Article pubs.acs.org/Biomac
Ionically Gelled Alginate Foams: Physical Properties Controlled by Operational and Macromolecular Parameters Therese Andersen,*,†,‡ Jan Egil Melvik,† Olav Gåserød,† Eben Alsberg,§ and Bjørn E. Christensen‡ †
FMC BioPolymer AS/NovaMatrix, Sandvika, Norway NOBIPOL, Department of Biotechnology, Norwegian University of Science and Technology (NTNU), Trondheim, Norway § Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, United States ‡
S Supporting Information *
ABSTRACT: Alginates in the format of scaffolds provide important functions as materials for cell encapsulation, drug delivery, tissue engineering and wound healing among others. The method for preparation of alginate-based foams presented here is based on homogeneous, ionotropic gelation of aerated alginate solutions, followed by air drying. The method allows higher flexibility and better control of the pore structure, hydration properties and mechanical integrity compared to foams prepared by other techniques. The main variables for tailoring hydrogel properties include operational parameters such as degree of aeration and mixing times and concentration of alginate, as well as macromolecular properties such as the type of alginate (chemical composition and molecular weight distribution). Exposure of foams to γ-irradiation resulted in a dose-dependent (0− 30 kGy) reduction in molecular weight of the alginate and a corresponding reduction in tensile strength of the foams.
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INTRODUCTION Hydrogel-forming biopolymers are being extensively explored within a wide range of pharmaceutical and biomedical applications, thereby allowing different types of scaffolds to be prepared and optimized for intended uses.1 One such polymer is alginate, which comprise a family of linear (unbranched) polysaccharides that can be extracted from brown seaweed and some bacteria. Alginates consist of residues of (1→4) linked β-D-mannuronate (M) and α-L-guluronate (G). Alginates are first biosynthesized as homopolymeric mannuronan, before being further processed by C5-epimerases converting M into its C5-epimer G.2 The enzymes tend to produce either G-blocks (..GGGG..) or alternating blocks (..MGMGMG..) in addition to the remaining M-blocks. The content and distribution of M and G residues vary considerably between different alginates, allowing a structural basis for tailoring alginate-based materials. The G-blocks are the key structural elements giving rise to gelation with divalent cations such as Ca2+,2,3 and the mechanical properties of alginate-based hydrogels will therefore depend on the monomer composition of the alginate, its molecular weight, gelling ions, and concentrations. As alginates form hydrogels under gentle conditions, the gels may be used to entrap cells and function as an extracellular matrix (ECM) with nonimmunogenic and adjustable bioresorption properties.4 Alginates are, therefore, of particular interest for cell encapsulation and implantation applications. Macroporous scaffolds as foams or sponges are recently recognized as materials providing improved cell invasion, vascularization, and improved mass transport of nutrients, © 2012 American Chemical Society
oxygen, and waste removal compared to nonporous hydrogels.5,6 Such characteristics may be beneficial for tissue engineering and regenerative medicine applications. Porous alginate scaffolds have been developed for different tissue engineering applications such as parenteral drug delivery7 and release of cell signaling factors for revascularization,8 and repair of cartilage,9,10 cardiac,11 bone,12 and liver.13 Additionally, dry porous alginate scaffolds have the ability to absorb high amounts of wound exudates and form conditions beneficial for wound healing.14 Lyophilization combined with different freezing regimes15,16 is currently the main method for preparation of alginate foams and is based either on ionic6 and covalent cross-linked hydrogels,16 covalent cross-linked hydrogels with porogen salts,17 ionic and covalent cross-linked hydrogels formed after in situ gas formation in alginate solution,18 or precipitates of alginate with oppositely charged polymers such as chitosan.7,12 Although a wide variety of foams can be prepared with known techniques, most of them are only applicable for smaller sample sizes, and large scale manufacture can not be accomplished in a cost and time-efficient manner. Swelling, mechanical strength, and degradation of alginate structures are known from the work of others to be influenced by the amount of gel forming ions.19−21 The amount and type of foaming agents are also important for preparation of porous alginate structures enabling incorporation of air by high shear mixing, stabilizing the wet foam after it is formed and modifying Received: July 27, 2012 Revised: September 18, 2012 Published: September 19, 2012 3703
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hydration properties due to differences in surface activity.22 Here, polysorbate and hydroxypropyl methylcellulose (HPMC) were used, but other foaming agents that may work well have been tested by others and includes poloxamers,18,23 bovine albumin,23 sodium dodecylsulfate,18 tyloxapol18 and triton.18 However, it is important to consider the foaming agent in relation to its end use as some of them may be toxic and not biocompatible. Active ingredients may be incorporated into alginate foams during preparation or added later before use. The use of alginate foams as carriers of curcumin was demonstrated by Hegge et al.24,25 as a potential delivery system for antimicrobial photodynamic therapy of topical wounds. Alginate foams that absorb high amounts of wound exudates and keep the wound bed moist may form a good healing environment and avoid tissue damage when removable in one piece. Other uses may include tissue engineering applications either as a biologic delivery device or as a cell scaffold material. Improved cell− matrix interaction may be achieved by using modified alginates with signaling molecules covalently attached such as peptides11,26 or sulfate.8 The present work describes the preparation of ionically gelled and air-dried alginate foams. Aerated alginate solutions were first homogenously gelled by slow internal gelling3,19,27 before air drying. The internal gelling of alginate, which is induced and controlled by calcium carbonate particles (CaCO3) and the slowly acidifying glucono-δ-lactone (GDL), enables mechanical incorporation of air and subsequent molding of the wet foam prior to onset of the gelation.24,25,28 Although the internal gelling has been used previously for preparation of homogeneous alginate hydrogels, here we present a new approach utilizing slow gelling to form and control the characteristics of gelled alginate foams. This technique for foam preparation enables new and additional operational parameters as well as utilization of alginate macromolecular properties for tailoring of foam properties. The characteristics of the dried foams such as average pore size, pore interconnectivity, absorbency properties, and mechanical properties were influenced by the amount of incorporated air (foam density), alginate type, and concentration, in addition to gamma irradiation dose (product sterilization).
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and Structure Probe, West Chester, PA, U.S.A.) trays with 4 mm high removable frames all made from acryl. Alginate Foam Preparation. Alginate foams (see Table S2 for details of composition and operational parameters) were made by preparing a dispersion of CaCO3 in MQ-water containing Polysorbate 20. Sorbitol was then added and dissolved by gentle swirling the mixing bowl before glycerol, HPMC and a premade aqueous alginate solution (4.1%, w/w) was added. All ingredients were mixed using a Hobart N50 mixer (Hobart) with a wire loop whip at intermediate speed (125 rpm) for 1 min to dissolve the HPMC and ensure a homogeneous blend. The blend was then mixed at high speed (259 rpm) for 1−7 min to incorporate air. A freshly made solution of GDL and MQ-water was added to the mixing bowl and high speed mixing continued for 15−60 s until the desired amount of air was incorporated. The wet foam density (WFD) was determined by weighing a 100 mL tared weighing boat filled with wet foam. The foam was then transferred to trays using a spatula and the top surface was leveled off with a ruler to ensure a consistent thickness. The foam was kept uncovered on the bench at ambient temperature for 1 h and dried in an air forced drying oven at 80 °C (60−90 min, depending on foam density). The water content of the dried foam was determined using an infrared drier by heating 15 foam discs of 1.0 cm in diameter to 115 °C for 30 min (triplicates). The dry foam discs were conditioned in advance in a closed container containing a saturated solution of sodium nitrite (Merck, Darmstadt, Germany) overnight to maintain a constant humidity (66% humidity at 20 °C). The alginates were saturated with 84% calcium where 100% saturation equals 1 mol divalent cations per 2 mol alginate monomers. The molar ratio between GDL and the CaCO3 were kept constant at 2 mols GDL per 1 mol CaCO3. Gelling Kinetics of Different Alginates. Formulations with 2% LVG, LVM, and MVG alginate were prepared as for the foams, but without plasticizers and foaming agents (Table S2). An aqueous dispersion containing alginate and CaCO3 was prepared by first dispersing the carbonate in MQ-water and sonicating for 3×15 s. The alginate powder was added to the dispersion and a stock solution of alginate (2.8−3.0%, w/w) and CaCO3 (0.48−0.53%, w/w) was prepared using a homogenizer Ultra-Turrax T25 (IKA Werke). A weighing boat was used to weigh and mix the suspension of alginate/ carbonate and a freshly made solution of GDL (7.7−12.4%, w/w) containing 4.0 or 5.0 g. The suspension was applied to a Physica MCR 300 rheometer (Anton Paar). The gelling kinetics of the different formulations were followed by oscillatory measurements at 1 Hz frequency and 0.001 strain with a 1.000 mm gap and serrated plates (PP50) at 20 °C. The measurements started 2 min after the GDL was first mixed with the alginate/carbonate suspension. The time required to reach the solution/gelation (sol/gel) transition point was registered. Sol/gel transition point occurs when loss modulus (G″) = storage modulus (G′) and the phase angle (δ) = 45°. The gelling rate was measured in triplicates ± standard error (SE). Pore Characterization. The average pore size distribution on the upper surface of the dry foam was determined by light microscopy (Leica DM 2500; camera, Leica DFC420). Software (Leica application suite 2.4.0 R1) was used to manually draw 50 lines of pore diameters on three images for each foam and the average value of the 150 lines was calculated. Pore interconnectivity was visually evaluated by light microscopy images and SEM (FEI Company Phenom, sputter-coated with gold/palladium) images of foam top surfaces and foam cross sections. Also, the level of transparency by hydrating the dried foams in MQ-water was evaluated qualitatively related to interconnectivity of the pores. Absorbency Properties and Dry Foam Density. The absorbency test method is based on BS EN 13726−1:2002.29 In brief, 5 × 5 cm foam pieces were conditioned at 66% humidity for at least 16 h. The dry foam weight was then recorded (W1) and the dry foam density (DFD) calculated. DFD is reported as grams of material per area, as this better shows the effect of different amounts of air incorporated. Volume calculations will be less practical here and provide less accurate results, as there were variations in dry foam thickness among the formulations. Differences in thicknesses were
MATERIALS AND METHODS
Materials. The chemical composition (fraction of G-monomers (FG) and average G-block length (NG>1)), weight average molecular weight (MW) and water content of the dry powders of the sodium alginates used are presented in Table S1 in the Supporting Information. Alginates of ultrapure grade (PRONOVA UP) were obtained from NovaMatrix (Sandvika, Norway). Calcium carbonate (CaCO3, HuberCal 500 Elite) was received from J.M. Huber Corp. (Quincy, IL, U.S.A.), hydroxypropyl methyl cellulose (HPMC, Hypromellose, Pharmacoat 603, substitution type 2910, 2% viscosity 3 mPas) was delivered by Shin-Etsu (Tokyo, Japan) and glucono-δlactone (GDL, Lysactone T) was provided by Roquette (Lestrem, France). D(−)-Sorbitol and glycerol were purchased from VWR (BDH, Prolabo, Leuven, Belgium) and Polysorbate 20 from SigmaAldrich (Fluka, Steinheim, Germany). The water content of the alginates were determined on 1 g material as loss on drying at 115 °C for 15 min using an infrared drier (Mettler LP16) with a balance (Mettler PM100), both from Mettler Toledo (Oslo, Norway). Model physiological solutions used were made from either 2.5 mM Ca2+ (CaCl2·2H2O, Sigma-Aldrich, Riedel-de Haën, Seelze, Germany) and 142 mM Na+ (NaCl, Merck, Darmstadt, Germany) or Hanks’ balanced salt solution (H8264, Sigma-Aldrich, Steinheim, Germany). The foam was molded in Teflon-coated (Bytac AF-21 FEP PTFE, SPI Supplies 3704
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G3000PWXL (Tosoh Bioscience LLC, King of Prussia, PA, U.S.A.), and mobile phase was 0.05 M Na2SO4 (Merck, Darmstadt, Germany) and 0.01 M EDTA (Calbiochem, Merck, Darmstadt, Germany) in MQ-water. Software for analysis from Wyatt Technology Corp. was Astra V SP, and constants used were refractive increment index (dn/ dc)μ of 0.150 mL/g31 and second virial coefficient A2 of 5.0 × 10−3 mL·mol/g2.
influenced by coalescence of wet foam before drying (all molded 4 mm thick) and slight compression during removal of the most fragile foams from the trays. The foam pieces were then immersed in 40 times their weight of a model physiological solution at 37 °C comprising 2.5 mM Ca2+ and 142 mM Na+ (solution A) and then kept at 37 °C for 30 min. The foam was then lifted in one corner with forceps and allowed to passively drip for 30 s before the weight of the wet foam was recorded (W2). The drained solution was collected and weighed (W3). Absorbency capacity and drainage was calculated by the following equations and reported as the average of six parallels ± SE:
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RESULTS AND DISCUSSION Dry Foam Composition. As the foam comprises hygroscopic alginate and plasticizers, the moisture content of the foam may change in contact with the surrounding humidity and the dry foam will become soft and pliable. The water content of dried alginate foam made from 2.0% MVG and conditioned at 66% humidity overnight was 18% one day after production. Neither the appearance nor the flexibility of the foams was notably changed after this treatment. See Table S3 for a detailed composition of the components in the dry foam. Foam Density and Structure. During processing of the wet alginate foam, the pore content could be controlled and varied by changing the mixing time. The maximum amount of air that could be incorporated into the foams depended on the formulation. The minimum amount of air incorporated was the amount that resulted in dry foams that still had the ability to absorb liquid, as this property is impaired as the foams become more compact. The wet foam density (WFD) measured immediately after mixing correlated well with the obtained dry foam density (DFD; Figure 2). DFD is reported as grams per area as all the wet foams were molded at the same thickness (4 mm).
absorbency(g/g) = (W2 − W1)/W1 drainage(%) = W3/(W3 + (W2 − W1)) × 100 Mechanical Properties of Rehydrated Foams. Mechanical properties of rehydrated foams were measured using a Texture Analyser with tensile grips (A/TG) and software TA-XTplus from Stable Micro Systems. The foams were cut as test specimens Type I according to ASTM test method D638−10 (Figure 1A)30 or a self-
Figure 1. Test specimens used to characterize the mechanical properties of the alginate foams. Type I ASTM D638−10 test specimen (A) where G is defined as the gage length (initial extensometer span for calculation of Young’s modulus) and selfdesigned dumbbell (B). designed dumbbell shape (Figure 1B). Three samples were placed in 100 g Hanks’ solution for 15 min. Excess solution was removed on three layers of one ply hand tissue paper and the foam was fixed to the tensile grips with a gap between the grips of 11.5 cm (Figure 1A) or 4.0 cm (Figure 1B). Tensile stress was applied at a constant strain rate of 0.50 mm/sec until foam rupture. Young’s modulus, tensile strength at break, and nominal strain at break were calculated. γ-Irradiation. Dry alginate foams made from 2% MVG and alginate powder (MVG) were γ-irradiated by doses of 10, 20, and 30 kGy obtained by a 60Co gamma source at the Institute for Energy Technology (Kjeller, Norway). Isolation of Alginate. Alginate was isolated from γ-irradiated foams by first dissolving a 5 × 5 cm foam piece in 30 mL 100 mM citrate (sodium citrate tribasic dehydrate, Sigma-Aldrich, Steinheim, Germany). This solution was then poured into 50 mL of ethanol (Kemetyl, Vestby, Norway) to precipitate the alginate. The precipitate was then washed twice in ethanol and kept at ambient conditions to let the ethanol evaporate. Determination of Molecular Weight. The molecular weight distributions (and the weight- and number average molecular weight) of alginates were determined as previously described by Vold et al.31 In short, the setup consisted of a Waters SEC (Waters 2695 Separations module), a MALS-detector (DAWN HELEOS; Wyatt Technology Corp., Santa Barbara, CA, U.S.A.), and an Optilab rEX RI-detector (Wyatt Technology Corp., Santa Barbara, CA, U.S.A.). The SECcolumns used were guard column, G6000PWXL, G5000PWXL, and
Figure 2. Dry foam density (DFD) as a function of the wet foam density (WFD). The thickness of the dried foams was in the range of 2.2−2.7 mm. The straight line was fitted by the method of leastsquares (R2: 0.92). Error bars depict SE when exceeding the size of the symbols.
Figure 3A and B present images of dry foams and foams rehydrated with MQ-water, respectively, obtained for both high and low density foams (26.4 and 13.2 g/cm2). The foam with low density contained visually larger pores when dry and became more transparent when rehydrated as compared to the foams with higher density. The difference in transparency may be attributed to an increased interconnectivity, where the low density foam has more interconnected pores and a more open pore network that enables absorption of more liquid as the space in closed pores is otherwise occupied by entrapped air. 3705
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alginate. DFD was controlled by varying the mixing time and hence, the amount of incorporated air. An essentially linear decrease in average pore size (from 400 to 225 μm) was observed when DFD increased from 12 to 28 mg/cm2 (Figure 5A). This change is attributed to various degrees of coalescence
Figure 3. Foams with different densities before (A) and after (B) rehydration in MQ-water. DFD of foams referred to as high and low DFD were 26.4 ± 0.7 g/cm2 and 13.2 ± 0.1 g/cm2, respectively. Scale bar: 250 mm.
The foam structures were further investigated by electron and optical microscopy (Figure 4). Cross section and top
Figure 5. Average foam pore size, fluid drainage, and absorbency capacity evaluated at different dry foam densities (DFD) at constant MVG alginate concentration of 2.0% (A, C, E) and alginate types and concentrations at constant DFD of 16 mg/cm2 (B, D, F). Error bars depict SE when exceeding the size of the symbols. Dotted lines are added as guides to the eye and are fitted by the method of leastsquares as straight lines or second degree lines. Symbols: ○ MVG, △ LVG, ▲ LVM.
of air bubbles prior to gelation, which is likely to be affected by the pore wall (lamellae) thickness, which again necessarily decreases when the amounts of air incorporated increases. Moreover, the thinner walls surrounding the air bubbles are probably more likely to break during gelling and drying, which may contribute to larger pores and also higher pore interconnectivity in the dried foam. Figure 5B shows average pore size data obtained at constant DFD (16 mg/cm2), but different concentrations of MVG alginate (1.5−2.5%), and for 2% also three different alginates (LVM, LVG, and MVG). Here, the largest pores were associated with low alginate concentrations and, hence, the lowest solution viscosities before gelation takes place. Large pores presumably are formed by coalescence of smaller bubbles, and high viscosity reduces the rate and extent of the process compared to lower viscosities. Results obtained for LVM, LVG, and MVG show, however, that the type of alginate plays an additional role, because LVM alginate gives smaller pores than
Figure 4. SEM images of top surface (A) and cross section (B) of dry alginate foams. Light microscope images of top surface (C) and cross section (D) of dry alginate foams.
surface SEM images of dry foams (Figure 4A, B) showed an interconnected pore network. Light microscope images of the upper surface and cross section of dried foams (Figure 4C, D) also illustrated substantial variation in pore size. Pore Size. The influence of DFD on the average pore size was first investigated for foams prepared from 2% MVG 3706
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LVG. These alginates have essentially the same solution viscosity (due to similar Mw), but LVG has a higher content of gelling G-residues and longer G-blocks, which are involved in the gel formation induced by calcium. The gels formed from LVG will be stronger than LVM gels, but the initial rate of gelation may be higher for the latter. This was confirmed by independently monitoring the gelation of LVM and LVG foam formulations (without air, plasticizer, and foaming agent) by oscillatory rheometry. The time to reach the sol/gel transition point was found to be 303 ± 10 s and 223 ± 9 s for LVG and LVM, respectively. The onset of gelation and establishment of a gel structure was also confirmed by the initially higher G′ of the LVM formulation (15.59 ± 1.05 Pa for LVM vs 10.04 ± 0.04 Pa for LVG at δ = 45°), although the LVG formulation at later stages formed a stronger gel due to more gelling sites. The role of factors other than solution viscosity is also illustrated by the fact that MVG, which gives more viscous solutions than LVG, does not give a corresponding decrease in average pore size. As no difference in the initial gelation kinetics could be observed using the approach mentioned in the paragraph above (data not shown), the viscosities may possibly be too high to influence coalescence. A larger difference may be expected with alginates of lower MW. Fluid Drainage. Fluid drainage refers to the immediate and passive loss of fluid when retrieving fully hydrated foams from the liquid bath. For 2% MVG foams with low DFD and, consequently, large pores, extensive drainage (35%) took place, whereas foams with small pores retained essentially all fluid (Figure 5C). The extent of drainage also decreased with increasing concentration of MVG alginate (at constant DFD; Figure 5D) and paralleled the concomitant decrease in average pore size (Figure 5B). The LVM alginate deviates from the general trend, having smaller pores but higher drainage. Absorbency. The absorbency capacity (retained fluid after draining) showed a bell-shaped curve with an optimum at about 16−20 mg/cm2 DFD for 2% MVG based foams (Figure 5E). In contrast, the capacity increased linearly with the concentration of MVG alginate (Figure 5F). Here, no difference was seen between LVG and MVG at 2% alginate, whereas LVM had significantly lower absorbency capacity (Figure 5F). The increased absorbency with increasing concentration demonstrates the high water-binding capacity of alginates. Another main factor is the presence of closed (not interconnected) pores, which increases by increasing DFD and are inaccessible to fluid during swelling. Swelling of alginate gels in a liquid environment is influenced by the concentration of ions and pH in addition to temperature.20,32 The testing of absorbency capacity by using a selected model physiologic solution (solution A, used in standard test methods for determination of absorbency capacity of wound dressings29) may be relevant for wound healing applications among others. The relatively high content of Ca2+ in the solution (2.5 mM) and in wound exudates compared to extracellular fluids (e.g., serum: 1.0−1.3 mM ionized calcium33) may have some impact on the mechanical properties of foams and absorbency profile, as it is known that surrounding Ca2+ retard the replacement of gelling calcium with nongelling sodium.20 Mechanical Properties of Rehydrated Foams. Figure 6 shows the mechanical properties of foams immersed in Hanks’ solution (1.3 mM Ca2+, test specimen, as in Figure 1A). The same set of foams, as shown in Figure 5, was used to evaluate how the mechanical properties are influenced by DFD and
Figure 6. Influence on mechanical properties Young’s modulus (A, D), tensile strength at break (B, E), and nominal strain at break (C, F) of foams rehydrated in Hanks’ solution (test specimen were as shown in Figure 1A, n = 10) evaluated for different dry foam densities (DFDs) at constant alginate concentration of 2% (A−C) and alginate types and concentrations at constant DFD at 16 mg/cm2 (D−F). Error bars depict SE when exceeding the size of the symbols. Dotted lines are added as guides to the eye and are fitted by the method of leastsquares as straight lines. Symbols: ○ MVG, △ LVG, ▲ LVM.
alginate type and concentration. No dimensional changes of the foams were seen by rehydration in Hanks’ solution, except for the foam made from LVM alginate that showed about a 5% increase in length and width. For this foam the tensile measurements and calculations was adjusted accordingly. Figure 6A−C show foams prepared with different DFDs and a constant alginate concentration of 2%. For rehydrated foams within the range of DFDs tested, the Young’s modulus (Figure 6A) and tensile strength (Figure 6B) increased with increasing DFD, whereas the nominal strain at break was slightly decreasing (Figure 6C). The data indicated a linear correlation and showed that, by doubling the DFD, the Young’s modulus and the tensile strength were increased about three times. Figure 6D−F show foams prepared with different alginate types and concentrations and a constant DFD. The influence on Young’s modulus of hydrated foams with different MVG alginate concentration showed similar values for 2.0 and 2.5% alginate, both being somewhat higher than for 1.5% alginate (Figure 6D). The tensile strength (Figure 6E) and nominal strain at break (Figure 6F) increased linearly with the alginate concentration. The data show that both the alginate molecular weight distribution and chemical composition were important for the mechanical properties. The foam with the highest and 3707
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elimination and acid hydrolysis, which may occur simultaneously under physiological conditions.39 Furthermore, including small amounts of periodate oxidized alginate in implantable structures has been shown to accelerate alginate degradation due to the presence of labile dialdehydes in the polymer chain.40−43 Alginate foams were γ-irradiated in the dry state and, subsequently, rehydrated after they were cut, as shown in Figure 1B. They exhibited decreasing tensile strengths with increasing irradiation doses (Figure 7A). Isolation of the
lowest Young’s modulus was obtained by using LVG and LVM alginate at 2.0% alginate, respectively (Figure 6D). Foams made from MVG alginate had a higher tensile strength (Figure 6E) and nominal strain at break (Figure 6F) than foams made from LVG alginate. The lowest values on all parameters describing mechanical properties were obtained for the foams made from LVM alginate (Figure 6D−F). The relations found by increasing DFD and alginate concentration on increasing mechanical properties are most likely due to the increased amount of alginate per area. The tensile foam strengths measured for foams made from different alginate types show that the mechanical strength increases by both weight average molecular weight (MW) and G-content (FG). Although the nominal strain at break in Figure 6C did not differ much over the range of densities, the data may indicate for the foams tested here, that the increased average foam pore size and pore interconnectivity allows the foam to stretch more before it ruptures (Figures 3 and 5A). This is opposed to the nominal strain at break in Figure 6F, which increased by increasing alginate concentration and decreasing average pore size (Figure 5B), showing the high importance of alginate concentration. Drury et al. obtained similar results as shown here by comparing the tensile properties of hydrogels from M-rich and G-rich alginates gelled with CaSO4.34 An increased compression strength of hydrogels from high-G alginates compared to high-M alginates have also been shown previously by Draget et al.27 and Kuo and Ma.19 In both cases an increased elasticity and strength by increasing the alginate concentration were demonstrated in a similar manner to what is demonstrated here for the foams. The foams tested here showed an increased deformability before rupture by increasing the alginate MW, FG, and concentration. The internal gelling technique used to prepare alginate foams will result in an almost homogeneous distribution of alginate and calcium.19,27 Some sedimentation of CaCO3 may, however, occur before the particles dissolve and the released calcium ions bind to the alginate, resulting in a slightly weaker upper part of the foam. This effect will depend on the viscosity of the wet foam. Sedimentation can therefore be reduced by increasing the alginate concentration, by increasing the alginate molecular weight, or by adding another viscosifier such as hyaluronate or xanthan.27 Increased particle surface area and increased gelling kinetics may also be obtained by using smaller particles of CaCO3 or additionally dispersing them by sonication.27,35 The temperature-dependent rate of GDL hydrolysis will also affect the gelling kinetics. Influence of γ-Irradiation. γ-Irradiation is a standard method for sterilizing biomaterials. However, exposing alginates to γ-irradiation is known to reduce the molecular weight in a dose-dependent manner36,37 and may, consequently, alter the properties of the biomaterial. During degradation of ionically gelled structures in vivo, alginate molecules will gradually leak out of the implantation site as a result of exchange of gelling ions with nongelling monovalent ions such as sodium ions. Irradiated alginates have been used to prepare implantable structures with different degradation properties.36,37 As mammals do not have the capability to degrade alginates enzymatically, it has been found to be beneficial to use alginates in the lower molecular weight range to increase degradation rates. For alginates above approximately 50 kDa, renal clearance may not be allowed.38 However, a slow but detectable cleavage of the glycosidic bonds will take place due to alkaline β-
Figure 7. Dependence on the irradiation dose on the tensile strength of γ-irradiated foams following immersion in solution A (test specimen as shown in Figure 1B, n = 3), and MW for the alginate isolated from the irradiated foams (A). Inverse MW of alginate isolated from foam and powder as a function of γ-irradiation doses (B). Correlation coefficients: R2 (powder), 0.994; and R2 (foam), 0.998. Error bars depict SE when exceeding the size of the symbols.
alginate followed by SEC-MALS analysis showed that the decrease in tensile strength was indeed closely associated with a corresponding decrease in weight average molecular weight (Mw; Figure 7A). The effect of irradiation on the foam tensile strength by changing the MW by γ-irradiation was here tested by leaving other parameters such as average pore size and foam density unchanged. As the irradiation did not affect the appearance of the foam, this may be used to prepare foams with the same structure but different physical properties. Foams may also be made initially from lower molecular weight alginates, 3708
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Biomacromolecules but then a higher alginate concentration is needed to obtain the same wet foam property before gelling and drying. Figure 7B shows the change in 1/Mw as a function of the irradiation dose. The linearity suggests that the alginates degrade randomly44 upon irradiation, with little or no preference for specific residues. The figure also includes data obtained for irradiated alginate powder, showing that the alginate degrades at approximately the same rate as in the dry foam. These data indicate that the radiation-induced degradation mechanism is independent of the alginate dry state format. In addition to producing sterile foams, irradiation treatment may be actively used to prepare foams with desirable physical properties without changing the foam appearance. Standard sterilization doses are 25 kGy or 15 kGy for materials with lower bioburden, but any dose ≤25 kGy derived to achieve a sterility assurance level of 10−6 can be used.45 However, as γ− irradiation significantly reduce the mechanical integrity of the foam, other sterilization techniques such as treatment with ethylene oxide and alcohol can be used to avoid the loss of initial mechanical strength. When using alternative sterilization techniques, the potential of having residual toxic components present or not full penetration by the sterilizing agent should be considered.
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CONCLUSION The presented method for preparation of dried alginate foams by ionotropic gelation of aerated alginate solutions followed by air drying, was found to enable high processing and formulation flexibility. This new method offers utilization of a combination of operational parameters such as aeration, mixing time and alginate concentration, as well as macromolecular properties such weight average molecular weight and chemical composition of the alginate. These parameters were found to influence the resulting average pore size distribution, hydration properties, and mechanical integrity that is not possible by other techniques. Based on the relations in foam properties described here, the optimization of foam preparation for a certain end use such as a wound dressing, the DFD and alginate type and concentration can be selected to give the highest possible absorbency capacity, an acceptable fluid drainage, and mechanical integrity, allowing one piece removal. Exposure of dried foams to γ-irradiation showed a dose-dependent reduction in foam strength and alginate molecular weight without affecting the foam appearance. ASSOCIATED CONTENT
S Supporting Information *
Tables of weight average molecular weight and chemical composition of alginates used for preparation of foams, formulations of wet foams before drying with different alginate concentrations and operational parameters, and final composition of dry alginate foam. This material is available free of charge via the Internet at http://pubs.acs.org.
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ACKNOWLEDGMENTS
The authors thank Joseph Lee for SEM images and Frode Magnussen, Christine Markussen, and Henrik B. Tomren for SEC-MALS and 1H NMR characterization of alginates.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 3709
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