Control of fibronectin displacement on polymer substrates to influence endothelial cell behaviour

Thin layered biomaterial surfaces of maleic anhydride copolymers are provided as a versatile platform for biomaterial applications. The provided comonomers define the character of the surface and its behaviour towards biomolecules and biosystems, such as proteins and cells. The kinetics of adsorption, desorption, and exchange of fibronectin and human serum albumin were investigated on different copolymer surfaces. Two different species of adsorbed proteins were found, a fast and a slow desorbing one. Furthermore, the exchange process depends on the kind of pre-adsorbed protein and the kind of exchange protein, as well as of the hydrophobicity of the copolymer surface. In this context adhesion, proliferation, and differentiation of endothelial cells from the umbilical cord vein onto fibronectin pre-coated surfaces were studied. Strong correlation between fibronectin exchange characteristics and the formation of focal adhesions, reorganisation of fibronectin, and generation of vascular-like structures by the cells was observed.     

Preliminary study on human protein adsorption and leukocyte adhesion to starch-based biomaterials

In this study, the adsorption of human serum albumin (HSA), fibronectin (FN) and vitronectin (VN) onto the surface of novel biodegradable materials was evaluated by immunostaining. Specifically, polymeric blends of corn starch with cellulose acetate (SCA), ethylene vinyl alcohol copolymer (SEVA-C), and polycaprolactone (SPCL) were immersed in unitary and competitive systems; that is, binary and more complex protein solutions. For binary solutions, different HSA and FN protein distribution patterns were observed depending on the starch-based surface. Furthermore, the relative amount of proteins adsorbed onto starch-based surfaces was clearly affected by protein type: a preferential adsorption of VN and FN as compared to HSA was observed. On tests carried out with unitary, binary and more complex solutions, it was found that vitronectin adsorption ability was enhanced in competitive systems, which was associated with a lower amount of adsorbed albumin. In order to assess the effect of these human proteins on cell behavior, a mixed population of human lymphocytes and monocytes/macrophages was cultured over pre-coated SEVA-C surfaces. Through anti-CD3 and CD-14 monoclonal antibody labeling and cell counting, leukocyte adhesion onto pre-coated SEVA-C surfaces was analyzed. Based on the results, it was possible to detect albumin long-term effects and fibronectin short-term effects on cell adhesion proving that previously adsorbed proteins modulate leukocyte behavior.     

Binding of ferritin molecules to a charged polypeptide layer of poly-1-benzyl-L-histidine

The binding of ferritin molecules to a charged polypeptide layer of poly-1- benzyl-L-histidine spread at the air-water interface has been studied. The films were transferred onto an alkylated silicon wafer and were observed with a scanning electron microscope without staining or metal shadowing. The purpose of the present study was to explore the formation of densely and/or regularly packed two- dimensional arrays of protein molecules by means of the Langmuir-Blodgett method. Compression of the ferritin-peptide film at the air-water interface was found to be highly effective in producing closely packed arrays of ferritin molecules. Control experiments with an uncharged interfacial film of poly-γ-benzyl-L- glutamate gave no evidence of binding or array formation.     

钛表面固定纤维连接蛋白及其体外细胞培养

通过低温等离子聚合的方法,以丙烯酸为单体在钛表面沉积含有羧基的薄膜,以羧基为接入点固定纤维连接蛋白。样品表面用X射线光电子能谱、傅里叶红外光谱仪进行表征。将固定了纤维连接蛋白的样品进行体外细胞培养,所用的细胞为MG63骨瘤细胞,对照样为纯钛。结果表明, 钛表面聚丙烯酸薄膜能有效地固定纤维连接蛋白,并且固定纤维连接蛋白的样品能促进骨瘤细胞的生长和黏附,具有更高的成骨活性。      

Preparation and characterization of protein Langmuir-Blodgett films

A protein monolayer of bovin serum albumin (BSA) was stabilized at the air- water interface using glutaraldehyde (GA). The GA-treated BSA film was transferred onto the glass surface, followed by immunochemical lamination of anti-BSA antibody. Protein A-β-galactosidase (pro A-β-gal) conjugate was then reacted with the antibody molecular layer. Biofunctional protein films can be prepared on the BSA base Langmuir-Blodgett film by intermolecular complementary affinity between the proteins concerned.     

BSA和FN在纳米化钛表面的蛋白吸附及释放行为

通过阳极氧化技术, 在钛表面制备一层管径为100nm左右的氧化钛纳米管. 选取小牛血清白蛋白(BSA)和纤维连接蛋白(FN)两种蛋白质进行蛋白吸附实验, 并在仿生条件下进行蛋白质的体外释放. 对吸附了蛋白的纳米管试样表面进行红外和荧光定性分析, 同时采用考马斯亮蓝法对纳米管表面的蛋白吸附进行定量检测, 实验发现纳米管试样更有利于蛋白质的吸附, 且FN在试样表面吸附时的吸附率大于BSA. 氧化钛纳米管的蛋白释放分为突释和缓释两个阶段, 其释放机制符合Fickian扩散.     

Revealing the dependence of cell spreading kinetics on its spreading morphology using microcontact printed fibronectin patterns

Since the dawn of in vitro cell cultures, how cells interact and proliferate within a given external environment has always been an important issue in the study of cell biology. It is now well known that mammalian cells typically exhibit a three-phase sigmoid spreading on encountering a substrate. To further this understanding, we examined the influence of cell shape towards the second rapid expansion phase of spreading. Specifically, 3T3 fibroblasts were seeded onto silicon elastomer films made from polydimethylsiloxane (PDMS), and micro-contact printed with fibronectin stripes of various dimensions. PDMS is adopted in our study for its biocompatibility, its ease in producing very smooth surfaces, and in the fabrication of micro-contact printing stamps. The substrate patterns are compared with respect to their influence on cell spreading over time. Our studies reveal, during the early rapid expansion phase, 3T3 fibroblasts are found to spread radially following a law; meanwhile, they proliferated in a lengthwise fashion on the striped patterns, following a law. We account for the observed differences in kinetics through a simple geometric analysis which predicted similar trends. In particular, a t² law for radial spreading cells, and a t¹ law for lengthwise spreading cells.     

Identification of fibronectin in tissue implant interfaces

This study demonstrates the presence of fibronectin in cells and extracellular deposits on the surface of a cellophane implant nine days after surgery. The mononuclear cells with epithelioid or fibroblastoid morphology were positive for fibronectin. A small quantity of this fibronectin contained fibrillar network-like extracellular deposits, which are typical of fibroblasts. Multinucleate foreign-body giant cells were only poorly positive for fibronectin. These results indicate the prevalence of macrophages on the surface of cellophane implants, contrasting with the low incidence of fibroblasts.     

Enhanced cellular activation with single walled carbon nanotube bundles presenting antibody stimuli

Efficient immunotherapy can be accomplished by expanding T cells outside the body using single walled carbon nanotube (SWNT) bundles presenting antibody stimuli. Owing to the large surface area of these bundles, which can reach 1560 m (2)/g, T cell stimulating antibodies such as anti-CD3, can be presented at high local concentrations inducing potent activation of T cells. We show that anti-CD3 adsorbed onto SWNT bundles stimulate cells more effectively than equivalent concentrations of soluble anti-CD3. Stimulation by antibody adsorbed onto SWNT is significantly higher than other high surface area materials (activated carbon, polystyrene, and C60 nanoparticles), suggesting unique properties of SWNT bundles for stimuli presentation. We demonstrate the surface area tunability of these bundles by chemical treatment and its effect on antibody adsorption and subsequent T cell activation. In addition, the T cell response varied with the concentration of SWNT in a concentration dependent manner. Antibody stimuli adsorbed onto SWNT bundles represent a novel paradigm for efficient activation of lymphocytes, useful for basic science applications and clinical immunotherapy.     

Optimization of the interaction between ethylenevinyl alcohol copolymers and human endothelial cells

To better understand and optimize the fine interactions that occur during adhesion events between human cells and synthetic materials, we seeded human umbilical vein endothelial cells (HUVEC) onto ethylene-vinyl alcohol (EVOH) copolymer films prepared by casting. Different adhesive proteins, e.g. fibronectin and gelatin, and the monoclonal antibody (MoAb) CLB-HEC19 specific for the endothelial cell membrane were used to coat the materials. We used atomic force microscopy (AFM) to analyse the EVOH film structure, to test its planarity and homogeneity, before seeding it with endothelial cells. The metabolic changes induced in the endothelial cells by interactions with the copolymer functional groups and the adhesive proteins were monitored by a micro-electronic pH sensor, positioned close to the HUVEC monolayer. We found that the adhesion of HUVEC onto various substrates was finely modulated by the MoAb CLB-HEC19 and that the endothelial cell metabolic rate was enhanced when cultured onto a CLB-HEC19 coating. Surface roughness seems also to play a role in the interaction with HUVEC. The AFM measurement analysis demonstrated that L6 surface is rougher than R20. These surface characteristics could favour cell adhesion; in fact HUVEC adhesion results on R20 were significantly lower than on L6.     

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

Abstract

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na⁺/K⁺-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.     

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na⁺/K⁺-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.     

Impedance endothelial cell biosensor for lipopolysaccharide detection

It is important to analyse endothelial cell adherence for the development of biomedical devices of antithrombogenic vascular grafts. Endothelial cells must be firmly attached to the biomaterials when cells are seeded in order to create a natural lining.

Polystyrene (PS) is presented as a reproducible implant model substrate for studying cell–material interactions. Polystyrene was deposited as a thin layer on a thiol functionalised gold electrode. Fibronectin, a protein promoting the endothelial cell adhesion was then adsorbed on PS surface. The different steps of this multilayer assembly were characterized by Electrochemical Impedance Spectroscopy (EIS). The charge transfer resistance and the capacitance of the total layer were modified at each step in agreement with the electrical properties of each layer. The electrical properties of the confluent layer of endothelial cells were determined: (i) a charge transfer resistance of 2 kΩ cm^− 2 shows no large defects in the cell layer, (ii) as the cells attach and spread on the gold electrode, the impedance increases.

EIS was used for testing behaviour of endothelial cells on substrate coated by fibronectin layer and in presence of cytotoxicants such as lipopolysaccharide (LPS). The impedance measurement may be a valuable method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. The results indicate that LPS causes a dose-dependent decrease in impedance of the endothelial cell monolayer, indicating widening of the paracellular pathways and increasing vascular endothelial permeability. This study is an increasing trend towards the development of impedimetric biosensors and designing cell sensor arrays for toxic and drug detection.     

Designing nanostructured block copolymer surfaces to control protein adhesion

The profile and conformation of proteins that are adsorbed onto a polymeric biomaterial surface have a profound effect on its in vivo performance. Cells and tissue recognize the protein layer rather than directly interact with the surface. The chemistry and morphology of a polymer surface will govern the protein behaviour. So, by controlling the polymer surface, the biocompatibility can be regulated. Nanoscale surface features are known to affect the protein behaviour, and in this overview the nanostructure of self-assembled block copolymers will be harnessed to control protein behaviour. The nanostructure of a block copolymer can be controlled by manipulating the chemistry and arrangement of the blocks. Random, A-B and A-B-A block copolymers composed of methyl methacrylate copolymerized with either acrylic acid or 2-hydroxyethyl methacrylate will be explored. Using atomic force microscopy (AFM), the surface morphology of these block copolymers will be characterized. Further, AFM tips functionalized with proteins will measure the adhesion of that particular protein to polymer surfaces. In this manner, the influence of block copolymer morphology on protein adhesion can be measured. AFM tips functionalized with antibodies to fibronectin will determine how the surfaces will affect the conformation of fibronectin, an important parameter in evaluating surface biocompatibility.     

Protein Corona Mediated Uptake and Cytotoxicity of Silver Nanoparticles in Mouse Embryonic Fibroblast

Medical applications of nanoparticles (NPs) require understanding of their interactions with living systems in order to control their physiological response, such as cellular uptake and cytotoxicity. When NPs are exposed to biological fluids, the adsorption of extracellular proteins on the surface of NPs, creating the so‐called protein corona, can critically affect their interactions with cells. Here, the effect of surface coating of silver nanoparticles (AgNPs) on the adsorption of serum proteins (SPs) and its consequence on cellular uptake and cytotoxicity in mouse embryonic fibroblasts are shown. In particular, citrate‐capped AgNPs are internalized by cells and show a time‐ and dose‐dependent toxicity, while the passivation of the NP surface with an oligo(ethylene glycol) (OEG)‐alkanethiol drastically reduces their uptake and cytotoxicity. The exposure to growth media containing SPs reveals that citrate‐capped AgNPs are promptly coated and stabilized by proteins, while the AgNPs resulting from capping with the OEG‐alkanethiol are more resistant to adsorption of proteins onto their surface. Using NIH‐3T3 cultured in serum‐free, the key role of the adsorption of SPs onto surface of NPs is shown as only AgNPs with a preformed protein corona can be internalized by the cells and, consequently, carry out their inherent cytotoxic activity.     

Establishment of the Co-C Eutectic Fixed-Point Cell for Thermocouple Calibrations at NIMT

In 2015, NIMT first established a Co-C eutectic temperature reference (fixed-point) cell measurement capability for thermocouple calibration to support the requirements of Thailand’s heavy industries and secondary laboratories. The Co-C eutectic fixed-point cell is a facility transferred from NPL, where the design was developed through European and UK national measurement system projects. In this paper, we describe the establishment of a Co-C eutectic fixed-point cell for thermocouple calibration at NIMT. This paper demonstrates achievement of the required furnace uniformity, the Co-C plateau realization and the comparison data between NIMT and NPL Co-C cells by using the same standard Pt/Pd thermocouple, demonstrating traceability. The NIMT measurement capability for noble metal type thermocouples at the new Co-C eutectic fixed point (\(1324.06\,{^{\circ }}\hbox {C}\)) is estimated to be within \(\pm 0.60\,\hbox {K}\) (\(k=2\)). This meets the needs of Thailand’s high-temperature thermocouple users—for which previously there has been no traceable calibration facility.     

Cellular Interactions and Modulated Osteoblasts Functions Mediated by Protein Adsorption

The rapid adsorption of proteins is one of the first few events that occur when a biomedical device is implanted and strongly affects attachment and proliferation of cells on the material surface. We describe here for the first time the favorable modulation of osteoblasts functions and cell–substrate interactions induced by protein adsorption (bovine serum albumin) on silicone elastomers. It was intriguing that the cell–substrate interactions on protein‐adsorbed silicone elastomer were significantly different from those observed in stand alone silicone. The origin and differences in cell–substrate interactions in terms of cell attachment, viability, and proliferation and assessment of proteins actin, vinculin, and fibronectin are addressed. Cell attachment and proliferation of cells were significantly enhanced on protein‐adsorbed surface in comparison to the bare surface. Similarly, the expression level of fibronectin, actin, and vinculin was appreciably greater on the protein‐adsorbed surface. The study underscores that protein adsorption on artificial biomedical devices can be envisaged as one of the methods to enhance the bioactivity on the surface and regulate biological functions.     

Engineered Fibrillar Fibronectin Networks as Three‐Dimensional Tissue Scaffolds

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre‐coatings in an attempt to create a cell‐supporting environment in both two‐ and three‐dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM‐constituting fibrillar protein networks naturally deposited by cells. Here, the cell‐free and scalable synthesis of freely suspended and mechanically robust three‐dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three‐phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial‐to‐mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre‐clinical diagnostics.     

Silk RGD融合蛋白修饰羟基磷灰石/丝素蛋白支架对成骨细胞生长的影响

通过浸渍吸附的方法, 用桑蚕丝素-RGD融合蛋白(简称Silk-RGD)对多孔状磷灰石/丝素蛋白(HA/SF)复合支架材料进行表面修饰, 研究了复合支架材料在不同浓度Silk-RGD蛋白溶液中浸渍后对两种不同成骨细胞MG-63和MC3T3-E1黏附、增殖和分化的影响。结果表明, Silk-RGD融合蛋白修饰的复合支架材料的细胞黏附性能显著高于未经修饰的对照组, 且其促黏附性能具有Silk-RGD浓度依赖性; 体外培养7天时, 细胞增殖能力较对照组更显著,当Silk-RGD的吸附量为11 μg/mg时, MG-63的增殖率较对照样提高了21%, MC3T3-E1提高了50%; 而碱性磷酸酶活性检测结果显示, 复合支架经Silk-RGD表面修饰后对MC3T3-E1细胞的分化有一定的促进作用, 但对MG-63细胞的影响不明显。