Self-Supported Fibrin-Polyvinyl Alcohol Interpenetrating Polymer

Sep 19, 2013 - The fibrin network was synthesized by the enzymatic hydrolysis of ... polymer network for all-solid self-standing polyelectrolyte mater...
1 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Biomac

Self-Supported Fibrin-Polyvinyl Alcohol Interpenetrating Polymer Networks: An Easily Handled and Rehydratable Biomaterial Laurent Bidault,†,‡ Marie Deneufchatel,†,‡ Cédric Vancaeyzeele,‡ Odile Fichet,*,‡ and Véronique Larreta-Garde† †

Equipe de Recherche sur les Relations Matrice Extracellulaire Cellules (Errmece), ‡Laboratoire de Physicochimie des Polymères et des Interfaces (LPPI), Institut des Matériaux, University of Cergy-Pontoise, 95000 Cergy-Pontoise, France S Supporting Information *

ABSTRACT: A fibrin hydrogel at physiological concentration (5 mg/mL) was associated with polyvinyl alcohol (PVA) inside an interpenetrating polymer networks (IPN) architecture. Previously, PVA has been modified with methacrylate functions in order to cross-link it by free-radical polymerization. The fibrin network was synthesized by the enzymatic hydrolysis of fibrinogen by thrombin. The resulting selfsupported materials simultaneously exhibit the properties of the fibrin hydrogel and those of the synthetic polymer network. Their storage modulus is 50-fold higher than that of the fibrin hydrogel and they are completely rehydratable. These materials are noncytotoxic toward human fibroblast and the fibrin present on the surface of PVAm-based IPNs favors cell development.

■

INTRODUCTION Among the large field of soft biomaterials, hydrogels1 occupy a major position, as they are usually biocompatible and occasionally biodegradable. Hydrogels may be synthesized from natural or synthetic compounds or from a mixture of them.2−4 Hydrogels made from biomolecules have the advantage to mimic the physiological microenvironment of tissues,5,6 to be enzyme-responsive.7 However, some of them are not easily handled, which limits their potential applications. This is the case of fibrin (Fb) hydrogel, frequently used as transitional material in tissue engineering.8 In vivo, fibrin indeed plays a key role in wound healing and hemostasis, where it forms a dynamic three-dimensional network that obstructs the vascular gap and serves as a provisional matrix. In vitro, fibrin hydrogels show promising biological properties for clinical applications in tissue engineering and damaged tissue regeneration.9−11 They are synthesized in vitro by hydrolysis of fibrinogen (Fg) by thrombin. However, they are very soft and not easily handled when they are synthesized at the physiological concentration (with a storage modulus at 20− 30 Pa12), which restricts their use in tissue engineering.13,14 Fibrin hydrogels with improved mechanical properties have been designed by increasing fibrinogen or thrombin concentrations.15,16 These commercially available systems, optimized for tissue sealing, display limited regenerative capacity due to dense fibrous networks that limit cell infiltration.17 In consequence, there is a real need to prepare in vitro a fibrin-based hydrogel at the physiological concentration that would be dimensionally stable and easy to handle. In this purpose, synthetic polymers were inserted into this hydrogel to bring about better mechanical properties. For thermodynamic © 2013 American Chemical Society

reasons, the simple polymer blends generally lead to a macroscopic phase separation. Therefore, synthetic polymers are usually grafted onto biological macromolecules.18 Another little investigated approach to associate these two types of polymers is the development of interpenetrating polymer networks (IPN).19,20 IPN is defined as a combination of two polymer networks cross-linked in the presence of the other. If one of the polymers is not cross-linked, the architecture is called semi-IPN. Physical entanglements between the polymers and the chemical cross-links prevent phase separation, and the material is, thus, dimensionally stable. The aim of this type of polymer associations is generally to obtain materials showing a combination of the properties of the associated partners. In addition, this type of architecture offers the possibility to easily change the proportion of the components in the materials and, thus, to modulate their properties. Some (semi-)IPNs based on biological macromolecules have been developed with the aim to create biomaterials.21 For instance, gelatin has been associated with silk fibroin in an IPN to improve its mechanical properties22 from 2.5 kPa for gelatin hydrogel to 70 kPa for IPN. However, the presence of the silk protein limits the cell growth on the IPN surface. Only few examples of native fibrin-based semi-IPN are described in the literature. Fibrin has been associated with type I collagen to improve the mechanical properties of collagen.23 Fibrin-alginate IPNs show storage moduli improved, between 370 and 235 Pa, compared to a single fibrin hydrogel.24 In Received: July 8, 2013 Revised: September 9, 2013 Published: September 19, 2013 3870

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

store them in a dry state and to rehydrate them just before use. In addition, PVA/Fb IPNs have to be of course nontoxic for cells. Due to these conditions, the PVA network has to be synthesized according to an innovative process in nondenaturing conditions for simultaneous enzymatic reaction of the fibrin network formation. Thus, PVA has been modified with methacrylate functions in order to cross-link it by freeradical polymerization. The synthesis of PVA/Fb IPN was monitored by real-time rheological measurements from which the gel time and the moduli of the resulting materials were determined. PVA and fibrin network formations were assessed by determination of a synthetic and protein soluble fraction. Particular attention was paid on the repartition on the surface and in the bulk of the fibrin in these IPNs and compared to that observed with the PEO-based IPN by confocal laser scanning microscopy (CLSM). The behavior of these materials during hydration/dehydration cycles was also studied. Finally, fibroblast behavior on the IPN surfaces was examined to demonstrate their biocompatibility.

addition, the inert slow-degradable alginate maintains the integrity of the matrix that provides the needed physical support during cell growth. Fibrin was also incorporated into a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-IPN.25,26 However, according to their synthesis protocol, these materials can be considered rather as bilayer materials.27 They can be used as scaffolds for the controlled and sustained release of bioactive pro-angiogenetic growth factors and cells.28 Recently, an IPN between fibrin and methacrylated hyaluronic acid was performed in order to obtain a full natural material with high angiogenic potential.29 However, this type of materials is too soft as are IPN combining collagen and methacrylated hyaluronic acid.30 In a recent paper, we have reported fibrin/polyethylene oxide (PEO) IPNs synthesis leading to the elaboration of naturalsynthetic biomaterials.31,32 Their properties can be modulated according to their composition. Contrary to the fibrin hydrogel, all the synthesized IPNs have storage moduli, which allow them to be easily handled. Finally, cell growth occurs on PEO/Fb IPN surfaces, indicating that they are noncytotoxic and suitable for use in tissue engineering. Thus, the new materials offer interesting properties combining those of the protein hydrogels and of the synthetic polymer. However, the rehydration of these biomaterials is not complete. It could be interesting for some applications that the materials are stored in a dry state and rehydrated just before use. Thus, we chose to exchange the PEO synthetic polymer with another one. The best candidate appears to be polyvinyl alcohol (PVA). Polyvinyl alcohol (PVA) is widely used to generate biomaterials because of its unique features. It can be processed into several forms, for instance, cryosponge33 or hydrogel34 for artificial cartilage35 or cornea36 applications. It is nontoxic for culture cell,32,33 but unfavorable to protein absorption.37 This semi-crystalline polymer is hygroscopic (solubility parameter = 22.8 MPa1/238). It swells in aqueous solution, but its mechanical properties are kept due to the existence of crystalline domains,39 the presence of which is promoted when the PVA hydrogel is prepared starting from concentrated solutions.40−43 In this work, a fibrin gel at physiological concentration (5 mg/mL) was associated with PVA inside an IPN architecture so that the resulting self-supported materials simultaneously exhibit the properties of the fibrin hydrogel and those of the synthetic PVA network. PVA network has already been associated with silk in an IPN in order to combine healing properties of skin lesions and good mechanical properties.44 The IPN has higher compression modulus than PVA single network and cells are able to adhere and proliferate on the IPN surface. Finally, in vivo tests with this material show a faster wound closure than with PVA single hydrogel (7 instead of 21 days) and a 50% increase of collagen synthesis by dermal fibroblasts. Kulkarni et al.45 has prepared alginate-PVA IPN by solvent casting for transdermal delivery of antihypertensive drug, and Finosh et al.46 recently reported the synthesis of the same type of IPN with good mechanical properties for tissue engineering of cardiac tissue. However, in this system, accurate quantification of cell growth and ECM synthesis from cells on the top or infiltrate inside the materials seems to be challenging. Contrary to these previous IPNs, the proposed PVA/fibrin IPNs have been designed to gather many properties. They must keep the in vivo key role of fibrin in wound healing and hemostasis47 with sufficient mechanical properties to be handled. Because the materials could become wound dressings,48 their rehydration has to be complete in order to

■

MATERIALS AND METHODS

Materials. Sodium chloride, sodium dodecylsulfate, and thrombin (BP 25432) were purchased from Fisher Reagents and bovine fibrinogen (Fg-341573) from Calbiochem. Tris(hydroxymethyl)aminomethane and urea were purchased from VWR. Calcium chloride was obtained from Riedel-de Haën, and magnesium chloride from Prolabo. Azide, Brillant Blue R, sodium carbonate, dietholamine, βmercaptoethanol, Tween 20, 2-isocyanatoethyl methacrylate (2ICEMA), paraformaldehyde (PFA, P-6148), 4,6-diamino-2-phenylindole (DAPI, D-9564), alkaline-phosphatase conjugated antirabbit IgG (A3687), and the α,ω-poly(ethylene oxide)dimethacrylate (Mn = 750 g/mol, density = 1.1) were obtained from Sigma. Antifibrinogen from rabbit (A0080) was purchased from Dako and p-nitrophenyl phosphate (pNPP, 71768) from Fluka. Polyvinyl alcohol (PVA 98%, M = 16000 g/mol) and hydroquinone were purchased from Acros. 2Hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (Irgacure, I2959) was obtained from Ciba. Dimethylsulfoxide (DMSO, Merck) was dried on calcium oxide for 24 h before distillation and stored in the dark on a molecular sieve under argon. Phalloidin-Alexa 532 (P-5282), Alamar blue (DAL 11000), n-succinimide ester Alexa 488 (A-20000) and LIVE/DEAD kit (L3224) were obtained from Invitrogen. Penicillin−streptomycin (1416), culture medium DMEM high glucose (31966021), trypsine EDTA, and FBS (fetal bovine serum, 10270−106) were obtained from Gibco. Synthesis of Polyvinyl Alcohol (PVAm) Modified with Methacrylate Groups. A 20% (w/v) PVA aliquot was solubilized in DMSO for 1 h at 80 °C. A total of 2.8 wt % hydroquinone with respect to 2-isocyanatoethyl methacrylate (2-ICEMA) was added to this solution cooled down to 60 °C. Then, 3 mol % 2-ICEMA (with respect to the PVA hydroxyl function) was added dropwise. The solution was mixed for 4 h at 60 °C and 12 h at 20 °C under argon. This solution was then diluted until 3 wt % with deionized water. Then the polymer was precipitated dropwise in a 10-fold acetone volume at room temperature. The modified PVA (denoted PVAm) was filtered and dried under vacuum at 30 °C for 48 h (yield: 87 ± 10%, [methacrylate]/[OH] = 1.95 ± 0.27%). FT-IR spectroscopy on PVAm confirmed the absence of residual isocyanate function. 1 H NMR in D2O: δ 6.12 (1H, s, CH2C), 5.71 (1H, s, CH2C), 3.99 (51H, m, C-CH(OR)-C), 1.64 (102 H, m, CH2). Gelation Procedure. All reactants were solubilized in a 0.05 mol/ L Tris-HCl buffer pH 7.4 and incubated at 37 °C for 15 min prior to mixing. Fibrin hydrogel, single PEO network, and PEO/Fb IPNs were synthesized as previously described.49,31 These materials were used for comparison. 3871

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

buffer (pH 6.8) containing 8 mol/L urea, 2 wt % sodium dodecyl sulfate (SDS), and 2% (v/v) β-mercaptoethanol. The fractions extracted from materials corresponding to 8 μg proteins were analyzed in a 10% polyacrylamide gel with molar weight standard ≪ Precision plus protein all blue standards ≫ (Biorad). Migration of proteins was occurred for 1 h at 30 mA and was revealed by use of silver stain kit (Biorad). Molar weights of the fragments were determined after gel scanning by scanner GS-800 calibrated Densitometer (Biorad) and the use of the software Quantity 1 V: 4.6.7 (Biorad). ELISA Test. Elisa test were realized with rabbit antihuman fibrinogen as the first antibody and alkaline-phosphatase conjugated antirabbit IgG as the second antibody following the previously described procedure.34 Dye-Labeled Fibrin Network. Staining of Materials by Coomassie Blue. Single PVAm(10%) network and IPN were incubated for 1.5 h in aqueous coloration solution containing Coomassie blue at 2.5 wt %, ethanol at 40% (v/v), and acetic acid 7% (v/v). Then the materials were rinsed in aqueous solution containing ethanol at 20% (v/v) and acetic acid at 10% (v/v) until removing of excessive coloration was done. Staining of Materials by Fluorescent Dye. For the fibrin hydrogel staining, the samples were rinsed with 1 mL of 0.2 mol/L carbonate buffer pH 8.5 for 12 h. Next, they were immersed in 1 mL of 2.2 × 10−3 wt % n-succinimide ester Alexa 488 in 0.2 mol/L carbonate buffer pH 8.5 for 12 h at 37 °C. Then they were rinsed three times with 0.2 mol/L carbonate buffer pH 8.5. Samples were observed with confocal laser scanning microscope (CLSM 710, Zeiss) with an 488 nm excitation wavelength with an objective ×63 (Plan-Apochromat 63x/ 1.40 Oil DIC M27). The average thickness of the fibrin fiber was measured using the ImageJ software. Rheology Measurements. Rheological measurements were performed with an Anton Paar Physica MCR 301 rheometer equipped with CTD 450 temperature control device and a cone−plate geometry (cone: diameter 25 mm, angle 2°; plate: polymerization system made from glass coupled with U.V Source Omnicure). A 1% deformation was imposed at 1 Hz. Storage modulus (G′) and loss modulus (G″) were recorded as a function of time. The gel time was determined at the intersection between storage and loss modulus curves. The solution of precursors of materials was put in the geometry and measurements begin immediately with U.V exposure (4.46 mW/cm2) at 37 °C. The average value of the storage modulus was measured for four times minimum. Thermal Analysis. All the thermal analyses were performed on samples dried under dynamic vacuum at 30 °C for at least 48 h. The remaining water amount in this dried samples was assessed between 1 and 3 wt % by TGA measurements (Q50, TA Instruments, from 20 to 200 °C, 5 °C/min heating rate). Then, the heat flows were measured by differential scanning calorimetry on a DSC Q100 (TA Instruments) from −50 to 250 °C with a 20 °C/min heating rate. Two cycles were carried out on each sample. Transitions were analyzed on the second cycle by using the software Universal Analysis V 4.3A (TA Instruments). Confocal Laser Scanning Microscopy of Stained Cell. For the live and dead tests, cell images were recorded on a Confocal Laser Scanning Microscope CLSM 710 (Zeiss, Germany) in sequential line mode (averaging 2) with a ×10 (EC Plan-Neofluar 10×/0.30 M27) objective for the live/dead assay. The living cells were stained with calcein AM (λex = 488 nm, λem = 490−573 nm) and the dead cells with ethidiumbromide dimer (λex = 561 nm, λem = 580−730 nm). Viability of cells was a mean value measured on three replicates. It was evaluated as

Single PVAm networks were synthesized as follows: 1 mL Tris-HCl buffer containing 10 wt % PVAm, 0.15 mol/L NaCl, 0.02 mol/L CaCl2, and 0.042 wt % Irgacure 2959 was prepared in a microvial. The solution was poured into a mold made from a glass plate and a Teflon plate separated by a 1 mm thick Teflon gasket. The mold was placed at 5 cm from a UV lamp (VL-6, Bioblock, 1.16 mW/cm2, 365 nm) for 1 h at 37 °C. White 1 mm thick hydrogel was obtained. The single PVAm networks are noted as a function of the precursor solution concentration (in w/v%); that is, single PVAm(10%) network for the typical recipe presented above. PVAm(10%)/fibrin IPN was synthesized as follows: 1 mL of TrisHCl buffer containing 10 wt % PVAm, 5 mg fibrinogen, 0.20 unit thrombin, 0.15 mol/L NaCl, 0.02 mol/L CaCl2, and 0.042 wt % Irgacure 2959 were stirred in a microvial. The mixture was placed into the previously described mold and the same photoinitiation process, as previously described for the single PVAm network was then applied. Self-supported materials were obtained with areas varying from 1.5 to 78.5 cm2 according to the mold size. The molar ratio of Irgacure/ PVA was kept constant when different fibrin/PVA weight proportions were used. The fibrinogen concentration was always equal to 5 mg/ mL. The synthesis of PVAm(10%)/Fb semi IPN in which PVA is not cross-linked was carried out with the same protocol than PVAm(10%)/Fb IPN, but Irgacure I2959 was not added. The PVAm(10%)/Fg semi-IPN, in which fibrinogen was not hydrolyzed, was synthesized without thrombin addition. The weight proportion (expressed in w/v%) of the synthetic partner is reported in brackets. For instance, IPN synthesized from 10 wt % PVAm and 5 mg fibrinogen mixed in a total volume of 1 mL is denoted as PVAm(10%)/Fb IPN. Spectroscopic Analysis. Nuclear magnetic resonance (NMR) analyses were performed on a 250 MHz NMR (Advance DPX-250FT-NMR Bruker) with the interface software TopSpin (Bruker). Products were dissolved in D2O and chemical shifts were measured relative to the chemical shift of the nondeuterated solvent fraction. Soluble Fraction. Unreacted PVAm contained in the single PVAm networks and IPNs were extracted for 48 h by Soxhlet extraction with deionized water. The resulting materials were then dried and weighed after extraction. The soluble fraction was calculated as a weight percentage:

SF(%) =

((wi − wsalt) − wf ) × 100 wi − wsalt

where wi and wf are the sample weight before and after extraction and wsalt is the weight of different salts present inside the material before extraction. All soluble fractions were measured at least three times for three samples produced by different syntheses, and the mean value was reported. Nonhydrolyzed fibrinogen was extracted from materials as follows: fibrin hydrogel and IPNs were incubated overnight in a 10-fold volume of buffer containing the same salt concentration as that in the material preparation. The extracted fibrinogen amounts were then quantified though the Elisa test. Hydration−Dehydration Cycles. Just after synthesis, the material was immersed for swelling in 20 mL of Tris-HCl buffer containing 0.02% sodium azide (NaN3), 0.15 mol/L NaCl, and 0.02 mol/L CaCl2 at 37 °C until equilibrium, and the sample weight was measured (w1). Then the material was dried at 37 °C until constant weight (w2). The material was submitted to a further same cycle of hydration− dehydration for which the weights of the wet and dry material are denoted w3 and w4, respectively. Each hydration−dehydration cycle was characterized by the swelling factor defined as SR1 = w1/w2 and SR2 = w3/w4. To compare the rehydration of the different materials, the swelling reversibility was defined as

swelling reversibility =

viability(%) =

SR 2 × 100 SR1

(full population of cells − dead cells) × 100 (full population of cells)

The cell density was observed with an objective ×20 (PlanApochromat 20×/0.8 M27). Their nuclei were stained with DAPI (λex = 405 nm, λem = 415−514 nm). Density of cell was a mean density of cell measured on three samples evaluated by counting nuclei with

SDS-PAGE Electrophoresis. Fibrin network in materials was disrupted by incubation for 72 h at 37 °C in 0.16 mol/L Tris-HCl 3872

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

imageJ (plug in cell counter) from morphology pictures taken on three different areas on the samples (nine replicates). For viability cell, the data were represented as the mean and the reported error corresponds to the standard deviation. Statistical comparisons were performed with Student’s t test, where p < 0.05 and below was accepted as statistically significant. Biocompatibility Test. Human fibroblast from foreskin (FB-BJ, ATCC CRL 2522) was cultured in DMEM high glucose medium supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin−streptomycin in a humidified 5% CO2 incubator. The materials were placed in a sterile culture plate (P24) and immersed in the culture medium without FBS for at least 24 h. The materials were then seeded with 1 mL of a cell suspension containing 1 × 105 cell/mL (cell density 5 × 104 cell/cm2). The materials were tested in triplicate at three different culture times (1, 3, and 7 days). The bottom plate of a P24 treated for cell culture was used as a positive control. A viability test was performed with a live/dead assay. Materials on which cells have been cultured were immersed in a 0.2 μM calcein AM and 0.2 μM ethidium bromide dimer in phosphate buffer for 30 min at 37 °C under 5% CO2 humid atmosphere. The staining of nuclei was carried out for cell density evaluation. The cell fixation was carried out with 3 wt % paraformaldehyde in phosphate buffer for 12 h at 37 °C. They were rinsed three times with phosphate buffer pH 7.4, permeabilized in a 200 mM phosphate and 0.1% (v/v) triton solution pH 7.4 at 4 °C for 30 min, and rinsed three times with phosphate buffer. Before immunostaining, cells were immersed for 45 min at room temperature in a saturation buffer composed of 200 mM phosphate buffer pH 7.4 with 10% (v/v) FBS. Then a 1 μg·mL−1 DAPI was spread on the samples at 37 °C. After 60 min, they were rinsed three times with 200 mM phosphate buffer pH 7.4.

high water swelling (for rehydration). Differential scanning calorimetry (DSC) analysis confirmed that the semicrystalline character of PVA was preserved after modification: the glass transition (Tg) and melting (Tm) temperatures were detected at 75 and 208 °C, respectively. PVAm Hydrogel. First, the PVAm network synthesis was carried out in buffer solution. Irgacure 2959 was selected as the photoinitiator because it is well tolerated by fibroblasts and it is not noxious toward different types of cells at concentrations lower than 0.1 wt %.52 The Irgacure concentration was thus set to 0.04 wt %. The cross-linking reaction was initiated under UV at 37 °C. The formation of the PVAm networks at different concentrations was monitored by rheology measurements (Figure 1).

■

RESULTS AND DISCUSSION In this work an original self-supported fibrin-based biogel was developed: a fibrin hydrogel was incorporated into Interpenetrating Polymer Networks (IPN) architectures according to a “one-pot” process. The fibrin gel was synthesized by the enzymatic hydrolysis of fibrinogen by thrombin (Tb, 0.20 unit), which necessitates a buffered aqueous medium. Thus, the synthetic polymer network partner must be able to be synthesized under these experimental conditions without hindering the enzymatic hydrolysis. PVA has been chosen as partner due to its good mechanical properties even when swollen with water. A highly hydrolyzed PVA (98%) with a molar weight of 16000 g/mol has been chosen as precursor. First, it was checked that the 0.5 wt % fibrin gel formation by enzymatic hydrolysis was effective in the presence of 5 wt % unmodified PVA. A fibrin gel was obtained but a syneresis was observed under mechanical stress. To avoid this phase separation, PVA must be cross-linked. PVA can be cross-linked with glutaraldehyde by aldehyde condensation with the alcohol functions leading to the formation of acetal ring.50 However, glutaraldehyde reacts indifferently on the alcohol functions of fibrin, fibrinogen, thrombin, and PVA leading to the formation of a single conetwork instead of IPN architecture. A photopolymerization of polyvinyl alcohol modified with methacrylate functions (PVAm) was thus chosen. Methacrylate groups were grafted on the PVA by alcohol-isocyanate addition between 2-isocyanatoethyl methacrylate and alcohol functions by adapting a protocol previously described.51 1H NMR analysis shows that 1.95 ± 0.27 mol % of the PVA hydroxyl function were modified. This modification ratio is a good compromise between the resulting PVA cross-linking density affecting the mechanical strength (for handle material) and the

Figure 1. Storage modulus G′ as a function of the irradiation time for (●) Fb 0.5% (w/v), and (■) 10, (◆) 7.5, or (▲) 5 wt % PVAm and (×) 10 wt % PEO under UV at 37 °C.

The gel time was detected after 2−3 min for single PVAm networks and Fb gel. The modulus of PVAm(5%) network reached 180 Pa after 45 min and remained stable. When the PVAm concentration is higher than 5 wt %, the PVAm networks have thus a storage modulus at least 10-fold higher than that of fibrin gel (60 Pa). Indeed, the storage modulus of PVAm(7.5%) and PVAm(10%) networks have reached 600 and 3160 Pa, respectively. For the same concentration (10%), the PVAm network forms quicker than the PEO network (28 min), and it has a higher storage modulus (3160 Pa) than that of PEO network (940 Pa). This gap in modulus values might be explained by the presence of crystalline domains in the single PVAm networks.39 Indeed, their presence has been confirmed by the Tm at 176 and 170 °C for the dried PVAm(10%) and PVAm(5%) networks, respectively, by DSC analysis. In addition, the corresponding melting enthalpy decreases from 21.9 J/g for the PVAm(10%) network to 5.3 J/g for the PVAm(5%) network showing that the proportion of crystalline domains decreases when the PVAm concentration decreases and it is related to the storage modulus of the network. To confirm the correct PVAm cross-linking, the soluble fractions of these single networks were determined. They were equal to 9.6 ± 0.6 and 16.4 ± 0.7 wt % for PVAm(10%) and PVAm(5%) networks, respectively. 1H NMR analysis of the soluble fractions shows that they were composed of unmodified 3873

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

mW/cm2). However, the light source power used for synthesis out of the rheometer is lower (1.16 mW/cm2). So the polymerization time was set at 60 min for the end of the study. These materials are homogeneous, transparent, and selfsupported (Figure 3).

PVA. Both PVAm single networks are correctly cross-linked as the soluble fractions remain reasonable for materials synthesized from low precursor concentration solutions. Finally, PVAm networks are more rapidly synthesized than the single PEO network and they show a three times higher storage modulus. PVAm/Fb IPNs as Biomaterials. Material Synthesis. The in situ syntheses of PVAm(10%)/Fb, PVAm(7.5%)/Fb, and PVAm(5%)/Fb IPNs have been worked out. The solution containing all the precursors of both networks was incubated at 37 °C and the photopolymerization was initiated under UV. The network formations were monitored by in situ rheology measurements. The storage moduli of IPNs with different PVAm concentrations were presented as a function of irradiation time in Figure 2.

Figure 3. Photograph of (A) PVAm(10%)/Fb IPN and (B) PVAm(5%)/Fb IPN.

The IPNs were dried and their thermal properties studied by DSC. IPNs keep a semicrystallyne behavior with Tg and Tm temperatures close to those detected on the corresponding single PVAm networks. These weak Tg and Tm variations suggest that Fb has a low influence on the conformation of PVAm network in the IPNs. The dried IPNs are ductil and selfsupported as PVAm networks. In consequence, their storage can be considered in the dried state. First, the effective formation of PVAm network in the IPNs has been verified. PVAm(10%)/Fb and PVAm(5%)/Fb IPNs were extracted for 48 h with water to determine the PVA soluble fraction found equal to 13 ± 2% and 20 ± 5%, respectively. 1H NMR analysis of the soluble fractions shows that unmodified PVA was extracted in agreement with the results obtained on single PVAm networks. Further analyses on the insoluble fraction reveal that IPNs are always positive to Coomassie blue staining showing that protein has not been totally extracted from IPNs under those conditions. Second, we verified that the fibrin network is also properly formed in the IPNs. The fibrin networks in the PVAm(10%)/ Fb and PVAm(5%)/Fb IPNs were disrupted by immersion in Tris buffer containing urea, SDS, and β-mercaptoethanol, for 72 h at 37 °C. The supernatants were then analyzed by electrophoresis (Figure 4). The results were compared to those obtained with a fibrin hydrogel and fibrinogen monomer (Fg). A PVAm(10%)/Fg semi-IPN was also analyzed to ensure that the presence of PVAm network does not prevent the protein extraction and the PVAm(10%) network was considered as negative control (no false positive revelation with silver nitrate). The γ−γ band specific of the action of thrombin on fibrinogen was clearly visible at 105 kDa for the fibrin hydrogel while it was absent for the fibrinogen monomer and for the PVAm(10%)/Fg semi-IPN. As the γ−γ band was detected for the PVAm(10%)/Fb and PVAm(5%)/Fb IPNs, the fibrin gel was effectively synthesized by the specific action of thrombin on fibrinogen monomer inside these IPNs. The extent of formation of the fibrin gel was evaluated by extraction of fibrin and fibrinogen not linked to the protein network by immersion of IPNs in a leaching solution (Tris

Figure 2. Rheology measurement (G′) during (■) PVAm(10%)/Fb IPN, (◆) PVAm(7.5%)/Fb IPN, (▲) PVAm(5%)/Fb IPN, and (×) PEO(10%)/Fb IPN formation.

A gel was obtained after 3 min for the PVAm(7.5%)/Fb and PVAm(10%)/Fb IPNs and after 6 min for the PVAm(5%)/Fb IPN. These gel times are similar to those of fibrin gel and single PVAm networks. They are short compared to that of PEO(10%)/Fb IPN (20 min). In comparison with the single networks, the irradiation times required to reach stable modulus values are unchanged. The storage modulus decreases when the PVAm proportion decreases in the IPN: they are equal to 3240, 900, and 330 Pa for IPNs containing 10, 7.5, and 5 wt % PVAm, respectively. So the PVAm(5%)/Fb IPN modulus is five times higher than that of a Fb hydrogel (60 Pa). Moreover, curiously, the addition of 0.5 wt % fibrin inside the PVAm(5%) network permits an increase by 2 of the storage modulus in comparison with the single PVAm(5%) network (180 Pa). Macroscopically, PVAm(5%)/Fb IPN is a selfsupported material, while the PVAm(5%) single network is not. For comparison, the PEO(10%)/Fb IPN has a storage modulus of 1320 Pa, in other words, 20 times higher than fibrin scaffold alone. So the replacement of PEO by PVAm in the fibrin-based IPN allows obtaining a material with a storage modulus 50 times higher than the proteic scaffold alone and 2 times higher than POE-based IPN. Based on these measurements, the formations of the PVAm based IPNs are completed after 45 min of UV irradiation (4.46 3874

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

concentration. These results confirm that the PVAm network does not inhibit the enzymatic hydrolysis of fibrinogen by thrombin inside the IPN. IPN Morphology. The distribution of fibrin amines of which were specifically labeled by N-succinimide ester Alexa 488, was imaged by confocal laser scanning microcopy (CLSM) on Fb hydrogel, PVAm(10%)/Fb, PVAm(5%)/Fb, and PEO(10%)/ Fb IPNs (Figure 5). It was checked that PVAm and PEO are not stained under those conditions. The large spherical fluorescence-free areas correspond to air bubbles, inherent to the formation of materials from viscous solutions. According to its partner, the fibrin distribution in the material is significantly different. While the Fb appears uniformly dense in the Fb hydrogel (Figure 5A), its distribution is less dense in the PVAm-based IPNs (Figure 5B,C). These fibrin repartitions remain similar when the observations were performed until 20 μm depth inside these materials. This difference between fibrin hydrogel and PVAm based IPNs can be explained by the rapid formation of the PVAm network in IPNs (about 2−5 min) that may require the Fb network to adopt a less dense conformation. Such a change in the density of the fibrin network has already been observed when the calcium and thrombin relative proportions increase.53 This hypothesis is strengthened by the significant increase of the Fb fiber thickness. Indeed, the average thickness of the fibers was about 360 ± 40 nm for Fb hydrogel and it was increased by 3 in the IPNs. However, the PVAm concentration does not significantly affect this thickness, which is 890 ± 80 and 970

Figure 4. SDS-PAGE electrophoresis gel of supernatants of (1) fibrin hydrogel, (2) fibrinogen monomer, (3) single PVAm(10%) network, (4) PVAm(10%)/Fb IPN, (5) PVAm(5%)/Fb IPN, and (6) PVAm(10%)/Fg semi-IPN after incubation for 72 h in chemical destruction buffer.

buffer pH 7.4 with 0.02% azide) for 24 h at 37 °C. Fibrin and fibrinogen were then quantified through an indirect ELISA protocol. Single PVAm(10%) network does not cause false positive (no absorbance measurement). Analysis of leaching solution of PVAm(10%)/Fg semi-IPN shows that 24 h of incubation are sufficient for the protein diffusion out of PVAm matrix without Fg confinement. The leaching solution of physiological Fb(0.5%) gel contains less than 0.1% soluble fraction. Similarly, the leaching solutions of IPNs contain less than or equal to 1% extractible protein whatever the polymer

Figure 5. Confocal microscopy (CLSM) images on the surface of (A) Fb hydrogel, (B) PVAm(5%)/Fb IPN, (C) PVAm(10%)/Fb IPN, and (D) PEO(10%)/Fb IPN. Fibrin was labeled by n-succinimide ester Alexa 488. Images E and F were recorded on PEO(10%)/Fb IPN at 2 and 10 μm depth, respectively. Objective ×63/1.4 (scale bar: 10 μm). 3875

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

Figure 6. (A) Swelling factors (SR1(gray) and SR2 (black)) and (B) swelling reversibility (SR2/SR1) of single PVAm(5%) and PVAm(10%) networks and of PVAm(5%)/Fb and PVAm(10%)/Fb IPNs.

Figure 7. Alive (green) and dead (red) cells on PVAm(10%) network, PVAm(5%)/Fb IPN, and PVAm(10%)/Fb IPN after 1, 3, and 7 days of culture. Cells staining with a live and dead cell kit; Objective 10×/0.30 (scale bar: 50 μm).

± 120 nm in PVAm(10%)/Fb and PVAm(5%)/Fb IPNs, respectively. Thus, the presence of PVAm, even if it does not prevent the formation of fibrin fiber, impacts their thickness. That can eventually explain the increase of storage modulus of PVAm(5%)/Fb IPN compared with those of each component. On the opposite, CSLM image recorded on the surface of PEO(10%)/Fb IPN reveals no fibrin presence (Figure 5D). However, fibrin was detected randomly at 2 μm depth (Figure 5E) and homogeneously at 10 μm depth (Figure 5F) of this IPN. Those three images reveal an important phase separation between POE and fibrin which leads to the absence of protein at the surface of IPN.31 The result explains a fairly limited CHO

cell growth observed on this material surface because their access to the protein network was not permitted. The comparison between the fibrin distribution in the PVAm(10%)/Fb and PEO(10%)/Fb IPNs shows that it depends on the synthetic polymer partner. Fibrin has to be present at the surface for biomaterial application. Dehydration/Rehydration Ability. To be able to store the materials in a dry state before use, they must both maintain their integrity and have an optimal swelling reversibility (SR2/ SR1). The last one is calculated after a maximum swelling (SR1) and one cycle of dehydration followed by rehydration (SR2). The ability to preserve the swelling capacity was evaluated on single PVAm(5%) and PVAm(10%) networks and on 3876

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

From these images, the cell viability on the different materials was calculated (Figure 8).

PVAm(10%)/Fb and PVAm(5%)/Fb IPNs (Figure 6). The Fb hydrogel has a very high swelling capacity (SR1 = 25), however, after dehydration, it becomes very rigid and brittle and shows poor swelling reversibility (22 ± 2%). The maximum swelling factor (SR 1 ) of the single PVAm(10%) network and PVAm(10%)/Fb IPN are close (10 and 11) and lower than those of the PVAm(5%)-based materials, which are equal to 22 and 17 for single network and IPN, respectively (Figure 6A). The swelling factor of all these hydrogels is important but lower than that of Fb one (SR1 = 25). In the studied proportion range, the more the material contains PVAm, the smaller the swelling factor. Except for PVAm(10%)/Fb IPN, the second swelling factor (SR2) is always lower than SR1. This decrease can be due to a higher PVAm crystalline domain amount after a drying step. Indeed, the salts in the materials promote PVA crystallization during the drying.54 This crystalline amount increase was confirmed by the melting enthalpy increase (DSC measurements) up to 28.0 and 24.5 J/g instead of 11.9 and 13.1 J/g initially for single PVAm(10%) network and PVAm(10%)/Fb IPN, respectively. However, whatever the PVAm proportion, the IPNs show better swelling reversibility (SR2/SR1) on one dehydration/ rehydration cycle than the corresponding single network (Figure 6B). In addition, the swelling reversibility increases from 73 to 100% when PVAm concentration in IPN increases from 5 to 10 wt %. From these results, it appears that PVAm based IPNs are more interesting materials than PEO-based IPN31 because they show a total rehydration as expected. Biocompatibility. Another important property for the potential applications of these materials is biocompatibility. All synthetic components (PVA, Irgacure) were chosen according to their recognized low toxicity. However, the absence of any cytotoxicity in the final materials was studied through a 2D cell culture of a fibroblast cell line of human dermis from newborn foreskin (FB-BJ). The used model has an adherent phenotype. First, the global metabolic activity of BJ fibroblast cells cultured on single PVA(10%) network, PVAm(10%)/Fb IPN, PVAm(5%)/Fb IPN, or cell culture plastic was measured. As the new materials do not induce a decrease in mitochondrial activity compared to the plastic support (Supporting Information, Figure S1), they do not leak any cytotoxic chemical toward FB-BJ. This test gives qualitative information. It does not discriminate between the mitochondrial activity of cells settled on the bottom plate from that of cells on the surface of materials. So, in addition, the cell viability was quantified using a live and dead cell kit (Figure 7). In living cells, the nonfluorescent calcein AM was converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases whereas ethidiumbromide dimer becomes red-fluorescent when it was inserted in DNA of proapoptic or dead cell. Few cells were observed on single PVAm(10%) network, whatever the culture time. They remain round and tend to form clusters commonly interpreted as a cell adhesion default on the surface. After 7 days, red apoptotic cells were observed either dispersed or in clusters. On the IPN surfaces, cell populations were composed of both spread cells, organized in cluster, and few isolated round cells. Isolated cells show specific coloration of dead cells. Contrarily, spread cells organized in cluster show a better viability. These results suggest that cells are able to adhere, spread and can survive on the surface of these materials.

Figure 8. (A) Cell viability and (B) cell density measured on the surface of PVAm(10%) network, PVAm(10%)/Fb IPN, and PVAm(5%)/Fb IPN after 1 (white), 3 (gray), and 7 days (black) of culture.

The cell ability to survive on the PVAm(10%) network is weak since after 1 day only 70% of the cells are alive. That is in agreement with cell density measurement (see below). The proportion of living cell decreases over the first week up to a mean value of 20%. On the fibrin based IPNs, the cell viability is interestingly higher than on the single network. After 3 days at least 80% of the initial cells survive; then this proportion decreases to 70% and 50% for IPNs containing 5 and 10% PVAm respectively. This indicates that the cells are sensitive to the polymer concentration and that the presence of fibrin on the surface of IPN (shown by CLSM) is necessary to ensure the cell viability on these materials. To quantify more accurately the cell growth on the surface, the DAPI stained nuclei were counted after 1, 3, and 7 days of cell incubation. On the surface of single PVAm(10%) network, the cell concentration decreases continuously for the week. Indeed, neutral hydrophilic polymer networks are known to limit protein adsorption55 as well as focal adhesion establishment56 that are both essential for cell survival. Their lack induces particular cell morphology followed by apoptosis (data not show).57 On the IPN surfaces, fibrin provides the RGD motif necessary to the cell adhesion, and thus ensures cell spreading; however no proliferation was observed (Supporting Information, Figure S2). The cell cultures carried out on the PVAm-based materials allowed demonstrating that this polymer does not interfere with cell metabolism. The presence of fibrin in the IPNs brings 3877

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

(5) Khetani, S. R.; Bhatia, S. N. Curr. Opin. Biotechnol. 2006, 17, 1−8. (6) Kirschner, C. M.; Anseth, K. S. Acta Mater. 2013, 61, 931−944. (7) Picard, J.; Doumeche, B.; Panouillé, M.; Larreta-Garde, V. Macromol. Syst. 2010, 291−292, 337−344. (8) Rosso, F.; Marino, G.; Giordano, A.; Barbarisi, M.; Parmeggiani, D.; Barbarisi, A. J. Cell. Physiol. 2005, 203, 465−470. (9) Ronfard, V.; Rives, J.-M.; Neveux, Y.; Carsin, H.; Barrandon, Y. Transplantation 2000, 70, 1588−1598. (10) Anitua, E.; Sanchez, M.; Nurden, A. T.; Nurden, P.; Orive, G.; Andia, I. Trends Biotechnol. 2006, 24, 227−234. (11) Linnes, M. P.; Ratner, B. D.; Giachelli, C. M. Biomaterials 2007, 28, 5298−5306. (12) Collet, J. P.; Shuman, H.; Ledger, R. E.; Lee, S.; Weisel, J. W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9133−9137. (13) Ahmed, T. A. E.; Dare, E. V.; Hincke, M. Tissue Eng., B 2008, 14, 199−215. (14) Shah, J. V.; Janmey, P. A. Rheol. Acta 1997, 36, 262−268. (15) Ho, W.; Tawil, B.; Dunn, J. C. Y.; Wu, B. M. Tissue Eng. 2006, 12, 1587−1595. (16) Rowe, S. L.; Lee, S.; Stegemann, J. P. Acta Biomater. 2007, 3, 59−67. (17) Schmoekel, H. G.; Weber, F. E.; Schense, J. C.; Grätz, K. W.; Schawalder, P.; Hubbell, J. A. Biotechnol. Bioeng. 2005, 89, 253−62. (18) Zhang, G.; Wang, X.; Wang, Z.; Zhang, J.; Suggs, L. Tissue Eng. 2006, 12 (1), 9−19. (19) Sperling, L. H.; Misha, V. IPNs around the World: Science and Engineering; Wiley: New York, 1997; pp 1−25. (20) Sperling, L. H. Adv. Chem. Ser. 1994, 239, 3−38. (21) Amoozgar, Z.; Rickett, T.; Park, J.; Tuchek, C.; Shi, R.; Yeo, Y. Acta Biomater. 2012, 8, 1849−58. (22) Xiao, W.; He, J.; Nichol, J. W.; Wang, L.; Hutson, C. B.; Wang, B.; Du, Y.; Fan, H.; Khademhosseinia, A. Acta Biomater. 2011, 7 (6), 2384−2393. (23) Rowe, S. L.; Stegemann, J. P. Biomacromolecules 2006, 7, 2942− 2948. (24) Shikanov, A.; Xu, M.; Woodruff, T. K.; Shea, L. D. Biomaterials 2009, 30, 5476−5485. (25) Losi, P.; Briganti, E.; Magera, A.; Spiller, D.; Ristori, C.; Battolla, B.; Balderi, M.; Kull, S.; Balbarini, A.; Di Stefano, R.; Soldani, G. Biomaterials 2010, 31, 5336−5344. (26) Soldani, G.; Losi, P.; Bernabei, M.; Burchielli, S.; Chiappino, D.; Kull, S.; Briganti, E.; Spiller, D. Biomaterials 2010, 31, 2592−2605. (27) Briganti, E.; Spiller, D.; Mirtelli, C.; Kull, S.; Counoupas, C.; Losi, P.; Senesi, S.; Di Stefano, R.; Soldani, G. J. Controlled Release 2010, 142, 14−21. (28) Lisi, A.; Briganti, E.; Ledda, M.; Losi, P.; Grimaldi, S.; Marchese, R.; Soldani, G. PLoS One 2012, 7, e34284. (29) Lee, F.; Kurisawa, M. Acta Biomater. 2013, 9 (2), 5143−5152. (30) Suri, S.; Schmidt, C. E. Acta Biomater. 2009, 5 (7), 2385−2397. (31) Larreta-Garde, V.; Fichet, O.; Akpalo, E. Eur. Patent No. WO2010058132, 2010. (32) Akpalo, E.; Bidault, L.; Boissière, M.; Vancaeyzeele, C.; Fichet, O.; Larreta-Garde, V. Acta Biomater. 2011, 7, 2418−2427. (33) Vrana, N. E.; Matsumura, K.; Hyon, S. H.; Geever, L. M.; Kennedy, J. E.; Lyons, J. G.; Higginbotham, C. L.; Cahill, P. A.; McGuinness, G. B. J. Tissue Eng. Regener. Med. 2012, 6 (4), 280−90. (34) Qi, M.; Gu, Y.; Sakata, N.; Kim, D.; Shirouzu, Y.; Yamamoto, C.; Hiura, A.; Sumi, V.; Inoue, K. Biomaterials 2004, 25 (27), 5885−92. (35) Kobayashi, M.; Hyu, H. S. Materials 2010, 3, 2753−2771. (36) Kobayashi, H. Adv. Sci. Technol. 2006, 53, 9−16. (37) Barrett, D. A.; Hartshorne, M. S.; Hussain, M. A.; Shaw, P. N.; Davies, M. C. Anal. Chem. 2001, 73, 5232−5239. (38) Moolman, F. S.; Meunier, M.; Labuschagne, P. W.; Truter, P.-A. Polymer 2005, 46, 6192−6200. (39) Hassan, C. M.; Peppas, N. A. Adv. Polym. Sci. 2000, 153, 37−65. (40) Stauffer, S. R.; Peppas, N. A. Polymer 1992, 33 (18), 3932− 3936. (41) Urushizaki, F.; Yamagushi, H.; Nakamura, K.; Numajiri, S.; Sugibayashi, K.; Morimoto, Y. Int. J. Pharm. 1990, 58, 135−142.

RGD peptides that support cell spreading and keep their viability close to 90% after 1 day. The cell population does not grow but cell death remains limited after 1 week.

■

CONCLUSION That is a challenge to create an easily handled biomaterial showing the exceptional properties of the fibrin. In this work, this protein hydrogel was associated into interpenetrating polymer networks (IPN) architecture with polyvinyl alcohol (PVA), which is highly hydrophilic for improved rehydration capability and semicrystalline for stronger mechanical properties. The PVA was first functionalized with methacrylate groups (PVAm) and was thus cross-linked by photoirradiation in an aqueous buffer medium. The PVAm/Fb IPNs were formed quickly (less than 6 min) according to a one-pot synthesis, and their storage modulus is 3- to 50-fold higher than that of the fibrin gel. These homogeneous materials can be easily stored in a dehydrated state because they are completely rehydratable afterward. In addition, contrary to polyethylene oxide/fibrin IPNs, protein is present on the surface of PVAm-based IPNs, which is important for cell development. These materials are noncytotoxic toward human fibroblast as targeted for the expected clinical application on wound healing. As a perspective, the full colonization in bulk of the IPN by cells could be envisioned. In analogy with the ECM, the cells would need remodeling these supports.

■

ASSOCIATED CONTENT

S Supporting Information *

The supporting documents describe the reduction of alamar blue by mitochondrial activity of cells after 1, 3, and 7 day culture times as well as the morphology of cells after 1 and 7 day culture times on PVAm-based samples. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: (33)134257050. E-mail: odile.fi[email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was sponsored by the Direction Générale de l′Armenent (DGA) of France. The authors thank more particularly Dr Bruno Mortaigne, responsible for “Materials and Chemistry” at DS MRIS. The authors would like to thank the Région Ile-de-France which has sponsored the SESAME project (Comicer project) that has allowed the acquisition of confocal laser scanning microscope with which were recorded the images reported in this work. The authors are also grateful to the “Fondation de l′UCP” for its financial support to Marie Deneufchatel.

■

REFERENCES

(1) (a) Peppas, N. A. Polymers; CRC Press: Boca Raton, FL, 1987; Vol. 2. (b) Hickey, A. S.; Peppas, N. A. J. Membr. Sci. 1995, 107, 229− 237. (2) Malafaya, P. B.; Silva, G. A.; Reis, R. L. Adv. Drug Delivery Rev. 2007, 59, 207−233. (3) Vert, M. Prog. Polym. Sci. 2007, 32, 755−761. (4) Shevchenko, R V; James, S. L.; James, S. E. J. R. Soc. Interface 2010, 7, 229−258. 3878

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879

Biomacromolecules

Article

(42) Hickey, A. S.; Peppas, N. A. J. Membr. Sci. 1995, 107, 229−37. (43) Peppas, N. A.; Scott, J. E. J. Controlled Release 1992, 18, 95−100. (44) Siritienthong, T.; Ratanavaraporn, J.; Aramwit, P. Int. J. Pharm. 2012, 439 (1−2), 175−186. (45) Kulkarni, R. V.; Sreedhar, V.; Mutalik, S.; Setty, C. M.; Sa, B. Int. J. Biol. Macromol. 2010, 47, 520−527. (46) Thankam, F. G. T.; Muthu, J.; Sankar, V.; Gopal, R.K.. Colloids Surf., B 2013, 107, 137−45. (47) Burnouf, T.; Goubran, H. A.; Chen, T.-M.; Ou, K.-L.; El-Ekiaby, M.; Radosevic, M. Blood Rev. 2013, 27, 77−89. (48) Moura, L. I. F.; Dias, A. M. A.; Carvalho, E.; de Sousa, H. C. Acta Biomater. 2013, 9, 7093−7114. (49) Akpalo, E.; Larreta-Garde, V. Acta Biomater. 2010, 6, 396−402. (50) Araujo, A. M.; Neves, M. T., Jr.; Azevedo, W. M.; Oliveira, G. G.; Ferreira, D. L., Jr.; Coelho, R. A. L.; Figueiredo, E. A. P.; Carvalho, L. B., Jr. Biotechnol. Technol. 1997, 11 (2), 67−70. (51) Bryant, S. J.; Davis-Arehart, K. A.; Luo, N.; Shoemaker, R. K.; Arthur, J. A.; Anseth, K. S. Macromolecules 2004, 37, 6726−6733. (52) Williams, C. G.; Malik, A. N.; Kim, T. K.; Manson, P. N.; Elisseeff, J. H. Biomaterials 2005, 26, 1211−1218. (53) Ryan, E. A.; Mockros, L. F.; Weisel, J. W.; Lorand, L. Biophys. J. 1999, 77, 2813−2826. (54) Patachia, S.; Florea, C.; Friedrich, C.; Thomann, Y. eXPRESS Polym. Lett. 2009, 3, 320−331. (55) Bačaḱ ová, L.; Filová, E.; Rypácě k, F.; Svorčík, V.; Starý, V. Physiol. Res. 2004, 53 (Suppl. 1), S35−S45. (56) Nuttelman, C. R.; Mortisen, D. J.; Henry, S. M.; Anseth, K. S. J. Biomed. Mater. 2001, 57, 217−223. (57) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425−1428.

3879

dx.doi.org/10.1021/bm400991k | Biomacromolecules 2013, 14, 3870−3879