Surface Modification of Polycaprolactone Membrane via Aminolysis

PCL Film Surfaces Conjugated with P(DMAEMA)/Gelatin Complexes for Improving ... Biomacromolecules 2010 11 (1), 68-75 ... Biomacromolecules 0 (proofing...
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Biomacromolecules 2002, 3, 1312-1319

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Surface Modification of Polycaprolactone Membrane via Aminolysis and Biomacromolecule Immobilization for Promoting Cytocompatibility of Human Endothelial Cells Yabin Zhu, Changyou Gao,* Xingyu Liu, and Jiacong Shen Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China Received June 26, 2002; Revised Manuscript Received August 15, 2002

Amino groups were covalently introduced onto a polycaprolactone (PCL) surface by the reaction between 1,6-hexanediamine and the ester groups of PCL. The occurrence of the aminolysis and the introduction of free NH2 groups were verified qualitatively by fluorescence spectroscopy, where rhodamine B isothiocyanate was employed to label NH2 groups, and quantitatively by absorbance spectroscopy, where ninhydrin was used to react with NH2 to generate a blue product. Due to the presence of deep pores on the PCL membrane, the aminolysis reaction could penetrate as deep as 50 µm to yield NH2 density as high as 2 × 10-7 mol/cm2. By use of the NH2 groups as active sites, biocompatible macromolecules such as gelatin, chitosan, or collagen were further immobilized on the aminolyzed PCL membrane via a cross-linking agent, glutaraldehyde. X-ray photoelectron spectroscopy (XPS) and surface wettability measurements confirmed the coupling of the biomacromolecules. The endothelial cell culture proved that the cytocompatibility of the aminolyzed PCL was improved slightly regardless of the NH2 amount on the surface. After immobilization of the biomacromolecules, however, the cell attachment and proliferation ratios were obviously improved and the cells showed a similar morphology to those on tissue culture polystyrene. Measurement of the von Willebrand factor (vWF) secreted by these endothelial cells (ECs) verified the endothelial function. Hence, a better EC-compatible PCL was produced. Introduction Many biodegradable synthetic materials such as polycaprolactone (PCL), poly(lactic acid) (PLA) and poly(lactideco-glycotide) (PLGA) have been used as scaffolds to support the regeneration of tissue-engineered organs such as cartilage, blood vessel, and skin. However, the poor cytocompatibility of the synthetic polymers leads to the inefficiency of the scaffold in constructing a friendly interface with living cells. Therefore, modification of the tissue-engineering polymeric materials to improve their cytocompatibility is necessary. Because the interaction between living cells and materials occurs mainly on the interfacial layer, many surface modification methods such as plasma treatment, γ-ray irradiation, ozone oxidization, end-grafting, or in situ polymerization have been developed to alter the surface properties of materials,1-8 for improving the cytocompatibility of the biomedical materials without alteration of the bulk properties. Among which end-grafting or in situ graft polymerization by photo or by radio frequency glow discharge deposition onto biomaterials has been widely employed to produce hydrophilic layer onto bulk biomedical polymers.9-11 The photoinduced grafting has been much studied to introduce hydrophilic groups onto poly(ester urethane) (PU), poly(L-lactic acid) (PLLA), and PCL membranes, resulting in better cell attachment, spreading, and proliferation.12-15 * Corresponding author: e-mail, [email protected]; Tel, +86-57187951108; fax, +86-571-87951948.

PCL, biodegradable aliphatic polyester,16,17 has been suggested for wide applications such as drug delivery systems,18,19 tissue-engineered skin (plain film), and scaffolds for supporting fibroblast and osteoblast growth.20,21 In PCL molecules, there exist abundant ester groups (-COO-). These ester groups can be hydrolyzed to carboxylic acid under alkaline condition. In addition, it is possible that the amino groups can be introduced onto the polyester surface by a reaction with diamine, providing that one amino group reacts with the -COO- group to form a covalent bond, -CONH-, while the other amino group is unreacted and free as shown in Scheme 1. It is worth noting that hydroxylterminated chains will also be yielded on the polyester surface during this process. Some advantages in tissue engineering can be expected by the steady introduction of these amino groups: (1) nontoxic to cells or tissues; (2) decreasing the surface hydrophobicity; (3) neutralizing the acid generated during the scaffold degradation and reducing the inflammation around the implanted scaffold; (4) providing active sites through which other biomolecules such as collagen, gelatin, or RGD peptides can be further immobilized, obtaining cytocompatible surface on which cells can grow well; (5) applying to three-dimensional (3-D) porous polyester scaffolds. 3-D porous scaffolds play an important role in supporting cell attachment, proliferation, and manipulating cell functions. However, the surface modification of 3-D porous scaffolds to improve their biocompatibility is difficult so far. Due to the easy performance, the aminolysis has been applied

10.1021/bm020074y CCC: $22.00 © 2002 American Chemical Society Published on Web 09/18/2002

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Scheme 1. The Schematic Representation of Aminolysis and Further Immobilization of Biomolecules on Polycaprolactone Membrane

to PLLA porous scaffolds to improve its cytocompatibility (unpublished). By aminolysis with 1,6-hexanediamine, free amino groups were introduced onto the PCL membrane surface. The existence of the ultralow concentration of amino groups was verified qualitatively and quantitatively by fluorescence spectroscopy and absorbance spectroscopy, respectively. Rhodamine B isothiocyanate or ninhydrin were used to label the amino groups to introduce fluorescent agent or to react with amino groups to produce a colored absorbance, respectively. Through a coupling agent, i.e., glutaraldehyde, gelatin, chitosan, or collagen was further immobilized onto a PCL surface. The culture of human endothelial cells (ECs) in vitro showed that the cytocompatibility of the aminolyzed and the biomacromolecules-immobilized PCL membranes is improved obviously. Experimental Section Aminolysis of PCL Membrane. PCL membrane was prepared by dissolving 5 g of PCL (Aldrich, Mn 80 000) in 50 mL of distilled 1,4-dioxane, and the mixture was then spread onto a stainless plate. The solvent was evaporated at 35 °C for 24 h and further dried under vacuum for another 24 h at 30 °C. A translucent PCL membrane with a thickness of 200 µm was obtained. The membrane was cut into pieces of 2 × 2 cm and immersed in alcohol/water (1/1, v/v) solution for 2-3 h to clean oily dirt and then washed with a large amount of deionized water. The membrane was subsequently immersed in distilled 1,6hexanediamine/2-propanol solution with suitable concentrations for given time at 37 °C, rinsed with deionized water for 24 h at room temperature to remove free 1,6-hexanediamine, and dried in a vacuum at 30 °C for 24 h to constant weight. Immobilization of Gelatin, Chitosan, or Collagen. The aminolyzed PCL membrane was immersed in 1 wt % glutaraldehyde (GA) solution for 3 h at room temperature, followed by rinsing with a large amount of deionized water for another 24 h to remove free GA. The membrane was then incubated in 3 mg/mL gelatin/phosphate buffered solution (PBS, pH ) 7.4) or 2 mg/mL chitosan solution (pH ) 3.5) or 3 mg/mL collagen solution (pH ) 3.4) for 24 h at 2-4 °C, respectively. The gelatin-immobilized membrane

Figure 1. The absorbance (at 538 nm) of ninhydrin-NH2 (with 1,6-hexanediamine) reaction products as a function of NH2 concentration.

was rinsed with deionized water for at least 24 h to remove free gelatin. The collagen- and chitosan-immobilized membranes were rinsed with 1.0% acetic acid solution and then rinsed with deionized water for 24 h to remove free chitosan or collagen.12-15 Determination of the Amino Groups. Rhodamine B isothiocyanate (RBITC) was used to label the amino groups on the aminolyzed PCL membrane for fluorescence measurement by immersing the PCL membrane in 0.1 mg/mL RBITC solution for 24 h at 2-4 °C. The obtained RBITClabeled PCL membrane was rinsed with deionized water for 24 h at room temperature to remove free RBITC and then dried under vacuum for another 24 h at 30 °C. The ninhydrin analysis method was employed to quantitatively detect the amount of NH2 groups on the aminolyzed PCL membrane. The membrane was immersed in 1.0 mol/L ninhydrin/ethanol solution for 1 min and then was placed into a glass tube, following with heating at 80 °C for 15 min to accelerate the reaction between ninhydrin and amino groups on PCL membrane. After the adsorbed ethanol had evaporated, 5 mL of 1,4-dioxane was added into the tube to dissolve the membrane when the membrane surface displayed blue. Another 5 mL of 2-propanol was added to stabilize the blue compound. The absorbance at 450-650 nm of this mixture was measured on a UV-vis spectrophotometer. A calibration curve was obtained with 1,6-hexanediamine in 1,4-dioxane/isopropane (1:1, v:v) solution (Figure 1). Human Endothelial Cell Culture. The endothelial cells (ECs) were isolated from the human umbilical cord veins of a new born baby with 1.0 mg/mL collagenase (type I, Sigma)/PBS for 20-25min at room temperature.22 The isolated ECs were routinely seeded on the beds prelaid with control or modified PCL membranes as well as on the tissue culture polystyrene (TCPS) (Nunc, Denmark) as control. The ECs were incubated in a culture medium consisting of 20% (v/v) fetal calf serum (FCS, Sijiqing Biotech. Co., China) and 80% RPMI1640 (Gibcobrl Co.) supplemented with 100 units/mL of penicillin and 100 µg/mL of streptomycin in humidified air containing 5% CO2 at 37 °C. After incubation for 24 h, the culture medium was changed, and then changed

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Figure 2. The survey XPS spectra of control PCL (a), PCL aminolyzed at a concentration of 10 wt % 1,6-hexanediamine for 10 min at 37 °C (b), PCL immobilized with gelatin (c), chitosan (d), or collagen (e).

Figure 3. The fluorescence intensity of the RBITC-immobilized PCL membranes as a function of 1,6-hexanediamine concentration. The aminolysis reaction took place at 37 °C for 10 min.

every 2 days. The ECs were fixed with 2.5% glutaraldehyde for 30 min for observation of cell morphology under a scanning electron microscope (SEM) after being cultured for 4 days. The cell attachment and proliferation ratio were averaged from five parallel measurements at 12 h and 4 days, respectively, by trypsinization of the ECs and counting the cell number under a hemocytometer. The cell proliferation ratio was defined as (N2 - N1)/N1, where N1 and N2 represent the cell number per well at 12 h and 4 days, respectively. Measurement of Released von Willebrand Factor (vWF). Secretion of vWF was measured in the supernatant of the cultured ECs by vWF enzyme-linked immunosorbent assay (ELISA) (Sun Diagnostics, Shanghai, China). Cell cultures were washed with the culture medium without serum when the ECs reached confluence (cultured for 72 h), followed by 2 h of incubation in culture medium with 20% FCS. Subsequently, supernatant medium was collected and centrifuged (10 min, 400g) for vWF measurement using vWF ELISA.23 Spectrophotometric readings were performed at 492 nm. Final results were obtained by comparison with a

Figure 4. The fluorescence intensity of the RBITC-immobilized PCL membrane as a function of aminolyzing time. The aminolysis reaction of PCL membrane took place at 37 °C in 10 wt % 1,6-hexanediamine/ 2-propanol solution.

standard curve constructed using dilutions of normal plasma. One milliliter of normal plasma was assumed to contain ∼10 µg of vWF (1 unit).24 Characterization. X-ray photoelectron spectroscopy (XPS) spectra were recorded on an ESCA LAB Mark II spectrometer employing Al KR excitation radiation. The charging shift was referred to the C1s line emitted from the saturated hydrocarbon. The fluorescence intensity was measured on a M850 fluorescence spectrophotometer and confocal laser scanning microscopy (CLSM, Radiance 2100, BIO-RAD). The UV-vis spectrum was measured on a UV-visible spectrophotometer (CARY 100 BIO, America). The water contact angle was measured at room temperature on a DSA10-MK2 contact angle measuring system from Kru¨ss, using the sessile drop and captive bubble methods. The atomic force microscopy (AFM) image was obtained by scanning probe microscopy (SPA400, Seiko) in the tapping mode. The cell morphology was observed under a scanning electron microscope (SEM, Stereoscan 260, Cambridge).

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Figure 5. The absorbance at 538 nm of the aminolyzed PCL membrane treated with ninhydrin as a function of aminolyzing time. The inset represents the absorbance spectra, where a, b, c, and d refer to PCL membrane aminolyzed for 5, 15, 45, and 120 min at 10 wt % 1,6hexanediamine/2-propanol solution, respectively.

Figure 6. The surface morphology of the control PCL membrane (a) and the PCL membrane aminolyzed at a concentration of 10 wt % 1,6hexanediamine/2-propanol for 10 min at 37 °C (b).

Results and Discussion Introduction of Amino Groups via Aminolysis. As shown in Scheme 1, a nitrogen element should appear on the PCL surface if the reaction of 1,6-hexanediamine with the ester groups occurs. However, from the XPS spectra of control and aminolyzed PCL (Figure 2 a, b), no obvious N1s peak was detected. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy characterization did not show the NH2 or -CONH- absorbance either. This might be the result of either the unreaction of amino groups with ester groups or the low sensitivity of the characterization methods. By the employment of more sensitive detecting methods, i.e., fluorescence spectroscopy after labeling the NH2 groups with RBITC, the existence of free NH2 groups was verified from the fluorescence emission data. Figure 3 showed that the fluorescence intensity from RBITC at 580 nm increased along with the concentration of 1,6-hexanediamine between 0 and 14 wt %. The intensity alteration proved that the amount of free NH2 groups on the PCL membrane surface increased with 1,6-hexanediamine concentrations at the given time. It is worth noting that free NH2 can react with -NCS in RBITC, resulting in a covalent coupling of RBITC on a PCL membrane surface that cannot be rinsed out, while the hydroxyl groups formed by the

aminolysis cannot react with RBITC under the same condition. It has to be indicated that the concentration of 1,6hexanediamine should be preferably lower than 14 wt % because of the poor solubility. The too strong basicity may also destroy the bulk mechanical property of PCL in higher concentration. Therefore, the optimal concentration of 1,6-hexanediamine was chosen as 10 wt % in the following study. At a given concentration of 10 wt % 1,6-hexanediamine, the influence of aminolyzing time on the surface amount of amino groups is shown in Figure 4. The fluorescence intensity increased rapidly with the increase of aminolyzing time, reached the maximum value at about 1 h, and then decreased to some extent. This may be caused by the further reaction with carboxyl of the free amino on the terminal chain or by the degradation of the superficial layer due to the high aminolyzing ratio at the longer time. In both cases the amount of the free amino groups on PCL membrane will be reduced; hence the fluorescence intensity decreases. The quantitative NH2 amount on aminolyzed PCL membrane surface was measured by the ninhydrin method. The blue reaction product of ninhydrin with free NH2 has a maximum absorbance at 538 nm in the solvent of 1,4dioxane/2-propanol (1:1) (Figure 5 inset). As shown in Figure 5, the alteration tendency of NH2 amount is similar to the

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Zhu et al. Table 1. The Water Contact Angle of the Control and the Modified PCL Membrane samples

SCA,a deg

CBCA,b deg

control PCL aminolyzed PCLc PCL-chitosan PCL-gelatin PCL-collagen

81.2 ( 2.4 68.4 ( 1.4 67.0 ( 0.8 59.5 ( 2.0 65.2 ( 1.9

67.9 ( 2.3 35.7 ( 2.3 28.5 ( 3.3 26.6 ( 2.3 27.9 ( 1.5

a Sessile contact angle. b Captive bubble contact angle. c The membrane was prepared in 10 wt % 1,6-hexanediamine solution at 37 °C for 10 min.

Table 2. The Dynamic Water Contact Angle and Hysteresis of the Control and the Modified PCL Membrane

Figure 7. CLSM fluorescence intensity of the control (a) and the aminolyzed PCL membrane (b) as a function of the depth in the z-direction. The aminolyzed PCL membrane was prepared in 10 wt % 1,6-hexanediamine solution at 37 °C for 10 min. Measuring area was 619 × 619 µm.

fluorescence result (Figure 4). The maximum NH2 density yielded at 1 h is ∼2 × 10-7 mol/cm2. From this result one can calculate the average area per amino-terminated chain, which is ∼0.1 Å2, supposing that the membrane surface was absolutely smooth and all the amino groups laid as a single layer. This is obviously unreasonable and impossible. Hence, either the surface is very rough or the aminolysis occurs in some depth in the z-direction instead of just on one layer, or the both. AFM measurement showed that the control PCL membrane was quite rough and there were many deep pores ranging from tens to hundred of nanometers, which were generated during the solvent evaporation process (Figure 6). After aminolysis, the PCL membrane surface became rougher and the pore size decreased as well. The existence of the deep pores provided also the probability that the 1,6hexanediamine molecules could penetrate into the inside of PCL membrane. To verify this hypothesis, a confocal laser scanning microscope was employed using the slice scanning function in the z-direction. The fluorescence intensity on a 619 × 619 µm as a function of depth in the z-direction is shown in Figure 7. It proves that the aminolysis could occur as deep as 50 µm in z-direction, and there existed a gradient distribution of the NH2 groups from the membrane surface to the inside, where more NH2 groups were located on

samples

ACA,a deg

RCA,b deg

hysteresis, deg (ACA-RCA)

control PCL aminolyzed PCLc PCL-chitosan PCL-gelatin PCL-collagen

81.2 ( 1.9 82.4 ( 2.4 62.1 ( 1.3 59.4 ( 2.1 63.7 ( 1.6

61.9 ( 5.1 28.7 ( 3.4 11.1 ( 2.6 9.8 ( 1.8 12.2 ( 2.4

19.3 53.7 51.0 49.6 51.5

a Advancing contact angle. b Receding contact angle. c The membrane was prepared in 10 wt % 1,6-hexanediamine solution at 37 °C for 10 min.

surface and less inside. It is worth noting that the NH2 groups were not homogeneously distributed in a given layer under the CLSM observation either. Stronger fluorescence intensity was observed around the area of pores. Figure 7 shows also that a physical adsorption of RBTIC on the control PCL membrane was possible, but the intensity was much lower. It is interesting that the fluorescence intensity in the control membrane exhibited a steplike decrease with the depth. The reason is not clear now. It may be caused by some physical structure formed during the membrane fabrication process. In conclusion, the density of amino groups on the superlayer of PCL membrane should be far less than 2 × 10-7 mol/cm2 due to the existence of surface roughness and deep pores. The aminolyzing reaction took place to a depth around 50 µm. The NH2 density on PCL membrane can be regulated and controlled through controlling the aminolyzing degree. Biomacromolecules Immobilization. The introduction of NH2 groups onto PCL membrane can not only modify the poor hydrophilicity but also provide the necessary active sites through which other biocompatible components such as proteins, polysaccharides, cell growth factors, or peptides can be further immobilized. To achieve the covalent coupling

Figure 8. The EC attachment (relative to TCPS) (9) and the proliferation ratio (0) cultured for 12 h and 4 days at 37 °C in humidified air with 5% CO2: (a) TCPS; (b) control PCL; (c) PCL aminolyzed for 3 min; (d) 10 min; (e) 30 min; (f) 120 min; (g) PCL immobilized with gelatin; (h) chitosan; (i) collagen. Cell seeding density was 15 × 104/cm2.

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Figure 9. The cell morphology cultured for 4 days on TCPS (a), PCL membrane aminolyzed for 3 min (b) and 120 min (c), and PCL membrane immobilized by gelatin (d), chitosan (e), or collagen (f), respectively. 1 refers to higher magnification while 2 refers to an overview under SEM with the same sample. Cell seeding density was 15 × 104/cm2.

of biomacromolecules, the aminolyzed PCL membrane was treated with a large amount of glutaraldehyde (GA) first as shown in Scheme 1. The reaction between NH2 and OHCs CHO yielded a bonding via sNdCHsCHO, and one free aldehyde group could react with NH2 groups existing in most biomacromolecules such as gelatin, chitosan, or collagen. The XPS analysis shows that N1s peaks at 400.2 eV appeared after immobilization of gelatin, chitosan, or collagen, which confirmed the coupling. The surface wettability alteration (Table 1) further confirmed the existence of the biomacromolecules. Though the water contact angles measured by the sessile drop method decreased only slightly after the

biomacromolecules immobilization, they obviously decreased from ∼70° to ∼30° when the captive bubble method was employed. This difference between the two measuring methods is a typical phenomena caused by the conformation alteration of the hydrophilic macromolecular chains.25 In the interface of biomacromolecules-immobilized PCL and air, the hydrophobic parts of the biomacromolecules tends to accumulate on the surface in order to reduce the interfacial energy, while in the interface of biomacromolecules-immobilized PCL and water, the hydrophilic parts of the biomacromolecules will reorganize their molecular structure to generate a hydrophilic parts dominating surface. This

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reorganization of the biomacromolecules was further proved by the dynamic water contact angle measurement through comparing the hystereses between the advancing and receding angles (Table 2). The larger contact-angle hystereses of the modified PCL surfaces (50-55 °C) than that of the control PCL (19.3 °C) demonstrated the occurrence of chain reorientation supposing no other disturbing factor.26 The slight decrease of ACA after aminolysis may be attributed to either the less amount incorporated or the easy movement of the amino groups with small molecular size which leads to the “hiding” of the hydrophilic parts under the hydrophobic substratum in air. The Cell Compatibility of Modified PCL Membrane. The increase of the surface hydrophilicity after aminolysis and the immobilization of the biocompatible macromolecules may provide the possibility of improving the cytocompatibility of PCL. The endothelial cell culture results showed that the cell attachment ratio of the aminolyzed PCL was not obviously changed compared with the control PCL regardless of the surface amino amounts (aminolyzing time) (Figure 8), although the water contact angles of these aminolyzed membranes have obviously been decreased. However, the cell proliferation ratios were improved clearly though the extent was still small compared with TCPS. The ECs on aminolyzed PCL also showed better cell morphology as shown in Figure 9. Hence, it is concluded that aminolysis of PCL to introduce NH2 groups has a positive effect on improving cytocompatibility in a limited extent. After immobilization of the biomacromolecules, however, the cytocompatibility was improved obviously (Figure 8 and Figure 9). The cell attachment and proliferation ratios of ECs on gelatin-, chitosan-, or collagen-immobilized PCL membranes were much larger than both of the control and the aminolyzed PCL membranes. The higher cell attachment ratios on the PCL membranes immobilized with gelatin or collagen that was even better than TCPS were yielded. The ECs grown on gelatin-, chitosan-, or collagen-immobilized PCL membranes presented also a flat and spreading morphology that was similar to those on TCPS. Considering the comprehensive results of cell attachment, proliferation, and morphology, the gelatin- or collagen-immobilized PCL has the best cytocompatibility. vWF Secretion. vWF is an adhesive glycoprotein synthesized exclusively in endothelial cells and megakaryocytes. Endothelial vWF is stored in rod-shaped organelles called Weibel-Palade bodies and affects the platelet adhesion and aggregation, blood coagulation, and fibrinolysis.27 Endothelial cell function thus directly affects the balance of hemostasis and thrombosis in the cardiovascular system. Therefore, we can investigate the ECs function through the measurement of the secretion of vWF by ECs growing on the modified PCL and on TCPS. Figure 10 showed that all cells seeded on TCPS or modified PCL membranes secreted vWF and maintained the endothelial function after cells were cultured in vitro for 72 h. Compared with control PCL, the ECs grown on aminolyzed PCL secreted more vWF regardless of the surface amino amounts (aminolyzing time), while the vWF concentrations on gelatin-, chitosan-, or collagen-immobilized PCL membranes were the highest. No big difference of the

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Figure 10. The vWF secretion (ng/10000 cells) by ECs on TCPS (a), control PCL (b), PCL membranes aminolyzed for 3 min (c), 10 min (d), 30 min (e), and 120 min (f), PCL membranes immobilized by gelatin (g), chitosan (h), or collagen (I), respectively. Cell seeding density was 15 × 104/cm2.

vWF between these biomacromolecules-immobilized PCLs was found. It was interesting that both amino groups and biomacromolecules could not only promote ECs growth but also maintain the endothelial function. Conclusion Free amino groups were introduced onto the PCL membrane surface through an aminolyzing reaction with 1,6hexanediamine. The NH2 density was measured by absorbance spectroscopy using ninhydrin. The introduced NH2 groups provided the opportunity to immobilize biomacromolecules such as gelatin, chitosan or collagen onto the PCL membrane surface. The introduction of the biocompatible macromolecules had a positive effect on modifying the cytocompatibility of PCL. The cell attachment and proliferation ratios were improved obviously, and the cells spread well and lived comfortably from the cell morphology observed under SEM. Moreover, the secreting function of ECs seeded on the modified PCL membrane also remained. In conclusion we have provided a novel technique, i.e., the aminolysis and the following biomacromolecules immobilization, through which a cytocompatible polymeric material can be easily fabricated. Acknowledgment. Financial support by the Major State Basic Research Program of China (G1999054305) is gratefully acknowledged. References and Notes (1) Ko, Y. G.; Kim, Y. H.; Park, K. D.; Lee, H. J.; Lee, W. K.; Park, H. D.; Kim, S. H.; Lee, G. S.; Ahn, D. J. Biomaterials 2001, 22, 2115. (2) Fujimoto K.; Takebayashi, Y.; Inoue, H.; Ikada, Y. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 1035. (3) Uchida, E.; Uyama, Y.; Ikada, Y. J. Appl. Polym. Sci. 1993, 47, 417. (4) Valuev, I. L.; Chupov, V. V.; Valuev, L. I. Biomaterials 1998, 19, 41. (5) Ozdemir, M.; Sadikoglu, H. Trends Food Sci. Technol. 1998, 9, 159. (6) Zhao, J. Q.; Geuskens, G. Eur. Polym. J. 1999, 35, 2115. (7) Geuskens, G.; Etoc, A.; Di Michele, P. Eur. Polym. J. 2000, 36, 265. (8) Aydinli, B.; Tincer, T. Radiat. Phys. Chem. 2001, 60, 237. (9) Uchida, E.; Uyama, Y.; Ikada, Y. Langmuir 1994, 10, 481. (10) Elbert, D. L.; Hubbell, J. A. Annu. ReV. Mater. Sci. 1996, 26, 365. (11) Kang, I. K.; Choi, S. H.; Shin, D. S.; Yoon, S. C. Int. J. Biol. Macromol. 2001, 28, 205.

Polycaprolactone Membrane Modification (12) Guan, J. J.; Gao, C. Y.; Feng, L. X.; Shen, J. C. J. Biomater. Sci., Polym. Ed. 2000, 11, 523. (13) Guan, J. J.; Gao, C. Y.; Feng, L. X.; Shen, J. C. J. Appl. Polym. Sci. 2000, 77, 2505. (14) Guan, J. J.; Gao, C. Y.; Feng, L. X.; Shen, J. C. Eur. Polym. J. 2000, 36, 2707. (15) Guan, J. J.; Gao, C. Y.; Feng, L. X.; Shen J. C. J. Mater. Sci.: Mater. Med. 2001, 12, 447. (16) Eldsa¨ter, C.; Erlandsson, B.; Renstad, R.; Albertsson, A. C.; Karlsson, S. Polymer 2000, 41, 1297. (17) Choi, E. J.; Kim, C. H.; Park, J. K. Macromolecules 1999, 32, 7402. (18) Zhong, Z. K.; Sun, X. Z. S. Polymer 2001, 42, 6961. (19) Allen, C.; Han, J. N.; Yu, Y. S.; Maysinger, D.; Eisenberg, A. J. Controlled Release 2000, 63, 275. (20) Woei, K.; Hutmacher, D. W.; Schantz, J. T.; Seng, C.; Too, H. P.; Chye, T.; Phan, T. T.; Teoh, S. H. Tissue Eng. 2001, 7, 441.

Biomacromolecules, Vol. 3, No. 6, 2002 1319 (21) Hutmacher, D. W.; Schantz, T.; Zein, I.; Ng, K. W.; Teoh, S. H.; Tan, K. C. J. Biomed. Mater. Res. 2001, 55, 203. (22) Jaffe, E. A.; Nachman, R. L.; Becker, C. G.; Minick, C. R. J. Clin. InVest. 1973, 52, 2745. (23) Favaloro, E. J.; Grispo, L.; Dinale, A.; Berndt, M.; Koutts J. Pathology 1993, 25, 152. (24) Lopes, A. A. B.; Peranovich, T. M. S.; Maeda, N. Y.; Bydlowski, S. P. Thromb. Res. 2001, 101, 291. (25) Roudman, A. R.; DiGiano, F. A. J. Membr. Sci. 2000, 175, 61. (26) Andrade, J. D.; Gregonis, D. E.; Smith, L. M. In Physico-chemistry Aspects of Polymer Surface; Plenum: New York, 1983; Vol. 2, p 911. (27) Wagner, D. D.; Olmsted, J. B.; Marder, V. J. J. Cell Biol. 1982, 95, 355.

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