Biomimetic strain hardening in interpenetrating polymer network hydrogels

Polymer 48 (2007) 5376e5387 www.elsevier.com/locate/polymer

Biomimetic strain hardening in interpenetrating polymer network hydrogels David Myung a,b, Wongun Koh c, Jungmin Ko a, Yin Hu a, Michael Carrasco d, Jaan Noolandi a,b, Christopher N. Ta b, Curtis W. Frank a,* a

Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stauffer III, Stanford, CA 94305-5025, United States Department of Ophthalmology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5080, United States c Department of Chemical Engineering, Yonsei University, 134 Shinchon-dong, Seodaemoon-ku, Seoul 120-749, South Korea d Department of Chemistry, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053, United States

b

Received 12 May 2007; received in revised form 27 June 2007; accepted 27 June 2007 Available online 13 July 2007

Abstract In this paper, we present the systematic development of mechanically enhanced interpenetrating polymer network (IPN) hydrogels with Young’s moduli rivaling those of natural load-bearing tissues. The IPNs were formed by synthesis of a crosslinked poly(acrylic acid) (PAA) network within an end-linked poly(ethylene glycol) (PEG) macromonomer network. The strain-hardening behavior of these PEG/PAA IPNs was studied through uniaxial tensile testing and swelling measurements. The interaction between the independently crosslinked networks within the IPN was varied by (1) changing the molecular weight of the PEG macromonomer, (2) controlling the degree of PAA ionization by changing pH, and (3) increasing the polymer content in the PAA network. Young’s moduli and the maximum stress-at-break of the swollen hydrogels were normalized on the basis of their polymer content. Strain hardening in the IPNs exhibited a strong dependence on the molecular weight of the first network macromonomer, the pH of the swelling buffer, as well as the polymer content of the second network. The results indicate that the mechanical enhancement of these IPNs is mediated by the strain-induced intensity of physical entanglements between the two networks. The strain can be applied either by mechanical deformation or by changing the pH to modulate the swelling of the PAA network. At pHs below the pKa of PAA (4.7), entanglements between PEG and PAA are reinforced by interpolymer hydrogen bonds, yielding IPNs with high fracture strength. At pHs above 4.7, a ‘‘pre-stressed’’ IPN with dramatically enhanced modulus is formed due to ionization-induced swelling of the PAA network within a static PEG network. The modulus enhancement ranged from two-fold to over 10-fold depending on the synthesis conditions used. Variation of the network parameters and swelling conditions enabled ‘‘tuning’’ of the hydrogels’ physical properties, yielding materials with water content between 58% and 90% water, tensile strength between 2.0 MPa and 12.0 MPa, and initial Young’s modulus between 1.0 MPa and 19.0 MPa. Under physiologic pH and salt concentration, these materials attain ‘‘biomimetic’’ values for initial Young’s modulus in addition to high tensile strength and water content. As such, they are promising new candidates for artificial replacement of natural tissues such as the cornea, cartilage, and other load-bearing structures. Ó 2007 Published by Elsevier Ltd. Keywords: Hydrogel; Artificial tissue; Double network

1. Introduction The fragility of most hydrogels poses a formidable obstacle to their application as substitutes for natural tissues, which have exceptional mechanical properties despite high water

* Corresponding author. Tel.: þ1 650 723 4573; fax: þ1 650 723 9780. E-mail address: [email protected] (C.W. Frank). 0032-3861/$ - see front matter Ó 2007 Published by Elsevier Ltd. doi:10.1016/j.polymer.2007.06.070

content (>75%). For instance, poly(ethylene glycol) (PEG) and poly(acrylic acid) (PAA) are relatively low-strength hydrogels with qualities such as biocompatibility, hydrophilicity, transparency, permeability, and resistance to protein adsorption [1,2] that would confer advantages if incorporated into implantable devices. PEG is widely utilized as a surface coating for intravenous and intraperitoneal catheters due to its ability to prevent the adhesion of thrombogenic and immunogenic proteins. PAA is the absorbent material used in infant diapers,

D. Myung et al. / Polymer 48 (2007) 5376e5387

and also has been investigated for use in ophthalmic medications [3]. To date, however, the fragility of PEG and PAA has precluded them from serving as the primary material for tissue replacement or augmentation applications that require high mechanical strength [4e6]. Although a number of strategies can be used to improve the strength of these hydrogels, including high crosslinking density, fiber reinforcement, and copolymerization, the strength enhancement afforded by these strategies often involves some compromise in the desired characteristics of the original material, such as hydrophilicity, transparency, or permeability [7]. For many tissue engineering applications, maintenance of these properties is critical to their performance in vivo. A recently described class of interpenetrating polymer networks (IPNs) has been shown to dramatically improve the mechanical properties of hydrogels [7]. These ‘‘double networks’’ (DNs), first described by Gong and coworkers [7], are distinguished by their extremely high fracture strength despite high water content (60e90%). As such, they are considered promising candidates for the replacement of a variety of load-bearing anatomical structures such as cartilage [7]. In Gong’s most extensively studied DN system, a densely crosslinked, ionizable first network consisting of poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) is interpenetrated with a flexible, loosely crosslinked, neutral second network consisting of poly(acrylamide) (PAAm). In recent work, they have characterized this material through compression tests [7], dynamic light scattering [8], and tear-testing to determine fracture energy [9,10], and have shed light on the role of the neutral PAAm second network in retarding crack propagation within ‘‘voids’’ present in the PAMPS first network [10]. We have developed and explored a double network system that is, in effect, the ‘‘inverse’’ of that prepared by Gong and colleagues: a neutral crosslinked polymer in the first network and an ionizable crosslinked polymer in the second network [11e14]. Specifically, the first network is composed of telechelic poly(ethylene glycol)-diacrylate (PEG-DA) macromonomers with defined molecular weight. The second network is, in contrast, a loosely crosslinked, ionizable network of poly(acrylic acid) (PAA). PEG-DA and PAA are both relatively fragile materials, so neither would be expected to make the sole contribution to mechanical enhancement. In previous work, interpenetrating networks of PEG and PAA have been explored as vehicles for drug delivery and as chemo-mechanical systems due to their reversible, pH-dependent swelling behavior [15e17]. The two polymers form complexes through hydrogen bonds between the ether groups on PEG-DA and the carboxyl groups on PAA [17]. This interpolymer hydrogen bonding enhances their mutual miscibility in aqueous solution, which, in turn, yields optically clear polymer blends. It is also a possible source of cooperative strength enhancement. By loosely crosslinking (instead of densely crosslinking) the ionizable network (PAA, pKa ¼ 4.7), large changes in its network configuration can be induced by changing the pH of the solvent without affecting the neutral PEG network. At a pH greater than 4.7, the PAA network is negatively charged and swollen; at a pH lower than 4.7, the PAA network is protonated and contracted.

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In recent work, we have reported studies of a PEG/PAA IPN as a promising material in an artificial cornea and other ophthalmic applications [11,12,14,18,19]. The controllable swelling of a PAA network within the confines of a more rigid, neutral first network provides a way to study the effect of relative chain configuration and topological interactions on the properties of an IPN. The use of defined, telechelic macromonomers in the first network facilitates tuning of the mesh size of the first network while placing a threedimensional constraint on the swelling of the second network. The experimental focus of this work is on the strain hardening observed in this system by testing how it manifests through uniaxial tensile tests under various conditions of first and second network crosslinking and swelling. Swelling data were used to calculate the equilibrium water and polymer content of the networks, which were correlated with Young’s modulus, true stress-at-break, and true strain-at-break. The results indicate that strain hardening is derived from physical entanglements between the PEG and PAA networks that are intensified by bulk deformation. Under conditions that promote hydrogen bonding (when the pH is at or below 4.7, the pKa value of PAA), these entanglements are reinforced by interpolymer complexes between PEG and PAA, leading to an increase in the fracture strength of the IPN. Under conditions that promote ionization of PAA (when the pH is above 4.7), intensified steric interactions between the swelling PAA network and static, telechelic PEG macromonomer network lead to a dramatic increase in Young’s modulus. 2. Experimental section 2.1. Chemicals Poly(ethylene glycol)-diacrylate (PEG-DA) (MW 575), poly(ethylene glycol) (PEG) (MW 1000 Da, 3400 Da, 4600 Da, 8000 Da, 14,000 Da), acryloyl chloride, acrylic acid (AA), 2-hydroxy-2-methyl-propiophenone, tetrahydrofuran, and triethylene glycol dimethacrylate (TEGDMA) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Buffers with varying pH (0.05 M potassium hydrogen phthalate) were obtained from VWR, Inc. (San Francisco, CA). 2.2. Preparation of hydrogels Interpenetrating polymer network hydrogels were synthesized by a (two-step) sequential network formation technique based on UV-initiated free-radical polymerization. The first hydrogel network was prepared from PEG-DA. Briefly, PEG-DA was synthesized by first dissolving the PEG macromonomer in anhydrous tetrahydrofuran at 50  C. Next, a molar excess of acryloyl chloride was added to the PEG solution and allowed to react for 5 h under a nitrogen atmosphere. The solution was then allowed to cool to room temperature and then recrystallized at 4  C. The PEG-DA was then purified by a second recrystallization step in fresh anhydrous tetrahydrofuran. Purified PEG-DA was then dissolved in deionized water. The water-soluble photoinitiator, 2-hydroxy-2-methyl-

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propiophenone (Sigma) was added to the PEG-DA solution at a concentration of 1% by weight with respect to the macromonomer. This precursor solution was cast within a Teflon spacer (250 mm thick, 2 cm inner diameter) positioned on a glass plate (1.0 mm thick), sandwiched with a second glass plate, and then reacted under a 75 W xenon ultraviolet (UV) light source (Oriel Instruments, Mountain View, CA) with a broad range of wavelengths (200e2500 nm) for 10 min. Upon exposure to UV light, the precursor solution underwent free-radical induced gelation and became insoluble in water. To incorporate the second network, the PEG-DA hydrogel was removed from the mold and immersed in an acrylic acid monomer solution containing 1% v/v photoinitiator solution (with respect to the monomer) and 1% v/v TEGDMA (with respect to the monomer) as a crosslinking agent for 24 h. The swollen gel was exposed to the same UV source for 5 min and a second network, PAA, was polymerized and crosslinked in the presence of the PEG-DA network to form the interpenetrating polymer network structure. The resultant IPN was washed extensively for a minimum of three days in deionized water to remove any unreacted components and to reach equilibrium swelling in deionized water. Copolymers of PEG and PAA (PEG/PAA) were synthesized by mixing PEG-DA, AA monomer, and deionized water in a 1:1:1 mixture by weight, adding 1% 2-hydroxy-2-methyl-propiophenone and 1% TEGDMA with respect to the macromonomers/monomers, and then proceeding with photopolymerization as described for the IPNs. After washing, the water content and mechanical strength were investigated. (From this point onward, the PEGDA networks will be referred to simply as PEG networks, and will be distinguished on the basis of the molecular weight (MW) of the PEG macromonomer. Single networks and IPNs will be designated PEG(X ) and PEG(X )/PAA, respectively, where X is the MW of the PEG network, while the copolymer will be designated as PEG-co-PAA.) In the case where the polymer content in the second network is changed, the IPN will be designated as PEG(X )/PAA[Y], where Y is the volume fraction of aqueous acrylic acid at the time of polymerization. The hydrogels were then washed and equilibrated in 500 mL of swelling solutions of varying pH and ionic strength and stored at room temperature until further use.

where Ws and Wd are the weights of swollen and dry hydrogels, respectively. The equilibrium swelling ratio (q) of the hydrogels was determined by the equation: q¼

Ws Wd

ð2Þ

The weight fraction of polymer (polymer content) of the swollen hydrogels was determined by dividing the dry weight of the hydrogel by its swollen weight: r¼

Wd Ws

ð3Þ

2.4. Mechanical testing

The water content of the hydrogels was evaluated in terms of the swelling ratio. Swelling studies were carried out by comparing the swollen and dry weights of the hydrogels in either deionized water or electrolyte buffers. The swollen gels were removed from the molds, patted dry, and weighed at regular intervals until equilibrium was reached. The percentage water content (WC) was calculated from the swollen and dry weights of the hydrogel:

The hydrogels were allowed to reach equilibrium in deionized water or pH buffers overnight prior to testing. The specimens were cut into dumbbell shapes that conform to ASTM D638-V standards using a dumbbell cutter (Ontario Die, Canada). Dumbbell shaped specimens (gauge length 9.5 mm, width 3.18 mm, and thickness 250e750 mm) of PEG, PAA, and PEG/PAA DN hydrogels were tested using an Instron 5844 materials testing apparatus equipped with a 10 N load cell and BioPuls bath, submersible pneumatic grips, and standard video extensometer system (Instron Corp., Norwood, MA). Thicknesses were measured by gently clamping a digital caliper (VWR International, Westchester, PA) over the samples sandwiched between 150 mm thick glass coverslips in order not to compress or damage the hydrogels. Prior to the tensile testing, a stresserelaxation test (data not shown) was conducted to verify that the stress data collected could be interpreted as ‘‘equilibrium stress’’ values as previously described by Mark and Sullivan [20]. The lack of observed stresse relaxation after stretching samples to a fixed strain indicated that the stress values obtained were equilibrium values. For the uniaxial tensile tests, the crosshead speed was set at 15 mm/min for all samples. Tensile tests were conducted under various pH and ionic strength conditions by submersing the gripped samples in 3.0 L of the solvent of interest. Load and extension measurements that take into account the thinning of the samples by extension were collected automatically by the load cell and video extensometer, respectively, and were used to obtain the true stress and strain values for each sample. Each material was tested in triplicate, and average values for Young’s moduli, true stress-atbreak (strue) and true strain-at-break (3break) of PEG, PAA, and PEG/PAA DNs were calculated. In order to normalize mechanical stress data among the samples with various degrees of swelling, we calculated the stress applied to the solid matter in the hydrogels (sr) as follows:

Ws  Wd WC ¼  100 Ws

    F 1 1 sr ¼ ¼ strue 2=3 2=3 A r r

2.3. Swelling studies

ð1Þ

ð4Þ

D. Myung et al. / Polymer 48 (2007) 5376e5387

where r is the weight fraction of solid in the sample (defined in Eq. (3) as the ratio of the dry weight and swollen weight of the hydrogel), taken to the two-third power to account for the mass fraction per unit area, F is the force (load), and A is the instantaneous cross-sectional area of the sample, which varies with extension.

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synthesized by polymerizing and crosslinking a PAA network within each type of PEG network. The resultant IPNs were transparent and had significantly greater mechanical strength compared with single network hydrogels. 3.2. Effect of changing the MW of the PEG-DA macromonomer

3. Results 3.1. Hydrogel synthesis and swelling All hydrogels were formed by photopolymerization with UV light using the water-soluble photoinitiator, 2-hydroxy-2methyl-propiophenone. Before the IPNs were prepared, single network hydrogels based on PEG and PAA were synthesized separately to confirm the formation of gels of each composition and to investigate the physical properties of the single networks. For the PEG single network, a range of hydrogels that varied between 575 Da and 14,000 Da (14k) for the MW of the PEG macromonomer were synthesized. It was found that low MW PEG macromonomers gave rise to gels that were transparent but brittle, whereas the hydrogels made from higher molecular weight PEG-DA (3.4k) were transparent and flexible when swollen in deionized water. Raman spectroscopy was used to monitor the photopolymerization of PEG-DA (data not shown). The vast majority of the terminal double bonds in PEG-DA were consumed during photopolymerization to produce crosslinked PEG-DA hydrogels. As a result, polymerization and crosslinking of acrylic acid monomers in aqueous solution within this preformed PEG-DA network effectively yielded an interpenetrating structure in which the two networks were independently crosslinked with little, if any, copolymerization between the two. Based on these results, a range of different MWs of PEG (3.4k, 4.6k, 8.0k, and 14k) were chosen as macromonomers for the first hydrogel network. A series of IPNs were

To explore the effect of the molecular weight of the telechelic PEG-DA macromonomer on IPN mechanical strength, the MW was changed from 8000 Da to 3400 Da, 4600 Da, and 14,000 Da while keeping the acrylic acid polymerization conditions constant (50% v/v in deionized water with 1% v/v crosslinker and 1% v/v photoinitiator with respect to the monomer). The resulting IPNs were characterized in terms of their water content and tensile properties in deionized water. Changing the MW of the PEG-DA macromonomer led to a change in the moduli of the PEG-DA single networks, as shown in Fig. 1a. This effect was magnified in the PEG/ PAA IPNs (Fig. 1b), where the IPNs initial and final moduli get increasingly higher as the networks are prepared from lower molecular weight PEG-DA macromonomers. Of note, there was little increase in strength when the PEG MW is increased above 8000 Da, indicating that a contrast in the mesh size of the PEG and PAA networks is important for strength enhancement. Moreover, the molecular weight of the PEG macromonomer was strongly correlated to the critical strain (3crit) at which the stressestrain curve makes the transition from the initial modulus to the strain-hardened final modulus (Fig. 1b). The 3crit was smaller for the IPNs prepared from lower MW PEG macromonomers, meaning that these networks strain harden more rapidly in response to deformation. The significance of forming an interpenetrating structure rather than a copolymeric structure was explored by synthesizing a PEG-co-PAA copolymer hydrogel and testing its tensile

Fig. 1. True stress versus true strain curves for (a) PEG-DA single networks of MW 3400 Da (:), 4600 Da (C), 8000 Da (-), and 14,000 Da (;) and (b) PEG/ PAA IPNs with PEG MW 3400 Da (:), 4600 Da (C), 8000 Da (-), and 14,000 Da (;). The intersection (*) between the initial and final tangents to each curve defines the critical strain (3crit) for strain hardening in each IPN.

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properties. Its stressestrain profile was then juxtaposed with those of the IPN and the PEG and PAA single networks. In Fig. 2a, a representative true stress (strue) versus true strain (3true) profile of the PEG(8.0k)/PAA IPN is compared to those of the PEG(8.0k)-co-PAA copolymer and their component PEG(8.0k) and PAA networks. The IPN exhibits strainhardening behavior with a stress-at-break that is greater than four times that of the copolymer and single network. However, since each of the materials tested has different water contents, the stress data were normalized on the basis of polymer content to determine the true stress per unit polymer in each hydrogel. In Fig. 2b, the true stress per unit polymer (strue per unit polymer) is plotted against true strain for PEG(8.0k)-DA, PAA, PEG(8.0k)/PAA, and the PEG(8.0k)-co-PAA copolymer. The initial moduli of the PEG single network, the copolymer, and IPN are identical (E0 per unit polymer ¼ 0.91 MPa), while that of the PAA single network is lower (E0 per unit polymer ¼ 0.55 MPa). Near the break point of the PEG network, 3true w 0.6, the copolymer continues to be elongated with a modulus that is intermediate between the PEG and PAA single networks, of which it is equally composed by weight. Ultimately, it fails at a strain that is also intermediate between the 3break values of the two single networks. In stark contrast, just beyond the failure point of the PEG network, the PEG/PAA IPN manifests a dramatic strain-hardening effect in which its modulus increases by 30-fold, and breaks at 3true w 1.0 under a mean maximum stress per unit solid of 10.6 MPa. Without normalization for polymer content, strue for the IPN (20% solid) and copolymer (51% solid) are 3.5 MPa and 0.75 MPa, respectively. 3.3. pH dependence of IPN and PAA physical properties Since the equilibrium swelling of PAA is sensitive to variations in pH, a change in the pH was expected to have an impact on the mechanical properties of PEG/PAA IPNs. After synthesis, the water-swollen PAA single networks and PEG(8.0k)/

PAA IPNs were placed in buffers of pH 3e6 and constant ionic strength (I ) of 0.05. In both the PAA network and the IPN, the equilibrium water content increased as the pH was increased from 3 to 6 (Fig. 3a, upper graphs). In the case of the PAA networks, those at pH 3 and 4 were moderately swollen, while those at pH 5 or 6 were highly swollen due to ionization of PAA above its pKa (4.7). The IPNs also achieved different levels of swelling depending on the pH; those at pH 3 and 4 were moderately swollen, while those at pH 5 or 6 were highly swollen due to ionization of PAA above its pKa (4.7). Of note, at both pH 3 and 4, the IPN achieved a lower equilibrium water content than PAA alone, which can be explained, in part, by the fact that PEG and PAA complex with each other via hydrogen bonds in an acidic environment, leading to a more compact, less hydrated interpenetrating network structure [17]. At pH above 4.7, the PEG and PAA chains dissociate as the PAA is ionized and counterions (along with water) enter the hydrogel to maintain charge neutrality, leading to a high degree of swelling. Nevertheless, the IPNs swell to a slightly lower extent (1.0e1.5%) than the PAA single networks due to the constraint that the PEG network places on PAA swelling. Fig. 3a also shows that the stress-at-break (sbreak), or tensile strength, of the PEG/PAA IPN is nearly an order of magnitude greater in its less-swollen state at pH 3 (sbreak ¼ 8.2 MPa) than in its more swollen state at pH 6 (sbreak ¼ 0.86 MPa). A similar phenomenon is observed in the PAA network, but the absolute values for sbreak are only 0.38 MPa at pH 3 and 0.05 MPa at pH 6. At every pH, then, the IPN has greater tensile strength than the PAA network, and this difference is intensified at lower pH. In contrast to the differences in the stress-at-break, the trends in the strain-at-break values of the IPN and PAA networks are roughly equivalent (Fig. 3b), changing from 3break values of w1.2 at pH 3 to w0.55 at pH 6. This result confirms the observation made in Fig. 2a and b, in which the extensibility of the IPN seems to be due to the presence of the PAA network, which has a higher 3break (0.9) than PEG (0.6). The mere presence of the PAA network in the IPN appears to enhance

Fig. 2. (a) True stressetrue strain curves for PEG(8.0k)/PAA IPN, PEG(8.0k)/PAA copolymer, PEG(8.0k), and PAA networks. (b) Normalized true stressetrue strain curves for PEG(8.0k)/PAA IPN, PEG(8.0k)-PAA copolymer, PEG(8.0k), and PAA networks.

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Fig. 3. pH dependence of (a) the stress-at-break (sbreak) and water content for PEG(8.0k)/PAA IPNs and PAA single networks, (b) the strain-at-break (3break) and water content for PEG(8.0k)/PAA IPNs and PAA single networks, and (c) the initial modulus (E0) and water content for PEG(8.0k)/PAA IPNs and PAA single networks.

the uniaxial extensibility of the network. In the context of the stress-at-break data (Fig. 3a), however, the load-bearing capacity at higher extensions is dramatically greater in the presence of hydrogen bonding at low pH than it is in the absence of hydrogen bonding at high pH. In contrast, Fig. 3c indicates that the pH dependence of the initial Young’s moduli (E0) of the IPN and PAA networks is less straightforward. The modulus of the PAA network exhibits a small drop from 0.09 MPa to 0.05 MPa as the pH is increased from 3 to 6. On the other hand, the modulus of the IPN does not appear to change significantly when going from pH 3 to pH 6. Of note, the pH dependence of the IPN does not follow the trend exhibited by the PAA single network, in which the modulus drops by approximately one-half when transitioning from pH 4 to pH 5. This decrease in modulus is correlated with an increase in water content of the PAA single network (Fig. 3c, upper plots). Moreover, the apparent preservation of the modulus in the IPN despite an increase in water content and loss of hydrogen bonding is paradoxical in the context of the sharp declines observed in the 3break and sbreak values.

The strain hardening per unit polymer is shown in the stressestrain profiles in Fig. 4a. Fig. 4a shows a plot of the true stress per unit polymer versus true strain in the PAA gels. In contrast to the non-normalized data in which the initial modulus of PAA at pH 3e4 is higher than it is at pH 5e6 (not shown), the initial moduli of PAA at all pHs converge when the stress data are corrected for differences in polymer content. This indicates that the reduction in mechanical strength that accompanies an increase in pH in PAA networks is largely due to the swelling of the network. Correcting the stress data for polymer content in the IPNs yields the graph shown in Fig. 4b. In this plot, the difference in the onset of strain hardening between the low pH and high pH regimes is accentuated. The stressestrain curves of the high pH IPNs proceed beyond their uncorrected maximum stresses and end parallel to those of the low pH regimes. This result suggests that, while the more swollen IPNs have lower tensile strength, this may be a side effect of the accelerated strain hardening that leads to greater load bearing at smaller strains (due to a higher modulus), and, in turn, an earlier strain-at-break.

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Fig. 4. True stress per unit polymer versus true strain curves for (a) PAA in pH 3e6 and (b) PEG(8.0k)/PAA in pH 3e6.

To investigate the consequence of relative network moduli even further, the swelling of PAA within the IPNs was maximized. The experimental data shown in Fig. 3c indicated that the modulus of the IPN was unaffected by the increased swelling. We hypothesized that the PEG network acts as a constraint on the swelling of PAA in a way that leads to additional interpolymer interactions and a corresponding increase in the IPN modulus. To test this hypothesis, the IPNs with first network MW PEG 3400 Da, 4600 Da, and 8000 Da and constant PAA network conditions were placed in phosphate buffered saline (PBS, pH 7.4, I ¼ 0.15) in order to induce maximal swelling under physiologic conditions. Table 1 shows the equilibrium water content and the corresponding swelling ratios for networks prepared from PEG macromonomers with each of these molecular weights, juxtaposed with the water content of the water-swollen and PBS-swollen IPNs. Increasing the size of the first PEG network from 3400 Da to 4600 Da and 8000 Da increases the degree to which Table 1 Equilibrium water content (%) and swelling ratio (q)a of PEG and PAA single networks and PEG/PAA interpenetrating networks under varying swelling conditions Specimen

Conditions

Equilibrium water content (%)

Swelling ratio (q)

PAA PAA PEG(3.4k) PEG(3.4k)/PAA PEG(3.4k)/PAA PEG(4.6k) PEG(4.6k)/PAA PEG(4.6k)/PAA PEG(8.0k) PEG(8.0k)/PAA PEG(8.0k)/PAA

dH20b pH 7.4, dH20 dH20 pH 7.4, dH20 dH20 pH 7.4, dH20 dH20 pH 7.4,

90.0  1.7 95.5  1.7 79.3  2.1 56.3  3.3 68.7  1.6 84.5  0.4 57.0  0.6 77.0  1.2 90.5  1.2 80.2  1.5 90.9  0.1

10.0 22.1 4.8 2.4 3.2 6.5 2.3 4.4 10.5 5.1 11.0

a b

I ¼ 0.15

I ¼ 0.15

I ¼ 0.15

I ¼ 0.15

q ¼ Ws/Wd ¼ ratio of swollen weight and dry weight. dH20 ¼ deionized water.

the IPN is able to swell. Specifically, while the PEG(3.4k)/PAA IPN swells to only 70% water when ionized, the PEG(4.6k)/ PAA IPN swells to 77% water and the PEG(8.0k)/PAA IPN swells to 90% water (nearly the same water content as the PEG(8.0k) single network) when ionized. Of note, the equilibrium water content values of the PEG(3.4k)- and PEG(4.6k)based IPNs do not approach those of their component PEG-DA networks (79.3% and 84.5%, respectively). The IPNs were also placed in phosphate buffered saline to examine them under physiologic conditions (pH 7.4, ionic strength ¼ 0.15) where the PAA network is greater than 99% ionized. Swelling of the PAA network in this way within the confines of a more densely crosslinked PEG network has dramatic consequences on the resulting IPN modulus. Fig. 5 shows that the accelerated strain hardening due to elevated pH, as demonstrated in Fig. 4b, is accentuated even further when an even tighter PEG network is used to constrain PAA. In this experiment, the PEG(4.6k)/PAA IPN e swollen to equilibrium in pure deionized water (pH 5.5, salt-free) e was switched to the ionizing conditions of phosphate buffered saline (pH 7.4, I ¼ 0.15) and again swollen to equilibrium. The increase in the pH to 7.4 and the addition of salt cause the PAA network (but not the PEG network) to swell. The result of this differential swelling within the IPN is a dramatic upward shift in the stressestrain profile that includes the initial portion of the curve. In other words, there is an increase in not only the rate of strain hardening, but also in the initial modulus. 3.4. Variation of the polymer content in the second network To increase the quantity of topological interactions between the PAA and PEG networks, the polymer content of PAA was varied inside of a PEG(3.4k) first network. The volume fraction of acrylic acid in solution at the time of the second network polymerization was varied between 0.5 and 0.8 prior to

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3.5. Hysteresis in PEG/PAA IPNs

Fig. 5. True stress versus true strain curves of the PEG(4.6k)/PAA IPN in PBS and deionized water, as well as the PEG and PAA single networks in PBS and deionized water. The PEG(4.6k) network is unaffected by the change from water to PBS.

polymerization. After polymerization, the IPNs were swollen to equilibrium in PBS, as is the IPN described in Fig. 5. The resultant hydrogels had different water contents, from 62% in the PEG(3.4k)/PAA[0.8] IPN to 65% in the PEG(3.4k)/PAA[0.7] IPN and 68.7% in the PEG(3.4k)/PAA[0.5] IPN. Of note, the IPNs with increased acrylic acid concentration had lower water content, which in light of the super-absorbency of PAA is a counterintuitive result. The true stressetrue strain profiles for these IPNs are shown in Fig. 6. The IPN with the highest PAA content had the highest stress-at-break and modulus, while the one with the lowest PAA content had the lowest stress-atbreak and strain-at-break. Notably, the initial modulus values for these samples varied significantly, from 3.6 MPa in the PEG(3.4k)/PAA[0.5] to 12 MPa in the PEG(3.4k)/PAA[0.7] IPN and 19.6 MPa in the PEG(3.4k)/PAA[0.8] IPN.

Fig. 6. Effect of varying the acrylic acid (AA) volume fraction in the preparation of PEG(3.4k)-based IPNs. Increasing the AA volume fraction from 0.5 (-) to 0.7 (C) and 0.8 (:) in the IPN leads to an increase in Young’s modulus.

PEG(8.0k)/PAA IPNs were subjected to progressively greater extensions until failure to see if a hysteresis effect would be observed (Fig. 7). In this figure, data is shown for eight successively higher extensions of a PEG(8.0k)/PAA IPN in deionized water. The first six extensions were between the strains of 0.2 and 0.7 and the stressestrain profiles overlapped each other. On the seventh extension, the IPN was brought to a strain of 1.1, far beyond the experimentally determined 3break of the PEG network but less than the 3break of the IPN. After allowing the sample to return to rest, the IPN was then extended again, but this time to failure. During this extension, a hysteresis effect was observed, where the stressestrain profile deviated from the trajectories exhibited during the first seven extensions (Fig. 7). The primary difference was a rightward curve shift, in which the onset of strain hardening was delayed to a higher extension. 4. Discussion Sequential IPNs of poly(ethylene glycol)-diacrylate and poly(acrylic acid) were synthesized and their swelling-dependent mechanical properties were characterized by uniaxial tensile measurements. Normalization for the fraction of polymer content in the swollen hydrogels was done to compare the mechanical behavior of IPNs with identical composition but with different degrees of swelling due to pH and ionic strength changes. The experiments were aimed at elucidating the mechanisms behind the synergistic strength enhancement in the PEG/PAA IPNs. To explore the role of interpolymer hydrogen bonding, the pH of the hydrogel swelling liquid was varied to

Fig. 7. Hysteresis in PEG(8.0k)/PAA stressestrain behavior. At low and intermediate extensions (curves 1e6), the stressestrain curve is reproduced. When the IPN is extended beyond the expected breaking point of the PEG network alone (curve 7), a hysteresis effect is observed the next time the sample is highly extended (curve 8). Curve 8 shows a deviation from the previous stressestrain profiles after intermediate deformation, followed by realignment just prior to fracture.

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change the ionization state of the PAA network. To explore the role of interpolymer entanglements, variations were made in the MW of the PEG macromonomer, the swelling of the PAA network, and the polymer content of the PAA network. 4.1. The impact of pH on IPN fracture properties Fig. 3aec shows that the modulus, stress-at-break (smax), and strain-at-break (3break) of the IPNs were found to be strongly dependent on the swelling liquid’s pH. The IPNs at pH 3 and 4 are able to elongate further and bear a greater load than at pH 5 and 6, which are greater than 50% ionized above the pKa of PAA (4.7). Because the four IPNs have the same polymer composition but are swollen to different extents (from 76.5% water at pH 4 to 95.7% water at pH 6), the stress data was normalized for polymer content (Fig. 4a and b). Normalizing in this way was done to reveal molecular-level information regarding the stress on the polymer chains, since a higher level of hydration renders the materials more sensitive to defects and crack propagation and possibly masks what is occurring at the molecular level. The polymer content correction in Fig. 4b demonstrates that, per unit area of polymer, the IPNs at pH 5 and 6 have half the tensile strength per unit polymer and extensibility of those at pH 3 and 4. The simplest explanation for the loss of extensibility at high pH is the fact that PAA alone at pH 5e 6 has a lower strain-at-break than it does at pH 3e4; therefore, the capacity of the ionized PAA to ‘‘support’’ PEG at high extensions is lost. The weakness of PAA at high pH is the consequence of the ‘‘polymer-diluting’’ effect of swelling. This loss of extensibility is propagated to the IPN, leading to failure at a lower strain. On the other hand, the dramatic loss of tensile strength (sbreak) at pH 5 and 6 indicates that a major contribution to the load-bearing capacity is lost with the absence of hydrogen bonding. When an IPN is placed in buffers of pH 5 and 6, the PAA network is ionized and the interpolymer complexes between it and PEG are broken. At pH 5, the PAA network is roughly 75% ionized and at pH 6 is 95% ionized. According to Bokias et al. [21], interpolymer complexes do not form beyond 10% PAA ionization, and thus at pH 6 and more, hydrogen bonds make no contribution to the mechanical properties. The stark difference in load bearing between the high and low pH regimes is consistent with the experimentally validated phenomenon of PEG/PAA complex formation [22], and as such, is evidence that internetwork hydrogen bonds are strength-enhancing elements in this system. Normalizing for polymer content (Fig. 4a and b) reveals that the major effect of lowering pH is to increase both sbreak and 3break by nearly two-fold. This can be explained by the fact that PEG and PAA are known to complex with each other via hydrogen bonds between ether oxygens in PEG and carboxylic acid groups in PAA [17]. Hydrogen bonds may make contributions on two levels: (1) in maintaining the integrity of the network through resistance to crack initiation during deformation and (2) as a source of strain hardening at high strains, since the functional groups of PEG and PAA are increasingly more

accessible as their chains are extended. The elongationdependent interpolymer complex formation may be analogous to the crystallization-based strain hardening observed in other elastomers [23]. 4.2. Strain hardening at low pH: the role of hydrogen bonding The hypothesis of ‘‘entanglement reinforcement’’ through interpolymer complexation is supported by extensive work done by other investigators on PEG/PAA interactions both in solution and in interpenetrating polymer networks. Iliopoulos and colleagues demonstrated the formation of complex aggregates of PEG and PAA at low pH [21,24,25] through potentiometric and viscometric measurements. Antipina and colleagues showed that this complexation only occurs above a PEG critical molecular weight of about 3000 Da [26], while Oyama and coworkers showed through measurement of excimer fluorescence that longer PAA chains enhanced complexation [22]. The latter group also showed that hydrogen bonds increased the local concentration of PEG in the vicinity of PAA [22]. Furthermore, Nishi and Kotaka showed that these interpolymer complexes impact the magnitude of the swelling/deswelling behavior of PEG/PAA IPNs [17]. Finally, in their theoretical model for polymer complexation, Iliopoulos and coworkers proposed that PEG and PAA complex together along the segments of complementary sequences [27]. They confirmed the consistency of this model with experimental data on polymer blends in solution [27,28]. These results lend support to the concept of strain hardening mediated by hydrogen bond reinforcement of physical entanglements in an IPN. Fig. 8 shows a schematic adapted from Iliopoulos’ work [27] that depicts how cooperative complex formation may serve to strengthen interpolymer entanglements during uniaxial deformation in a way that is consistent with both our experimental data and those of the aforementioned works. When the PEG/PAA IPN is at low pH, the two networks interact through hydrogen bonds between PAA carboxylic acid groups and PEG ether oxygens. Interpolymer hydrogen bonding (H) between monomer units at entanglement junctions (E) may serve to reinforce these physical crosslinks. The result is a series of

Fig. 8. Schematic of cooperative reinforcement of interpolymer entanglements by hydrogen bond complexes, adapted from Iliopolous [27]. During uniaxial deformation, chain stretching leads to greater accessibility of hydrogen bonding functional groups, and sequences of complementary ether oxygens/carboxylic acids are able to anneal as shown. This leads to ‘‘enhanced entanglements’’ (E þ H), where existing physical entanglements (E) are reinforced by hydrogen bonds (H). As the network continues to elongate, a greater number of the entanglements (E) become enhanced entanglements (E þ H).

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entanglements enhanced by hydrogen bonds (E þ H). Continued stretching of the IPN leads to increased accessibility of PEG ether oxygens as well as increased availability of PAA carboxylic acids. The pronounced strain hardening at low pH may arise because the intramolecular hydrogen bonding between carboxylic acid groups in the compacted, unperturbed state of PAA is broken during uniaxial deformation and progressively replaced by hydrogen bonding between carboxylic acid groups and the ether oxygens on PEG chains. We hypothesize that the increased accessibility and availability of hydrogen bonding pairs at these entanglement junctions are the primary source of the cooperative, deformation-dependent enhancement in the effectiveness of the crosslinks and in turn, the material’s strain-hardening behavior. Intra-chain interactions within the IPN may also play a role, but in light of the inferior properties of the single networks, their contribution is likely to be small in comparison to inter-chain interactions. 4.3. Strain hardening at high pH: ‘‘pre-stress’’ through PAA swelling To explore whether the apparent increase in moduli in the ionized IPNs (Figs. 2 and 3) is legitimate or simply an artifact of polymer content normalization, deionized water-swollen IPNs with more densely crosslinked PEG networks were placed in physiologic aqueous conditions (pH 7.4, I ¼ 0.15) in which the PAA network is 99.9% ionized. The hypothesis driving this experiment was that increasing the constraining effect of the neutral PEG network on PAA swelling would increase the intensity and/or the number of interpolymer physical entanglements and, in turn, the strain-hardening behavior of the IPN. IPNs with PEG macromonomer MWs of 4600 Da and 3400 Da were prepared and swollen in this fashion. Table 1 shows that the equilibrium water content and swelling ratio of the IPNs in PBS decreased as the MW of the PEG macromonomer in the IPN decreased. This result implies that the modulus of the more crosslinked PEG-DA networks in these IPNs resists the osmotic pressure resulting from PAA ionization. In contrast, the PEG(8.0k) network, with its relatively lower modulus (nearly three-fold less than the others), does not offer much resistance and allows swelling to continue to the equilibrium water content mandated by the ionized PAA. The PEG network, therefore, acts like a three-dimensional constraint to limit the swelling of the second network. The swelling of PAA is more constrained when it is interpenetrated with a stiffer PEG network. For a given network, however, an increase in water content due to ionization produces a paradoxical increase in the modulus. For the PEG(3.4k)/PAA IPN, there was a 50% increase (data not shown) and in the PEG(4.6k)/PAA IPN there was a 300% increase (Fig. 5). In Fig. 5, the stressestrain profile of the PEG(4.6k)/PAA IPN is juxtaposed with its constituent PEG(4.6k) and PAA networks individually in both deionized water and at pH 7.4 in the presence of salt (I ¼ 0.15). As expected, ionization and swelling of the PAA network alone leads to a decrease in modulus, stress-at-break, and strain-atbreak. The PEG(4.6k) is mechanically unaffected by the

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change in buffer conditions. In contrast, the IPN exhibits a three-fold increase in initial modulus while undergoing only a moderate reduction (w15%) in its stress-at-break. The enhanced strain hardening observed under the fully ionized condition is likely due to intensified intermolecular interactions caused by swelling of the PAA network against the static PEG network. These additional interactions yield a higher overall network modulus, leading to the accelerated strain hardening. In effect, swelling of the PAA network against the static PEG network leads to a pre-stressed state in which more (or more intensified) interactions e that would ordinarily be induced by mechanical deformation e are built into the IPN. The result is a hydrogel with significantly enhanced initial modulus rather than a gradual, deformationdependent enhancement. In contrast to the low pH condition, at high pH, hydrogen bonding plays little or no role in strength enhancement. At high pH, the PAA network is ionized and will swell to accommodate the entry of counterions and water. The steric hindrance to PAA swelling presented by the PEG network leads to intensified entanglements between the two networks. Under high pH, ionization leads to swelling of PAA within the unresponsive PEG network and isotropic strain throughout the IPN. This differential swelling creates a ‘‘pre-stressed’’ IPN that has enhanced Young’s moduli due not only to hydrogen bonding, but also to more intense PEG/PAA entanglements in the unperturbed (i.e. not mechanically deformed) state. It is important to note, however, that the pre-stress introduced into the network by PAA swelling should also have an impact on intra-chain entanglements. Activation of intra-chain entanglements may also make a contribution to the increased modulus of the IPN, but this contribution is likely to be small (as it is in the low pH regime) in light of the poor mechanical properties of the single networks alone. Because of the pH-induced pre-stressed condition, internetwork entanglements enhance the modulus of the IPN at smaller strains than they do at low pH. The initial modulus is increased at high pH because either the number, functionality, or effectiveness of interpolymer physical crosslinks is increased by the three-dimensional expansion of the PAA coils that had been residing dormantly within the tight mesh of the PEG network. This increase in the number of activated entanglements with the PEG network leads to an increase in the initial modulus of the IPN. The interpolymer tension is increased because of the active ‘‘tug of war’’ between the PAA network, which prefers to swell to a larger volume, and the PEG network, which does not. Specifically, while under physiologic conditions (pH 7.4, I ¼ 0.15), the PAA network is ionized and swells, while the neutral PEG network remains in place and exerts an opposing force on the PAA chains at interpolymer entanglement points. The ‘‘active entanglements’’ produced by the pre-stressed condition of the unperturbed IPN at high pH are accompanied by an increased effectiveness of PEG/PAA physical crosslink junctions. This is observed experimentally by the dramatically enhanced Young’s modulus of the PEG(4.6k)/PAA IPNs in phosphate buffered saline relative to that in water (Fig. 5). The stressestrain profile of

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the PBS-swollen IPN has a similar shape and trajectory to that of the water-swollen IPN at 3true values greater than about 0.4. The phenomenon of a pre-stressed IPN can be observed in Fig. 4b, where the true stress per unit polymer is plotted against the true strain. In this figure, the shapes of the normalized stressestrain curves of PEG(8.0k)/PAA IPNs at pH 5 and 6 closely resemble the intermediate region of their counterparts at pH 3 and 4, from 3true values from about 0.5 to roughly 1.0. The similarity of the shapes of these curves suggests that the swelling produced under high pH leads to a ‘‘pre-stressed’’ version of the IPNs in the low pH regime. These pre-stressed IPNs at high pH have enhanced modulus but reduced fracture stress compared to those observed in the low pH condition. The magnitude of the pre-stressing effect also depends on the relative amount of PAA present in the second network. Fig. 6 shows PEG(3.4k)/PAA IPNs with polymer content of PAA swollen to equilibrium in PBS at pH 7.4. The PEG(3.4k)/PAA[0.5] IPN has an initial modulus of 3.6 MPa. This modulus is higher than that found for the IPN prepared from the PEG(4.6k) first network (Fig. 5) with the same second network polymer content and swelling condition. Increasing the volume fraction of AA in the second network to 0.7 and 0.8 yielded large changes in the initial modulus of the PEG(3.4k)/PAA IPN (12 MPa in the case of the former and 19.6 MPa in the case of the latter). This result indicates that an increase in the polymer content of the swelling PAA network increases the magnitude of its interaction with the PEG network, leading to a greater degree of pre-stress in the IPN, and a significantly higher initial modulus. The modulus increased nearly three-fold (from 3.6 MPa to 12.0 MPa) when the monomer volume fraction was increased to 0.7, and over five-fold (from 3.6 MPa to 19.6 MPa) when it was increased to 0.8.

material may also have relevance to other load-bearing applications. The PEG(3.4k)/PAA IPNs with increased PAA content shown in Fig. 6 have moduli greater than 10 MPa. In particular, both the water content (w65%) and modulus (12 MPa) of the PEG(3.4k)/PAA[0.7] system are comparable to those previously measured for cartilage e which contains approximately 70% water and a modulus of about 13 MPa on its surface [34] e making this material potentially useful in orthopaedic applications. Both the cornea and the cartilage are ‘‘natural’’ interpenetrating networks comprised of collagen fibrils and negatively charged proteoglycans [34,35]. Just as natural tissues with varying water content and modulus (like the cornea and various types of cartilage) differ in terms of their specific collagen to proteoglycan content, so do the range of IPNs prepared in this study differ as far as their relative content of end-linked PEG macromonomers and PAA. Swelling a PAA network through ionization within an existing telechelic PEG macromonomer network yields a material that mimics the ‘‘pre-stressed’’ condition present in natural load-bearing tissues.

4.4. Significance

Acknowledgments

The PEG/PAA IPNs prepared in this study are able to achieve values for Young’s moduli that are over an order of magnitude higher than other IPNs and double networks reported in the literature. Increasing the modulus from w0.3 MPa to w20.0 MPa while maintaining >60% water content effectively places these materials in the truly ‘‘biomimetic’’ regime. For instance, the human cornea consists of 78% water, has an initial modulus of roughly 3.9 MPa, and has a maximum tensile strength in the order of several megapascals [29e33]. The PEG(4.6k)/PAA[0.5] IPN has 77% water, an initial modulus of 3.4 MPa, and maximum tensile strength of approximately 4.0 MPa. The modulus of this IPN is within the range of literature values for corneal moduli of various species [29e33] and measured using several different techniques, including uniaxial extensometry, biaxial extensometry, and rigidity experiments. In recent work, we have reported our studies on the refractive index, transparency, glucose permeability, and cellular biocompatibility of PEG/PAA hydrogels [12,14,18,19]. In addition, we have presented in vivo corneal implantation studies of this combination of materials, demonstrating ocular tolerance and sustained optical clarity for up to two months [11]. This

This research was supported by the Bio-X Interdisciplinary Initiatives Program and Bio-X Graduate Student Fellowship at Stanford University and the Office of Technology Licensing at Stanford University. Instrument support was provided by the shared facilities at the Center on Polymer Interfaces and Macromolecular Assemblies (CPIMA) at Stanford University. Additional external support was also received from VISX, Incorporated (now VISX Technology) and the Fight for Sight Foundation. The authors thank Per Linse at Lund University for his input on approaching mechanical property comparisons on the basis of polymer content.

5. Conclusions Uniaxial tensile measurements were used to study PEG/PAA IPN hydrogels with varying first network mesh size and second network, pH-mediated swelling. The strain hardening of PEG/ PAA IPNs was determined to be primarily a product of straininduced, interpolymer entanglements, which can be further reinforced at low pH through hydrogen bonds between PEG and PAA. IPNs with Young’s moduli, tensile strength, and water content comparable to natural tissues were produced that show promise as replacement materials for the cornea, cartilage, and other load-bearing anatomical structures.

References [1] Czeslik C, Jackler G, Hazlett T, Gratton E, Steitz R, Wittemann A, et al. Physical Chemistry Chemical Physics 2004;6:5557e63. [2] Wittemann A, Haupt B, Ballauff M. Physical Chemistry Chemical Physics 2003;5:1671e7. [3] De TK, Hoffman A. Journal of Bioactive and Compatible Polymers 2001;16(1):20e31. [4] Kim D, Seo K, Park K. Journal of Biomaterials Science, Polymer Edition 2004;15(2):189e99.

D. Myung et al. / Polymer 48 (2007) 5376e5387 [5] Lutolf MP, Lauer-Fields JL, Schmoekel HG, Metters AT, Weber FE, Fields GB, et al. Proceedings of the National Academy of Sciences of the United States of America 2003;100(9):5413e8. [6] Nguyen KT, West JL. Biomaterials 2002;23(22):4307e14. [7] Gong JP, Katsuyama Y, Kurokawa T, Osada Y. Advanced Materials 2003;15(14):1155e8. [8] Na YH, Kurokawa T, Katsuyama Y, Tsukeshiba H, Gong JP, Osada Y, et al. Macromolecules 2004;37(14):5370e4. [9] Tanaka Y, Kurwabara R, Na YH, Kurokawa T, Gong JP, Osada Y. Journal of Physical Chemistry B 2005;109:11559e62. [10] Tsukeshiba H, Huang M, Na YH, Kurokawa T, Kuwabara R, Tanaka Y, et al. Journal of Physical Chemistry B 2005;109:16304e9. [11] Bakri A, Farooqui N, Myung D, Koh WG, Noolandi J, Carrasco M, et al. Investigative Ophthalmology and Visual Science 2006;47. E-Abstract 3592. [12] Myung D, Koh W, Ko J, Noolandi J, Carrasco M, Smith A, et al. Investigative Ophthalmology and Visual Science 2005;46. E-Abstract 5003. [13] Koh WG, Myung D, Ko J, Noolandi J, Carrasco M, Smith A, et al. Investigative Ophthalmology and Visual Science 2005;46. E-Abstract 4994. [14] Myung D, Koh W, Bakri A, Zhang F, Marshall A, Ko J, et al. Biomedical Microdevices 2007. [15] Choi HK, Kim OJ, Chung CK, Cho CS. Journal of Applied Polymer Science 1999;73:2740e54. [16] Kim IS, Kim SH, Cho CS. Archives of Pharmacal Research 1996; 19(1):18e22. [17] Nishi S, Kotaka T. Polymer 1989;21(5):393e402. [18] Myung D, Zheng L, Bakri A, Marshall A, Duhamel P, Carrasco M, et al. Investigative Ophthalmology and Visual Science 2007;48. E-Abstract 1864. [19] Myung D, Koh W, Ko J, Noolandi J, Carrasco M, Frank CW, et al. Investigative Ophthalmology and Visual Science 2006;47. E-Abstract 3931. [20] Mark JE, Sullivan JL. The Journal of Chemical Physics 1976;66(3): 1006e11.

5387

[21] Bokias G, Staikos G, Iliopoulos I, Audebert R. Macromolecules 1994; 27(2):427e31. [22] Oyama HT, Tang WT, Frank CW. Macromolecules 1987;20(3):474e80. [23] Rubinstein M, Colby RH. Polymer Physics. New York: Oxford University Press; 2003. p. 440. [24] Iliopoulos I, Halary JL, Audebert R. Journal of Polymer Science Part A: Polymer Chemistry 1988;26(1):275e84. [25] Iliopoulos I, Audebert R. Polymer Bulletin 1985;13(2):171e8. [26] Antipina AD, Baranovs Vy, Panisov IM, Kabanov VA. Vysokomolekulyarnye Soedineniya Section A 1972;14(4):941. [27] Iliopoulos I, Audebert R. Journal of Polymer Science Part B: Polymer Physics 1988;26(10):2093e112. [28] Jiang M, Li M, Xiang ML, Zhou H. Interpolymer complexation and miscibility enhancement by hydrogen bonding. In: Advances in polymer science: polymer synthesis/polymerepolymer complexation, vol. 146. Berlin: Springer; 1999. p. 121e96. Available from: http://www.springerlink.com/content/d63m6ynct8wg51mv/. [29] Hoeltzel DA, Altman D, Buzard K, Choe K. Journal of Biomechanical Engineering 1992;114:202e15. [30] Zeng Y, Yang J, Huang K, Lee Z, Lee X. Journal of Biomechanics 2001;34(4):533e7. [31] Woo SL, Kobayashi AS, Lawrence C, Schlegel WA. Annals of Biomedical Engineering 1972;1(1):87e98. [32] Woo SLY, Kobayashi AS, Schlegel WA, Lawrence C. Experimental Eye Research 1972;14:29e39. [33] Nash IS, Greene PR, Foster CS. Experimental Eye Research 1982; 35(5):413e24. [34] Mow VC, Radcliffe A. Structure and function of articular cartilage and meniscus. In: Mow VC, Hayes WC, editors. Basic Orthopaedic Biomechanics. Philadelphia: Lippincott-Raven Press; 1997. p. 113e77. [35] Speziale P, Bardoni A, Balduini C. The Biochemical Journal 1980; 187(187):655e9.