Plasma surface modification of chitosan membranes: characterization and preliminary cell response studies

Surface modification of biomaterials is a way to tailor cell responses whilst retaining the bulk properties. In this work, chitosan membranes were prepared by solvent casting and treated with nitrogen or argon plasma at 20 W for 10-40 min. AFM indicated an increase in the surface roughness as a result of the ongoing etching process. XPS and contact angle measurements showed different surface elemental compositions and higher surface free energy. The MTS test and direct contact assays with an L929 fibroblast cell line indicated that the plasma treatment improved the cell adhesion and proliferation. Overall, the results demonstrated that such plasma treatments could significantly improve the biocompatibility of chitosan membranes and thus improve their potential in wound dressings and tissue engineering applications.     

Surface free energy and bacterial retention to saliva-coated dental implant materials--an in vitro study

The aim of the present investigation was to compare the in vitro bacterial retention on saliva-coated implant materials (pure titanium grade 2 (cp-Ti) and a titanium alloy (Ti–6Al–4V) surfaces), presenting similar surface roughness, and to assess the influence of physico-chemical surface properties of bacterial strain and implant materials on in vitro bacterial adherence. Two bacterial strains (one hydrophilic strain and one hydrophobic strain) were used and the following were evaluated: bacterial cell adherence, SFE values as well as the Lifshitz-van-der Waals, the Lewis acid base components of SFE, the interfacial free energy and the non-dispersive interactions according to two complementary contact angle measurement methods: the sessile drop method and the captive bubble method.

Our results showed similar patterns of adherent bacterial cells on saliva-coated cp-Ti and saliva-coated Ti–6Al–4V. These findings could suggest that bacterial colonization (i.e. plaque formation) is similar on saliva-coated cp-Ti and Ti–6Al–4V surfaces and indicate that both materials could be suitable for use as transgingival abutment or healing implant components. The same physico-chemical properties exhibited by saliva-coated cp-Ti and TA6V, as shown by the sessile drop method and the captive bubble method, could explain this similar bacterial colonisation. Therefore, higher values of total surface free energy of saliva-coated cp-Ti and saliva-coated TA6V samples (γ_SV ≈65 mJ/m²) were reported using the captive bubble method indicating a less hydrophobic character of these surfaces than with the sessile drop method (γ_S ≈44.50 mJ/m²) and consequently possible differences in oral bacterial retention according the theory described by Absolom et al.

The number of adherent hydrophobic S. sanguinis cells was two-fold higher than that of hydrophilic S. constellatus cells. Our results confirm that physico-chemical surface properties of oral bacterial strains play a role in bacterial retention to implant materials in the presence of adsorbed salivary proteins.     

Blood compatibility of thermoplastic polyurethane membrane immobilized with water-soluble chitosan/dextran sulfate

Water-soluble chitosan (WSC)/dextran sulfate (DS) was immobilized onto the surface of thermoplastic polyurethane (TPU) membrane after ozone-induced graft polymerization of poly(acrylic acid) (PAA). The surface was characterized with contact angle measurement and X-ray photoelectron spectroscopy (XPS). The adsorption of human plasma fibrinogen (HPF) followed the Langmuir adsorption isotherm. The results showed that the surface density of peroxides generated and poly(acrylic acid) (PAA) grafted reached the maximum value at 20 min of ozone treatment. It was found that the WSC- and DS-immobilized amount increased with pH and the molecular weight of WSC. The membrane/water interfacial free energy increased with PAA-grafting and WSC/DS-immobilization, indicating the increasing wettability of TPU membrane. The adsorption of HPF on TPU-WSC/DS membranes could be effectively curtailed and exhibited unfavorable adsorption. Moreover, WSC/DS immobilization could effectively reduce platelet adhesion and prolong the blood coagulation time, thereby membrane improving blood compatibility of TPU membrane. In addition, the in vitro cytotoxicity test of PEC modification was non-cytotoxic according to much low growth inhibition of L929 fibroblasts. Furthermore, TPU-WSC/DS membranes exhibited higher cell viability than native TPU membrane.     

Surface physicochemical properties of Pseudomonas fluorescens and impact on adhesion and transport through porous media

The physiological state of an examined Pseudomonas fluorescens strain had a significant impact on its adhesion to glass surfaces and transport through glass-bead columns. In both batch and column studies collision efficiencies, , for exponential phase cells were much larger (≈2–3) than for stationary or decay phase cells (≈0.5–0.7). Centrifugation of exponential phase cells substantially reduced collision efficiencies (≈0.8). Over the examined range (0.02–0.2 M), ionic strength had no impact on cell attachment. The Lewis acid/base (A/B) character of the cell surface varied with physiological states: exponential phase cells exhibited larger values of the electron–donor parameter of the polar surface tension component, γ_S⁻, than stationary or decay phase cells, resulting in larger calculated cell hydrophilicities. A reduction in exponential phase cell ζ-potential was observed upon centrifugation. Traditional Derjaguin–Landau–Verwey–Overbeek (DLVO) interaction energy profiles (between cells and glass surfaces) indicated energy maxima of the order of 90–130kT, and secondary energy minima of less than 10kT. Extended DLVO modeling predicted infinite energy barriers attributable to repulsive A/B interactions, and similar magnitude secondary energy minima. A pseudo-chemical kinetic approach was used to calculate activation energies of adhesion from experimental collision efficiencies. Collision efficiencies were also predicted from a diffusion-governed mass transport model incorporating interacting force fields. Predicted energy barriers underestimated cell collision efficiencies, suggesting that secondary energy minimum interactions governed initial attachment of cells. The partial reversibility of adhesion upon ionic strength reduction supported the secondary minimum interaction hypothesis.     

Electrophilic aromatic substitution regioselectivity for benzene derivatives in terms of cationic localization energies from semiempirical quantum chemical computations

Cationic localization energies (Λ⁺) for the interaction of monosubstituted benzenes with nitronium cation have been computed by the PM3 method. Yields (Y) of nitration isomeric products correlate linearly with the Λ⁺ values. The lnY=a+bΛ⁺ expressions agree both with Arrhenius equation and with linear free energy relationships.     

壳聚糖膜固定生物活性短肽RGDS的研究

为改善壳聚糖对细胞的特异性吸附,采用水溶性碳二亚胺将生物活性短肽精氨酸-甘氨酸-天冬氨酸-丝氨酸(RGDS)固定到壳聚糖膜的表面,采用X射线光电子能谱检测固定多肽前后的壳聚糖膜表面,发现反应后壳聚糖膜表面氮元素含量增大,Nls和Cls曲线拟合谱中酰胺键增多,表明RGDS短肽已固定到壳聚糖膜的表面;人角膜缘上皮细胞体外培养实验表明,固定RGDS后壳聚糖膜的细胞黏附率有了明显提高,固定RGDS后的壳聚糖膜在角膜组织工程支架等方面有更好的应用潜力。     

Temperature‐sensitive chitosan membranes as a substrate for cell adhesion and cell sheet detachment

A series of temperature‐sensitive poly(CSA‐co‐NIPAAm) membranes that were suitable for cell culture and confluent cell sheets detachment were prepared. The membranes with thermo‐responsive surface properties were synthesized by the copolymerization of acrylic acid‐derivatized chitosan (CSA) and N‐isopropylacrylamide (NIPAAm) in aqueous solution. Characterization of the membranes were carried out by means of the Fourier transform infrared (FTIR), X‐ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), and water contact‐angle (WCA) measurements. The adhesion and detachment of mouse fibroblast (L929) cells on these membranes have been investigated. The study showed that poly(CSA‐co‐NIPAAm) membranes could not only enhance fibroblasts attachment but also harvest confluent cell sheets by simply lowering the temperature. Furthermore, the detached cells retained high viability and could proliferate again after transferred to a new culture surface. Copyright © 2011 John Wiley & Sons, Ltd.     

Photosensitive chitosan to control cell attachment

An approach to control cell adhesion using a photocleavable molecule on chitosan has been developed and studied. Photocleavable 4,5-dimethoxy-2-nitrobenzyl chloroformate (NVOC) was introduced into chitosan to control the surface properties. The two UV illuminations with a photomask controlled the cleavage of NVOC and the presentation of deprotected amines on one chitosan surface spatially and temporally. The following immobilizations of cell repulsive poly(ethylene glycol) after the first illumination and cell adhesive sequence Arg-Gly-Asp-Ser (RGDS) after the second illumination on the surface helped create surface heterogeneity. Fourier transform infrared spectroscopy (FTIR), water contact angle, and UV-visible spectroscopy were used to characterize the surfaces and photoactivation during the process. To study the cell attachment and morphology on our designed surfaces, NIH/3T3 fibroblast cell was used. Cell number and morphology on the surfaces were investigated. The cell study demonstrated the feasibility of the surfaces on the control of cell adhesion and the formation of cell patterns by UV illuminations and the following immobilizations of different biomolecules.     

The emulsifying properties of β-lactoglobulin genetic variants A, B and C

The emulsifying properties of three genetic variants of β-lactoglobulin (β-lac) (the A, B and C variants) are investigated as a function of protein concentration. Differences in the emulsifying properties and emulsion stability are explained in view of the known differences in physico-chemical and structural/conformational properties of the β-lac variants. β-lac A forms the finest emulsion droplets, and β-lac C the largest droplets. The order of decreasing emulsifying ability (A>B>C) can be explained in terms of differences in the molecular structure, and conformational stability of the variant proteins. The creaming stability, when compared at the same particle size, is greatest for β-lac C, with β-lac A and B having a similar and lower stability. The differences in creaming stability may arise from a higher surface coverage for the β-lac C droplets at an equivalent particle size. The storage stability is lower for β-lac A than for β-lac B and C, which both show a similar behaviour. Storage stability differences are discussed in terms of differences in molecular structure, conformational stability, interfacial viscosity and surface coverage for the three variants.     

Adhesion contact dynamics of fibroblasts on biomacromolecular surfaces

Biomacromolecules like gelatin and chitosan have emerged as highly versatile biomimetic coatings for applications in tissue engineering. The elucidation of the interfacial kinetics of cell adhesion on biomacromolecular surfaces will pave the way for the rational design of chitosan/gelatin-based systems for cell regeneration. Biomacromolecular ultra-thin films, chemically immobilized on fused silica are ideal experimental models for determining the effect of surface properties on the biophysical cascades following cell seeding. In this study, confocal reflectance interference contrast microscopy (C-RICM), in conjunction with phase contrast microscopy and fluorescence confocal microscopy, was applied to detect the adhesion contact dynamics of 3T3 fibroblasts on chitosan and gelatin ultrathin films. X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of chitosan or gelatin on the silanized glass surface. Both the initial cell deformation rate and the change of two-dimensional spread area of the 3T3 fibroblasts are higher on gelatin-modified surfaces than on chitosan surfaces. The steady-state adhesion energy of 3T3 fibroblasts on gelatin film is three times higher than that on chitosan film. Immuno-staining of actin further demonstrates the different organization of cytoskeleton, likely induced by the change in cell signaling mechanism on the two biomacromolecular surfaces. The better attachment of 3T3 fibroblast to gelatin is postulated to be caused by the presence of adhesive domains on gelatin.     

New Prediction Model of Surface and Interfacial Energies Based on COSMO-UCE**

The nature and strength of intermolecular and surface forces are the key factors that influence the solvation, adhesion and wetting phenomena. The universal cohesive energy prediction equation based on conductor-like screening model (COSMO-UCE) was extended from like molecules (pure liquids) to unlike molecules (dissimilar liquids). A new molecular-thermodynamic model of interfacial tension (IFT) for liquid-liquid and solid-liquid systems was developed in this work, which can predict the surface free energy of solid materials and interfacial energy directly through cohesive energy calculations based on COSMO-UCE. The applications of this model in prediction of IFT for water-organic, solid (n-hexatriacontane, polytetrafluoroethylene (PTFE) and octadecyl-amine monolayer)-liquid systems have been verified extensively with successful results; which indicates that this is a straightforward and reliable model of surface and interfacial energies through predicting intermolecular interactions based on merely molecular structure (profiles of surface segment charge density), the dimensionless wetting coefficient R_A/C can characterize the wetting behavior (poor adhesive (non-wetting), wetting, spreading) of liquids on the surface of solid materials very well.     

环形嵌段共聚物的胶束化行为模拟

利用Monte Carlo模拟, 对比了相同组成下环形二嵌段共聚物AB和线形三嵌段共聚物ABA在选择性溶剂中的胶束化行为. 结果发现, 相同链组成的环形和线形嵌段共聚物的临界胶束浓度(cmc)的差别与A嵌段的比例(f_A)及B嵌段间的吸引强度(ε)密切相关. 在f_A较小、 ε较大的情况下, 相应环形嵌段共聚物的cmc值更小; 而在f_A较大、 ε较小的情况下, 线形嵌段共聚物的cmc值更小. 为了进一步理解胶束化行为同f_A及ε的关系, 计算了胶束化过程中熵和势能部分对自由能的贡献. 结果表明, 在所研究的f_A和ε范围内, 环形嵌段共聚物形成胶束时的熵损失更小, 因而从熵贡献角度来看, 环形嵌段共聚物更易发生胶束化. 而从势能贡献角度来看, 当f_A较小、 ε较大时, 环形嵌段共聚物形成胶束时势能有较大程度的降低, 对自由能的贡献更大, 因而此时环形嵌段共聚物更易发生胶束化. 而当f_A较大、 ε较小时, 线形嵌段共聚物形成胶束时势能有较大程度的降低, 对自由能的贡献更大, 因而此时线形嵌段共聚物更易发生胶束化. 由此可见, 对体系的胶束化自由能进行系统分析, 有助于更好地理解环形和线形嵌段共聚物的胶束化行为.     

交联结构对肝素/壳聚糖层层组装多层膜内皮细胞相容性的影响

采用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺交联技术对具有抗凝血抗菌作用的肝素/壳聚糖多层膜进行交联, 研究了交联结构对多层膜稳定性和血管内皮细胞亲和性的影响. QCM-D结果显示, 交联可有效地提高多层膜的稳定性, 在模拟人体血液流速(3.0 cm/s)下保持良好的稳定. 体外内皮细胞的研究结果显示, 多层膜的交联可有效地调节肝素/壳聚糖多层膜表面粘弹性, 并显著增加内皮细胞的粘附与生长. 交联的肝素/壳聚糖多层膜有望成为理想的心血管功能界面涂层材料.     

Grain boundaries as barrier for oxygen transport in perovskite-type membranes

Perovskite-type membranes of (Ba_0.5Sr_0.5)(Co_0.8Fe_0.2)O_3−δ (BSCF) and (Ba_0.5Sr_0.5)(Fe_0.8Zn_0.2)O_3−δ (BSFZ) were successfully prepared via liquid-phase sintering using BN as sintering aid. The obtained membranes were examined via powder X-ray diffraction pattern (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and oxygen permeation experiments. It has emerged that the use of BN as sintering aid lowers sintering temperatures in order to obtain dense membranes with relative densities in the range of 93–96% as proven by the Archimedes method. It was further shown that the perovskite structure could be maintained after sintering with BN. Additionally, BN was completely removed from the sintered membranes. Investigation of the microstructure revealed that the average grain size of the membranes was influenced by the amount of BN added prior the sintering process. It was found that large amounts of BN effectively lower the average grain size. Oxygen permeation experiments have shown that the lower the average grain size the lower the oxygen permeation performance, particularly in the case of BSCF. Transmission electron microscopy revealed that no evidence for an amorphous layer or any other interfacial phase in the grain boundary is present.     

Polysaccharide films built by simultaneous or alternate spray: a rapid way to engineer biomaterial surfaces

We investigated polysaccharide films obtained by simultaneous and alternate spraying of a chitosan (CHI) solution as polycation and hyaluronic acid (HA), alginate (ALG), and chondroitin sulfate (CS) solutions as polyanions. For simultaneous spraying, the film thickness increases linearly with the cumulative spraying time and passes through a maximum for polyanion/CHI molar charge ratios lying between 0.6 and 1.2. The size of polyanion/CHI complexes formed in solution was compared with the simultaneously sprayed film growth rate as a function of the polyanion/CHI molar charge ratio. A good correlation was found. This suggests the importance of polyanion/polycation complexation in the simultaneous spraying process. Depending on the system, the film topography is either liquid-like or granular. Film biocompatibility was evaluated using human gingival fibroblasts. A small or no difference is observed in cell viability and adhesion between the two deposition processes. The CHI/HA system appears to be the best for cell adhesion inducing the clustering of CD44, a cell surface HA receptor, at the membrane of cells. Simultaneous or alternate spraying of CHI/HA appears thus to be a convenient and fast procedure for biomaterial surface modifications.     

The physicochemical surface characteristics of Brevibacterium linens

The physicochemical surface characteristics and interfacial behavior of two strains of Brevibacterium linens (BL-MGE and BL-9174), that may enhance cheese flavor, were assessed. Cell surface hydrophobicity was determined by measuring the contact angle of a variety of polar and non-polar wetting agents on lawns of the bacterial cells. The contact angles obtained were used to calculate the cell surface free energy components γ^LW, γ^AB, γ⁺ and γ⁻. The Lifshitz van der Waals component and the Lewis acid-base component were approximately 35 mJ m⁻² and 22 mJ m⁻², respectively, for both strains. Under conditions of physiological pH and low ionic strength, neither strain exhibited affinity for an octyl ligand in hydrophobic interaction chromatography. This occurred despite a favorable free energy of interfacial interaction which was attributable almost entirely to favorable acid-base interactions between cells and octyl-sepharose. The nature of surface functional groups was evaluated using X-ray photoelectron spectroscopy. Excluding H, the mean percentage of atomic fraction for C, O, N and P for BL-MGE was 57.7, 37.8, 3.9 and 0.6%, respectively and 61.0, 31.8, 7.0 and 0.2%, respectively for BL-9174.     

Impact of Peptide Micropatterning on Endothelial Cell Actin Remodeling for Cell Alignment under Shear Stress

HSVEC behavior under physiological shear stress in vitro is investigated on PET surfaces micropatterned with both RGDS and WQPPRARI peptides. This technique allows (i) creating geometries on surface to guide cell orientation under shear stress and (ii) controlling surface chemical composition in order to modulate cell behavior. Under shear stress, endothelial cells adhere on patterned PET surfaces and present a more rapid orientation in flow direction in comparison to cells cultured on homogeneous surfaces. Micropatterned surfaces presenting a large surface area ratio of RGDS/WQPPRARI peptides induce fibrillar adhesion, while surfaces presenting an equal RGDS/WQPPRARI peptides surface area ratio preferentially induce focal adhesion.

     

Development of polyelectrolyte multilayer-coated electrospun cellulose acetate fiber mat as composite membranes

Electrostatic multilayers of chitosan (CHI)/sodium alginate (SA) and CHI/poly(styrene sulfonate) sodium salt (PSS) were alternatively coated on electrospun cellulose acetate (CA) fiber mat. Morphologies of the composite membranes were characterized by scanning electron microscopy. The morphology of the CHI/SA-coated membrane was denser than the CHI/PSS-coated one. The top layers consisted of carboxyl and sulfonic functional groups for SA and PSS layers, respectively. Amino groups of CHI were only presented in slight quantity. X-ray photoelectron spectroscopy (XPS) confirmed the deposition of the amino groups of CHI on the multilayer membrane surface. These composite membranes were characterized for its water permeability where the water flux decreased with an increase in the number of the bilayers. The water flux was in the range of 60 and 40 L m⁻² h⁻¹ for 15 and 25 bilayered membranes, respectively. The sodium chloride (NaCl) solution flux was lower than the pure water flux due to the effect of osmotic pressure, and it decreased with an increase in the NaCl concentration. The rejection of NaCl increased substantially with the number of the bilayers of the polyelectrolytes multilayers. The level of NaCl rejection from this work was in the range of 6% and 15% for 15 and 25 bilayered membranes, respectively.     

白山毛桃根抗脑胶质瘤活性成分及其纳米胶束的制备

采用溶剂提取和萃取方法得到白山毛桃根乙酸乙酯、 正丁醇和水层萃取物. 通过噻唑蓝(MTT)比色法考察了各萃取物对脑胶质瘤细胞U87MG活性的抑制作用. 采用高效液相色谱(HPLC)指纹图谱法比对确认活性部位的主要化学成分为2α,3α,24-三羟基-12-烯-28-乌苏酸. 采用溶剂挥发法制备了2α,3α,24-三羟基-12-烯-28-乌苏酸纳米胶束, 对其包封率、 粒径及ζ电位等进行了表征, 并考察了其抗肿瘤活性. 体外细胞实验结果表明, 白山毛桃根乙酸乙酯萃取物对脑胶质瘤细胞U87MG具有抑制作用, 通过与标准品比对确认2α,3α,24-三羟基-12-烯-28-乌苏酸为乙酸乙酯部位的主要成分, 且该成分对脑胶质瘤细胞活性具有较强抑制作用. 通过溶剂挥发法制备的2α,3α,4-三羟基-12-烯-28-乌苏酸纳米胶束包封率为64.7%, 粒径为20 nm, 粒径分布宽度(PDI)为0.246, ζ电位为-5.7 mV. 细胞实验结果进一步证明, 与单体化合物相比, 2α,3α,24-三羟基-12-烯-28-乌苏酸纳米胶束对脑胶质瘤细胞活性具有更强的抑制作用, 为白山毛桃根在脑胶质瘤治疗中的应用提供了实验依据.     

Spreading of oil from protein stabilised emulsions at air/water interfaces

Spreading of a drop of an emulsion made with milk proteins on air/water interfaces was studied. From an unheated emulsion, all oil molecules could spread onto the air/water interface, indicating that the protein layers around the oil globules in the emulsion droplet were not coherent enough to withstand the forces involved in spreading. Heat treatment (90 °C) of emulsions made with whey protein concentrate (WPC) or skim milk powder reduced the spreadability, probably because polymerisation of whey protein at the oil/water interface increased the coherence of the protein layer. Heat treatment of emulsions made with WPC and monoglycerides did not reduce spreadability, presumably because the presence of the monoglycerides at the oil/water interface prevented a substantial increase of coherence of the protein layer. Heat treatment of caseinate-stabilised emulsions had no effect on the spreadability. If proteins were already present at the air/water interface, oil did not spread if the surface tension (γ) was <60 mN/m. We introduced a new method to measure the rate at which oil molecules spread from the oil globules in the emulsion droplet by monitoring changes in γ at various positions in a ‘trough’. The spreading rates observed for the various systems agree very well with the values predicted by the theory. Spreading from oil globules in a drop of emulsion was faster than spreading from a single oil drop, possibly due to the greater surface tension gradient between the oil globule and the air/water interface or to the increased oil surface area. Heat treatment of an emulsion made with WPC did not affect the spreading rate. The method was not suitable for measuring the spreading rate at interfaces where surface active material is already present, because changes in γ then were caused by compression of the interfacial layer rather than by the spreading oil.