2-甲基丙烯酰羟乙基磷酰胆碱聚合膜的研究现状及应用前景

2-甲基丙烯酰羟乙基磷酰胆碱(2-methacryloyloxyethyl phosphorylcholine,MPC)聚合膜是由MPC与其它乙烯基化合物聚合而得的具有优良血液渗透性和生物相容性的生物医学材料。该聚合膜侧链上含有类似细胞膜结构的磷脂极性基团,这在其良好的血液和组织相容性上起了重要作用。MPC聚合膜能有效抑制蛋白吸附和血小板黏附作用,能有效抑制凝血作用,它不仅是人造器官的主要材料来源,还可对生物医学设备进行表面修饰以增加其血液相容性和生物相容性。因而,MPC聚合膜被广泛应用于血液透析、人造器官、膜充氧器和一次性临床设备等生物医学领域。MPC聚合膜在血液相容性上很具发展空间,但关于它的很多研究工作还都处于初期或中级阶段,本文介绍了MPC聚合膜的研究现状及应用前景。     

Modification of polysulfone with phospholipid polymer for improvement of the blood compatibility. Part 2. Protein adsorption and platelet adhesion.

Protein adsorption and platelet adhesion from human plasma on polysulfone (PSf) membranes modified with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were studied. The modification was carried out by blending of the MPC polymer in the PSf. The amount of protein adsorbed on the PSf/MPC polymer blend membrane was significantly decreased with an increase in the composition of the blended MPC polymer. The distribution of the specific proteins adsorbed on the membrane surface was also determined by a gold-colloid immunoassay. Albumin, gamma-globulin and fibrinogen were observed on every membrane surface after contact with plasma. However, in the case of the blended membrane, the density of the adsorbed proteins decreased compared with that of original PSf membrane. That is, the MPC polymer blended in the membrane could function as a protein-adsorption-resistant additive. The number of platelets adhered on the PSf membrane was reduced, and change in the morphology of adherent platelets was also suppressed by the modification with the MPC polymer. Therefore, the PSf/MPC polymer blend membrane had improved blood compatibility compared with the PSf membrane.     

Asymmetrically functional surface properties on biocompatible phospholipid polymer membrane for bioartificial kidney

To obtain a bioartificial kidney composed of a porous polymer membrane and renal cells, a polysulfone (PSf) membrane (PSM) blended with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer was prepared. The PSM flat membrane with a porous structure could be prepared from the polymer blend containing 1 wt % of the MPC polymer in PSf by the phase inversion technique in a dry-wet process. Asymmetrical surface properties were observed on both sides of the membrane surfaces. That is, the sponge layer formed at the substrate-contacting surface of the membrane had 10-20 microm pores, but the pores in the micrometer range could not be observed for a skin layer formed at the air-contacting surface of the membrane. At the sponge layer surface, the MPC unit composition was 7 times larger than that at the skin layer surface. The amount of proteins adsorbed on the surface corresponded to the MPC unit composition. On the skin layer, a small amount of adsorbed proteins and platelet adhesion could be suppressed compared with those on the sponge layer. However, the skin layer had a moderate protein adsorption, so it showed a sufficient cytocompatibility to enable renal tubule epithelial cells to adhere and proliferate in the membrane. Thus, it functioned well as a renal tubule. Therefore, because of both its hemocompatibility and cytocompatibility, we could conclude that the PSM membrane is useful for as a renal tubule device for a bioartificial kidney.     

Preparation and performance of protein-adsorption-resistant asymmetric porous membrane composed of polysulfone/phospholipid polymer blend

To obtain protein-adsorption-resistant membrane for hemodialysis, we prepared a polymer blend composed of polysulfone and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer (PSf/MPC polymer). The content of the MPC polymer in the PSf was 7 and 15 wt%. The asymmetric porous membrane was obtained by the dry/wet membrane processing method. The surface characterization of the PSf/MPC polymer membrane by X-ray photoelectron spectroscopy revealed that the MPC polymer located at the surface. The mechanical strength of the PSf/MPC polymer membrane did not change compared with that of the PSf membrane. On the other hand, the permeability of solute below a molecular weight (Mw) of 2.0 x 10(4) through the PSf membrane increased with the addition of the MPC polymer, which is considered to be an effect of the hydrophilic character of the MPC polymer. The amount of protein adsorbed on the PSf membrane from plasma was reduced by the addition of the MPC polymer. The permeability of low-molecular-weight protein (Mw = 1.2 x 10(4)) did not change even after the PSf/MPC polymer membrane was contacted with plasma protein solution for 4 h, whereas it decreased dramatically in the case of the PSf membrane. Platelet adhesion was also effectively suppressed on the PSf/MPC polymer membrane. Based on these results, the MPC polymer could serve as a doubly functional polymeric additive, that is, to generate a protein-adsorption-resistant characteristic and to render the membrane hydrophilic.     

New modified polyetheretherketone membrane for liver cell culture in biohybrid systems: adhesion and specific functions of isolated hepatocytes

There has been growing interest in innovative materials with physico-chemical properties that provide improved blood/cell compatibility. We propose new polymeric membranes made of modified polyetheretherketone (PEEK-WC) as materials with potential for use in biohybrid devices. PEEK-WC exhibits high chemical, thermal stability and mechanical resistance. Owing to its lack of crystallinity this polymer can be used for preparing membranes with cheap and flexible methods. We compared the properties of PEEK-WC membranes to polyurethane membranes prepared using the same phase inverse technique and commercial membranes. The physico-chemical properties of the membranes were characterised by contact angle measurements. The different parameters acid (gamma+), base (gamma-) and Lifshitz-van der Waals (gammaLW) of the surface free energy were calculated according to Good-van Oss's model. We evaluated the cytocompatibility of PEEK-WC membranes by culturing hepatocytes isolated from rat liver. Cell adhesion and metabolic behaviour in terms of ammonia elimination, urea synthesis and protein synthesis were evaluated during the first days of culture. Liver cells adhered and formed three-dimensional aggregates on the most tested membranes. PEEK-WC membranes promoted hepatocyte adhesion most effectively. Urea synthesis, ammonia elimination and protein synthesis improved significantly when cells adhered to PEEK-WC membrane. The considerable metabolic activities of cells cultured on this membrane confirmed the good structural and physico-chemical properties of the PEEK-WC membrane that could be a promising biomaterial for cell culture in biohybrid devices.     

Cell adhesion on phase-separated surface of block copolymer composed of poly(2-methacryloyloxyethyl phosphorylcholine) and poly(dimethylsiloxane)

We investigated the morphological effect of phase-separated block copolymer surfaces composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) and poly(dimethylsiloxane) (PDMS) on protein adsorption and cell adhesion behavior. We observed three different types of phase-separated surface morphologies by TEM and AFM. The elemental composition of phosphorus on the surface increases with the PMPC composition. Furthermore, the polymer surface formed by a block copolymer-containing a higher MPC unit composition shows a slightly lower static water contact angle. This result indicates that the elemental surface ratio of the surface depends on the MPC composition in the block copolymer. Protein adsorption tests revealed that only hydrophobic PDMS domains showed selective protein adsorption. Cell adhesion tests revealed that the number of adhered cells increased with increasing hydrophobic PDMS domain size of block copolymers in serum-containing media. In contrast, no cells adhered onto block copolymer surfaces in serum-free media, whereas a large amount of adhered cells were observed on the hydrophobic PDMS surface. This result indicates that segregated hydrophobic domains on a biocompatible PMPC surface strongly affect serum protein adsorption, thereby promoting considerable cell adhesion, although the surface is hydrophilic. Thus, both the composition of MPC units and the segregated hydrophobic surface morphology are important considerations in biomaterial surface design.     

Durable modification of segmented polyurethane for elastic blood-contacting devices by graft-type 2-methacryloyloxyethyl phosphorylcholine copolymer

We propose a novel application of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers for enhancing the performance of modified segmented polyurethane (SPU) surfaces for the development of a small-diameter vascular prosthesis. The SPU membranes were modified by random-type, block-type, and graft-type MPC polymers that were prepared using a double-solution casting procedure on stainless steel substrates. Among these MPC polymers, the graft-type poly(MPC-graft-2-ethylhexyl methacrylate [EHMA]), which is composed of a poly(MPC) segment as the main chain and poly(EHMA) segments as side chains, indicated a higher stability on the SPU membrane after being peeled off from the stainless steel substrate, as well as after immersion in an aqueous medium. This stability was caused by the intermiscibility in the domain of the poly(EHMA) segments and the soft segments of the SPU membrane. Each SPU/MPC polymer membrane exhibited a dramatic suppression of protein adsorption from human plasma and endothelium cell adhesion. Based on these results, the performance of SPU/poly(MPC-graft-EHMA) tubings 2?mm in diameter as vascular prostheses was investigated. Even after blood was passed through the tubings for 2?min, the graft-type MPC polymers effectively protected the blood-contacting surfaces from thrombus formation. In summary, SPU modified by graft-type MPC polymers has the potential for practical application in the form of a non-endothelium, small-diameter vascular prosthesis.     

Improvement of blood compatibility on cellulose hemodialysis membrane: IV. Phospholipid polymer bonded to the membrane surface

To improve the surface blood compatibility on a cellulose hemodialysis membrane, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers with a phospholipid polar group were immobilized on the surface through covalent bonding. The MPC polymers had a carboxylic group, which can react with hydroxyl groups on the cellulose membrane, and were synthesized by conventional radical polymerization. The reaction between the MPC polymers and the cellulose membrane was carried out in a heterogeneous system using a condensation reagent. Surface analysis of the modified membrane by X-ray photoelectron spectroscopy revealed the immobilization of the MPC polymer on the surface. The mechanical strength and permeability for a solute of the membrane did not change even after the modification. The modified cellulose membrane was blood-compatible, as determined by the prevention of adhesion, deformation, and aggregation of platelets after contact with platelet-rich plasma. Based on these results, it is concluded that the MPC polymers may be a useful material for improving the blood compatibility of cellulose hemodialysis membranes.     

Gas transfer and blood compatibility of fluorinated polyimide membranes

Fluorinated polyimide derived from 2,2'-bis(3,4-dicarboxyphenyl) hexafluoropropane dianhydride (6FDA) and bis[4-(4-aminophenoxy) phenyl]sulfone (APPS) was synthesized to develop a novel membrane oxygenator combining excellent gas transfer and blood compatibility. The asymmetric gas exchange membranes of 6FDA-APPS made by a dry/wet process consisted of an ultrathin and defect-free skin layer supported by a porous substructure. O₂ transfer through the 6FDA-APPS membrane was extremely augmented as compared with that of the presently available membrane, poly(dimethylsiloxane), and the previously reported 6FDA-DDS membrane. Since CO₂ transfer through the 6FDA-APPS membrane increased with a decrease in CO₂ pressure according to dual-mode transport theory, CO₂ from the membrane was selectively removed at low CO₂ pressure. For the evaluation of in vitro blood compatibility, the platelet adhesion and the plasma protein adsorption on the surface of the 6FDA-APPS membrane were observed by using scanning electron microscopy and the amounts of platelet and plasma protein were determined by an amino acid analyzer. The results indicated that the fluorinated polyimide membranes showed excellent blood compatibility.     

Design of novel biointerfaces (II). Fabrication of self-organized porous polymer film with highly uniform pores

Application of porous polymer materials to novel bio-interfaces for tissue engineering scaffold and artificial organs including blood filters, dialyzer, and oxygenator membranes have been in progress. The present study describes the fabrication and characterization of self-organized highly regular porous polymer films with uniform pore sizes are prepared by simple casting technique. Various fabrication parameters affecting the pore size such as polymer concentration, boiling point of solvent, cast volume and substrate are studied. The pore size can be controlled in the range from 1 to 50 microm by changing the evaporation rate of the polymer solutions. The porous film with uniform pore size is used for tissue engineering scaffold and cell separation membrane. To simulate the leukocyte eliminating from human blood, the porous film was attached to a module. The films with 5-9 microm pores provided the complete selectivity of separation for the leukocyte from the whole blood. The leukocyte elimination ratio depends on pore structures (size and depth) as well as recovery of platelet and erythrocyte.     

Antifouling blood purification membrane composed of cellulose acetate and phospholipid polymer

The ideal surface of an artificial blood purification membrane needs hemocompatibility and durability of high performance; it should not adsorb any proteins or cells but should still have high permeability in the desired range of solute size. To improve the anti-fouling property of cellulose acetate (CA) membranes, a CA membrane blended with poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (PMB30) was designed as a blood purification membrane. The polymer solutions for preparing the membrane were prepared using a solvent mixture composed of N, N-dimethylformamide, acetone, 2-propanol or water. The CA and CA/PMB30 blend membranes with an asymmetric and porous structure were prepared by a phase inversion process.The characteristics of the CA/PMB30 blend membrane, such as structural properties, mechanical properties, and solute permeability were examined with attention to changes in the preparation conditions of the membrane. The CA/PMB30 blend membrane had good water and solute permeability and a sharp molecular weight cut-off property. Moreover, the amount of proteins adsorbed on the CA/PMB30 blend membrane surface was less than that of the original CA membrane and a conventional polysulfone membrane. Adhesion and activation of platelets on the CA/PMB30 blend membrane were reduced compared with that on a CA membrane. In addition, the CA/PMB30 blend membrane showed good permselectivity and an antifouling property during a long time ultrafiltration experiment with protein solutions.     

Modification of polysulfone with phospholipid polymer for improvement of the blood compatibility. Part 1. Surface characterization.

To improve the surface blood compatibility of polysulfone (PSf) membranes, we prepared novel polymeric additives which have suitable blood compatibility. They were polymers with a phosphorylcholine group, a 2-methacryloyloxyethyl phosphorylcholine (MPC) unit. The MPC polymer could be blended with polysulfone by a solvent evaporation method during membrane processing, and a transparent membrane could be obtained. The mechanical properties of the blend membrane were similar to that of the original PSf membrane. Surface analysis of the blend membrane by X-ray photoelectron spectroscopy and dynamic contact angle measurement revealed that the MPC unit in the polymeric additive was concentrated on the surface of the membrane. The blend membrane significantly reduced plasma protein adsorption compared with that of the PSf membrane.     

HSP 47 and collagen mRNA expression in L929 cells adhered to lipid films

L929 cell adhesion on various lipid films prepared by Langmuir-Blodgett methods (LB method) were studied. L929 cells adhered to every lipid film similar to tissue culture poly(styrene) (TCPS). The mRNA expression of both collagen and HSP47 in adherent cells were evaluated by the RT-PCR method. mRNA expression of collagen was not altered during adhesion and proliferation duration, while HSP47 mRNA expression changed depending on culture time. L929 cells adhered to L-alpha-dipalmitoylphosphatidylcholine (DPPC)-films showed little HSP47 mRNA expression. It was suggested that DPPC films regulate L929 cell function via unique serum protein adsorption.     

组织因子途径抑制因子对生物材料表面血小板粘附的影响

组织因子途径抑制因子（tissue factor pathway inhibitor,TFPI）是组织因子凝血途径的主要抑制因子，具有抑制组织因子、凝血因子VIIa、和Xa的功能。我们以前的研究显示TFPI在体外可以明显延长被修饰材料表面的凝血时间，在体内显著减少材料表面的血栓形成。本文观察了TFPI包被对聚乙烯和聚氯乙烯材料表面血小板粘附的影响。结果显示，通过TFPI处理后，上述两种材料表面的血小板粘附数目较对照组明显减少，提示TFPI可通过抑制血小板在材料表面的粘附起到改善生物材料血液相容性的作用。     

Surface modification of poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactic acid) affects initial cell attachment,cell morphology,and cell growth

The object of this study was to develop a highly porous scaffold to be used in regeneration of blood vessels, nerves, and other hollow tissues with small openings. Using the phase-inversion method and a mixture of water and methanol as a coagulating agent, we prepared highly porous flat membranes from poly(l-lactic acid) (PLLA) with numerous pores both on the surface and in the interior of the membranes. Chinese hamster ovary (CHO) cells were cultured on the membranes to evaluate initial cell adhesion, cell proliferation, and cell morphology. Adhesion of CHO cells to PLLA was poor: the cells adhered at approximately half the rate observed with a tissue culture polystyrene dish (TCPS). In contrast, adhesion of cells to PLLA treated with a low-temperature oxygen plasma was good; the adhesion rate was the same as that on TCPS. The rate of cell proliferation on the treated membranes was no different from that on the nontreated membranes, but cell morphologies were quite different. The cells on the nontreated membranes were small and round and proliferated separately from one another. In contrast, the cells on the plasma-treated membranes proliferated in close contact with other cells, spreading out extensively in sheet-like formations. Since the plasma treatment not only accelerated cell adhesion but also enabled cells to proliferate in the form of sheets resembling biological tissue, we believe that oxygen-plasma treatment is extremely effective for modifying surfaces of materials used for tissue regeneration.     

Biocompatibility evaluation of ePTFE membrane modified with PEG in atmospheric pressure glow discharge

ePTFE membranes were modified by poly(ethylene glycol) having a molecular weight of 600 (PEG-600) in atmospheric pressure glow discharge (APG) plasma treatment. ePTFE membranes were immersed in 1%, 3%, or 5% (w/v) PEG-600 in dehydrated ethanol. PEG-600-penetrated ePTFE membranes were dried in a vacuum to immediately remove ethanol, then treated with APG at 20 kHz and 60-70 W for 15 min and thoroughly washed with ethanol and water. PEG-600-modified ePTFE membranes were analyzed using contact angle measurement, Fourier transform infrared attenuated total reflectance (FTIR-ATR), and scanning electron microscopy (SEM). ePTFE membrane contact angles were reduced after PEG-600 plasma treatment. FTIR-ATR spectra showed an absorption band due to a PEG hydroxyl group (-OH). SEM showed that ePTFE fiber surfaces were uniformly immobilized with PEG-600 and retained their porous structure. A general biological evaluation of the PEG-modified ePTFE membranes showed no cytotoxicity on CHO-K1 cell lines and no hemolytic action. Albumin adsorption on the PEG-modified ePTFE membranes increased with increasing PEG-600 deposited on ePTFE membranes. Fibrinogen adsorption decreased with increasing PEG-600 deposited on ePTFE membranes. gamma-Globulin adsorption did not change before or after PEG plasma modification. 1% and 3% PEG-600 plasma-treated ePTFE only slightly increased platelet adhesion, but adhering platelets evidenced no pseudopod formation. 5% PEG-600-modified ePTFE showed relatively large numbers of platelet adhesion. We concluded that 3% PEG-600-modified ePTFE membrane had the best physical properties and biological compatibility, indicating 3% PEG-600-modified ePTFE membranes exhibit the potential for blood filter application.     

Membrane thickness is an important variable in membrane scaffolds: Influence of chitosan membrane structure on the behavior of cells

Cell and tissue responses to polymeric materials are orchestrated in part by the conformations of adsorbed plasma proteins. Thus, the chemical properties of a polymer membrane that govern protein adsorption behavior can play an important role in determining the biological properties of tissue engineered scaffolds derived from that polymer. In this study, we explored the role of membrane thickness as a factor influencing cell adhesion and proliferation on chitosan membranes with and without covalently attached glycosaminoglycans. Rat mesenchymal stem cells (MSCs) cultured on chitosan membranes of various thicknesses demonstrated significantly improved cell adhesion, spreading and proliferation as membrane thickness was increased. Rat hepatocytes displayed increased spreading on the substrate with increasing membrane thickness, similar to MSCs. Increased thickness reduced the overall crystallinity of the membrane, and the data indicate that the improved cellular responses were likely due to enhanced adsorption of serum vitronectin, presumably due to reduced membrane crystallinity. These results demonstrate that membrane thickness is an important design variable that can be manipulated in chitosan-based scaffolds to achieve enhanced cell spreading, proliferation and function.     

Preparation of blood-compatible hollow fibers from a polymer alloy composed of polysulfone and 2-methacryloyloxyethyl phosphorylcholine polymer

Blood-compatible hollow fibers were successfully prepared from a polymer alloy composed of polysulfone (PSf) and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer. To improve the hydrophilicity, fouling-resistance characteristics, and blood compatibility of the PSf hollow fiber in a hemodialyzer, an MPC polymer that can be blended with PSf was synthesized in order to prepare the polymer alloy (PSf/MPC polymer). The contents of the MPC polymer blended in the PSf were 7 and 15 wt%. The PSf/MPC polymer hollow fiber could be prepared by both wet and dry-wet processing methods. The hollow fiber took an asymmetric structure, that is, the hollow-fiber membrane had a dense skin layer on the porous sponge-like structure. The mechanical strength was higher than that of conventional PSf hollow fibers for hemodialysis. The surface characterization of the PSf/MPC polymer hollow fiber by x-ray photoelectron spectroscopy revealed that the MPC units were concentrated at the surface. The permeability for solutes through the PSf/MPC polymer hollow fibers was measured for 4 h. The permeabilities of both a low-molecular-weight compound and protein were greater than those of the PSf hollow fibers. The amount of adsorbed protein was lower on the PSf/MPC polymer hollow fiber when compared to that of the PSf hollow fiber. Moreover, platelet adhesion was also effectively inhibited on the PSf/MPC polymer hollow fiber. Based on these results, the addition of the MPC polymer to the PSf is a very useful method to improve the functions and blood compatibility of the hollow fiber.     

Effects of hydroxyapatite in 3-D chitosan-gelatin polymer network on human mesenchymal stem cell construct development

Human mesenchymal stem cells (hMSCs) have great potential in bone tissue engineering, and hydroxyapatite (HA), a natural component of human hard tissues, is believed to support hMSC growth and osteogenic differentiation. In this study, two types of biomimetic composite materials, chitosan-gelatin (CG) and hydroxyapatite/chitosan-gelatin (HCG), were fabricated and compared to examine the effects of HA on hMSC adhesion and 3-D construct development. The 2-D membranes were prepared to examine the influence of HA on adhesion efficiency of hMSCs, while 3-D porous scaffolds were produced to investigate the effects of HA on material adsorption properties and 3-D hMSC construct development. HA was found to promote protein and calcium ion adsorption of the 3-D porous scaffolds in the complete tissue culture media. HMSCs exhibited higher initial cell adhesion efficiency to 2-D HCG membranes, and maintained higher proliferation rates in the 3-D porous HCG than CG scaffolds with 3.3 times higher final DNA amount in HCG scaffolds over a 35-day period. Colony forming unit-fibroblast (CFU-F) assays showed that higher percentages of cells maintained their progenicity in the 3-D porous HCG scaffolds over the 35-day culture period. Differentiation assays indicated that the multi-lineage differentiation potential of the hMSCs was preserved in both 3-D porous scaffolds. However, higher alkaline phosphate activity was detected in the 3-D porous HCG scaffolds upon osteogenic induction indicating improved osteogenic differentiation potential. The results demonstrate that enhanced protein and calcium ion adsorption properties of HA in the CG polymer network improve initial cell adhesion and long-term growth, favor osteogenic differentiation upon induction, as well as maintain the progenicity of the 3-D hMSC constructs.