Transaction of the Materials Research Society of Japan
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Antithrombogenic/Cytocompatible Heterogeneous Functioned Polymer Membrane for Hybrid Artificial Organs Hirotaka Kawai1,3, Ryosuke Matsuno2,3, Tomohiro Konno2,3,4, Madoka Takai2,3 and Kazuhiko Ishihara1,2,3,4
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Department of Bioengineering, 2Department of Materials Engineering, 3Center for NanoBio Integration (CNBI), The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, 4 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan Fax: +81-3-5841-8647, e-mail:
[email protected]
To develop hybrid artificial organs, we prepared a heterogeneous functioned polymer membrane as an interface between blood and tissue. The membrane was constructed by three polymer layers, that is, cellulose acetate membrane as a base membrane, photo-reactive gelatin and a phospholipids polymer (poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)](PMB)). Gelatin and PMB layers were introduced on each side of the cellulose acetate membrane. The gelatin layer played as cell adhesive, but PMB layer prevented cell adhesion. We observed good cell proliferation on the gelatin side and blood compatibility on PMB layer. The heterogeneous functioned polymer membrane possessed permeability of insulin. The heterogeneity of this membrane represented the two conflicting surface properties in one: cytocompatible surface on one side and an antithrombogenic surface on the other side. We considered that the membrane has the solute permeability to transport the products of cells. It could be concluded that the membrane is excellent hybrid of polymer and cells for realizing hybrid artificial organs. Key words: polymer membrane, phospholipid polymer, photo-reactive gelatin, hybrid artificial organs 1. INTRODUCTION Artificial organs were developed for the purpose of the supporting a part of functions of organs to prolong patients’ lifetime. Many researches on artificial organs have been done to achieve the functions including lung, blood vessel, kidney, and liver. Some devices are used in clinical practice. Current therapy with hemodialysis is well known as artificial kidney. However, it is difficult to achieve functions as sophisticated as internal organs with artificial organs made of artificial materials alone. To overcome this problem, approach from not only materials engineering but also cell engineering is becoming a new way of thinking. This approach leverages the materials abilities of cells to artificial materials and makes it possible to obtain capacity as high as in the living organisms. The combination of the materials engineering and the cell engineering can lead to development of hybrid materials. For example, hybrid materials are studied to apply to hybrid artificial kidney. Current renal substitution therapy for acute or chronic renal failure with hemodialysis or hemofiltration is replacing the function of glomeruli incompletely. This therapy is not complete renal replacement. It results in serious complications and high mortality rates. The reason is that it does not perform the reabsorption, excretion, metabolic and endocrine functions of renal tubules. To achieve the functions, the development of hybrid artificial kidney using cells and artificial scaffolds is essential. Saito et al. researched cell adhesion and cell proliferation on synthetic membranes [1,2]. They also constructed a bioartificial renal tubule device by using renal tubule
epithelial cells in an artificial membrane [3], and evaluated transport properties of the device for 2 weeks [4]. Humes et al. constructed a bioartificial renal tubule assist device by seeding porcine renal proximal tubule cells into the intralumenal spaces of hollow fibers, and then studied the metabolic and endocrine functions of these tubule cells [5,6]. In this way, the properties are necessary to functionalize the adhered cells and maintain their activities for the materials of the hybrid artificial organs. Add to it, the materials must possess a high hemocompatibillity to prevent blood coagulation without anticoagulant use. This is because blood transports substances including metabolic products and wastes. We designed a novel membrane as an interface between blood and tissue to apply to the artificial organs. It has asymmetrical surfaces with conflicting properties; one surface has a high antithrombogenic property and the other side has a cytocompatibility in order to adhere and properly proliferate enough to form a cell-layer. It has also the ability to transport products of the immobilized cells by membrane permeability. A hollow fiber membrane (HFM) module made of cellulose acetate (CA) mounted in the extracorporeal circulation like a hemodialysis system has been widely used in last decades. However, the CA membrane dose not have a sufficient hemocompatibility. Therefore, we modified the CA membrane with a phospholipid polymer, that is, the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer. The MPC polymers have been proved that they are useful for the surface modification of conventional materials to improve their
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Antithrombogenic/Cytocompatible Heterogeneous Functioned Polymer Membrane for Hybrid Artificial Organs hemocompatibility by effectively reducing protein adsorption, platelet adhesion. Dialysis membranes modified with the MPC polymers possess high hemocompatibility [7,8,9]. On the other hand, proteins are needed to obtain cytocompatible surface. It leads to enhance cells adhering to the membrane and maintaining the metabolic activities. In tissue engineering, a scaffold for cells is important and it potentially mimics the many roles of extracellular matrices (ECMs) in tissue. We use gelatin to achieve the property. Gelatin is a derivative of collagen, which is the major constituent of skin, bones and connective tissue. It is widely studied and used as a biomaterial for wound dressing or as a scaffold for tissue engineering [10,11]. Furthermore, we added a photo-reactive function to the gelatin for convenient fabrication. This photo-reactive ability enabled us to control immobilizing area of gelatin and obtain a covalent bonding between a substrate and gelatin. The aim of this study was to develop a hybrid membrane with dual function of antithrombogenicity and cytocompatibility using CA membrane, photo-reactive gelatin and MPC polymer; a heterogeneous functioned polymer membrane. 2. EXPERIMENTAL 2.1 Preparation of photo-reactive gelatin 2.1 Preparation of photo-reactive gelatin Photo-reactive gelatin (Fig. 1) was prepared as previous reported [12,13]. Gelatin (50.0 mg) dissolved in 10 mL phosphate-buffered solution (pH=7.0) was added to a dimethylformamide (DMF) solution (20 mL) of 5-azido-2-nitorobenzoic acid N-hydroxysuccinimide ester (30.7 mg) while being stirred on ice. After a stirred incubation at 4°C for 24 h, the solution was ultrafiltrated using Millipore ultrafiltration membrane (molecular weight, 1.0 x 104). The dialyzed solution was 500 mL as a photo-reactive gelatin standard solution for coating.
Fig. 1 Preparation of photo-reactive gelatin
Fig. 2 Chemical structure of PMB
Fig. 3 Preparation of HFP membrane
2.2 Synthesis of phospholipid polymer (PMB) PMB (Fig. 2) was synthesized by conventional radial polymerization of MPC, BMA by α, α’-azobisisobutyronitrile (AIBN) as an initiator reported in a previous study [14]. The ratio of monomer unit composition in PMB was determined by 1H-NMR (JEOL JNM-NR30, Tokyo, Japan). The molecular weight of PMB was evaluated by gel-permeation chromatography (GPC) (OHpak SB-804 HQ column, Shodex, Tokyo, Japan) in a mixture of water and methanol (3:7). 2.3 Preapration of heterogeneous functioned polymer membrane (HFP membrane) A CA membrane purchased from Sartorius Stedium (Tokyo, Japan) was placed on a filter holder and the ethanol solution containing PMB was flowed through the membrane. The solvent was removed in the atmosphere for one night and then under reduced pressure. Photo-reactive gelatin solution was cast on top of the dried membrane. After drying in the atmosphere, UV rays (16 W/cm2) were irradiated for 10 seconds by SPOT-CURE (USHIO INC, Tokyo, Japan). The gelatin was immobilized to the surface of PMB coated CA membrane (Fig. 3). 2.4 Chemical composition of HFP membrane surface The prepared HFP membrane was dried in a vacuum, and both surface of the membrane; the gelatin side and PMB side, were analyzed by X-ray photoelectron spectroscopy (XPS, AXIS-HSi, Shimadzu/KRATOS, Kyoto, Japan) to confirm the chemical composition of the each surface. 2.5 Renal cells culture on the each surface of HFP membrane; the gelatin side and the PMB side The heterogeneous property was evaluated by cell adhesion and proliferation. The pig-derived renal proximal tubule epithelial cells (Lewis lung cancer-porcine kidney 1, LLC-PK1) were cultured on the each surface. The round-shaped HFP membranes were placed onto a 12-well tissue culture plate and fixed with a silicon rubber ring so as the surface to evaluate was located upward. LLC-PK1 cells were seeded (1.0 x 104/ cm2) onto the gelatin side and the PMB side, and cultured at 37°C in a humidified atmosphere of air containing 5% CO2. The number of adhered cells was then measured at 1 day, 2 days, and 3 days after cell seeding by a cell-counting kit (Cell Counting Kit-8; Dojindo LABORATORIES, Kumamoto, Japan). Briefly, a 10 % volume of WST-8 (200 µL) was added to the culture medium. 180 minutes later, absorbance at 450 nm was read with a spectrophotometer. These data were compared with the number of cells cultured on a nonporous polystyrene plate (12-well culture plate; IWAKI, Tokyo, Japan). The morphology of the adhered cells at 3 days after the cell seeding was observed by a scanning electron microscope (SEM, SM-200, TOPCON, Tokyo, Japan) after the cells were fixed with 10% formalin for 60 min, dehydrated with a series of graded ethanol (30, 50, 70, and 99.5%) for 15 min each, freeze-dried overnight, and sputtered with gold.
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2.6 Permeability evaluation of polypeptide through the membrane The solute permeability performance of the HFP membrane was conducted. A diffusion set-up shown in Fig. 4 was used to evaluate solute permeation through the HFP membrane. Fluorescein (FITC)-labeled insulin (Sigma-Aldrich, Corp, St. Louis, USA) was used as a solute. The cell-cultured HFP membrane for 3 days after cells were seeded (1.0 x 104/ cm2) was placed between the chambers so as the cell-culture side was located in receiving chamber. Insulin solution was injected into a donor chamber (3.0 mL), while medium was injected into a receiving chamber (3.0 mL). A 100 µL of solution was extracted from both chambers, and the solution from the receiving chamber placed in a 96-well plate. The membrane permeability to insulin was evaluated by the fluorescence of the solution with a multilabel counter (Wallac ARVOsx1420, Perkin Elmer). 3. RESULTS AND DISCUSSION 3.1 Surface characterization of the HFP membrane The MPC unit mole fraction in the PMB was 0.28 and the weight average molecular weight was 3.4 x 10 4. Fig. 5 shows the results of XPS analysis. On the gelatin side and the PMB side, the XPS signals observed at 133 eV and 402 eV that are attributed to phosphorus atom and nitrogen atom of the PMB, respectively. On the gelatin side, the XPS signals observed at 400 eV that is attributed to nitrogen atom of gelatin. The intensity of gelatin side at 402 eV from phosphorus atom was at the same level as that of PMB side. We considered that the surface on the gelatin side was a coexistence state of the gelatin and the PMB. The gelatin and the PMB were introduced on the surface of the membrane. 3.2 Effects of polymer on cell adhesion On the gelatin side, the morphology of the adhered cells to the surface is shown in Fig. 6. The cells adhered effectively and cell layer was observed. It means that enough gelatin for LLC-PK1 to adhere could be immobilized on the PMB surface. On the other hand, cells could not adhere to the PMB side (Fig. 7). There were no cells on the surface and so we observed the structure of the CA surface. Judging from the results of cell adhesion and XPS analysis, we concluded that photo-reactive gelatin was immobilized on the PMB surface certainly and the prepared membrane act successfully as heterogeneous functioned polymer membrane. That is, cell adhesion was occurred on the
Fig. 4 Solute permeation cell-cultured membrane
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gelatin side as cytocompatible surface and cells could not adhere on the PMB side as antithrombogenic surface. 3.3 Cell proliferation on membranes In Fig. 8, cell proliferation of LLC-PK1 cultured on each surface is shown. By investigation of the time course of the cell number, the cell proliferation on the gelatin side was significantly higher than that on the CA membrane. Cells were grown in the presence of the
Fig. 5 XPS spectra of the HFP membrane and CA membrane on their surfaces
Fig. 6 SEM pictures of the gelatin side observed 3 days after cell seeding
Fig. 7 SEM pictures of the PMB side observed 3 days after cell seeding
Fig. 8 Cell proliferation on the HFP membrane (gelatin side () and PMB side ()) and CA membrane ()
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Antithrombogenic/Cytocompatible Heterogeneous Functioned Polymer Membrane for Hybrid Artificial Organs
Fig. 9 Change in the percentage of permeated insulin to the receiver chamber compared with the initial concentration in the donor chamber using the cell-cultured HFP membrane (), the cell-cultured CA membrane (), and CA membrane () gelatin. On the other hand, cell number did not increase on the PMB surface because the surface was non-adhesive surface. We could functionalize the surface for cell adhesiveness. 3.4 Permeation of insulin The solute permeation of the cell-cultured HFP membrane was confirmed (Fig. 9). The permeability of the cell-cultured HFP membrane was as high as that of the CA membrane and the cell-cultured CA membrane. The solute of insulin may penetrate through the cells because cell-cultured membrane surface for 3 days was covered with a cell layer (Fig. 6). The HFP membrane maintained solute permeation of cell adhesion state. 4. CONCLUSION We succeeded in developing the antithrombogenic/cytocompatible heterogeneous functioned polymer membrane with PMB, photo-reactive gelatin, and cellulose acetate membrane. Cell adhesion was induced on one side (gelatin immobilized surface) and inhibited on the other side (PMB coated surface). Solute permeability was maintained after cell adhesion on the heterogeneous functioned polymer membrane. This membrane as an interface between blood and tissue can be excellent for hybrid artificial organs. 5. ACKNOWLEDGEMENT The authors thank Prof. Akira Saito, Tokai University, for providing many helpful comments and cells. References [1] N. Kanai, Y. Fujita, T. Kakuta, and A. Saito, Artif Organs, 23, 114-118,1999 [2] Y. Sato, M. Terashima, N. Kagiwada, T. Aung, M. Inagaki, T. Kakuta, and A. Saito, Tissue Eng, 11, 1506-1515, 2005 [3] M. Terashima, Y. Fujita, K. Sugano, M. Asano, N. Kagiwada, S. Y, S. Nakamura, A. Hasegawa, T. Kakuta, and A. Saito, Artif Organs, 25, 209-212, 2001 [4] N. Ozgen, M. Terashima, T. Aung, Y. Sato, C. Isoe, T. Kakuta, and A. Saito, Nephrol Dial Transplant, 19, 2198-2207, 2004
[5] H. Humes, S. MacKay, A. Funke, and D. Buffington, Kidney Int, 55, 2502-2514, 1999 [6] H. Humes, D. Buffington, S. MacKay, J. Funke, and W. Weitzel, Nat Biotechnol, 17, 451-455, 1999 [7] K. Ishihara, K. Fukumoto, Y. Iwasaki, and N. Nakabayashi, Biomaterials, 20, 1553-1559, 1999 [8] S-H. Ye, J. Watanabe, M. Takai, Y. Iwasaki, and K. Ishihara, Biomaterials, 27, 1955-1962, 2006 [9] H. Ueda, J. Watanabe, T. Konno, M. Takai, A. Saito, and K. Ishihara, J Biomed Mater Res, 77A, 19-27, 2006 [10] A. Ito, A. Mase, Y. Takizawa, M. Shinkai, H. Honda, K. Hata, M. Ueda, and T. Kobayashi, J Biosci Bioeng, 95, 196-199, 2003 [11] H. Kang, Y. Tabata, and Y. Ikada, Biomaterials, 20, 1339-1344, 1999 [12] Y. Ito, G. Chen, and Y. Imanishi, Bioconj Chem, 9, 277-282, 1998 [13] Y. Ito, H. Hasuda, T. Yamauchi, N. Komatsu, and K. Ikebuchi, Biomaterials, 25, 2293-2298, 2004 [14] K. Ishihara, H. Oshida, T. Ueda, Y. Endo, A. Watanabe, N. Nakabayashi, J Biomed Mater Res, 26, 1543-52, 1992 (Received January 5, 2009; Accepted February 23, 2009)
