Nerve cells culture from lumbar spinal cord on surfaces modified by plasma pyrrole polymerization

Currently, there are several techniques for modified cell culture surfaces under research to improve cell growth and adhesion. Recently, different methods have been used for surface coating, using biomolecules that enhance cell attachment and growth of nerve cells from spinal cord, such as the use of Poly-DL-Ornithine/Laminin. Plasma-polymerized pyrrole (PPy)-treated surfaces have showed improvement on surfaces biocompatibility with the cells in culture since they do not interfere with any of the biological cell functions. In the present work, we present a novel mouse nerve cell culture technique, using PPy-treated cell culture surfaces. A comparative study of cell survival using Poly-DL-Ornithine/Laminin-treated surfaces was performed. Our results of cell survival when compared with data already reported by other investigators, show that cells cultured on the PPy-modified surface increased survival up to 21?days when compared with Poly-DL-Ornithine/Laminin-coated culture, where 8?days cell survival was obtained. There were electrical and morphological differences in the nerve cells grown in the different surfaces. By comparing the peak ion currents of Poly-DL-Ornithine/Laminin-seeded cells for 8?days with cells grown for 21?days on PPy, an increase of 516% in the Na⁺ current and 127% in K⁺ currents in cells seeded on PPy were observed. Immunofluorescence techniques showed the presence of cell synapses and culture viability after 21?days. Our results then showed that PPy-modified surfaces are an alternative culture method that increases nerve cells survival from lumbar spinal cord cell culture by preserving its electrical and morphological features.     

Blood compatibility of surfaces with superlow protein adsorption

In this work, five self-assembled monolayers (SAMs) and three polymeric brushes with very low fibrinogen adsorption were prepared. The five SAMs are oligo(ethylene glycol) (OEG), phosphorylcholine (PC), oligo(phosphorylcholine) (OPC), and two mixed positively and negatively charged SAMs of SO(3)(-)/N(+)(CH(3))(3) (SA/TMA) and COO(-)/N(+)(CH(3))(3) (CA/TMA). Three polymer brushes were prepared on gold surfaces via surface-initiated atom transfer radical polymerization (ATRP) using three monomers, sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), and oligo(ethylene glycol) methyl ether methacrylate (OEGMA). Surface plasmon resonance (SPR) measurements show that although all of these surfaces are "nonfouling" to fibrinogen adsorption from buffer solution, their protein adsorption from undiluted human blood plasma varies widely. Polymer brushes exhibit much lower protein adsorption from plasma than any of the five SAMs tested. However, platelet adhesion measurements on plasma-preadsorbed surfaces show that all of these surfaces have very low platelet adhesion. Clotting time measurements using recalcified platelet poor plasma (PPP) incubation with the eight types of surfaces show that they do not shorten clotting times. Linear polymers of polySBMA and polyCBMA with similar molecular weights were also synthesized and characterized. In the presence of polyCBMA linear polymers, the clotting time of PPP was prolonged and increased with the concentration of the polymer, while no anticoagulant activity was observed for the polySBMA or PEG polymers. The unique anticoagulant activity of polyCBMA, as well as its high plasma protein adsorption resistance, makes polyCBMA a candidate for blood-contacting applications.     

不同分子量的PEG改性聚氨酯以及表面的血液相容性研究

目的 研究经不同分子量PEG修饰后聚氨酯(PU)膜的血液相容性.方法 采用两步法将不同分子量的PEG(400、800、1 000和2 000)修饰到PU膜上,获得了不同分子量PEG修饰的PU膜,进行凝血因子试验、复钙时间的测定、血小板计数和血细胞计数,实验的数据行统计分析.结果 随着修饰剂PEG分子量的增加,复钙时间延长、凝血因子消耗量和血小板的吸附量降低.结论 经过修饰的PU膜和血液相容性得到了明显改善,并且随着分子量的增加,这种相容性也在增加,PEG的分子量为1 000时,这种效果达到最好.     

Biomedical applications of plasma polymerization and plasma treatment of polymer surfaces

Thin polymer films obtained by plasma polymerization usually show good biocompatibility when compared to classical biomaterials such as Silastic. The thicknesses of these films (from several hundreds of Å to several μm) make them suitable for the purpose of changing the surface properties of the substrate without altering its bulk properties. Both the above features together indicate the possible Biomedical use of plasma polymerization as well as plasma treatment of polymer surfaces processes. The purpose of this review is to present the most significant efforts to develop such applications with the specification of particular fields where these efforts are directed.     

Blood compatibility of polypropylene surfaces in relation to the crystalline-amorphous microstructure

Blood-contacting properties of polypropylene surfaces with different crystalline states at the surface layer were examined in terms of plasma protein adsorption and changes in cytoplasmic free Ca²⁺ levels in platelets. Though the wettability of polypropylene surfaces was almost constantly independent from the surface layer crystallinity and interlamellar spacing, an increase in adhesiveness was observed with decreasing surface layer crystallinity and interlamellar spacing. It is suggested that the surface properties of the sheets varied in relation to the crystalline-amorphous microstructure. Minimum magnitudes in albumin and fibrinogen adsorption were observed on the polypropylene surface with a particular surface layer crystallinity (c. 55 wt%). A decrease in interlamellar spacing resulted in enhancing albumin adsorption and diminishing fibrinogen adsorption. Transient phenomena in plasma protein adsorption were observed on their surfaces with a plasma concentration. It is considered that the polypropylene surface with a particular crystalline-amorphous microstructure reduces the denaturation of adsorbed proteins. An increase in cytoplasmic free Ca²⁺ levels in platelets was prevented at the polypropylene surface with a surface layer crystallinity of 55 wt%: the particular crystalline-amorphous microstructure of such apolar surfaces as polypropylenes acts to reduce platelet activation. Thus, it is concluded that the blood compatibility of polypropylene surfaces is greatly improved by controlling a crystalline-amorphous microstructure at the surface layer.     

Fouling and non-fouling surfaces produced by plasma polymerization of ethylene oxide monomer

This paper presents the results of plasma polymerization using diethylene glycol dimethyl ether as a precursor in a capacitively coupled radio frequency system. The chemical structure of the coatings was characterized using several analysis techniques (X-ray photoelectron spectroscopy, Fourier transform-infrared spectroscopy, ellipsometry), while the biological response of these coatings has been tested by protein adsorption and cell culture experiments. The modulation of the input plasma power controls the concentration of polyethylene oxide groups in the coatings and allows the production of films with opposite protein and cell repellent properties. The study of the stability of these coatings in different media (water, acetone, phosphate-buffered saline) reveals that these films could be involved in classical lift-off processes for the production of patterned surfaces.     

The chemiluminescence response of neutrophils on polymer surfaces made by glow discharge plasma polymerization

Polypropylene tubes were coated with different polymers made by glow discharge plasma polymerization. Isolated human blood neutrophils were allowed to interact with the polymer surface and the chemiluminescence response of the cells was recorded as a measure of oxidative activation. The polymers represented surfaces that differed markedly with respect to charge, hardness, and wettability. We found that all polymers stimulated the chemiluminescence response in neutrophils differently; when preincubation with human serum albumin (HSA) there was a general reduction of the chemiluminescence response particularly on one of the positively charged surface 1, 2-diamino-cyclohexane (DACH). Addition of a soluble stimulus, the chemoattractant formylmethionyl-leucylphenylalanine (FMLP), to the cells caused a dramatic increase in the response on one of the hydrophobic surface hexamethylenedisiloxane (HMDSO). However, there was also a pronounced reduction in the response on polymers with acrylic acid (AA). The response was normalized after addition of HSA. Taken together, the chemiluminescene response of the neutrophils interacting with the polymer surfaces differed with regard to the type of surfaces. When HSA and FMLP were added a larger difference in the response was found. Our results showed that the activation of human neutrophil granulocytes influenced by different polymer surfaces, followed unspecific different patterns which were someway related to the specific characteristics of the polymer and from this point we came to similar conclusions made by Kaplan et al. (J. Biomater. Res. 28, 377 (1994)), that it is difficult to extrapolate any activation mechanisms from one material to another. The reproducibility of the reaction patterns, the importance of neutrophil activation, and the relative simplicity of the method indicates, however, that the mechanisms of neutrophil activation at biomaterial surface is well worth further research.     

Blood cell and plasma protein repellent properties of star-PEG-modified surfaces

The implantation of biomaterials, medical devices or prostheses can instigate a rejection response or initiate an undesirable adsorption of plasma proteins, as well as blood cells on the implant surface, thus triggering diverse defense mechanisms against the supposed pathologic invader. The extent of this inflammatory reaction depends in part on the biocompatibility of the used materials or coatings. Although adsorption and coagulation responses can appear during the total in vivo lifetime of the implant, they are initially and crucially formed within the first 2-4 weeks of implantation. This early phase is of decisive importance for the consecutive in-growth and healing process. The present study was intended to elucidate the effects of blood contact to surfaces modified with reactive six-arm star-shaped poly(ethylene glycol-stat-propylene glycol) pre-polymers (Star PEG). Taken together, for Star-PEG-covered substrates we could demonstrate a profound reduction of various blood-biomaterial interactions compared to non-coated substrates, indicating the promising potential of this material as future coating for biomaterials with blood contact.     

Blood cell and plasma protein repellent properties of Star-PEG-modified surfaces

The implantation of biomaterials, medical devices or prostheses can instigate a rejection response or initiate an undesirable adsorption of plasma proteins, as well as blood cells on the implant surface, thus triggering diverse defense mechanisms against the supposed pathologic invader. The extent of this inflammatory reaction depends in part on the biocompatibility of the used materials or coatings. Although adsorption and coagulation responses can appear during the total in vivo lifetime of the implant, they are initially and crucially formed within the first 2–4 weeks of implantation. This early phase is of decisive importance for the consecutive in-growth and healing process. The present study was intended to elucidate the effects of blood contact to surfaces modified with reactive six-arm star-shaped poly(ethylene glycol-stat-propylene glycol) pre-polymers (Star PEG). Taken together, for Star-PEG-covered substrates we could demonstrate a profound reduction of various blood–biomaterial interactions compared to non-coated substrates, indicating the promising potential of this material as future coating for biomaterials with blood contact.     

Competitive plasma protein adsorption on modified polymer surfaces monitored by quartz crystal microbalance technique

This paper describes the effects of photochemical modifications of polymer surfaces on the competitive adsorption of serum proteins and cell adhesion (hepatoma cell line HepG2, L929 fibroblasts and others). The UV modification of polystyrene, poly(methylmethacrylate) and polycarbonate alters the physico-chemical properties of these polymers in a way that allows the formation of micrometer scaled cellular patterns in vitro by controlling the composition and properties of the protein adsorbate. Using a quartz microbalance technique, capable to extract viscoelastic data in addition to the mass load of the polymer coated sensor, we have demonstrated the importance of the thickness and the viscosity of an albumin adsorbate for the observed cell adhesion in vitro. The quantity and viscosity of surface bound albumin on polystyrene, being a cell repellent material in its native state, is lowered when the surface is exposed to UV of λ = 185 nm in air prior to the contact with albumin solutions or cell culture media. This promotes the deposition of cell adhesion proteins and explains the observed cell patterns. Apart from this special application the described quartz microbalance with dissipation monitoring provides a useful tool for general biocompatibility studies based on surface phenomena of biomaterials.     

Competitive plasma protein adsorption on modified polymer surfaces monitored by quartz crystal microbalance technique

This paper describes the effects of photochemical modifications of polymer surfaces on the competitive adsorption of serum proteins and cell adhesion (hepatoma cell line HepG2, L929 fibroblasts and others). The UV modification of polystyrene, poly(methylmethacrylate) and polycarbonate alters the physico-chemical properties of these polymers in a way that allows the formation of micrometer scaled cellular patterns in vitro by controlling the composition and properties of the protein adsorbate. Using a quartz microbalance technique, capable to extract viscoelastic data in addition to the mass load of the polymer coated sensor, we have demonstrated the importance of the thickness and the viscosity of an albumin adsorbate for the observed cell adhesion in vitro. The quantity and viscosity of surface bound albumin on polystyrene, being a cell repellent material in its native state, is lowered when the surface is exposed to UV of lambda = 185 nm in air prior to the contact with albumin solutions or cell culture media. This promotes the deposition of cell adhesion proteins and explains the observed cell patterns. Apart from this special application the described quartz microbalance with dissipation monitoring provides a useful tool for general biocompatibility studies based on surface phenomena of biomaterials.     

Blood compatibility of hydrophilic polymers

Blood compatibility has been studied for hydrophilic polymers such as poly(vinyl alcohol) (PVA), its derivative, and polyethylene grafted with water-soluble monomers. The surfaces in contact with electrolyte solutions have been characterized by measuring the zeta potentials. The study of plasma protein adsorption on these polymers has revealed that bovine serum albumin as well as bovine serum fibrinogen adsorbs to a lesser extent as the hydrophilicity of the polymers increases. Platelet deposition and fibrin formation, examined using platelet-rich plasma, have been found to take place less significantly on PVA as well as sodium acrylate- and acrylamide-grafted polyethylene than on nongrafted and acrylic acid-grafted polyethylene. Ex vivo experiments with canine whole blood have shown that formation of thrombus on PVA is less than on siliconized glass but increases upon heat treatment which reduces the hydrophilicity. When PVA tubes of about 1 mm diameter are anastomosed to the carotid artery of rat, the patency rate is found to depend strongly on the anastomotic technique. From the results on the zeta potential and the experiments in vitro and ex vivo it can be concluded that the material having a surface from which solvated, neutral chains are extended into the outer aqueous phase may exhibit excellent resistance to thrombus formation.     

Blood compatibility of photografted hydrogel coatings

In this work, we have evaluated the haemocompatibility of different surface modifications, intended for biomaterials and bioanalytical applications. Polystyrene slides were coated with thin hydrogel films by self-initiated photografting and photopolymerization (SIPGP) of four different monomers. The hydrogel surface modifications were thoroughly characterized and tested for their protein resistance and ability to resist platelet adhesion and activation of the coagulation system. There was very little protein adsorption from human plasma on the hydrogels prepared from poly(ethylene glycol) methacrylate and 2-hydroxyethyl methacrylate. Platelet adhesion tests performed under both static and flow conditions showed that these coatings also demonstrated very high resistance towards platelet adhesion. A small amount of platelets were found to adhere to hydrogels formed from ethylene glycol methyl ether methacrylate and 2-carboxyethyl methacrylate. The polystyrene substrates themselves facilitated large amounts of platelet adhesion under both static and flow conditions. Utilizing a novel setup for imaging of coagulation, it was confirmed that none of the hydrogel surfaces activated the coagulation system to any great extent. We suggest that this simple fabrication method can be used to produce hydrogel coatings with unusually high blood compatibility, suitable for demanding biomaterials applications.     

Blood plasma proteins on polyurethane and alkylsiloxane plasma-treated polyurethane surfaces. Dynamic approach by stimulus-response technique

In this study, interactions of blood proteins (i.e. albumin and fibrinogen) with polyurethane biomaterial surfaces were investigated in an in vitro bead column test circuit using a stimulus-response technique. The dynamic sorption process of radiolabelled proteins on the surfaces was followed by detecting the radioactivity at the exit stream of the column, which was the response of a pulse stimulus at the inlet. The mathematical model was described and solved using ‘parameter estimation by cybernetic moment technique’, and the adsorption rate constants of plasma proteins on different biomaterial surfaces were calculated. By evaluation of the response curves with standard and cybernetic moment techniques, the following results were obtained. Albumin and fibrinogen adsorption is competitive, and the competition is strongly dependent upon the surface characteristics of the biomaterial. Preadsorption or preferential adsorption of albumin decreases the fibrinogen adsorption, and there-fore increases the biocompatibility of material surface. Adsorption of blood plasma proteins are irreversible. The moment technique can also be used for the evaluation of stimulus-response data of biological systems, to determine the process parameters.     

Blood compatibility of surfaces with immobilized albumin-heparin conjugate and effect of endothelial cell seeding on platelet adhesion.

Endothelial cell (EC) seeding significantly improves the blood compatibility of artificial surfaces. Although a coating consisting of albumin and heparin (alb-hep) is a suitable substrate for seeded ECs, binding of ECs to the substrate further improves when small amounts of fibronectin are present in the alb-hep coating. Alb-hep conjugate was immobilized on carbon dioxide gas plasma-treated polystyrene (PS-CO(2)), thereby significantly increasing the recalcification time of blood plasma exposed to this surface. Furthermore, surface-immobilized alb-hep conjugate inhibited exogenous thrombin. Heparin activity was reduced by adding fibronectin on top of a monolayer of alb-hep conjugate, but not by simultaneous coating of fibronectin and alb-hep conjugate. Coating of PS-CO(2) with alb-hep conjugate significantly decreased contact activation (FXII activation). The number of platelets deposited from blood plasma on PS-CO(2) coated with alb-hep conjugate was twice as high as on PS-CO(2) coated with albumin. Addition of fibronectin to alb-hep conjugate-coated PS-CO(2) had no significant effect on the number of adhered platelets. Seeding of the substrates with ECs significantly reduced the number of adhered platelets under stationary conditions. Platelets deposited onto endothelialized surfaces were primarily found on endothelial cell edges, and sparingly on areas between ECs. In conclusion, alb-hep conjugate-coated surfaces display anticoagulant activity. ECs adhering to and proliferating on this coating significantly decrease the number of platelets which adhere to the surface. Therefore, alb-hep conjugate-coated surfaces form a suitable substrate for seeding of ECs in low density. Although application of fibronectin on top of the coating decreases the anticoagulant activity to some extent, it might be useful in view of the improved adherence of ECs to the coating.     

Micropatterning of biomedical polymer surfaces by novel UV polymerization techniques

The "living" radical polymerization with an iniferter was used to create micropatterned biomedical surfaces. Novel, photosensitive biomedical polymers were created by the incorporation of dithiocarbamate groups from iniferters. A second monomer layer was then irradiated onto the photosensitive polymer substrate created with the iniferter to form a copolymer. Patterns were created on the films by application of modified microfabrication-based photolithographic techniques. The technique was used to create patterns with depths from 5 to 80 microm. In addition, various polymers were incorporated, including polyethylene glycol methacrylates, styrene, and methacrylic acid, to synthesize regions with different physico-chemical properties. Applications include novel surfaces for biosensors and biomaterials for the selective adhesion of cells and proteins.     

胆红素吸附柱的血液相容性

背景：胆红素吸附柱在临床上用于清除患者体内高浓度的胆红素,在使用过程中与血液有大面积长时间的接触,对其血液相容性的评价具有重要意义。 目的：评价一个树脂类胆红素吸附柱的血液相容性。 方法：将待测胆红素吸附柱及对照已经上市胆红素吸附柱中的树脂微粒置于硅化玻璃管中,以空硅化玻璃管作为空白对照。向3组硅化玻璃管中加入人新鲜血浆,于37 ℃隔水培养箱的旋转培养器上以30 r/min的速率旋转,3 min后吸取出血浆,用于凝血酶原时间、活化部分凝血活酶时间、纤维蛋白原含量及总补体活性CH50的检测,实验重复3次。 结果与结论：与空白对照组比较,对照组与实验组凝血酶原时间、活化部分凝血活酶时间延长,纤维蛋白原含量减少,CH50减小,但随着树脂微粒与新鲜血浆接触次数的增多,这种变化越来越小。与对照组比较,实验组凝血酶原时间、活化部分凝血活酶时间、纤维蛋白原含量的3次检测数值更接近于空白对照组;实验组CH50在3次检测中先大于对照组,再与对照组相当,最后小于对照组。表明：①随着与新鲜血浆接触次数的增多,树脂微粒的血液相容性得到改善。②待测胆红素吸附柱中树脂微粒对凝血系统的影响小于对照,对补体系统的影响在预吸附过程中小于对照,但其相容性的改善慢于对照。