Stereocomplex Formation by Enantiomeric Poly ... - ACS Publications

Jul 4, 2002 - Kiyoaki Ishimoto , Maho Arimoto , Tomoya Okuda , Syuhei Yamaguchi , Yuji Aso , Hitomi Ohara , Shiro Kobayashi , Masahiko Ishii , Koji Mo...
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Biomacromolecules 2002, 3, 1109-1114

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Stereocomplex Formation by Enantiomeric Poly(lactic acid) Graft-Type Phospholipid Polymers for Tissue Engineering 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 March 18, 2002; Revised Manuscript Received May 29, 2002

A porous scaffold as a cell-compatible material was designed and prepared using a phospholipid copolymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and enantiomeric macromonomers, the poly(L-lactic acid) (PLLA) macromonomer, and poly(D-lactic acid) (PDLA) macromonomer. On the basis of the wide-angle X-ray diffraction and differential scanning calorimetry measurements, the formation of a stereocomplex between the PLLA and PDLA segments of the copolymer was observed on the porous scaffold. The porous structure was prepared by a sodium chloride leaching technique, and the pore was linked to the scaffold. The pore size was confirmed by scanning electron microscopy and found to be ca. 200 µm. These observations suggest that the porous scaffold makes it possible to produce cell-compatible materials, which may involve the following advantages for tissue engineering: (i) cell compatibility using phospholipid copolymer, (ii) adequate cell adhesion by poly(lactic acid), and (iii) complete disappearance of scaffold by dissociation of stereocomplex. The cell experiment using the porous scaffold will be the next subject and reported in a forthcoming paper. Introduction Tissue engineering scaffolds have been widely investigated in terms of cell specificity1,2 and matrix degradability.3-6 The preparation of cell-compatible materials is important for the designing of tissue engineering scaffolds as well as biomedical devices. Generally, excellent cell growth was observed on tissue culture poly(styrene) (TCPS). TCPS might be a cell-compatible biointerface based on this observation, however, serious problems have been reported. From the evaluation of the mRNA expression of inflammatory cytokines, an inflammatory reaction was observed not only on TCPS but also on conventional polymer materials such as polyurethane and poly(ethylene terephthalate).7 From this consideration, cell-compatible materials, which do not induce any inflammatory reactions, have to be designed as a tissue engineering scaffold. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was designed based on the inspiration from the structure of the biomembrane which consists of a phospholipid, and was copolymerized with n-butyl methacrylate (BMA).8,9 The MPC copolymers were widely investigated and used as excellent biomaterials which suppress not only protein adsorption8,9 and cell adhesion10,11 but also the inflammatory reaction for attached cells.7,12 Our next concern is the MPC copolymer targets during the preparation of a cell-compatible scaffold for tissue engineering. Poly(lactic acid) (PLA) was incorporated into the MPC copolymer for enhancement of cell adhesion, due to poor cell adhesion on the MPC copolymer composed of MPC and BMA.13 PLA has been * To whom correspondence should be addressed. TEL: +81-3-58417124. FAX: +81-3-5841-8647. E-mail: [email protected].

widely utilized as base materials for biomedical matrixes,14-17 especially, poly(L-lactic acid) (PLLA) and poly(D-lactic acid) (PDLA) have been investigated for the formation of a stereocomplex and its application as a biomedical matrix.18-21 Generally, the degradation of a stereocomplex is quite low in comparison with PLLA and PDLA under physiological conditions. However, Park et al. reported the anomalous degradation behavior of the stereocomplex using a series of enantiomeric triblock copolymers composed of PLLAterminated poly(ethylene glycol) (PEG) and PDLA-terminated PEG.20 This report described that the degradation was estimated by comparing the relative molar ratio between the lactic acid (LA) unit and ethylene glycol (EG) unit for 50 days, and then the LA/EG ratio of the stereocomplex was significantly lower than that of the triblock copolymers. This result shows one of the reasons for the quick stereocomplex degradation. Kimura et al. discovered a thermoresponsive hydrogel by the formation of a stereocomplex which consists of the PLLA-terminated PEG and PDLA-terminated PEG and discussed it in relation to biomedical application.21 The polymer shows an interesting sol-gel transition which was induced by the stereocomplexation around 37 °C. Next, the toxicity of PLLA and PDLA must be considered for biomedical applications. Incomplete hydrolysis was induced by decreasing the water intrusion into the PLLA and the PDLA crystallite. The remaining crystallite then caused chronic inflammatory reactions at the implanted sites.22 In this study, PLLA and PDLA were used for the purpose of enhancing for cell adhesion and the formation of a porous scaffold for tissue engineering. The favorable characteristics of the enantiomeric PLA graft-type phospholipid copolymer may involve (i) adequate cell adhesion by PLA, (ii) prepara-

10.1021/bm025586r CCC: $22.00 © 2002 American Chemical Society Published on Web 07/04/2002

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Figure 1. Synthetic route of enantiomeric macromonomers, poly(Llactic acid) (PLLA) and poly(D-lactic acid) (PDLA) macromonomers.

tion of porous scaffold by stereocomplexation, (iii) stereocomplex dissociation by hydrolysis of PLA, and (iv) suppression of inflammatory cellular response by the MPC unit. A novel phospholipid copolymer with graft-type PLA was synthesized. The copolymer (PMBLLA and PMBDLA) composed of MPC, BMA, and the PLLA or PDLA macromonomer was utilized for the preparation of a porous scaffold by stereocomplexation. This paper reports the preparation and characterization of the porous scaffold. 2. Experimental Section 2.1. Materials. L-Lactide and D-lactide were kindly supplied by Dainippon Ink and Chemicals, Inc. (Tokyo, Japan). n-Butyl methacrylate (BMA, Wako Pure Chemical Co., Ltd., Osaka, Japan) and 2-isocyanate ethyl methacrylate (IEMA, Showa Denko Co., Tokyo, Japan) were distilled at reduced pressure (50 °C/20 mmHg for BMA, 60 °C/2.5 mmHg for IEMA). n-Dodecanol, stannous octoate (Sn(oct)2), and dibutyltin dilaurate (DBTL) were purchased from Wako Pure Chemical Co., Ltd., and used without further purification. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was synthesized and purified by a method from a previous report.8 Sodium chloride (mesh size 80) was purchased from Aldrich Chem. Co., WI. The other reagents were commercially available and used without further purification. 2.2. Macromonomer Synthesis. Poly(L-lactic acid) (PLLA) and the poly(D-lactic acid) (PDLA) macromonomer were prepared by the following two steps: (i) ring-opening polymerization of L-lactide and D-lactide at the terminal hydroxyl group of n-dodecanol, and (ii) methacrylation of the terminal hydroxyl group in the PLLA and PDLA macromonomers via urethane bond formation with IEMA. The preparative route is shown in Figure 1, and a typical example for the synthesis of the PLLA macromonomer is as follows. A total of 7.6 g (52 mmol) of L-lactide and 0.65 g (3.5 mmol) of n-dodecanol were added to a round-bottomed flask with a magnetic stirring bar, and the flask was evacuated by a vacuum pump for 3 h. After removal of the water, 100 µL (0.1 g/mL, 0.045 mol % of the L-lactide) of a toluene solution of Sn(oct)2 was added, and the flask was evacuated again for 3 h. After removal of the toluene, and the flask was then heated at 150 °C for 1 h. After cooling, the mixture was dissolved in chloroform and then poured into excess methanol to obtain PLLA as a white powder. The obtained PLLA was confirmed by FT-IR and 1H NMR. The FT-IR spectra were recorded using a VALOR-III (Jasco,

Watanabe et al.

Tokyo, Japan). The dried samples were powdered, ground with dried KBr powder, and pressed into pellets. Each sample was recorded between 4000 and 400 cm-1 by 16 scans. The 1 H NMR (JEOL R-500, Tokyo, Japan) spectra were measured using CDCl3 (Merck). Approximately 30 mg of each sample was dissolved in 600 µL of the solvent. The measurement of 16 scans was carried out. Yield: 92%. IR (KBr, cm-1): 3511 (hydroxyl), 1756 (ester). 1H NMR (CDCl3, ppm): δ 0.81 (t, 3H, CH3 for n-dodecanol), 1.19 (m, 2H ×11, CH2 for n-dodecanol), 1.51-1.59 (m, 3H ×30, CH3 for PLLA), 2.67 (s, 1H, OH), 4.29 (q, 1H, CH for terminal PLLA), 5.08-5.16 (m, 1H ×29, CH for PLLA). For the methacrylation, 6.3 g (2.7 mmol) of the PLLA was dissolved in 40 mL of dioxane, and IEMA (1.9 mL, 13.7 mmol) and DBTL were added to the solution. The mixture was stirred at 65 °C for 6 h. After the reaction had finished, toluene was evaporated under reduced pressure, and the residual fluid was poured into excess methanol to obtain PLLA macromonomer as a white powder. The obtained PLLA was confirmed by FT-IR, 1H NMR, and gel permeation chromatography (GPC) (column was KF-803, Shodex, Tokyo, Japan) relative to the poly(styrene) standards with tetrahydrofuran (THF) as the eluent. Yield: 90%. IR (KBr, cm-1): 1758 (ester), 1638 (double bond), 1560 (urethane). 1 H NMR (CDCl3, ppm): δ 0.85 (t, 3H, CH3 for n-dodecanol), 1.23 (m, 2H ×11, CH2 for n-dodecanol), 1.52-1.55 (m, 3H ×30, CH3 for PLLA), 1.92 (s, 3H, CH3 for methacryloyl), 3.48-4.20 (t, 2H ×2, CH2CH2 for IEMA), 4.72 (s, 1H, urethane), 5.11-5.20 (m, 1H ×30, CH for PLLA), 5.506.12 (s, 1H ×2, double bond). 2.3. Polymer Synthesis. A typical example of the copolymerization is as follows (Figure 2). The desired amounts of MPC, the PLLA macromonomer, and 2,2′-azobisisobutyronitrile (AIBN) were placed in a glass tube, and the mixture was diluted with ethanol-THF (1/1 by volume). BMA was added to the solution, and then the final concentrations of the monomer and the initiator were 0.5 mol/L and 2 mmol/L, respectively. The glass tube was cooled in liquid nitrogen with Ar bubbling. The glass tube was sealed, and kept at 60 °C for 24 h. After the polymerization, the product was poured into an excess diethyl etherhexane-ethanol (4/4/1 by volume) mixture to obtain the copolymer. The precipitate was filtered and dried in vacuo. The chemical structures of the copolymers were confirmed by FT-IR and 1H NMR. 2.4. Preparation of Porous Stereocomplex. To prepare a porous scaffold composed of PMBLLA and PMBDLA, 0.125 g of each polymer was dissolved in 0.6 mL of mixed solvent (methanol/methylene chloride 2:1, by volume). The solution was poured into a Teflon spacer (14 mm diameter, 5 mm depth) which was filled with 0.5 or 1.0 g of sodium chloride (mesh size 80), 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. To remove the sodium chloride, the scaffold was immersed in distilled water for one night. The water was substituted for the methanol, and then the scaffold was lyophilized overnight. The obtained

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Phospholipid Polymers for Tissue Engineering

Figure 2. Chemical structure of phospholipid random copolymer (PMBLLA and PMBDLA).

Figure 3. FT-IR spectra of (a) PLLA and (b) PLLA macromonomers.

scaffold was characterized by wide-angle X-ray diffraction (WAXD) and differential scanning calorimetry (DSC). A WAXD measurement (M18XHF-SRA, MAC Science, Yokohama, Japan) was performed from 5 to 30° using a Cu KR. source (40 kV and 200 mA). The DSC (DSC 6100, Seiko Instruments Co., Chiba, Tokyo) measurement was scanned at 5 °C/min over the temperature range of 20-200 °C. The porous structure of the scaffold was characterized using a scanning electron microscope (SEM, JSM-5400, JEOL, Tokyo, Japan). 3. Results and Discussion 3.1. Synthesis of PLLA and PDLA Macromonomers. The PLLA and PDLA macromonomers were first synthesized for copolymerization with MPC and BMA and were obtained in high yield (ca. 80%). n-Dodecanol was used as the initiator for the ring-opening polymerization of D(L-)lactide and was stable under the reaction conditions. After the synthesis of PLLA and PDLA, the methacryloyl group was incorporated into a terminal hydroxyl group (Figure 1). Figure 3 shows the IR spectra of the (a) PLLA and (b) PLLA macromonomer. For PLLA, a hydroxyl group and an ester group were observed at 3511 and 1756 cm-1, respectively. After the methacrylation by IEMA, the presence of a double bond at 1638 cm-1 and a urethane group at 1560 cm-1 was observed (Figure 3b). On the other hand, the disappearance of a hydroxyl group was clearly observed after the methacrylation. This observation indicates that IEMA reacted with the terminal hydroxyl group in PLLA. Furthermore, no significant difference between the PLLA and PDLA macromonomers in the IR spectra was observed. From the results

Figure 4. mers.

1H

NMR spectra of (a) PLLA and (b) PLLA macromono-

Table 1. Characterization of Enantiomeric Poly(lactic acid) Macromonomers code

DPa,b

Mnb

Mnc

Mw/Mnc

PLLA macromonomer PDLA macromonomer

23 27

2000 2300

5400 6400

1.11 1.11

a DP: degree of polymerization in poly(lactic acid) segment. b Determined by 1H NMR. c Determined by gel permeation chromatography (poly(styrene) standard).

of the IR spectra, the chemical structure of the PLLA and PDLA macromonomers was confirmed. Figure 4 shows the 1H NMR spectra of (a) PLLA and (b) PLLA macromonomer. The degree of polymerization (DP) of lactic acid in the PLLA macromonomer was determined from the ratio of the integrals of the methine protons (5.115.20 ppm) of lactic acid and the methyl proton (0.85 ppm) of n-dodecanol and was found to be ca. 23. Characterization of the PLLA and PDLA macromonomers is summarized in Table 1. DP was tailored by changing the ratio of the lactide monomer (L(D)-lactide) and initiator (n-dodecanol) as described by other researchers.24 In this study, the PLLA and PDLA macromonomers with a DP of 23 and 27 were

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Watanabe et al. Table 2. Preparative Conditions of Enantiomeric Polymers, PMBLLA and PMBDLA in feed (mol %) code PMBLLA PMBDLA

in copolymer (mol %)

macromacroyield MPC BMA monomer MPC BMA monomer DPa (%) 5 5

85 85

10 10

16 16

72 68

12 16

23 27

69 55

a DP: degree of polymerization in poly(lactic acid) segment determined by 1H NMR.

Figure 5. FT-IR spectra of PMBLLA.

Figure 6.

1H

NMR spectra of PMBLLA.

synthesized for the preparation of the scaffold by the formation of a stereocomplex. The methacrylation conversion was also determined for a comparison between the methyl proton (0.85 ppm) and methylene proton of the double bond (5.50-6.12 ppm) and was over 90%. The concentration of IEMA in the feed was 5 times higher than that of PLLA and PDLA, and the methacrylation quantitatively proceeded. From these results, a series of PLLA and PDLA macromonomers was confirmed. Table 1 shows the molecular weight of the PLLA and PDLA macromonomers determined by 1H NMR and GPC. The molecular weights of the synthesized macromonomers were within the confined range between 2000 and 2300 (by 1 H NMR). For the GPC measurement, the molecular weights relative to the poly(styrene) standard samples were estimated between 5400 and 6400 with a narrow Mw/Mn distribution. The reason the difference in the molecular weight exists between the 1H NMR and GPC was unexpected, and it could be considered to be due to the aggregation of the macromonomer. 3.2. Synthesis of Copolymers (PMBLLA and PMBDLA). Polymerization was carried out for 24 h because the reactivity of the PLLA and PDLA macromonomers is considered to be lower than that of the comonomers (MPC and BMA). THF and ethanol were used as a mixed solvent. Here, the copolymers are designated by the following code

Figure 7. Differential scanning colorimetry charts of (a) PMBLLA, (b) PMBDLA, and (c) SC.

such as PMBLLA where M, B, and LLA refer to the MPC unit, BMA unit, and PLLA macromonomer unit, respectively. Figure 5 shows the IR spectra of PMBLLA. PMBLLA was confirmed by the presence of an ester group for PLLA at 1760 cm-1, an ester group for the methacryloyl group at 1730 cm-1, a urethane bond at 1560 cm-1, and a choline group at 966 cm-1 and by the disappearance of the double bond at 1638 cm-1. In the case of PMBDLA, a similar chart of IR spectra was obtained. To determine the chemical composition in the copolymer, the 1H NMR spectrum of PMBLLA was measured (Figure 6). The DP of the lactic acid segment in PMBLLA and PMBDLA was determined from the ratio of the integrals of the methine protons (5.10-5.22 ppm) of lactic acid and the methyl proton (0.86 ppm) of n-dodecanol, and were ca. 23 and 27, respectively. The chemical composi-

Phospholipid Polymers for Tissue Engineering

Figure 8. Wide-angle X-ray diffraction profiles of (a) PMBLLA, (b) PMBDLA, and (c) SC.

tions of PMBLLA and PMBDLA were determined as shown in Table 1. The DP values of PMBLLA and PMBDLA were estimated from the ratio of the integrals of the methylene group for the polymer backbone at 1.40 ppm. The mole fraction of each monomer within the copolymer, the MPC and PLLA macromonomer, was estimated from the choline group at 3.44 ppm and the methylene group at 1.25 ppm, respectively. The rest of their mole fraction was considered to be BMA. From Table 1, the reactivity of the PLLA and PDLA macromonomers was quite high despite the large molecular weight (ca. 2000). The mole fraction of the PLLA and PDLA macromonomers in the copolymer showed almost a quantitative polymerization. This result indicates that PMBLLA and PMBDLA could be tailored by changing the monomer ratio. From these results, PMBLLA and PMBDLA were confirmed. 3.3. Preparation of Porous Scaffold. To prepare a porous scaffold, a mixed solvent composed of methanol and methylene chloride was used. PMBLLA and PMBDLA were

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easily and homogeneously dissolved in the mixed solvent. In this study, sodium chloride (80 mesh) was used for the purpose of forming a porous structure. The sodium chloride was easily and completely removed from the stereocomplex. After each copolymer solution was mixed, stirring of the solution was necessary to homogeneously disperse the sodium chloride. The solution turned turbid for a few minutes, and then the mixed dispersion was stored for 3 h to form the stereocomplex. To remove the free copolymer (not forming the stereocomplex), the spacer was immersed in the mixed solvent, and then the porous scaffold from the formation of the stereocomplex was removed from the spacer. The porous scaffold was washed with distilled water and lyophilized. The obtained scaffold was labeled using the following code such as SC or SC/P. Here, SC and P refer to the stereocomplex and porous structure, respectively. The obtained scaffold was brittle. To estimate the thermal properties, a DSC measurement was performed over the temperature range of 100-200 °C (Figure 7). For PMBLLA, two kinds of endothermic peaks were observed at 125 and 138 °C after the second scan (Figure 7a). Generally, an endothermic peak, which is attributed to the melting point of the PLLA, was observed around 130 °C.18,24 Tsuji reported that the melting subpeak of the homocrystallites might be ascribed to those crystallized during the DSC measurement. This result indicates that the PMBLLA showed two peaks, a main peak (crystal, 138 °C) and a subpeak (crystallite, 125 °C). This phenomenon was observed for PMBDLA. To confirm the scaffold by the formation of a stereocomplex, the endothermic peak of SC was measured by DSC (Figure 7b). Only the one melting peak was observed at 195 °C (SC10). This result indicates that the melting point of the scaffold (SC) was ca. 60 °C higher than that of the copolymers (PMBLLA and PMBDLA). It is generally reported that the melting point increased with the formation of the stereocomplex.18,24 As an alternative characterization of the stereocomplex formation, the crystal structure was characterized by WAXD. Figure 8 shows the X-ray diffraction profiles of the scaffolds (SC) and their copolymers (PMBLLA and PMBDLA). Intense peaks of the PMBLLA were observed at the 2θ values of 17 and 19°. These results were in good agreement with a previous report.23-25 For the stereocomplex, three additional peaks were observed at 2θ values of 12, 21, and 24°. The peaks of the copolymers were also observed at 17° for the major peak

Figure 9. Scanning electron microscope pictures of (a) SC, (b) SC/P by 0.5 g of NaCl, and (c) SC/P by 1.0 g of NaCl.

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and 19° for the trace peak. The former three peaks were attributed to the formation of the stereocomplex as previously reported.23-25 The latter two peaks were considered to be the PMBLLA and/or PMBDLA crystal structure, which could not form a perfect stereocomplex between the PLLA and PDLA segment in the copolymers. Taking these results into account, the scaffolds by the formation of the stereocomplex were obtained. 3.4. Characterization of Porous Scaffold. The freezedried scaffold was characterized by scanning electron microscopy (SEM) in order to observe the porous structure formed by sodium chloride. The scaffold was cut in liquid nitrogen to observe the cross section. Figure 9 shows the SEM observations of the SC and SC/P. The aggregation of small particles (2 µm particle size) was observed on all of the scaffolds. The small particles formed a network in the scaffold. Woerly reported that this aggregation was based on the formation of a heterogeneous phase separation system, which involved a large amount of nonsolvating solvent for the formed polymer.26 From this consideration, the mixed solvent (methanol and methylene chloride) was thought to be a nonsolvating solvent after the formation of the stereocomplex, and then phase separation of the stereocomplex occurred. Actually, the copolymers were easily dissolved in the mixed solvent, and then a heterogeneous (opaque) precipitation was obtained after their mixing. From the SEM pictures of SC/P, a porous structure formed by sodium chloride was observed. The pore size (200 µm) was in good agreement with the size of the sodium chloride. The formed pores (200 µm) penetrated into the scaffold by small pores for the aggregation of small particles. Taking these observations into account, the porous scaffold from the MPC copolymer grafted with poly(lactic acid) was obtained. Quite recently, cell adhesion of a coating surface by PMBLLA and PMBDLA and the porous scaffold was evaluated using fibroblast cells. The cell adhesion and proliferation on the surfaces were observed and enhanced by the introduction of the PLA macromonomer unit into the MPC copolymer. These results on the cell adhesion will be reported in our forthcoming paper. Conclusions The formation of a porous scaffold from a phospholipid copolymer with enantiomeric poly(lactic acid) (PLA) was examined. The phospholipid copolymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), butyl methacrylate, and enantiomeric macromonomers (PLLA macromonomer or PDLA macromonomer) was synthesized as cellcompatible materials. The degree of polymerization of the lactic acid in the PLLA and PDLA macromonomers was tailored and found to be ca. 23 and 27, respectively. The mole percentage of the PLLA and PDLA macromonomers in the copolymer was determined by 1H NMR and to be the range of 12-16. A porous scaffold was prepared by the

Watanabe et al.

formation of a stereocomplex between the PLLA and PDLA segments. The obtained porous scaffold was confirmed by wide-angle X-ray diffraction and differential scanning calorimetry. The pore size was found to be ca. 200 µm based on scanning electron microscopy and was in good agreement with the size of sodium chloride as the template. The formed pores had penetrated into the scaffold. The porous scaffold from the phospholipid copolymer is of great importance as novel cell-compatible materials for tissue engineering. Acknowledgment. The authors thank Dr. Yasuhiko Iwasaki and Mr. Akihiko Watanabe, Tokyo Medical and Dental University, for their help in SEM observation. A part of this study was financially supported by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS: 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.; Vacanti, C. A.; Atala, A.; Freed, L. E.; Vunjak-Novakovic, G. Tissue Eng. 1995, 1, 151. (2) Tabata, Y.; Ikada, Y. AdV. Drug DeliVery ReV. 1998, 31, 287. (3) Rashkov, I.; Manolova, N.; Li, S. M.; Espartero, J. L.; Vert, M. Macromolecules 1996, 29, 50. (4) Pitt, C. G. Biodegradable Polymers as Drug DeliVery Systems; Marcel Dekker: New York, 1990; p 71. (5) Heller, J.; Sparer, R. V.; Zentner, G. M. Biodegradable Polymers as Drug DeliVery Systems; Marcel Dekker: New York, 1990; p 121. (6) Domb, A. J.; Gallardo, C. F.; Langer, R. Macromolecules 1989, 22, 3200. (7) Sawada, S.; Shindo, Y.; Sasaki, S.; Watanabe, A.; Iwasaki, Y.; Kato, S.; Akashi, M.; Ishihara, K.; Nakabayashi, N. Trans. Soc. Biomater. 1999, 25, 231. (8) Ishihara, K.; Ueda, T.; Nakabayashi, N. Polym. J. 1990, 22, 355. (9) Ishihara, K.; Tsuji, T.; Sakai, Y.; Nakabayashi, N. J. Polym. Sci., Part A: Polym. Chem. 1994, 32, 859. (10) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323. (11) Ishihara, K.; Ishikawa, E.; Watanabe, A.; Iwasaki, Y.; Kurita, K.; Nakabayashi, N. J. Biomater. Sci., Polym. Ed. 1999, 10, 1047. (12) Sawada, S.; Sakaki, S.; Iwasaki, Y.; Nakabayashi, N.; Ishihara, K. J. Biomed. Mater. Res., in press. (13) Watanabe, J.; Ishihara, K. Submitted for publication in Artif. Organs. (14) van Dijk-Wolthuis, W. N. E.; Tsang, S. K. Y.; Kettenes-van den Bosch, J. J.; Hennink, W. E. Polymer 1997, 38, 6235. (15) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860. (16) Tasaka, F.; Ohya, Y.; Ouchi, T. Macromolecules 2001, 34, 5494. (17) Yamaoka, T.; Takahashi, Y.; Fujisato, T.; Lee, C. W.; Tsuji, T.; Ohta, T.; Murakami, A.; Kimura, Y. J. Biomed. Mater. Res. 2001, 54, 470. (18) Yui, N.; Dijkstra, P. J.; Feijen, J. Macromol. Chem. 1990, 191, 481. (19) 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. (20) Lim, D. W.; Park, T. G. J. Appl. Polym. Sci. 2000, 75, 1615. (21) Fujiwara, T.; Mukose, T.; Yamaoka, T.; Yamane, H.; Sakurai, S.; Kimura, Y. Macromol. Biosci. 2001, 1, 204. (22) Kimura, Y. In Biomedical Applications of Polymeric Materials; Tsuruta, T., Hayashi, T., Kataoka, K., Ishihara, K., Kimura, Y., Eds.; CRC Press: New York, 1993; pp 163-189. (23) Tsuji, H.; Ikada, Y. Polymer 1999 40, 6699. (24) Tsuji, H. Polymer 2000, 41, 3621. (25) Kister, G.; Cassanas, G.; Vert, M. Polymer 1998, 39, 267. (26) Woerly, S. Mater. Sci. Forum 1997, 250, 53.

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