Surface engineering of biomaterials with plasma techniques

In this study, 3 types of plasma techniques, i.e. plasma modification, plasma deposition and plasma followed by grafting reaction, are used for the fabrication of tools, medical devices and biomaterials. Depending on purpose, bioadhesion of cells and biomolecules is either looked for or avoided. Since the mechanisms of bioadhesion depend on the characteristics of the surface (hydrophilic or hydrophobic), modifying the surface by a treatment will alter the bioadhesion. These treatments are developed for the anti-fouling process, the sterilisation and the improvement of the formation of biofilms. They have also proved useful for the synthesis of biomimicking devices.     

Comparison of Bone Marrow Stromal Cell Behaviors on Poly(caprolactone) with or without Surface Modification: Studies on Cell Adhesion,Survival and Proliferation

Poly(caprolactone) (PCL) is a promising biodegradable polymer for tissue engineering. However, intrinsically poor cell-adhesive properties of PCL may limit its application. In this study, the PCL film surface was modified with RGDC peptide by a chemical immobilization procedure. Furthermore, bone marrow stromal cell (BMSC) behaviors including attachment, spreading, focal adhesion formation, focal adhesion kinase (FAK) activation, apoptosis and proliferation when cultured on the modified PCL films were investigated. Our results demonstrated that PCL with RGD modification promoted initial BMSC attachment, spreading and focal adhesion formation. At a later time point (12 h), BMSC attachment on both RGD peptide-modified PCL and PCL-NH₂ films significantly increased compared to untreated PCL films. Importantly, FAK phosphorylation was significantly increased only on the films with RGD-modified films, not on the PCL-NH₂ films, demonstrating that PCL with RGD modification had an advantage in initiating the specific integrin-mediated signal transduction and might play an important role in the subsequent retardation in cell death and enhancement in cell proliferation. The present results provide more evidence that functionalizing PCL with RGD peptides may be a feasible way to improve the interaction between BMSC and PCL substrate, which is important in tissue engineering.     

聚二甲基硅氧烷／聚氨酯共混体系的表面结构和抗凝血性能

本文用光电子能谱（ＸＰＳ）、衰减全反射红外光谱（ＡＴＲ）及扫描电子显微镜（ＳＥＭ），研究了一系列不同结构聚二甲基硅氧烷／聚氨酯（ＰＤＭＳ／ＰＵ）共混体系的表面化学组成和表面形态结构．研究结果表明：共混物具有织态状微相分离结构；在垂直表面方向上化学组成具有不均匀性，软段和聚二甲基硅氧烷在表面富集，氧原子优先在表面层中分布．对吸附血小板形态观察及再钙化时间的测定表明，聚二甲基硅氧烷／聚氨酯共混物的抗凝血性能优于聚氨酸，和共混物的表面形态结构和化学组成相关。     

Plasma modification of PMMA films: surface free energy and cell-attachment studies

The surface of a material is the most important part determining the acceptance by and compatibility with the environment. In many cases, although the bulk properties are excellent for a specific application, the surface may require to be modified and engineered in the desired direction. This is especially important for materials used in biological media, since the surface charge, hydophilicity and wettability are important for thrombosis formation, cell attachment or cell proliferation. In this study, poly(methyl methacrylate) films were prepared by solvent casting and their surfaces were modified by oxygen plasma treatment by applying powers of 20, 100 and 300 W. The effects of surface chemistry alterations on hydophilicity, work of adhesion, surface free energy and cell adhesion were examined. Cell attachment and proliferation are especially important for the materials used for tissue-engineering purposes. The results demonstrated that there is an optimum value for hydrophilicity and surface free energy which enhance cell attachment.     

Effects of an Intervention Aimed at Reducing Night Waking and Signaling in 6- to 12-Month-Old Infants

This quasi-experimental one-group pre- and posttest pilot study evaluated an intervention aimed at reducing night waking and signaling for infants between 6 and 12 months of age. Thirty-nine healthy infants and their parents were recruited. Thirty-five infants completed the intervention and data collection. Both parents participated in a group teaching session with telephone follow-up for 2 weeks. Actigraphy and sleep diary data were collected at baseline and 6 and 16 weeks postintervention. We hypothesized a decrease in length and number of infant waking and crying periods and an increase in longest night sleep and nap time. Following the intervention, infants had significantly reduced length of night crying and number of wakes and longer night sleep periods. The intervention warrants evaluation with a randomized controlled design.     

单壁碳纳米管无纺膜表面的PEG修饰及蛋白质吸附研究

碳纳米管是一种纳米尺度的新型碳材料，具有独特的物理、化学性质。近年来，碳纳米管在生物医学领域的潜在应用前景已经引起科学界和产业界的极大兴趣与关注。与蛋白质分子的非特异性结合是碳纳米管应用于生物系统中必须考虑的基本问题之一。本研究应用扫描电镜和酶联免疫法作为评价方法，定性和定量地分析了血浆中重要凝血因子纤维蛋白原在单壁碳纳米管薄膜表面的非特异性吸附行为；同时，采用聚乙二醇（PEG）分子对单壁碳纳米管薄膜（SWNT膜）进行了表面修饰，通过x．光电子能谱（XPS）对材料表面的化学组成进行了表征，并初步探讨了PEG修饰对纤维蛋白原分子在SWNT膜表面非特异性吸附的阻止作用。实验结果表明，纤维蛋白原分子在SWNT膜表面有强烈的非特异性结合，吸附于薄膜表面的纤维蛋白原分子仍然保有自身的免疫原性。SWNT膜表面可以被PEG分子修饰，连接在薄膜表面的PEG分子可以在一定程度上抑制一定浓度范围内的纤维蛋白原分子的非特异性结合。     

Effect of Ammonia Plasma Treatment on the Properties and Cytocompatibility of a Poly(L-Lactic Acid) Film Surface

Ammonia plasma treatment is an efficient method to modify the surface of polymeric biomaterials to improve their hydrophilicity and biocompatibility. In this study, poly(L-lactic acid) (PLLA) films were treated with ammonia plasma to investigate the effects on the surface properties and cytocompatibility. Surface morphologies of the films were observed with atomic force microscopy (AFM) and the surface roughness was analyzed with the software attached to the AFM. Mass loss density and contact angles associated to plasma treatment power and time were also studied. The stability of the treated films was evaluated by testing the contact angle change. The cytocompatibiliy was evaluated by cell adhesion, proliferation and cell cycle. The results showed that the surface morphology and roughness of the treated PLLA surfaces increased with treatment power. The mass loss density increased with plasma treatment power and time. With increasing treatment power and time, the resulting amino group density on treated PLLA film surface increased first and decreased later, while the contact angle showed an opposite trend. The contact angle of the treated films increased with storage time and returned to its original value after about 2 weeks. The cell experiments indicated that promotion of cell adhesion and proliferation were significantly improved on the treated PLLA surfaces.     

Bioactivity and Platelet Adhesion Study of a Human Thrombomodulin-Immobilized Nitinol Surface

Nitinol is a newly developed biomaterial that is gaining popularity in many biomedical applications. It has been reported that nitinol would not induce an inflammatory response and repulsion by the immunization after implantation in the human body. Besides, nitinol is a kind of shape memory alloy, which can memorize shapes at different temperatures. This can improve the convenience in surgery. However, nitinol has poor blood compatibility, so that further modification was needed to improve the antithrombogenicity. Human thrombomodulin (hTM), an endothelial-cell-associated glycoprotein, can be considered as a natural potent anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. In this study, the surface of nitinol was pre-activated by utilizing silanization with amino-terminated silane. The incorporated amino groups were available for the subsequent covalent immobilization of hTM by 2,4,6-trichloro-1,3,5-triazine (TCT), the coupling reagent. The surface density of immobilized hTM was determined by the Bradford method. The bioactivity of immobilized hTM and blood compatibility of various nitinol substrates were evaluated by the protein C activation assay and platelet adhesion test. It was observed that the immobilized hTM still had the ability to enhance protein C activation, though its activity was lower than the free hTM in solution. Furthermore, the platelet adhesion test showed that only a few platelets were adhered on the hTM-immobilized nitinol substrate. Therefore, the immobilization of thrombomodulin onto nitinol substrate could improve the blood compatibility of nitinol and might have the potential of application in antithrombogenic medical applications.     

Correlation between In Vitro Complement Deposition and Passive Mouse Protection of Anti-Pneumococcal Surface Protein A Monoclonal Antibodies

The shortcomings of the licensed polysaccharide-based pneumococcal vaccine are driving efforts toward development of a protein-based vaccine that is serotype independent and effective in all age groups. An opsonophagocytic killing assay (OPKA) is used to evaluate the antibody response against polysaccharide-based pneumococcal vaccines. However, the OPKA is not reliable for noncapsular antigens. Thus, there is a need to develop an in vitro surrogate for protection for protein vaccine candidates like pneumococcal surface antigen A (PspA). PspA is a serologically variable cell surface virulence factor. Based on its sequence, PspA has been classified into families 1 (clade 1 and 2), 2 (clades 3, 4 and 5), and 3 (clade 6). Here, we report the characterization of 18 IgG anti-PspA monoclonal antibodies (anti-PspA^hkR36A MAbs) generated from mice immunized with heat-killed strain R36A (clade 2). An enzyme-linked immunosorbent assay (ELISA)-based analysis of the reactivity of the MAbs with recombinant PspAs from the 6 clades indicated that they were family 1 specific. This was confirmed by flow cytometry using a hyperimmune serum generated against PspA from R36A. Eight MAbs that bind at least one clade 1- and clade 2-expressing strain were evaluated for complement deposition, bactericidal activity, and passive protection. The anti-PspA^hkR36A MAb-dependent deposition of complement on pneumococci showed a positive correlation with passive protection against strain WU2 (r = 0.8783, P = 0.0041). All of our protective MAbs showed bactericidal activity; however, not all MAbs that exhibited bactericidal activity conferred protection in vivo. The protective MAbs described here can be used to identify conserved protection eliciting B cell epitopes for engineering a superior PspA-based vaccine.     

Surface characterization and platelet compatibility evaluation of surface-sulfonated chitosan membrane

The effect of various sulfonated derivatives of chitosan on platelet activation and blood coagulation was examined. The surface properties of artificial biomaterials have been thought as the key factors which mediate the interactions between the biological environment and biomaterial itself. In this study, the sulfonation was directly performed on the chitosan membrane surface. The chitosan membrane was surface-sulfonated by reactions with sulfur-pyridine trioxide complex (SO₃/pyridine) in H₂O solution and N,N-sulfur-dimethylformamide trioxide complex (SO₃/DMF) in DMF.Blood compatibility was evaluated by an in vitro platelet adhesion assay. The surface reaction of SO₃/pyridine in aqueous acid medium yields N,O-sulfated chitosan with cationic NH⁺ ₃ groups. After neutralization, this surface has been shown to induce a low degree of platelet adhesion and activation. When the surface-sulfonation is performed in an aqueous alkaline medium, although the degree of sulfonation is lower than the samples above, the N-sulfated chitosan significantly reduced the adhesion and activation of platelets. For the acidic SO₃/DMF reaction system, N,O-sulfated chitosan was obtained with a high extent of sulfonation and cationic NH3+groups. On this surface fully spread platelets and some platelet aggregates were found instead. This may be attributed to the ionic interactions between the platelets membrane surface and the cationic groups on the modified chitosan membrane.     

Plasma lithography — thin-film patterning of polymeric biomaterials by RF plasma polymerization I: Surface preparation and analysis

Plasma lithography, combining plasma deposition with photolithography, is described as a versatile method to manufacture all-polymeric substrates with thin-film patterns for applications in biomedical engineering. Patterns of a hydrophobic fluorocarbon plasma polymer with feature sizes between 5 and 100 μm were deposited on a base substrate in a lift-off process; an intermediate tetraglyme plasma polymer layer provides non-fouling properties to the base substrate. Careful analysis of critical process parameters identified the narrow window of process conditions that led to the formation of functional surface patterns. High pattern fidelity, aspect ratios, and resolution of the patterns are demonstrated by atomic force microscopy. Electron spectroscopy for chemical analysis (ESCA) and secondary ion mass spectroscopy (SIMS) were used to characterize the surfaces, showing good retention of the original chemical structure of the pattern components throughout the process. SIMS imaging was used for specific chemical imaging of the components. Potential applications for the patterned polymer films, e.g., for studying cell behavior in vitro in dependence of shape and size of adhering cells, are discussed.     

Rapid and Efficient Assembly of Functional Silicone Surfaces Protected by PEG: Cell Adhesion to Peptide-Modified PDMS

While silicone elastomers generally have excellent biomaterials properties, their hydrophobicity can elicit undesired local biological responses through adsorption and denaturation of proteins. Surface-bound poly(ethylene glycol) (PEG) can ameliorate the situation by preventing contact between the external biology and the silicone elastomer. It is further possible to manipulate the biocompatibility of the surface by linking peptides, proteins or other biological entities to the PEG. Previous synthetic approaches to PEG-protected surfaces are compromised by issues of reproducibility. We describe two rapid and efficient approaches to silicone surface modification by PEG-linked adhesion peptides that overcome this problem: SiH groups are introduced throughout a silicone elastomer during elastomer synthesis or only at the surface after cure; then, in either case, protein-repellent PEG brushes at the surface are introduced by hydrosilylation to give surfaces that can be stored for extensive periods of time without degradation. Activation of the free alcohol with an NSC group followed by immediate conjugation to relevant biological molecules occurs in high yields, as shown for RGDS and GYRGDS. High surface grafting density of the peptides was demonstrated using radiolabeling techniques. Biological activity was demonstrated by a 5-fold increase in cell adhesion on the peptide-modified surfaces when compared to unmodified PDMS control surfaces.     

HPVL1保守序列肽段诱导多型别HPV抗体的初步研究

为探索一段人类乳头瘤病毒(HPV)L1蛋白中长12个氨基酸残基的保守序列是否能诱导产生多型别HPV抗体,我们通过B细胞表位预测和多序列对比,筛选出一条HPV L1蛋白保守肽段,并人工合成此肽段,加弗氏佐剂后免疫家兔,对照组只用弗氏佐剂。先用ELISA的方法检测免疫家兔血清中抗体滴度;再用免疫细胞化学、免疫细胞荧光、Western blot和免疫组织化学的方法检测此抗血清与HPV阳性的宫颈癌细胞株和宫颈组织的反应情况。结果发现,用ELISA法检测抗血清效价在1∶25600以上,而免疫细胞化学、细胞免疫荧光、Western blot和免疫组织化学等方法均检测出抗血清能与多型别HPV进行反应,而对照组呈阴性。此结果表明,该多肽可诱导产生针对多型别HPV的抗体,对于研发制备HPV、HPV L1诊断试剂盒有重要意义。     

Modification of gold surface by grafting of poly(ethylene glycol) for reduction in protein adsorption and platelet adhesion

Gold surfaces were first treated in an alkanethiol solution to form self-assembled monolayers (SAMs). The thiolated Au surface was then subjected to Ar plasma pretreatment, followed by air exposure and UV-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA) macromonomer. In comparison with the 3-mercaptopropionic acid-2-ethylhexyl ester (MPAEE) SAM, the (3-mercaptoproply)trimethoxysilane (MPTMS) SAM on Au exhibited higher stability under the conditions of Ar plasma pretreatment. The graft concentration of the PEGMA polymer on SAMmodified Au surface increased with increasing PEGMA macromonomer concentration and UV-graft polymerization time. The modified-Au surfaces were characterized by X-ray spectroscopy (XPS), atomic force microscopy (AFM), and water contact angle measurement. The Au surface with a high concentration of grafted PEGMA polymer could completely repel protein adsorption and platelet adhesion.     

Surface plasma modification and tropoelastin coating of a polyurethane co-polymer for enhanced cell attachment and reduced thrombogenicity

Polymers currently utilized for dermal and vascular applications possess sub-optimal biocompatibility which reduces their efficacy. Improving the cell-binding and blood-contacting properties of these polymers would substantially improve their clinical utility. Tropoelastin is a highly extensible extracellular matrix protein with beneficial cell interactive and low thrombogenic properties. We transferred these benefits to the polyurethane block copolymer Elast-Eon E2A through a specific combination of surface plasma modifications and coating with human tropoelastin. The cell-binding activity of bound tropoelastin was modulated by ion implantation of the underlying polymer, and correlated with surface hydrophobicity, carbon and oxygen content. This combined treatment enhanced human dermal fibroblast (HDF) and human umbilical vein endothelial cell (HUVEC) attachment, cytoskeletal assembly and viability, combined with elevated PECAM-1 staining of HUVEC cell junctions. The thrombogenicity of the polymer was ameliorated by tropoelastin coating. We propose that a combination of metered plasma treatment and tropoelastin coating of Elast-Eon can serve to improve the biological performance of implantable devices such as vascular conduits.     

Changes in the Content of Protein and Lipid Oxidative Modification Products in Tumor Tissue at Different Stages of Lung Cancer

The content of LPO products (conjugated dienes, MDA) and products of oxidative modification of proteins (protein carbonyl derivatives) is reduced in tumor tissue in comparison with normal tissue and varies depending on the disease stage.     

Surface modification of the polymers present in a polysulfone hollow fiber hemodialyser by covalent binding of heparin or endothelial cell surface heparan sulfate: Flow characteristics and platelet adhesion

The present study addresses the problem of simultaneous surface modification of various polymers, i.e. polysulfone (PSU), polycarbonate (PC), and polyurethane (PU), which constitute the Ultraflux AV 600 S® hollow fibre hemodialyser. An investigation was first made into six different chemical routes aimed at introducing carboxyl groups onto the surface of PSU, PC, and PU model polymers to which heparin (HE) or endothelial cell surface heparan sulfate (ESHS) was covalently bound via the reaction of residual amino groups and a coupling reagent. Carboxyl groups were introduced using three specific reactions based on their nucleophilic or electrophilic introduction into aromatic repeating units of the polymers and three non-specific carboxylation reactions, i.e. UV, heat or redoxactivation via nitrene or radical species. Concentrations of 1-20 nmol COOH groups per cm^-2 led to HE or ESHS surface concentrations corresponding to one or several layers. Two nonspecific carboxylation reactions followed by HE- or ESHS-coupling provided the lowest change in membrane pore structure according to cut off, clearance (urea, phosphate, maltose), ultrafiltration, and diafiltration assessments. In some cases the introduction of excess negatively-charged carboxyl groups and HE improved the flux properties of the modified membranes. The various methods were applied to the dialysis module. Platelet adhesion was not observed in the case of the ESHS-coating of PSU membrane at shear rates of 1050 s^-1, whereas HE and subendothelial matrix showed 56 and 100% coverage, respectively, under similar conditions. The coating of PSU or of other highflux membranes by ESHS appears a promising method for improving membrane properties and to generate biocompatibility characteristics similar to those of natural blood vessels, i.e. inertness to platelet adhesion and no level effects for complement and intrinsic coagulation cascade activation. The ESHS coating may be used without anticoagulants.     

Electrophoretic Deposition (EPD) of Lectin in the Presence of New Glycopolymers Aiming at Facile Detection of Carbohydrate–Protein Interactions

Atom transfer radical polymerization of allyl methacrylate followed by a thiol‐ene click reaction with 1‐thio‐β‐D‐glucose tetraacetate is performed to afford a new type of glycopolymers via deacetylation. Their interactions with a sugar‐binding protein are evaluated by electrophoretic deposition (EPD) procedures as well as fluorescence measurements. The deposition amount of concanavalin A (conA) in the presence of glucose or galactose measured by EPD is remarkably different from that in the presence of the synthesized glycopolymer, even though the concentration of sugar residue is the same. The deposition amount, as well as the electric current that flows during the EPD, is higher in the presence of the polymer, which confirms that interaction of conA with the glycopolymer is detected by the EPD procedure.     

三嵌段高分子骨组织工程支架材料的表面多肽修饰与细胞粘附性研究

目的用甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸对三嵌段高分子骨组织工程支架材料进行表面修饰，并检测其细胞粘附性。方法利用异双官能交联剂将甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸多肽固定在聚丙交酯／乙交酯／天冬氨酸-聚乙二醇材料表面，并进行X线光电子分光镜检测和表面接触角测定；体外培养骨髓基质细胞，接种至表面修饰的材料上，测定细胞粘附力，并和未修饰材料对比。结果固定交联剂和多肽后X线光电子分光镜检测示硫元素的含量分别为0．3％和0．2％；硫元素的结合能是164eV和163．9eV；表面接触角为60．2±2．364度；细胞粘附力为（521．45±134．98）×10^-10牛顿。结论甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸能共价固定在聚丙交酯／乙交酯／天冬氨酸-聚乙二醇材料表面；多肽修饰后的材料能特异性的介导骨髓基质细胞粘附，增强其粘附力。     

Surface Modification of Polystyrene Films. Depth Profiling and Mapping by Raman Microscopy

The chemical modification of polymer films was studied using Raman microscopy. The nitration of polystyrene films (PS) in a non‐swelling reaction medium was compared with the nucleophilic substitution of chlorine atoms by cyano groups in poly(p‐chloromethylstyrene) (PSC) films in mixtures of a good and a non‐solvent for the polymer. Using Raman microscopy in the confocal mode, depth profiles of films modified in different conditions were recorded. In both reactions, conditions could be found which allowed the polymer films to be preferentially modified at the surface. The gradient of the degree of modification and the surface selectivity are functions of reaction time, temperature and solvent quality. The homogeneity of the modification reactions was compared by mapping of surface areas using Raman spectroscopy in the conventional mode.