Adhesion Behavior of Peritoneal Cells on the Surface of Self

stage of cultivation with murine peritoneal cells, cell adhesion on the hydrogels of PEO and PBLA with 18. (20K18) and 25 (20K25) monomeric units was ...
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Biomacromolecules 2004, 5, 2447-2455

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Adhesion Behavior of Peritoneal Cells on the Surface of Self-Assembled Triblock Copolymer Hydrogels Shinji Tanaka,†,‡ Atsuhiko Ogura,†,‡ Tatsuo Kaneko,†,§ Yoshishige Murata,†,‡ and Mitsuru Akashi*,†,§ Department of Nanostructured and Advanced Materials, Graduate School of Science and Engineering, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065, Japan, Tsukuba Research Laboratory, NOF Corporation,5-10, Tokodai, Tsukuba, Ibaraki, 300-2635, Japan, and Department of Molecular Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871, Japan Received June 15, 2004; Revised Manuscript Received August 2, 2004

Adhesion behavior of cells to the surface of physical hydrogel membranes prepared by water-induced selforganization of precisely synthesized ABA-triblock copolymers comprised of poly(β-benzyl L-aspartate) (PBLA) as A segment and poly(ethylene oxide) (PEO, molecular weight ) 20 000) as the B segment were investigated. The cast film from the methylenechloride solution of these copolymers swelled in water very rapidly forming hydrogels (100-400% water content of total weight). The content of PBLA affected the strength, the hydrophobicity, and the amount of water involved in the hydrogel surface. During the early stage of cultivation with murine peritoneal cells, cell adhesion on the hydrogels of PEO and PBLA with 18 (20K18) and 25 (20K25) monomeric units was not observed, while adhesion on the hydrogels of PEO and PBLA with 32 (20K32) and 55 (20K55) monomeric units was successful, suggesting more than 12 mol % in PBLA content is necessary for adhesion of these cells. Although cell spreading on the hydrogels of 20K18, 20K25, and 20K32 was not sufficient, the hydrogel of 20K55 allowed cell adhesion and spreading to be bipolar with leading edge whose raffling is active with pseudopodium and lamellipodium as well as PBLA homopolymer, suggesting active motility of these cells. Remarkably, prolonged incubation restored adhesiveness onto the films at 20K18 in contrast to adhesion with 20K25 despite low hydrophobicity. It is conceivable that adaptation of proteins and chemical changes to the surface during the culture period may participate in these phenomena. Mechanical properties and interaction between cell and these copolymer hydrogels could be controlled by composition of block segments, and optimization for implants could also be attainable. Introduction Implant polymeric materials frequently elicit an inflammatory response. After implantation, immediate adsorption of proteins, including albumin, immunoglobulin, complement, and of other adhesive proteins such as fibrinogen and fibronectin from body fluid, may take place. These elicit further adhesion of leukocytes such as monocytes/macrophages and neutrophils1,2 depending on the amount of binding, composition, orientation, or conformational change of proteins.3-10 Adherent macrophages became activated and fused to form multinucleate foreign body giant cells (FBGC) on the surface by the coordination of other immune cells. FBGC persistently secrete cytokines and prostanoids to recruit proinflammatory and mesenchymal cells, which contribute to the encapsulation of the polymer surface with an extracellular matrix. Adhesion and morphological changes of these early inflammatory cells on the surface are closely associated with their function, such as migration, activation, * Correspondence author. E-mail: [email protected]; tel: +81-6-6879-7356; fax: +81-6-6879-7359. † Kagoshima University. ‡ NOF Corporation. § Osaka University.

fusion, and excretion of chemical mediators.11-19 The interactions between these cells and the material surface are thought to be decisive for the subsequent chronic inflammation processes and disintegration of materials by proteolytic enzymes and radicals.20 Various attempts have been made to regulate the adhesion of these proteins and cells, including the modification of surface physiochemical, biochemical, and biological properties. Poly(ethylene oxide) (PEO) (which has a high degree of steric exclusion by segment flexibility) is chemically stable and biologically compatible when used for the PEGylation of bioactive compounds to avoid metabolism. PEO, possessing a molecular weight less than 20 000, can be dissolved in body fluids and excreted through the kidneys, although PEO is not biodegradable.21,22 Biodegradable homo-poly(L-amino acid) exhibits distinct mechanical and biological properties including flexibility, toughness, nontoxicity, and lower antigenicity. To investigate higher-order structures, poly(β-benzyl Laspartate) (PBLA) and poly(γ-benzyl L-glutamate) (PBLG) were widely investigated as typical hydrophobic polypeptides. Kugo et al. synthesized and characterized ABA-triblock copolymers comprised of PBLG or PBLA and PEO (Mw 4000), showing that adhesion of Hela cells to ABA triblock

10.1021/bm049653o CCC: $27.50 © 2004 American Chemical Society Published on Web 09/25/2004

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Scheme 1

Tanaka et al. Scheme 2

copolymers was controlled by their composition.23 Yokoyama et al. reported that B-cells and T-cells were easily separated using their difference in adhesiveness against ABA-triblock copolymers comprised of PBLG and PEO (Mw 1000 and 4400), showing that adhesion of the cells could be controlled by balancing these segments.24 In the preceding study, we precisely synthesized ABAtriblock copolymers consisting of PBLA as A segment and poly(ethylene oxide) (PEO) as B segment with a high molecular weight of greater than 20 000. We demonstrated that these copolymers yield flexible and tough film casts that have rubberlike properties.25 It also has been shown that these polymers formed a stable hydrogel in water with a hierarchical structure and that the intermolecular relationship between hydrophobic segments was strengthened during swelling in water.26 Hence, hydrogels are highly interesting as biomaterials. To obtain appropriate biocompatible hydrogels for implants, here we investigated the effect of molecular weight of PBLA on the swelling behavior, mechanical strength, surface properties, and cell anchoring behavior of the cells on ABAtype copolymer hydrogels containing high molecular weight PEO (Mw ) 20 000). Adhesion and morphology of murine peritoneal infiltrate cells, mainly, monocytes/macrophages, were applied because these cells are worthwhile for estimating the fate of materials in the living body where inflammatory cells with intensive activity of adhesion in comparing with immortalized cell lines by transformation. Methods Synthesis of PBLA Homopolymer. PBLA was obtained by ring-opening polymerization of β-benzyl L-aspartate N-carboxyanhydride (BLA-NCA) as shown in Scheme 1.25 The reaction was initiated by methoxyethylamine in N,Ndimethylformamide (DMF) for 24 h at 40 °C in an argon atmosphere. Polymerization with BLA-NCA with a molar ratio of 26.3 to initiator yielded PBLA with an average molecular weight of 6 × 103 and Mw/Mn of 1.14. The reaction solution was evaporated and the residual solid was washed with diethyl ether and then dried in vacuo at room temperature, yielding the PBLA homopolymers (yields: over 98 wt %). Synthesis of PBLA-PEO-PBLA Triblock Copolymers. The ABA block copolymers consisting of PEO and PBLA were precisely synthesized in the following procedure. The ring-opening polymerization of BLA-NCA was performed without any catalyst for 24 h at 40 °C in a nitrogen atmosphere. Initiation from the amine-terminated PEO (10

w/v %) in the mixture of dichloromethane/DMF (9/1, v/v), varied the molar composition of BLA-NCA to the ethylene oxide (EO) units in the feed from 39 to 119 (mol %) as shown in Scheme 2.27 The polymerization proceeded homogeneously and the reaction solution was poured into a large quantity of hexane/ ethyl acetate (1/1, v/v) to precipitate the white matter. The products were washed with hexane and then dried in vacuo at room temperature, yielding the PBLA-PEO-PBLA triblock copolymers (yields: over 98 wt % based on PEO recovery). The structures were confirmed by infrared (IR) spectroscopy and proton nuclear magnetic resonance (1H NMR) and also by gel permeation chromatography. Hydrogel Preparation. The dry films were prepared by casting from a methylenechloride solution (1%, w/v) of PBLA-PEO-PBLA triblock copolymers on Teflon film and were desiccated spontaneously. The dry films, which were weighed in advance, Wdry, were swollen in distilled water at 25 °C for 1 h, yielding the hydrogels. After the excess water on the surface of the hydrogels was removed, the swollen films were weighed at specific time intervals, Wgel. The swelling ratio was determined according to the following equation: swelling ratio (wt%) ) Wgel/Wdry × 100 Tensile Test of Hydrogels. The stress-strain curve of a strip of film 2 mm in width and 20 mm in length was recorded on a Yamaden creep meter RE3305 at room temperature with a tensile rate of 10 mm/min. Young’s modulus was determined from the slope of the stress-strain curve for a strain of less than 1%. Preparation of Murine Peritoneal Cells. Peritoneal cells from sacrificed mice were obtained by injecting 5 mL of ice-cold calcium and magnesium ion free phosphate-buffered saline (PBS(-)) into the peritoneal cavity of 6-week old mice. After gentle massage, the fluid was harvested. Peritoneal cells were washed with cold PBS (-) and resuspended in RPMI medium supplemented with 10% FCS, immobilized by treatment at 55 °C for 1 h; 100 u/mL penicillin G, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B. After the number of white blood cells was determined using a hematology analyzer (Sysmex KX21), cells (8 ∼ 11 × 105) in 500 µL medium were placed in 24-well plates containing preswollen polymers for specific intervals in a 5% CO2 atmosphere.

Cell Adhesion on Self-Assembled Hydrogels

Morphological Observation of Cells. For morphological observation, polymer films were cultivated with murine peritoneal cells at 37 °C for 1 day. After being rinsed with PBS (-) twice, they were fixed with glutaraldehyde solution (2.5 w/v %) in 0.2 M phosphate buffer (pH 7.4) for 2 h at 4 °C and stained with 1% osmium tetroxide for 12 h. They were then washed with PBS and gradually dehydrated by treatment with ethanol solution from 70 to 90 v/v %. These specimens were freeze-dried for 6 h after replacement of ethanol with tert-butyl alcohol and then Pt-coated using a sputter coater. The surface was examined with scanning electron microscopy (SEM, HITACHI N-300). Cell densities were determined from 5 to 7 objective fields for each sample, and cell areas were measured using Scion Image software. Cytoskeletal Organization. The organization of F-actin stress fibers in the cells on the polymer substratum was demonstrated on the first day and seventh day after seeding by staining with rhodamine-conjugated phalloidin reagent. Cells were fixed in 1 w/v % glutalaldehyde solution on ice for 45 min and permeabilized by treatment with 0.1 w/v % Triton X-100 in PBS (-) for 10 min, followed by incubation with 900 ng/mL of rhodamine-conjugated phalloidin solution. Cells were washed sufficiently with PBS (-), and fluorescence of cells was documented using an Olympus OMmicroscope under a xenon lamp. Measurements. Gel-permeation chromatography was carried out using a TOSOH SG-820 liquid chromatograph equipped with a PEO-calibrated TSK gel column (G30,000H HR or G4,000H HR) and an internal RI detector (RI-8022). 1H NMR was measured at 80 °C in a solvent of DMSO-d 6 on a JEOL JNM-EX270. The surface of the hydrogels was characterized by measuring the water contact angle using a contact angle meter CA-A (Kyowa Scientific co. Tokyo, Japan) after swelling in distilled water. Surface analysis of the films as cast and dehydration after swelling with distilled water were carried out by X-ray photoelectron spectroscopy (XPS, Shimadzu ESCA 3300). Results and Discussion Synthesis and Characterization of PBLA Homopolymer and ABA Copolymers. To obtain homopolymer of β-benzyl-L-aspartate (PBLA), we performed the ring-opening polymerization of β-benzyl L-aspartate N-carboxyanhydride (BLA-NCA) initiated with methoxyethylamine. The synthesized polymers gave a broad peak with one top in the GPC curve (Mw/Mn )1.17). To prepare the ABA triblock copolymers with varying degrees of polymerization of the BLA-NCA, we also performed the ring-opening polymerization of BLA-NCA initiating from amine terminal group of PEO with Mn of 20 000 and a monodisperse molecular weight distribution (Mw/Mn < 1.03). The synthesized copolymers gave a sharp peak with one top in the GPC curve (data not shown), indicating no contamination of PBLA and AB diblock copolymers. FT-IR and 1H NMR data indicated that polymers obtained contained two PBLA blocks attached at both PEO terminals. Swelling of ABA Triblock Copolymer Films. The ABAtriblock copolymers comprised of PBLA, polymerization

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Figure 1. Swelling behavior of P(BLA-block-EO-block-BLA) films. (A) Time course of the swelling ratio. (B) The equilibrated swelling ratio plotted against PBLA content.

degree ) 18, 25, 32, and 55 as A segment and poly(ethylene oxide) (PEO, molecular weight ) 20 000, polymerization degree ) 454) as B segment, abbreviated as 20K18, 20K25, 20K32, and 20K55, respectively, were synthesized as previously reported. The polydispersity of the molecular weight ranged from 1.04 to 1.37. Casting of methylenechloride solution copolymers successfully yielded translucent films, which were then immersed in distilled water resulting in the formation of hydrogels. The process of hydrogel formation was investigated by measuring the time course of the change in swelling ratio (Figure 1A). The swelling was equilibrated within 10 min in every film and the swelling speed was 2048 wt % per minute. Swelling decreased with an increase in the degree of polymerization of PBLA. The swelling was rapid relative to conventional hydrogels presumably because of the thinness of the study hydrogels. The swelling ratio in the equilibrated state ranged from 240 to 570 wt % and showed a negative linear correlation with the degree of polymerization of PBLA, which ranged from 18 to 55, and corresponded to the PBLA unit molar composition, which ranged from 7.3 to 19.5 mol %. (Figure 1B, R2 ) 0.9944). As shown in the previous paper, the PEO segment was hydrated while hydrophobic PBLA segments showed an interchain self-organization forming a physical cross-linking junction (26). An increase in the PEO composition reduced the apparent cross-linking density to enhance the swelling speed and the equilibrated swelling degree. Mechanical Properties of ABA Copolymer Hydrogels. Tensile testing of ABA copolymer hydrogels with a width of 2 mm and a length of 20 mm was performed in the equilibrated water-swollen state. As shown in Figure 2, the hydrogels of 20K25 and 20K32 showed a stress-strain curve near elastic deformation and broke at a low strain of 35% and 55%, respectively, while 20K55 hydrogel showed slight yielding at a strain around 30% and was not broken at a strain of as much as 500%, which is the upper limit of the apparatus. Young’s moduli, E, of the hydrogels ranged from

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Figure 2. Stress-strain curve of the hydrogels prepared by the immersion of P(BLA-block-EO-block-BLA) films in water until an equilibrated swollen state was reached.

Figure 3. Contact angles of the hydrogels prepared by immersion of P(BLA-block-EO-block-BLA) films in water until an equilibrated swollen state was reached.

Table 1. Synthetic Results of Polymers abb. 20K18 20K25 20K32 20K55 PBLA

BLA/PEO BLA units Mw Mn in feed in copolymera ∼10-4 ∼10-4 Mw/Mn 39 53 67 119 -

36 (7.3) 50 (9.9) 64 (12.4) 110 (19.5) - (100)

2.9 2.8 3.8 5.2 0.7

2.3 2.7 3.6 3.8 0.6

1.04 1.04 1.07 1.36 1.17

Eb (MPa) wet dry ND 0.8 1.4 4.8 ND

25 27 22 17 ND

a Determined by 1H NMR spectroscopy. Unit molar compositions (mol %) of BLA to EO are shown in parentheses. b Young’s modulus determined from the slope of stress-strain curve recorded by tensile test. ND means that E values were not determined since the samples were too weak.

0.8 to 4.8 MPa, increasing with an increase in the degree of polymerization of the PBLA segments (Table 1). This range is lower than those values found in the dry state but is higher than those of conventional physical hydrogels. As described in the preceding paper, the hydrogel robustness may result from the hydrophobic interaction between PBLA segments in water.26 20K55 hydrogel showed extremely high E values presumably because of the combined effects of the low water content and the enhanced hydrophobic aggregation of the long PBLA segments, leading to efficient physical crosslinking junctions. Specific maximal strength of the hydrogel films was estimated by dividing the maximum strength recorded from the stress-strain curves by the copolymer weight in the hydrogel samples which were 1.0, 2.3, and >5.5 MPa/g for 20K25, 20K32, and 20K55, respectively. These values were plotted against PBLA composition as shown in Figure 2. The specific maximum strength increased with an increase in PBLA composition, indicating that the intrinsic strength of the copolymer chains was enhanced by the PBLA segment hydrophobicity organizing in the presence of water. Although 20K18 is the same molecular structure except for polymerization degree of PBLA and makes a selfsustainable hydrogel, it was too fragile to obtain exact mechanical data. Characterization of the Polymer Surface. The water poorly spread on the surface of the hydrogels with a PBLA content over 9.9 mol % at 20K25 although they contained a large amount of water. The 20K55 hydrogel shed a large amount of water. These findings are quite unique since it is contrary to the usual observation that water dropped on conventional hydrogels easily spreads and unifies within the hydrogel. In the analogous ABA-triblock copolymer of polylactide-PEO-polylactide, such a phenomenon has not been reported.28 To quantify these phenomena, we measured

Figure 4. Elemental analyses of the surface of the dried hydrogel for P(BLA-block-EO-block-BLA) films. (A) Spectra of carbon binding energy recorded by X-ray photoelectron spectroscopy (XPS). (B) Nitrogen atomic composition of as-cast (closed marks) and the dried hydrogel films (open marks), calculated using XPS spectra.

the water contact angle on the surface of these hydrogels, which increased drastically from 16 ( 3° at 20K18, 7.3 mol %, to 55 ( 1° at 20K25, 9.9 mol % . Copolymer 20K55, 19.5 mol %, showed a value of 76 ( 7°, which is comparable with that of PBLA homopolymer (80 ( 2°) as shown in Figure 3. Contact angles of 20K25 and 20K32 were 55 ( 1° and 60 ( 8°, respectively, which were almost the same as for a glass cover slip or a plastic plate for cell culture.29-31 These phenomena may be associated with the vertical microphase separation of the hydrophobic PBLA segment and the PEO segment from the observation that microphase separation of poly(dimethylsiloxane)-polyamide multiblock copolymer has been directly detected by TEM and SIMS examination.32 To investigate this hypothesis, we carried out elemental analysis on the surface of the dried hydrogel (1 GPa) contained both spherical and spread with polar indicating mobility (Figure 9I-L). Figure 10 represents morphology and staining characteristics after 7 days of continuous incubation. The cells on the surface of 20K18 drastically spread and focal contacts developed at marginal cell sites, indicating relatively strong adherence with the substratum (Figure 10B). The cells on 20K25 remained globular and no subcellular localization was observed, indicating poor interaction between cells and materials (Figure 10C, D). In 20K32 and 20K55, the cells were flat and bipolar or tripolar in morphology and formed lamellipodia, suggesting a relatively strong adhesion ability and high mobility (Figure 10E-H). On the surface of homo PBLA, two types of cells were observed: subround cells that were 50 µm or more in diameter and fibroblast-like bipolar cells with pseudopodia. This suggested the existence of two populations of cells with different mobilities (Figure 10I, J). Accumulation of F-actin

Figure 9. Morphological (left) and cytoskeletal (right) changes to murine peritoneal cells cultured on the hydrogels for 1 day. A and B, 20K18; C and D, 20K25; E and F, 20K32; G and H, 20K55; I and J, PBLA homopolymer; K and J, coverglass; bar ) 50 µm.

filaments in the raffle was observed in the peripheral region of these subround cells. Cells were elongated 100 µm in length on the surface of the glass cover slip, some exhibiting stress fiber formation. This spread of cells was not observed for cells grown on the polymer surface. In some cases, round cells 10 µm in diameter were found on the apical surface (Figure 10K, L), similar to SEM findings. Flat and subround cells grew to 70 µm in diameter and did not show extensive

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Conclusions ABA-triblock copolymers comprising PEO as a hydrophilic block segment and PBLA as a hydrophobic block segment were synthesized by a ring-opening reaction. The films cast from methylenechloride solution successfully swelled in water yielding physical hydrogels. Saturation of swelling was rapid (within 10 min), with water content correlating to the magnitude of the PBLA component. Young’s modulus, maximal strength, and elongation at fractures were within the range of 0.8-4.8 MPa, 1-5 MPa/ g, and 3.5-500%, respectively, depending on the hydrophobic domain. Contact angles changed drastically (depending on PBLA content) from 16 ( 3 for 20K18 to 76 ( 7° for 20K55. After incubation with murine peritoneal macrophages on 20K32 (12 mol % PBLA) hydrogels, adherence of cells was noted without spreading. Twenty mol % PBLA (20K55) allowed the cells to spread, but the cells did not show stress fiber formation. The cells extruded pseudopodia and filopodia with adhesion plaques in cases where the PBLA content was 20 mol % or more, suggesting dynamic mobility. PBLA content of less than 10% (20K25) suppressed cell adhesion drastically. Interestingly, adhesion and spreading of cells recovered after prolonged incubation on the surface of 7 mol % PBLA (20K18) in contrast with 10 mol % (20K25). It is thought that sequential adaptation of proteins and chemical changes to the hydrogel may occur during incubation. Acknowledgment. This study was supported financially in part by NOF Corporation, Japan. References and Notes

Figure 10. Morphological (left) and cytoskeletal (right) changes to murine peritoneal cells cultured on the hydrogels for 7 days. A and B, 20K18; C and D, 20K25; E and F, 20K32; G and H, 20K55; I and J, PBLA homopolymer; K and J, coverslip; bar ) 50 µm.

raffle on the marginal region. These data may suggest that these cells lost their motility during the long period of cultivation on glass. The reason that cells cultured on hydrophilic 20K18 displayed enhanced cellular adhesion when compared to those cultured on 20K25 is under investigation. We can theorize that the roughness of the surface of the hydrogel, protein adsorption to it, or chemical modification of the gel composition may be involved.

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