Cell Adhesion and Morphology in Porous Scaffold Based on

Biomacromolecules 2002, 3, 1375-1383

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Cell Adhesion and Morphology in Porous Scaffold Based on Enantiomeric Poly(lactic acid) Graft-type Phospholipid Polymers Junji Watanabe, Takahisa Eriguchi, and Kazuhiko Ishihara* Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received August 28, 2002; Revised Manuscript Received September 18, 2002

Poly(D-lactic acid) (PDLA) and poly(L-lactic acid) (PLLA) macromonomers were synthesized for preparation of a novel cytocompatible polymer. The cytocompatible polymer was composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), and the enantiomeric PLLA (or PDLA) macromonomer. The degree of polymerization of the lactic acid in the PLLA and PDLA segments was designed to be ca. 20. The copolymer-coated surface was analyzed with static contact angle by water. From the result, the PLLA (or PDLA) segment and MPC unit were located on the coated surface, and the monomer unit in the copolymer was reconstructed by contacting water. Fibroblast cell culture was performed to evaluate cell adhesion on the coated surface, and the cell morphology was observed. The number of cell adhesion is correlated with the PL(D)LA content, and the cell morphology is correlated with the MPC unit content. The porous scaffold was prepared by the formation of a stereocomplex between the PLLA and PDLA, and the cell adhesion and following cell intrusion was then evaluated. The fibroblast cells adhered on the surface and intruded into the scaffold through the connecting pores after 24 h. The cell morphology became round shape from spreading with the decreasing PLLA (or PDLA) content in the copolymer. It is considered that the change in the cell morphology would be induced by the MPC unit as cytocompatible unit. These findings suggest that the porous scaffold makes it possible to have cytocompatibility and to produce three-dimensional tissue regeneration. Introduction Tissue engineering is a multidisciplinary science that utilizes basic principles of engineering and life sciences to create new tissues from their cellular components. Langer et al. defined the strategy of the tissue engineering as the creation of new tissue from isolated cells, bioactive molecules, and polymer scaffold.1 Furthermore, Langer et al. showed the methodology and concrete examples concerning three-dimensional tissue regeneration using biodegradable polymeric scaffolds.1-4 Biodegradable polymers such as poly(lactic acid) (PLA) are the most attractive candidates because of their well-known physical properties.5-6 Furthermore, the degradation products are natural metabolites and are easily removed from the body. The polymer provides a three-dimensional porous scaffold to transplant the cells and to organize the tissues. Several approaches have been examined to fabricate the porous scaffold, including a woven mesh,7 fiber bonding,8 phase inversion,9 gas foaming,10 and salt leaching.11 These porous scaffolds provide suitable properties for the exchange of gas and metabolites from the tissue. Required properties for porous scaffold may involve (i) adequate cell adhesion and cell proliferation, (ii) cell intrusion into the scaffold, and (iii) cytocompatibility. Many researchers have done investigations in terms of the cell * To whom correspondence should be addressed. Tel: +81-3-58417124. Fax: +81-3-5841-8647. E-mail: [email protected].

adhesion and cell intrusion.12-13 Recently, cytocompatible materials have focused on the field of cellular response.14 An inflammatory reaction was observed on conventional polymer materials such as polyurethane and poly(ethylene terephthalate) by evaluation of the mRNA expression of inflammatory cytokines.15 Based on this report, one of the promising approaches to overcome these requirements is incorporation into the materials following dual function, cytocompatibility, and cell adhesiveness. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was designed because of its resemblance to the chemical structure of phospholipids in biomembranes.16 The MPC could polymerize with alkyl methacrylate such as n-butyl methacrylate (BMA).17 The MPC copolymers have been widely investigated aiming at biomedical applications.18-20 The MPC copolymer effectively suppressed the secretion of inflammatory cytokine in comparison with conventional polymers.15 Our next concern of the MPC copolymer is the target on the cytocompatible scaffold, and a preliminary study has already been issued.21-22 Enantiomeric macromonomers, which have a poly(L-lactic acid) (PLLA) segment and poly(D-lactic acid) (PDLA) segment, were incorporated into the MPC copolymer for enhancement of cell adhesion because the number of cell adhesion decreased with increasing MPC unit content in the copolymer.23 Furthermore, the enantiomeric polymer segments, PLLA and PDLA, would be utilized for preparation of the scaffold by formation of a

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Figure 1. Chemical structure of enantiomeric macromonomers.

stereocomplex. PLLA and PDLA, which are forming stereocomplex, are enantiomers. A driving force of the stereocomplexation is van der Waals force; especially, complementary fitting of the polymer segments is the most important factor to form the stereocomplex. For example, poly(γ-benzyl L-(D-)glutamate), such as enantiomeric poly(amino acid), were also forming stereocomplex. The formation of the stereocomplex has been widely investigated as a biomedical matrix.24-25 The advantage of the enantiomeric PL(D)LA graft-type phospholipid copolymer would be that it is expected to have the following characteristics: (i) adequate cell adhesion by the PL(D)LA segment on the coated surface, (ii) easy preparation of porous scaffold by stereocomplexation, and (iii) suppression of inflammatory reaction by the MPC unit. In this study, cell adhesion on the PMBL(D)LA and cell intrusion into the stereocomplex were examined, and adherent cell morphology was discussed. 2. Experimental Section 2.1. Materials. L-Lactide and D-lactide were kindly supplied by Dainippon Ink and Chemicals, Inc. (Tokyo, Japan). 2-Isocyanate ethyl methacrylate (IEMA, Showa Denko Co., Tokyo, Japan) was distilled at reduced pressure (60 °C, 2.5 mmHg). n-Butyl methacrylate (BMA, Wako Pure Chemical Co., Ltd., Osaka, Japan) was distilled under reduced pressure (50 °C, 20 mmHg). 2-Methacryloyloxyethyl phosphorylcholine (MPC) was synthesized and purified using a method from a previous report.16 Sodium chloride (+80 mesh) was purchased from the Aldrich Chemical Co., WI. All other reagents were commercially available and used as received. 2.2. Synthesis of Enantiomeric Poly(lactic acid) Grafttype Copolymer. The poly(L-lactic acid) (PLLA) and poly(D-lactic acid) (PDLA) macromonomers (Figure 1, MaPL(D)LA) were prepared according to a previously reported method.22 The preparative method was briefly described as follows: (i) ring-opening polymerization of L-lactide or D-lactide using n-dodecanol as an initiator and (ii) methacrylation of the terminal hydroxyl group in PLLA or PDLA

Figure 2. Chemical structure of random copolymers.

Watanabe et al.

with IEMA. The chemical structure of the obtained MaPL(D)LA was confirmed by FT-IR (VALOR-III, Jasco, Tokyo, Japan) and 1H NMR (JEOL R-500, Tokyo, Japan). Yield: 90%. FT-IR (KBr, cm-1): 1758 (ester bond), 1638 (double bond), 1560 (urethane bond). 1H NMR (CDCl3, ppm): δ 0.85 (t, 3H, CH3 for n-dodecanol), 1.23 (m, 2H × 10, CH2 for n-dodecanol), 1.52-1.55 (m, 3H × 23, CH3 for PLLA), 1.92 (s, 3H, CH3 for methacryl group), 3.48-4.20 (t, 2H × 2, CH2CH2 for IEMA), 4.03-4.07 (m, 2H, CH2 in terminal of n-dodecanol), 4.72 (s, 1H, urethane), 5.11-5.20 (m, 1H × 23, CH for PLLA), 5.50 (s, 1H, CH2d for locating opposite side with COO), 6.12 (s, 1H, CH2d for locating same side with COO). Gel permeation chromatography (GPC) was performed using a column of KF-803 (Shodex, Tokyo, Japan). A GPC system (Jasco, Tokyo, Japan) was used at a flow rate of 1.0 mL/min. Tetrahydrofuran (THF) was used as the eluent, and the column was calibrated with poly(styrene) standard samples (Tosoh, Tokyo, Japan) using an RI detector. The enantiomeric PLLA (or PDLA) graft-type phospholipid copolymer (Figure 2) was synthesized by conventional radical polymerization. A typical procedure is briefly described. The desired amounts of MPC, BMA, the MaPL(D)LA, and 2,2′-azobisisobutyronitrile (AIBN) were placed in a two-necked round-bottom flask, and the mixture was then dissolved with ethanol-THF (1/1 by volume). The final concentration of the monomers and the AIBN was 0.5 mol/L and 2 mmol/L, respectively. The flask was kept at 60 °C for 24 h with N2 bubbling. After the polymerization, the product was poured into an excess amount of diethyl ether-hexaneethanol (4/4/1 by volume) mixture to obtain the copolymer. The precipitate was filtered and dried in vacuo. PMBLLA and PMBDLA were obtained. Here, the copolymers are designated by the following code, for example, PMBLLA10 where M, B, and LLA refer to the MPC unit, BMA unit, and MaPL(D)LA unit, respectively. The chemical structure of the copolymers was confirmed by FT-IR and 1H NMR. Yield: 69%. FT-IR (KBr, cm-1): 1760 (ester for PLLA), 1722 (ester for BMA), 1543 (urethane), 1088 (phosphate), 970 (choline). 1H NMR (CDCl3, ppm): δ 0.85 (t, 3H, CH3 for n-dodecanol), 0.96 (s, 3H, R-methyl), 1.21 (m, 2H × 10, CH2 for n-dodecanol), 1.50-1.54 (m, 3H × 23, CH3 for PLLA), 1.88 (br, 2H, CH2 for polymer backbone), 3.323.40 (m, 3H × 3, choline), 3.88-4.13 (m, 2H × 2, methylene for side chain), 4.03-4.06 (m, 2H, CH2 in terminal of n-dodecanol), 5.05-5.18 (m, 1H × 23, CH for PLLA).

Cell Adhesion and Morphology in Porous Scaffold

2.3. Preparation of Enantiomeric Poly(lactic acid) Graft-type Copolymer-Coated Surface. A series of enantiomeric copolymers, PMBLLA (or PMBDLA), were coated on cell culture poly(ethylene terephthalate) (PET) films (diameter 14 mm, Wako Pure Chemical Co. Ltd., Osaka, Japan). The PET films were first washed with chloroform and then immersed in a chloroform solution containing 1 w/v % of the copolymer. The solvent was slowly evaporated under a chloroform atmosphere at room temperature, and the films were dried in vacuo. The coated surface was analyzed by static contact angle by water using an automatic contact angle meter apparatus (CA-W, Kyowa Interface Science Co., Ltd., Saitama, Japan) at 25 °C. Direct methods of measuring contact angles involve measurement on a bubble resting on a solid surface. This is referred to as the sessile drop method. The drop (10 µL) of pure water was introduced on the surface by using a microsyringe. As a reference sample for characterization of the polymer, poly(MPC-co-BMA) (MPC unit composition in copolymer 5 mol %, PMB5) was synthesized and coated on the PET films using ethanol solution (0.5 w/v %). Poly(D,L-lactic acidco-glycolic acid) (PLGA) was purchased from Aldrich Chemical Co., WI, and was coated on the PET films using chloroform (1 w/v %) as a commercially available reference. 2.4. Cell Culture and Fibronectin Adsorption on Copolymer-Coated Films. L-929 mouse fibroblasts were routinely cultured in Eagle’s Minimum Essential Medium (E-MEM, Nissui Co., Tokyo Japan) supplemented with 10% fetal bovine serum (FBS, Gibco, U.S.A.) at 37 °C in a 5% CO2 incubator. After treatment with 0.25% trypsin (Gibco, U.S.A.), the cell density was adjusted to 3 × 104 cells/mL. The copolymer-coated films were placed in a 24-well cell culture plate (Falcon, NJ) using a silicone ring; the wells were then filled with sterilized Dulbecco’s phosphate buffered saline (D-PBS, Gibco, U.S.A.), and the 24-well plate was sterilized with UV. After the sterilization, 1 mL of the fibroblast suspension was seeded on each well (cell density on the copolymer-coated surface: 3 × 104 cells/cm2). After 24 and 48 h, the cell culture medium was removed by an aspirator, and the copolymer-coated film was removed from the 24-well plate. The films were washed three times with D-PBS, and 0.5 mL of Triton X-100 (0.05 wt %) was added to the films to determine the adhered fibroblasts using the lactate dehydrogenase (LDH) cytotoxic test kit (Wako Pure Chemical Co., Ltd., Osaka, Japan). The cell morphology was evaluated by using a phase-contrast microscope (BX60, Olympus, Tokyo, Japan). The amount of fibronectin adsorbed on the copolymercoated surfaces from E-MEM (including 10% FBS) was determined by the antigen-antibody reaction using the enzyme-immunoglobulin conjugate.23 The copolymercoated films were placed in a 24-well plate and then equilibrated with D-PBS. After equilibration, the films were immersed in E-MEM containing 10% FBS for 3 h at 37 °C. The copolymer-coated films were also immersed into EMEM (without FBS) as the negative control. The medium was removed, and antibovine fibronectin rabbit polyclonal antiserum (YU-B0004, Yagai, Yamagata, Japan) was added as the primary antibody. After treatment with the blocking

Biomacromolecules, Vol. 3, No. 6, 2002 1377

reagent (1 wt % of ovalbumin D-PBS solution), horseradish peroxidase (HRP)-conjugated immunoglobulins ((antirabbit IgG) goat IgG) (A-6154, Sigma, MI) were added to the films as a secondary antibody. After rinsing, o-phenylenediamine hydrochloride (ML-1120T, Sumitomo Bakelite Co., Ltd., Tokyo, Japan) was added as a substrate for HRP, and then the absorbance of the solution was measured at 450 nm. The relative value of the absorbance at 450 nm was evaluated for the fibronectin adsorption. 2.5. Preparation of Porous Scaffold by Formation of Stereocomplex. A porous scaffold composed of PMBLLA and PMBDLA was prepared according to a previously reported method.22 A typical example is briefly described as follows: 0.125 g of each copolymer, PMBLLA and PMBDLA, was dissolved in 0.6 mL of mixed solvent (methanol-methylene chloride, 2:1 by volume). The solution was poured into a Teflon spacer (diameter ) 14 mm, depth ) 5 mm), which was filled with 1.0 g of sodium chloride, and the solution was stirred using a polyethylene stick at room temperature. After the formation of the stereocomplex, the scaffold in the spacer was immersed into a large amount of the mixed solvent to remove the free PMBLLA and PMBDLA copolymers; then the solvent was changed from the mixed solvent to methanol. To remove the trapped sodium chloride, the scaffold was immersed in distilled water for one night, and the water was then substituted for the methanol. After the substitution, the scaffold was lyophilized overnight. The porous structure of the scaffold was golddeposited for observation using a scanning electron microscope (SEM, JSM-5400, JEOL, Tokyo, Japan). 2.6. Cell Culture on Porous Scaffold. The porous scaffold was sterilized overnight by 99.5% ethanol, and the scaffold was then washed five times in sterilized water. After the sterilization, the scaffold was placed in a 24-well plate with silicone ring and was equilibrated by D-PBS. L-929 mouse fibroblasts were routinely cultured in E-MEM supplemented with 10% FBS at 37 °C in a 5% CO2 incubator. After treatment with 0.25% trypsin, the cell density was adjusted to 3 × 105 cells/mL because of the large surface area of the porous scaffold. The fibroblast suspension (1 mL) was seeded in each well. After 24 h, the cell culture medium was removed by an aspirator, and the scaffold was washed three times with D-PBS. The attached cells were fixed with 2.5% glutaraldehyde-PBS solution (Wako Pure Chemical Co., Ltd., Osaka, Japan) for 24 h. After the fixation, the scaffold was washed with distilled water, and staining of the scaffold with osmium tetraoxide (OsO4) was carried out for 60 min. The stained scaffold was dehydrated with a graded ethanol series (30%, 50%, 70%, 90%, 99.5%) for 15 min, and then lyophilization was carried out. The cell adhesion was observed by SEM after gold deposition. 3. Results and Discussion 3.1. Synthesis of Enantiomeric Copolymers (PMBLLA and PMBDLA). The enantiomeric PLLA (or PDLA) macromonomer (Figure 1) was designed not only as hydrophobic domains but also as physical cross linker by stereocomplexation. The ring-opening polymerization of L-lactide (or

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Table 1. Preparative Conditions of Enantiomeric Copolymers PMBLLA and PMBDLA in feed (mol %)

in copolymer (mol %)

code

MPC

BMA

macromonomer

MPC

BMA

macromonomer

DPa

yield (%)

PMBLLA10 PMBLLA20 PMBDLA10 PMBDLA20

5 5 5 5

85 75 85 75

10 20 10 20

16 19 16 23

72 53 68 51

12 28 16 26

23 23 27 27

69 60 55 64

a

Degree of polymerization in poly(L-lactic acid) (or poly(D-lactic acid)) segment determined by 1H-NMR.

D-lactide)

was initiated from the hydroxyl group of ndodecanol. After the polymerization, the terminal hydroxyl group was reacted with IEMA. The addition reaction quantitatively proceeded using a catalyst, dibutyltin dilaurate, at 60 °C. The MaPL(D)LA was obtained in high yield (over 80%). The chemical structure of the MaPL(D)LA was confirmed by FT-IR and 1H NMR. In the case of FT-IR, CH2 of the methacryl group at 1638 cm-1 and the urethane bond at 1560 cm-1 were observed. The degree of polymerization (DP) of the lactic acid in the PLLA and PDLA segment was determined to be ca. 23 and 27, respectively, from the ratio of the integration of the signals using 1H NMR. The enantiomeric poly(lactic acid) graft-type copolymer was designed and synthesized for the purpose of a cytocompatible scaffold. A series of copolymers are summarized in Table 1. Here, the last number in PMBLLA10 denotes the composition of the MaPLLA concentration (10 mol %) in the feed. As shown in Table 1, the copolymer composition was almost dependent on the monomer content. This result suggests that the monomer content in the PMBLLA and PMBDLA copolymers can be controlled by the feeding ratio of the monomers. The chemical structure of the PMBLLA and PMBDLA copolymers was also confirmed by FT-IR and 1 H NMR. From the FT-IR spectra of the copolymers, the IR absorptions of two ester groups attributed to PLLA (or PDLA) and the methacryloyl group were observed. Furthermore, the IR absorptions of the urethane bond and choline group were also observed. The DP of the PLLA (or PDLA) segment was also determined using 1H NMR, and a significant difference was not observed in comparison with the MaPL(D)LA. These results suggest that the monomer content and DP were tailored by the preparative conditions. 3.2. Surface Characterization of Copolymer-Coated Films. For the polymer coating, 1 w/v % of the copolymerchloroform solution (PMBLLA and PMBDLA) was prepared, and the PET films were then immersed into the polymer solution. The coated surface was transparent, and an interference fringe was observed. To estimate the swelling surface, the films were first immersed overnight in distilled water at 37 °C and the swelled films were then lyophilized. From the X-ray photoelectron spectroscopy, the nitrogen peaks attributed to the urethane bond in the macromonomer and choline group in the MPC were observed after (or before) contact with water (data not shown). Furthermore, phosphorus peak attributed to the phosphate in the MPC was also observed. From this, the PET films as a substrate were definitely covered with a series of copolymers. The mobility of the segment in the copolymer was estimated with static contact angle measurement as shown in Table 2. The contact angle of the polymer-coated surface before contact with water

Table 2. Results of Static Contact Angle Measurement on Polymer-Coated Surfacea code

before contact with water

after contact with water

PMBLLA10 PMBLLA20 PMBDLA10 PMBDLA20 PMB5 PLGA PET

77.6 ( 0.8 79.4 ( 1.0 78.4 ( 1.4 77.6 ( 1.0 77.8 ( 1.2 97.8 ( 2.0 82.4 ( 1.8

70.0 ( 2.3b 68.3 ( 3.0b 68.1 ( 1.3b 63.9 ( 2.1c 60.9 ( 4.5 101.4 ( 2.2 76.4 ( 1.4

a n ) 10, mean ( SD. b Significantly different from PMB5 at P < 0.05 calculated using the t-test. c Significantly different from PMB5 at P > 0.05 calculated using the t-test.

was found to be ca. 78° (PMBL(D)LA and PMB5). After the contact with water, their contact angle decreased with a range of 7°-17°. This result indicated that spontaneous reorganization of the hydrophilic-hydrophobic component occurred to reduce surface free energy. In our previous report, the contact angle decreased with increasing the MPC unit in the copolymer because of strong hydrophilicity by phosphorylcholine group.26 The contact angle of the PMBL(D)LA was larger than that of PMB5, although that MPC unit was found to be 16°-23°. This result suggested that PL(D)LA segment was located on the surface with forming domain structure. In the case of PLGA, the contact angle was not changed by contacting water. The contact angle was high in comparison with PMBL(D)LA and PMB5. This result indicated that the mobility of the PLGA segment was lower than that of PMBL(D)LA and PMB5. Furthermore, the stability of the PLLA (or PDLA) segment was estimated by a hydrolysis experiment. PMBLLA (or PMBDLA) powder (10 mg) was incubated in 10 mL of PBS (pH 7.4) at 37 °C. After 48 h, the PBS solution was evaporated, and the residue was dissolved in THF (2 mL). The THF solution was characterized by GPC (eluent THF), and no significant molecular weight loss was observed. It is suggested that the PLLA (or PDLA) segment was stable during hydrolysis for at least 48 h. These results indicate that the monomer content of the surfaces correspond with the copolymer composition. It was suggested that the dual functions of cytocompatibility and cell adhesiveness were expected for the cultured cells. 3.3. Biological Characterizations on Copolymer-Coated Films. L-929 fibroblast cells were used for the characterization of the preliminary cell adhesion. The number of fibroblast cells after 24 and 48 h on the films is shown in Figure 3. After 24 h, the number of fibroblast cells on both PMBLLA10 and PMBLLA20 was found to be ca. 5.0 × 103 cells/cm2. On PMBDLA10 and PMBDLA20, a fibroblast cell adhesion of 5.0 × 103 cells/cm2 was also observed. There is no significant difference among the four copolymers. In the case of PMB5, the cell number was significantly lower


Figure 3. Fibroblast cell adhension on copolymer-coated films after 24 h (open bar) and 48 h (filled bar). Mean values of three measurements and standard deviation are indicated. Entries marked with / and // were significantly different from PMB5 (24 h) at P < 0.01 and PMB5 (48 h) at P < 0.01, respectively, calculated using the t-test.

than the others. This result indicates that the PLLA (or PDLA) segment effectively enhanced the cell adhesion, and the number of cell adhesion was similar to the PLGA, which was a commercially available sample. This phenomenon is quite important for material design. Ishihara et al. reported a relationship between the MPC unit content in the copolymer and the number of cell adhesions (or amount of fibronectin adsorption).23 On the basis of this report, the cell adhesion is found to be suppressed by the incorporation of MPC units. The MPC unit in a series of enantiomeric PMBLLA and PMBDLA copolymers is 16-23 mol %. Because of this high MPC content, the fibroblast cells could not attach to the copolymer-coated films without the PLLA (or PDLA) segment. After 48 h, the number of cell adhesions increased to be ca. 1.3 × 104 cells/cm2 on a series of enantiomeric PMBLLA and PMBDLA copolymers, for which the cell number had doubled after 24 h. On the other hand, the cell number on PMB5 was only 6.0 × 103 cells/ cm2 after 48 h, though the cell number had almost doubled.


These results suggest that the number of cell adhesions was regulated by the content of PLLA (or PDLA) segment in the copolymer. The cell morphology was studied using a phase contrast microscope after 48 h (Figure 4). In the case of the copolymers with MPC units (PMBLLA, PMBDLA, and PMB5), some of the cells were observed as round shape and the others were a little bit spread. Though the photographs in Figure 4a-e were not very clear, round shape was the relatively predominant morphology. On the other hand, spread cells are predominantly observed on PLGA and PET (data not shown) surfaces. From the viewpoint of cell morphology, it is suggested that the interaction between the cells and the copolymer-coated surface is lower than that of the PLGA and PET. Therefore, the morphology of the cells would be round shape. Ishihara et al. reported the fibroblast cell morphology would change to round shape by increasing the MPC content in the copolymer.23 It is considered that the change in the cell morphology would be induced by the MPC unit. In the case of PMB5, complete round shape, which was regulated by MPC unit, was observed (Figure 4e). On the other hand, spread shape was observed in addition to the round shape on PMBLLA and PMBDLA because PL(D)LA segment was incorporated into the copolymer as a hydrophobic domain. Taking these cell adhesion and cell morphology into account, the number of cell adhesion is correlated with the PL(D)LA content and the cell morphology is correlated with the MPC unit content. The cells that show round shape were still alive after 48 h, if the cells were transfered to another cell culture dish. The materials that show lower interaction between cells and surface were considered to be cytocompatible materials. These observational results were appropriate phenomena as we designed the copolymer. Generally, protein adsorption such as absorption of fibronectin is a key role of the cell adhesion. The results of the fibronectin adsorption from the cell culture medium are shown in Figure 5. The amount of fibronectin adsorption was compared with the negative control by the relative intensities of the absorbance at 450 nm. A relatively high

Figure 4. Phase-contrast microscopic pictures of fibroblast cell adhesion on copolymer-coated films after 48 h incubation.

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Watanabe et al. Table 3. Copolymer and NaCl Combination for Preparation of Stereocomplex code SC10 SC10/P SC20 SC20/P

PMBLLA10a PMBLLA20a PMBDLA10a PMBDLA20a NaClb + +

+ + + +

+ + +

+

20 w/v % of mixed solvent (methanol-methylene chloride ) 2:1 by volume). b 1.0 g/well (diameter ) 14 mm, depth ) 5 mm). a

Figure 5. Amount of relative fibronectin adsorption on copolymercoated films from cell culture medium. Mean values of three measurements and standard deviation are indicated. Entries marked with / and // were significantly different from PMBDLA10 at P < 0.01 and PMBDLA20 at P < 0.01, respectively, calculated using the t-test.

fibronectin adsorption was observed on PMBLLA10, PMBLLA20, PMB5, and PLGA. On the other hand, low fibronectin adsorption was observed on PMBDLA10, PMBDLA20, and PET. A significant difference between PMBDLA10 and PET was not observed in terms of fibronectin adsorption. Considering the number of cell adhesion, cell number on PMBLLA, PLGA, and PET was well-correlated with amount of fibronectin adsorption. In the case of PMBDLA and PMB5, anomalous correlation was observed between the cell number and the fibronectin adsorption. Fibronectin adsorption on the PMBDLA should increase because a large number of cell adhesion was observed. However, the amount of fibronectin adsorption was almost the same level in comparison with PET surface as a cell culture film. This result suggested that a minimum requirement of fibronectin adsorption was considered over 0.2 of absorbance at 450 nm. Furthermore, the difference of fibronectin adsorption on PMBLLA and PMBDLA might be complexation between PMBDLA and fibronectin. Domb et al. reported that interesting phenomenon regarding stereocomplexation.27 PDLA and insulin, which is made of L-amino acid formed a stereocomplex in the solution. In this case, insulin behaved like a PLLA. Taking this report into account, fibronectin would form stereocomplex on the PMBDLA-coated surface. The three-dimensional structure of fibronectin changed to fit the PDLA segment in the PMBDLA, and then the binding constant to antibovine fibronectin polyclonal antibody would decrease because of the conformational change of the fibronectin. As a result, the amount of fibronectin adsorption was under-estimated. In the case of PMB5, the fibronectin adsorption was quite high despite the lower cell adhesion. In this case, a large number of the fibronectin adsorption would occur. However, the number of cell adhesion was found to be low because of the weak interaction between cells and surface. Its weak interaction was regulated by MPC unit content in the copolymer. When we evaluate the fibronectin adsorption and the cell adhesion, the specimen was washed by PBS or cell culture medium, respectively. In this procedure, the remov-

Figure 6. Scanning electron microscope pictures of SC10/P using 1.0 g of NaCl.

able cells would be higher than that of the fibronectin. From this, fibronectin adsorption on PMB5 was so high although the number of cell adhesion was the lowest of all of the coated samples. 3.4. Preparation of Porous Scaffold and Characterization of Cell Intrusion. In this study, a stereocomplex formation was observed between the enantiomers of PLLA and PDLA, which were located on the side chain. A detailed characterization has previously been reported in terms of differential scanning calorimetry and wide-angle X-ray diffraction.22 The combination of the copolymers and sodium chloride used to prepare the porous scaffold is summarized in Table 3. Here the stereocomplexes are designated by the following codes: for instance, SC10/P where SC10 refers to stereocomplex composed of PMBLLA10 and PMBDLA10 and P refers to the porous structure by sodium chloride. The sodium chloride was trapped in the stereocomplex, and the sodium chloride easily leaked out by immersion in the distilled water. Thomson et al. reported that a large surface area of the scaffold should be attained by preparation of high porosity with small diameter pores (10-103 µm).28 In this study, sodium chloride, which is a 200 µm cube (+80 mesh), was selected for the purpose of cell intrusion into the scaffold. The obtained scaffold was slightly brittle, and no significant



Figure 7. Scanning electron microscope pictures at 24 h.

difference was observed among the scaffold. A cross section of the scaffolds was observed with SEM (Figure 6). To observe the porous structure formed by the sodium chloride, the scaffold was cut in liquid nitrogen. From the SEM pictures of SC10/P, many pores were formed by the sodium chloride leaking out (Figure 6a). The pore size was found to be ca. 200-250 µm, which was attributed to the size of the sodium chloride. The pores were homogeneously dispersed in the scaffold. The scaffold was formed by the aggregation of small particles that had a size of 2 µm in diameter (Figure 6b). The particles were connected to each other, and a porous structure was observed. Paige et al. reported that interfiber distance of 100-200 µm was optimal for cellular attachment using polymer scaffold by nonwoven meshes, and the fibers were 14-15 µm in diameter.29 Taking these reports into account, the porous scaffold by stereocomplexation has adequate surface area for large number of cells to attach. Cell adhesion (surface) and intrusion (cross section) of the scaffold was observed by SEM (Figure 7). For the SC10 and SC10/P, cell adhesion was observed on both surfaces after 24 h (Figure 7a,c). From the cross-sectional view of SC10 and SC10/P, the cells intruded into the SC10/P (Figure 7d)). On the other hand, there were no cells in the SC10 (Figure 7b, without pores by sodium chloride). This result indicates that the pores formed by sodium chloride were effective for the cell intrusion into the scaffold. Furthermore, the cell morphology was round on the scaffold. From this

observation, it was considered that there was weak interaction between the cells and the scaffold by the MPC unit. Generally, the cell intrusion into the porous scaffold depends on chemical properties and physical structures. Physical structures such as porous scaffold could be good guide for cell intrusion into the scaffold in comparison with closepore scaffold. In the case of chemical properties, surface and bulk properties are dominant factor for cell adhesion and intrusion. For example, a porous hydrogel composed of poly(ethylene glycol) was good candidate for tissue scaffold; however, cell adhesion and intrusion were at quite a low level because of their high water content.30 Taking these reports into account, physical and chemical properties play an important role for cell adhesion and intrusion into the scaffold. Figure 8 shows the SEM pictures of SC20 and SC20/P. On the surface, cell adhesion was observed after 24 h. The cell morphology gradually spread out in comparison with SC10 or SC10/P. As for the monomer content, the ratio of the macromonomer/MPC in SC20 (or SC20/P) was higher than that of SC10 (or SC10/P). Therefore, a large amount of crystalline domain was formed by stereocomplexation. It is considered that the monomer content would regulate the cell morphology. In the cross-sectional view, the cell intrusions were observed on SC20/P, and their morphology was spread (Figure 8d). This result suggests that the cells intruded through the pores that were formed by sodium chloride. Paige et al. described the cell morphology as an important key factor to perform differentiated func-

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Figure 8. Scanning electron microscope pictures at 24 h.

tion.29 In the case of chondrocyte, the adherent cells show round shape, which has been correlated with a differentiated phenotype. Taking these observations and reports into account, the MPC unit content in the copolymer would be a dominant factor to regulate the cell morphology, and the pore size and its connection in the scaffold would be an enhancement for cell intrusion. To evaluate the cell functions and inflammatory reaction will be the next subject and reported in a forthcoming paper. Conclusions The enantiomeric poly(lactic acid) graft-type copolymer was designed and synthesized for tissue scaffold. The copolymer was composed of MPC, BMA, and the enantiomeric macromonomers with a PLLA segment (or PDLA segment). The degree of polymerization of the lactic acid in the PLLA and PDLA segments was tailored and found to be ca. 23 and 27, respectively. The content of the PLLA and PDLA macromonomers in the copolymer were in the range of 12%-28%. The fibroblast cell adhesion and morphology on a copolymer-coated surface was evaluated. From the cell culture results, the number of cell adhesion increased with incorporation of the PLLA (or PDLA) segment, though the reference copolymer composed of the MPC and BMA units showed low cell adhesion. As for the cell morphology, a round shape was observed as an effect of the MPC units. These results suggest that the PLLA (or PDLA) segment was effective for cell adhesion, and the cell

morphology was regulated by MPC content. A porous scaffold was prepared by the stereocomplexation of PLLA and PDLA. The cell adhesion and intrusion were observed on the porous scaffold. The fibroblast cells adhered to the surface and intruded into the scaffold despite over 16 mol % of MPC unit. The spread cell morphology was observed on the porous scaffold by increasing the PLLA (or PDLA) macromonomer content in the scaffold. These results suggest that the cell adhesion and the cell intrusion could be regulated by the macromonomer content and the pore size, respectively. Acknowledgment. The authors thank Dr. Yasuhiko Iwasaki, Mr. Akihiko Watanabe, and Mr. Shin-ichi Sawada, Tokyo Medical and Dental University, for their help in the cell culture and SEM observations. A part of this study was financially supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS Grant 13780672) and a Grant-in-Aid from The Kurata Memorial Hitachi Science and Technology Foundation, Japan. References and Notes (1) Langer, R.; Vacanti, J. P. Science 1993, 260, 920. (2) Hubbell, J. A.; Langer, R. Chem. Eng. News 1995, March 13, 42. (3) Saltzman, W. M. In Principles in Tissue Engineering; Lanza, R., Langer, R., Chick, W., Eds.; Academic Press: New York, 1995; pp 225-246. (4) Langer, R. S.; Vacanti, J. P. Sci. Am. 1999, April, 86. (5) Li, S. J. Biomed. Mater. Res. Appl. Biomater. 1999, 48, 342. (6) Ikada, Y.; Tsuji, H. Macromol. Rapid Commun. 2000, 21, 117. (7) Freed, L. E.; Marquis, J. C.; Nohria, A.; Emmanual, J.; Mikos, A. G.; Langer, R. J. Biomed. Mater. Res. 1993, 27, 11.

Cell Adhesion and Morphology in Porous Scaffold (8) Mikos, A. G.; Bao, Y.; Cima, L. G.; Ingber, D. E.; Vacanti, J. P.; Langer, R. J. Biomed. Mater. Res. 1993, 27, 183. (9) Park, Y. J.; Lee, Y. M.; Park, S. N.; Lee, J. Y.; Ku, Y.; Chung, C. P.; Lee, S. J. J. Biomed. Mater. Res. 2000, 51, 391. (10) Harris, L. D.; Kim, B. S.; Mooney, D. J. J. Biomed. Mater. Res. 1998, 42, 396. (11) Mikos, A. G.; Thorsen, A. J.; Czerwonka, L. A.; Bao, Y.; Langer, R. Polymer 1994, 35, 1068. (12) Zund, G.; Hoerstrup, S. P.; Schoeberlein, A.; Lachat, M.; Uhlschmid, G.; Vogt, P. R.; Turina, M. Eur. J. Cardio-thorac. Surg. 1998, 13, 160. (13) Gao, J.; Niklason, L.; Langer, R. J. Biomed. Mater. Res. 1998, 42, 417. (14) Kishida, A.; Kato, S.; Ohmura, K.; Sugimura, K.; Akashi, M. Biomaterials 1996, 17, 1301. (15) Sawada, S.; Shindo, Y.; Sasaki, S.; Watanabe, A.; Iwasaki, Y.; Kato, S.; Akashi, M.; Ishihara, K.; Nakabayashi, N. Trans. Soc. Biomater. 1999, 25, 231. (16) Ishihara, K.; Ueda, T.; Nakabayashi, N. Polym. J. 1990, 22, 355. (17) Ueda, T.; Oshida, H.; Kurita, K.; Ishihara, K.; Nakabayashi, N. Polym. J. 1992, 24, 1259. (18) Iwasaki, Y.; Nakabayashi, N.; Ishihara, K. J. Biomed. Mater. Res. 2001, 57, 72.

Biomacromolecules, Vol. 3, No. 6, 2002 1383 (19) Hasegawa, T.; Iwasaki, Y.; Ishihara, K. Biomaterials 2001, 22, 243. (20) Nam, K. W.; Watanabe, J.; Ishihara, K. Biomacromolecules 2002, 3, 100. (21) Watanabe, J.; Ishihara, K. Artif. Organs, in press. (22) Watanabe, J.; Eriguchi, T.; Ishihara, K. Biomacromolecules 2002, 3, 1109. (23) Ishihara, K.; Ishikawa, E.; Watanabe, A.; Iwasaki, Y.; Kurita, K.; Nakabayashi, N. J. Biomater. Sci., Polym. Ed. 1999, 10, 1047. (24) de Jong, S. J.; De Smedt, S. C.; Wahls, M. W. C.; Demeester, J.; Kettenes-van den Bosch, J. J.; Hennink, W. E. Macromolecules 2000, 33, 3680. (25) Fujiwara, T.; Mukose, T.; Yamaoka, T.; Yamane, H.; Sakurai, S.; Kimura, Y. Macromol. Biosci. 2001, 1, 204. (26) Ueda, T.; Oshida, H.; Kurita, K.; Ishihara, K.; Nakabayashi, N. Polym. J. 1992, 24, 1259. (27) Slager, J.; Domb, A. J. Biomaterials 2002, 23, 4389. (28) Thomson, R. C.; Wake, M. C.; Yaszemski, M. J.; Mikos, A. G. In AdVances in Polymer Science; Peppas, N. A., Langer, R. S., Eds.; Springer-Verlag: Berlin, Heidelberg, 1995; pp 245-274. (29) Paige, K. T.; Vacanti, C. A. Tissue Eng. 1995, 1, 97. (30) Han, D. K.; Hubbell, J. A. Macromolecules 1997, 30, 6077.

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Cell Adhesion and Morphology in Porous Scaffold Based on

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