. 2007, 90, 1703–1708.

(15) Yildirim, E. D.; Yin, X.; Nair, K.; Sun, W. J. Biomed. Mater. Res.2008, 87B, 406–414.

(16) Leea, C. S. D.; Gleghornb, J. P.; Choi, N. W.; Cabodic, M.; Stroockd,A. D.; Bonassarb, L. J. Biomaterials 2007, 28, 2987–2993.

(17) Freeman, I.; Kedem, A.; Cohen, S. Biomaterials 2008, 29, 3260–3268.(18) Balakrishnan, B.; Jayakrishnan, A. Biomaterials 2005, 26, 3941–3951.(19) Kuo, C. K.; Ma, P. X. Biomaterials 2001, 22, 511–521.(20) Wase, J.; Foster, C. Biosorbents for Metal Ions; Taylor and Francis

Ltd.: London, 1997.(21) Chen, J.; Tendeyong, F.; Yiacoumi, S. EnViron. Sci. Technol. 1997,

32, 1433–1439.(22) Shapiro, L.; Cohen, S. Biomaterials 1997, 18, 583–590.(23) Hashimoto, T.; Suzuki, Y.; Yamamoto, E.; Tanihara, M.; Kakimaru,

Y.; Suzuki, K. Biomaterials 2004, 25, 1407–1414.(24) Bouhadir, K. H.; Lee, K. Y.; Alsberg, E.; Damm, K. L.; Anderson,

K. W.; Mooney, D. J. Biotechnol. Prog. 2001, 17, 945–950.(25) Mierisch, C. M.; Cohen, S. B.; Jordan, L. C.; Robertson, P. G.; Balian,

G.; Diduch, D. R. J. Arthrosc. Relat. Surg. 2002, 18, 892–900.(26) Alsberg, E.; Anderson, K. W.; Albeiruti, A.; Franceschi, R. T.;

Mooney, D. J. J. Dent. Res 2001, 80, 2025–2029.(27) Chung, T. W.; Yang, J.; Akaike, T.; Cho, K. Y.; Nah, J. W.; Kim,

S. I.; Cho, C. S. Biomaterials 2002, 23, 2827–2834.(28) Dar, A.; Shachar, M.; Leor, J.; Cohen, S. Biotechnol. Bioeng. 2002,

80, 305–312.(29) Klock, G.; Pfeffermann, A.; Ryser, C.; Grohn, P.; Kuttler, B.; Hahn,

H. J.; Zimmermann, U. Biomaterials 1997, 18, 707–713.(30) Bratthall, G.; Lindberg, P.; Havemose-Poulsen, A.; Holmstrup, P.; Bay,

L.; Sonderholm, G.; Norderyd, O.; Andersson, B.; Rickardsson, B.;Hallstrom, H.; Kullendorff, B.; Skold Bell, H. J. Clin. Periodontol.2001, 28, 923–929.

(31) Bent, A. E.; Tutrone, R. T.; McLennan, M. T.; Lloyd, L. K.; Kenelly,M. J.; Badlani, G. Neurourol. Urodyn. 2001, 20, 157–165.

(32) Dentini, M.; Rinaldi, G.; Barbetta, A.; Risica, D.; Anselmi, C.; Skjak-Braek, G. Carbohydr. Polym. 2007, 67, 465–473.

(33) Dentini, M.; Caucci, D.; Barbetta, A.; Crescenzi, V.; Skjk-Brk, G.;Capitani, D.; Mannina, L.; Viel, S. Biomacromolecules 2006, 7, 54–63.

(34) Dentini, M.; Rinaldi, G.; Risica, D.; Barbetta, A.; Skjak-Braek, G.Carbohydr. Polym. 2005, 59, 489–499.

(35) Holtan, S.; Bruheim, P.; Skjak-Braek, G. Biochem. J. 2006, 395, 319–329.

(36) Bjerkan, T. M.; Bender, C. L.; Ertesvag, H.; Drablos, F.; Fakhr, M. K.;Preston, L. A.; Skjak-Braek, G.; Valla, S. J. Biol. Chem. 2004, 279,28920–28929.

(37) Barbetta, A.; Cameron, N. R. Macromolecules 2004, 37, 3188–3201.(38) Barbetta, A.; Cameron, N. R. Macromolecules 2004, 37, 3202–3213.(39) Barbetta, A.; Cameron, N. R. J. Mater. Chem. 2000, 10, 2466–2472.(40) Barbetta, A.; Dentini, M.; Leandri, L.; Ferraris, G. React. Funct. Polym.

2009, submitted for publication.(41) Jerabek, K.; Pulko, I.; Soukupova, K.; Stefanec, D.; Krajnc, P.

Macromolecules 2008, 41 (10), 3543–3546.(42) Brun, N.; Lopez, B. J.; Hesemann, P.; Laurent, G.; Deleuze, H.;

Sanchez, C.; Achard, M.-F.; Backov, R. Chem. Mater. 2008, 20, 7117–7129.

(43) Ungureanu, S.; Deleuze, H.; Sanchez, C.; Popa, M. I.; Backov, R.Chem. Mater. 2008, 20, 6494–6500.

(44) Barbetta, A.; Massimi, M.; Di Rosario, B.; Nardecchia, S.; De Colli,M.; Conti Devirgiliis, L.; Dentini, M. Biomacromolecules 2008, 9,2844–2856.

(45) Palocci, C.; Barbetta, A.; La Grotta, A.; Dentini, M. Langmuir 2007,23, 8243–8251.

(46) Barbetta, A.; Massimi, M.; Conti Devirgiliis, L.; Dentini, M. Biom-acromolecules 2006, 7, 3059–3068.

(47) Barbetta, A.; Dentini, M.; Zannoni, E. M.; De Stefano, M. E. Langmuir2005, 21, 12333–12341.

(48) Barbetta, A.; Dentini, M.; De Vecchis, M. S.; Filippini, P.; Formisano,G.; Caiazza, S. AdV. Funct. Mater. 2005, 15, 118–124.

(49) Bokhari, M.; Carnachan, R. J.; Przyborski, S. A.; Cameron, N. R. J.Mater. Chem. 2007, 17, 4088–4094.

(50) Carnachan, R. J.; Bokhari, M.; Przyborski, S. A.; Cameron, N. R.Soft Matter 2006, 2, 608–616.

(51) Barrett, E. P.; Joyner, L. G.; Halenda, P. H. J. Am. Chem. Soc. 1951,73, 373–380.

(52) Gottschalk, J.; Husemann, K. Powder Technol. 1989, 58, 131–136.(53) Grasdalen, H.; Larsen, B.; Smidsrød, O. Carbohydr. Res. 1979, 68,

23–31. Crescenzi, V.; Dentini, M.; Bernalda, M. S.; Masci, G.; Rori,V.; Skjaak-Brk, G. Biomacromolecules 2001, 2, 958–964.

(54) Cameron, N. R.; Sherrington, D. C.; Albiston, L.; Gregory, D. P.Colloid Polym. Sci. 1996, 274, 592–595.

2336 Biomacromolecules, Vol. 10, No. 8, 2009 Barbetta et al.

(55) Valentin, R.; Molvinger, K.; Viton, C.; Domard, A.; Quignard, F.Biomacromolecules 2005, 6, 2785–2792.

(56) Valentin, R.; Horga, R.; Bonelli, B.; Garrone, E.; Di Renzo, F.;Quignard, F. Biomacromolecules 2006, 7, 877–882.

(57) Pierre, A. C.; Pajonk, G. M. Chem. ReV. 2002, 102, 4243–4265.(58) Draget, K. I.; Skjak-Braek, G.; Smidsrod, O. Carbohydr. Polym. 1994,

25, 31–8.

(59) Dentini, M.; Rinaldi, G.; Barbetta, A.; Risica, D.; Skjak-Braek, G.Carbohydr. Polym. 2006, 63, 519–526.

(60) Buzzacchi, M.; Schmiedel, P.; von Rybinski, W. Colloids Surf., A2006, 273, 47–54.

BM900517Q

Porous Alginate Hydrogels Biomacromolecules, Vol. 10, No. 8, 2009 2337