[show abstract][hide abstract] ABSTRACT: Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the cytokine, leukaemia inhibitory factor (LIF). Following LIF withdrawal, mESCs differentiate, and this is accompanied by an increase in cell-substratum adhesion and cell spreading. The purpose of this study was to investigate the relationship between cell spreading and mESC differentiation. Using E14 and R1 mESC lines, we have restricted cell spreading in the absence of LIF by either culturing mESCs on chemically defined, weakly adhesive biomaterial substrates, or by manipulating the cytoskeleton. We demonstrate that by restricting the degree of spreading by either method, mESCs can be maintained in an undifferentiated and pluripotent state. Under these conditions, self-renewal occurs without the need for LIF and is independent of nuclear translocation of tyrosine-phosphorylated STAT3 or β-catenin, which have previously been implicated in self-renewal. We also demonstrate that the effect of restricted cell spreading on mESC self-renewal is not mediated by increased intercellular adhesion, as evidenced by the observations that inhibition of mESC adhesion using a function blocking anti E-cadherin antibody or siRNA do not promote differentiation. These results show that mESC spreading and differentiation are regulated both by LIF and by cell-substratum adhesion, consistent with the hypothesis that cell spreading is the common intermediate step in the regulation of mESC differentiation by either LIF or sell-substratum adhesion.
The international journal of biochemistry & cell biology 07/2013; · 4.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: External parameters (RF power and precursor flow rate) are typically quoted to define plasma polymerization experiments. Utilizing a parallel-plate electrode reactor with variable geometry, it is shown that these parameters cannot be transferred to reactors with different geometries in order to reproduce plasma polymer films using four precursors. Measurements of ion flux and power coupling efficiency confirm that intrinsic plasma properties vary greatly with reactor geometry at constant applied RF power. It is further demonstrated that controlling intrinsic parameters, in this case the ion flux, offers a more widely applicable method of defining plasma polymerization processes, particularly for saturated and allylic precursors.
[show abstract][hide abstract] ABSTRACT: Film thickness and functional group retention are routinely measured parameters for plasma polymers. It is known that other parameters such as density, solubility and mechanical properties can affect the performance of the plasma polymer film, however such parameters are not often measured; nor is there any understanding of the link between the mechanisms of film growth and these properties. In this investigation we produced thin films from three classes of commonly used plasma polymers (hydrocarbons, glymes and carboxylic acids). By choosing the monomer structure and applied RF power, the dominant mechanism of film growth was varied between ionic deposition and neutral grafting. The density, solubility and modulus of the resulting films were then measured by atomic force microscopy. Films grown from saturated monomers had higher moduli, were less soluble, and surprisingly had lower density compared to their unsaturated analogues. The results demonstrate that cognizance of the mechanism of film growth allows the physical properties of the film to be tailored for specific applications.
[show abstract][hide abstract] ABSTRACT: Plasma polymerisation is a technologically important surface engineering process capable of depositing ultra-thin functionalised films for a variety of purposes. It has many advantages over other surface engineering processes, including that it is completely dry, can be used for complex geometries, and the physico-chemical properties of the film can be tailored through judicious choice of processing conditions. Despite this, the mechanisms of film growth are largely unknown, and current models are based on purely chemical arguments. Consideration of some basic plasma physics shows that some species can arrive at surfaces with energies greater than 1000 kJ mol−1 (>10 eV), and thus open a range of surface reactions that have not been considered previously. This review aims to close the gap between the physics and chemistry of reactive plasma systems.
[show abstract][hide abstract] ABSTRACT: This paper reports the first systematic investigation on the effects of atmospheric-pressure helium microplasma array treatment on the surface chemistries of model organic materials and a biological coating. These materials include polystyrene (PS), low-density polyethylene (LDPE) and ethylene tetrafluoroethylene (ETFE), and bovine serum albumin (BSA). The plasma treatment introduced a range of oxygen functionalities into the surface of the polymers, with oxygen incorporation reaching “saturation” after relatively short treatment times. PS and LDPE surfaces were more readily oxidised and to a greater depth compared to ETFE. The polymer surfaces became smoother at short plasma treatment times due to removal of adventitious hydrocarbon, but became rougher at longer treatment times as a result of etching of low molecular weight, volatile material from the surface. Atmospheric-pressure helium microplasma array treatment of a BSA layer resulted in the majority of the protein being removed from the underlying (PS) surface. The plasma treatment reduced the surface roughness of the BSA coating at short treatment times, but at longer treatment times, the surface roughness increased and the surfaces exhibited granular structures. All of the hydrophobic polymers became hydrophilic after the plasma treatment. The hydrophilicity of the surfaces decreased upon storage (hydrophobic recovery) and none of the polymers reverted to their original hydrophobic state, even after 500 h of storage. The knowledge presented in this paper may be useful in the development of new manufacturing processes based on atmospheric-pressure plasma and in the field of plasma medicine, particularly with respect to cleaning and sterilisation methods. In addition, it provides a foundation for future efforts to establish the mechanisms behind interactions of atmospheric-pressure plasmas with materials.
[show abstract][hide abstract] ABSTRACT: A synthetic biological sensor was developed to monitor the interaction of plasma with soft, hydrated biological material. It comprises phospholipid vesicles in a hydrated proteinaceous environment comprising 5% (w/v) gelatin. The vesicles contained a self-quenched dye, which was activated by vesicle destruction giving a clear fluorescent switch on. The interaction of bacterial toxins with the sensor was measured in a proof of principle experiment, then the effect of atmospheric plasma jets with the sensor, was studied in order to assess the cytolytic effect of plasma jets in biological systems. When the plasma contacted the gelatin surface perpendicular to the surface, the treatment resulted in the formation of a star-shaped pattern of microchannels that radiated out from the centre of the treatment area within the gelatin matrix, and locally damaged vesicles within the microchannels at a depth of 150 µm below the gelatin surface. Plasma jets applied in parallel to the surface of the matrix resulted in the formation of a single microchannel with damage to the vesicles only evident at the walls of the channel, and a much reduced penetration depth within the gelatin. Our data show that the effects of plasma can be deep in the gelatin material and that the angle of treatment significantly influenced the nature and level of damage to the gelatin and vesicles. Potentially this gelatin model can be used to unravel the roles of different plasma species and the direct effect of whole plasma contact, from those of primary and secondary species—i.e. primary, those emanating directly from the plasma and secondary, those species created in the 'target' tissue. This type of insight could be useful in the future development of safe and effective plasma medical technologies.
Journal of Physics D Applied Physics 04/2013; 46(18):185401. · 2.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: High conductivity poly(3,4-ethylene dioxythiophene) (PEDOT) was synthesised using vacuum vapour phase polymerization (VVPP). The process produces PEDOT composites which incorporate glycol within the polymer. To assess biocompatibility, a suite of analytical techniques were utilised in an effort to characterise the level of glycol present and its impact on cell attachment and proliferation. A small decrease in fibroblast cell attachment and proliferation was observed with increasing glycol content within the PEDOT composite. Keratinocyte cell attachment and proliferation by comparison showed an increase. As such, the results herein indicate that cell attachment and proliferation depends on the individual cell lines used and that the impact of glycol within the PEDOT composite is negligible. This positive outcome prompted investigation of this polymer as a platform for electro-stimulation work. Application of oxidising and reducing potentials to the PEDOT composite were utilised to examine the effect on biocompatibility. Significant effects were seen with altered protein presentation on the reduced surface, and lower mass adsorbed at the oxidised surface. Keratinocytes interacted significantly better on the reduced surface whereas fibroblasts displayed dependence on protein density, with significantly lower spreading on the oxidised surface. Understanding how proteins interact at electrically biased polymer surfaces and in turn affect cell behaviour, underpins the utilisation of such tunable surfaces in biomedical devices.
[show abstract][hide abstract] ABSTRACT: It has been shown that both ions and neutral species may contribute to plasma polymer growth. However the relative contribution from these mechanisms remains unclear. We present data elucidating the importance of considering monomer structure with respect to which growth mechanism dominates for non-fouling PEG-like plasma polymers. The deposition rate for saturated monomers is directly linked with ion flux to the substrate. For unsaturated monomers, the neutral flux also plays a role, particularly at low power. Increased fragmentation of the monomer at high power reduces the ability of unsaturated monomers to grow via neutral grafting. Chemical characterization by X-ray Photoelectron Spectroscopy (XPS) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) confirm the role plasma phase fragmentation plays in determining the deposition rate and surface chemistry of the deposited film. The simple experimental method used here may also be used to determine which mechanisms dominate plasma deposition for other monomers. This knowledge may enable significant improvement in future reactor design and process control.
[show abstract][hide abstract] ABSTRACT: Gradient surfaces are highly effective tools to screen and optimize cell- surface interactions. Here, the response of embryonic stem (ES) cell colonies to plasma polymer gradient surfaces is investigated. Surface chemistry ranged from pure allylamine (AA) plasma polymer on one end of the gradient to pure octadiene (OD) plasma polymer on the other end. Optimal surface chemistry conditions for retention of pluripotency were identified. Expression of the stem cell markers alkaline phosphatase (AP) and Oct4 varied with the position of the ES cell colonies across the OD-AA plasma polymer gradient. Both markers were more strongly retained on the OD plasma polymer rich regions of the gradients. The observed variation of expression across the plasma polymer gradient increased with duration of stem cell culture. While maximum cell adhesion to the gradient substrate occurred at a nitrogen- to-carbon (N/C ratio) of approximately 0.1, Oct4 and AP expression was best retained at an N/C ratio < 0.04. Stem cell marker expression correlated with colony size and morphology: more compact, multilayered colonies with prominent F-actin staining arose as the N/C ratio decreased. Disruption of actin polymerization using Y-27632 ROCK inhibitor resulted in a collapse of the multilayer colony structure into monolayers with limited cell-cell contact. A corresponding decrease in expression of AP and Oct4 was observed. Oct4 expression along with 3D colony morphology was partially rescued on the OD plasma polymer rich regions of the gradient.
[show abstract][hide abstract] ABSTRACT: Vapor phase polymerization (VPP) is at the forefront for synthesizing high conductivity poly(3,4-ethylenedioxythiophene) (PEDOT) as an alternative to indium tin oxide (ITO). Little attention, however, has been directed to the oxidant layer used in the polymerization process. In this study the observation of an oxidant layer (oxidant + PEG–PPG–PEG) possessing liquid-like properties during the vacuum synthesis of PEDOT is reported. This is in contrast to the other oxidant layer variants studied which are observed as solid (pristine oxidant) or gel-like (oxidant + pyridine). Tailoring of the liquid-like properties leads to confluent PEDOT films with a conductivity of 2500 S cm−1, placing this PEDOT within the conductivity range of commercially available ITO. Building on the liquid-like observation, XPS and ToF-SIMS experiments reveal that PEDOT growth is via a bottom-up mechanism with transportation of new oxidant up to the forming PEDOT layer.
Journal of Materials Chemistry 07/2012; 22(30):14889-14895. · 5.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe a pH responsive drug delivery system which was fabricated using a novel approach to functionalize biodegradeable porous silicon (pSi) by initiated chemical vapor deposition (iCVD). The assembly involved first loading a model drug (camptothecin, CPT) into the pores of the pSi matrix followed by capping the pores with a thin pH responsive copolymer film of poly(methacrylic acid-co-ethylene dimethacrylate) (p(MAA-co-EDMA)) via iCVD. Release of CPT from uncoated pSi was identical in two buffers at pH 1.8 and pH 7.4. In contrast, the linear release rate of CPT from the pSi matrix with the p(MAA-co-EDMA) coating was dependent on the pH; release of CPT was more than four times faster at pH 7.4 (13.1 nmol/(cm(2) h)) than at pH 1.8 (3.0 nmol/(cm(2) h)). The key advantage of this drug delivery approach over existing ones based on pSi is that the iCVD coating can be applied to the pSi matrix after drug loading without degradation of the drug because the process does not expose the drug to harmful solvents or high temperatures and is independent of the surface chemistry and pore size of the nanoporous matrix.
[show abstract][hide abstract] ABSTRACT: The deposition of a thin film layer by plasma polymerization enables the surface functionalization of a wide range of substrate materials for biointerfacial interactions. Plasma polymers can surface-bind proteins specifically via covalent linkages or nonspecifically through other irreversible adsorption mechanisms; key questions are whether covalent chemisorption has indeed occurred, and whether the protein retains functionality. Here the mode of surface binding of streptavidin and the biotin binding functionality of the bound streptavidin layer are studied on plasma polymer (pp) surfaces deposited using propionaldehyde and ethanol that were plasma polymerized at different powers (P) to investigate possible mechanisms for protein binding to a range of different surface chemistries. As expected, with pp surfaces composed principally of aldehyde groups, protein conjugation appears to be specific (chemisorption) allowing the immobilization of streptavidin (SAV) molecules retaining the ability to bind biotinylated probes. To contrast with this, we present the first study of protein adsorption to ethanol pp surfaces prepared at different P. This provides an investigation into retention of the hydroxyl functionality in the pp at low P and its effect on protein adsorption. Adsorption of human serum albumin (HSA) to ethanol pp was similar to that on propionaldehyde pp except at low P (5 W) where hydroxyl group retention and hydration presumably has a role in reducing protein adsorption. Although we observed SAV adsorption to ethanol pp surfaces at all P, interestingly, the protein lost its ability to bind biotinylated probes. Thus we suggest that irreversible, nonspecific adsorption of protein on ethanol pp surfaces results in apparent protein denaturation despite the hydrophilic nature of the ethanol pp surface. We conclude by making inferences between the pp structure as measured by X-ray photoelectron spectroscopy (XPS) and the related protein adsorption mechanisms.
[show abstract][hide abstract] ABSTRACT: The control of cell-material interactions is the key to a broad range of biomedical interactions. Gradient surfaces have recently been established as tools allowing the high-throughput screening and optimization of these interactions. In this paper, we show that plasma polymer gradients can reveal the subtle influence of surface chemistry on embryonic stem cell behavior and probe the mechanisms by which this occurs. Lateral gradients of surface chemistry were generated by plasma polymerization of diethylene glycol dimethyl ether on top of a substrate coated with an acrylic acid plasma polymer using a tilted slide as a mask. Gradient surfaces were characterized by X-ray photoelectron spectroscopy, infrared microscopy mapping and profilometry. By changing the plasma polymerization time, the gradient profile could be easily manipulated. To demonstrate the utility of these surfaces for the screening of cell-material interactions, we studied the response of mouse embryonic stem (ES) cells to these gradients and compared the performance of different plasma polymerization times during gradient fabrication. We observed a strong correlation between surface chemistry and cell attachment, colony size and retention of stem cell markers. Cell adhesion and colony formation showed striking differences on gradients with different plasma polymer deposition times. Deposition time influenced the depth of the plasma film deposited and the relative position of surface functional group density on the substrate, but not the range of plasma-generated species.
[show abstract][hide abstract] ABSTRACT: Surface density gradients of streptavidin (SAV) were created on solid surfaces and demonstrated functionality as a bioconjugation platform. The surface density of immobilized streptavidin steadily increased in one dimension from 0 to 235 ng cm(-2) over a distance of 10 mm. The density of coupled protein was controlled by its immobilization onto a polymer surface bearing a gradient of aldehyde group density, onto which SAV was covalently linked using spontaneous imine bond formation between surface aldehyde functional groups and primary amine groups on the protein. As a control, human serum albumin was immobilized in the same manner. The gradient density of aldehyde groups was created using a method of simultaneous plasma copolymerization of ethanol and propionaldehyde. Control over the surface density of aldehyde groups was achieved by manipulating the flow rates of these vapors while moving a mask across substrates during plasma discharge. Immobilized SAV was able to bind biotinylated probes, indicating that the protein retained its functionality after being immobilized. This plasma polymerization technique conveniently allows virtually any substrate to be equipped with tunable protein gradients and provides a widely applicable method for bioconjugation to study effects arising from controllable surface densities of proteins.
[show abstract][hide abstract] ABSTRACT: Maskless microplasma treatment of passivated surfaces has been developed for the micropatterning of materials surfaces. The micropatterned surfaces are used in the fabrication of arrays for protein and cell-based assays. The advantage of this micropatterning approach is that it can be easily integrated into current manufacturing practices and the resultant micropatterned surfaces used with existing life sciences techniques and instrumentation.
[show abstract][hide abstract] ABSTRACT: In this paper we describe the spatial surface chemical modification of
bonded microchannels through the integration of microplasmas into a
microfluidic chip (MMC). The composite MMC comprises an array of
precisely aligned electrodes surrounding the gas/fluid microchannel.
Pairs of electrodes are used to locally ignite microplasmas inside the
microchannel. Microplasmas, comprising geometrically confined
microscopic electrically-driven gas discharges, are used to spatially
functionalise the walls of the microchannels with proteins and enzymes
down to scale lengths of 300 μm inside 50 μm-wide microchannels.
Microchannels in poly(dimethylsiloxane) (PDMS) or glass were used in
this study. Protein specifically adsorbed on to the regions inside the
PDMS microchannel that were directly exposed to the microplasma. Glass
microchannels required pre-functionalisation to enable the spatial
patterning of protein. Firstly, the microchannel wall was functionalised
with a protein adhesion layer, 3-aminopropyl-triethoxysilane (APTES),
and secondly, a protein blocking agent (bovine serum albumin, BSA) was
adsorbed onto APTES. The functionalised microchannel wall was then
treated with an array of spatially localised microplasmas that reduced
the blocking capability of the BSA in the region that had been exposed
to the plasma. This enabled the functionalisation of the microchannel
with an array of spatially separated protein. As an alternative we
demonstrated the feasibility of depositing functional thin films inside
the MMC by spatially plasma depositing acrylic acid and 1,7-octadiene
within the microchannel. This new MMC technology enables the surface
chemistry of microchannels to be engineered with precision, which is
expected to broaden the scope of lab-on-a-chip type applications.
[show abstract][hide abstract] ABSTRACT: Glycosaminoglycans play an important role in tissue organisation through interactions with a diverse range of proteins, growth factors and other chemokines. In this report, we demonstrate the GAG-binding 'fingerprint' of two important GAG-binding proteins - osteoprotogerin and TIMP-3. The technique uses a straightforward method for attaching GAGs to assay surfaces in a non-covalent manner using plasma polymerization that leaves the adsorbed GAG able to participate in subsequent ligand binding. We show that OPG and TIMP-3 bind preferentially to different GAGs in a simple ELISA and that this binding does not correlate directly with simple GAG properties such as degree of sulfation. The methods outlined in this report can be easily applied to tissue engineering scaffolds in order to exploit the potential of surface-bound GAGs in influencing the structure of engineered tissues.
[show abstract][hide abstract] ABSTRACT: Nanoporous alumina (PA) arrays produced by self-ordering growth, using electrochemical anodization, have been extensively explored for potential applications based upon the unique thermal, mechanical and structural properties, and high surface-to-volume ratio of these materials. However, the potential applications and functionality of these materials may be further extended by molecular-level engineering of the surface of the pore rims. In this paper we present a method for the generation of chemical gradients on the surface of PA arrays based upon plasma co-polymerization of two monomers. We further extend these chemical gradients, which are also gradients of surface charge, to those of bound ligands and number density gradients of nanoparticles. The latter represent a highly exotic new class of materials, comprising aligned PA, capped by gold nanoparticles around the rim of the pores. Gradients of chemistry, ligands and nanoparticles generated by our method retain the porous structure of the substrate, which is important in applications that take advantage of the inherent properties of these materials. This method can be readily extended to other porous materials.
[show abstract][hide abstract] ABSTRACT: New data shed light on the mechanisms of film growth from low power, low pressure plasmas of organic compounds. These data rebalance the widely held view that plasma polymer formation is due to radical/neutral reactions only and that ions play no direct role in contributing mass at the surface. Ion reactions are shown to play an important role in both the plasma phase and at the surface. The mass deposition rate and ion flux in continuous wave hexamethyl disiloxane (HMDSO) plasmas have been studied as a function of pressure and applied RF power. Both the deposition rate and ion flux were shown to increase with applied power; however, the deposition rate increased with pressure while the ion flux decreased. Positive ion mass spectrometry of the plasma phase demonstrates that the dominant ionic species is the (HMDSO-CH(3))(+) ion at m/z 147, but significant fragmentation and subsequent oligomerization was also observed. Chemical analysis of the deposits by X-ray photoelectron spectroscopy and secondary ion mass spectrometry show that the deposits were consistent with deposits reported by previous workers grown from plasma and hyperthermal (HMDSO-CH(3))(+) ions. Increasing coordination of silicon with oxygen in the plasma deposits reveals the role of ions in the growth of plasma polymers. Comparing the calculated film thicknesses after a fixed total fluence of 1.5 × 10(19) ions/m(2) to results for hyperthermal ions shows that ions can contribute significantly to the total absorbed mass in the deposits.