Elaboration of highly hydrophobic polymeric surface--a potential strategy to reduce the adhesion of pathogenic bacteria?

Different polymeric surfaces have been modified in order to reach a high hydrophobic character, indeed the superhydrophobicity property. For this purpose, polypropylene and polystyrene have been treated by RF or µwaves CF4 plasma with different volumes, the results were compared according to the density of injected power. The effect of pretreatment such as mechanical abrasion or plasma activation was also studied. The modified surfaces were shown as hydrophobic, or even superhydrophobic depending of defects density. They were characterized by measurement of wettability and roughness at different scales, i.e. macroscopic, mesoscopic and atomic. It has been shown that a homogeneous surface at the macroscopic scale could be heterogeneous at lower mesoscopic scale. This was associated with the crystallinity of the material. The bioadhesion tests were performed with Gram positive and negative pathogenic strains: Listeria monocytogenes, Pseudomonas aeruginosa and Hafnia alvei. They have demonstrated an antibacterial efficiency of very hydrophobic and amorphous PS treated for all strains tested and a strain-dependent efficiency with modified PP surface being very heterogeneous at the mesoscopic scale. Thus, these biological results pointed out not only the respective role of the surface chemistry and topography in bacterial adhesion, but also the dependence on the peaks and valley distribution at bacteria dimension scale.

How to control the recombinant prion protein adhesion for successful storage through modification of surface properties.

Depletion of neuroproteins on the inner walls of storage tubes influences the accuracy of tests used for identification of various neurodegenerative disorders. In this paper, a strategy is described for surface modification of Eppendorf tubes leading to non-adhesive properties towards the recombinant human prion proteins (PrPrec(hum)). Tubes were pre-activated by helium plasma and grafted with three diverse coatings: pure poly(N-isopropylacrylamide) (PNIPAM), PNIPAM admixed with either neutral PEG(20)sorbitan monolaurate (PEG(20)) or positively charged cetyl trimethylammonium bromide (CTAB) at varying plasma activation times and polymer to surfactant ratios. New functionalized surfaces were analyzed by goniometry, streaming potential measurement and X-ray photoelectron spectroscopy, whereas the protein adhesion was monitored by enzyme linked immunosorbent assays and confocal microscopy. The mapping of PrPrec(hum) adhesion associated with surface analyses enabled us to determine that no or negligible depletion of PrPrec(hum) can be obtained by surfaces possessing basic component in the range between 50 and 60 mJ m(-2) and streaming potential ζ(7.4) - -50 mV.

Improvement of the detection of neurodegenerative Alzheimer's disease through a specific surface chemistry applied onto the inner surface of the titration well.

The main objective of this paper was to illustrate the enhancement of the sensitivity of the ELISA titration of Tau proteins while reducing other non-specific adsorptions that could increase the optical densities and could lead to false positives. This goal was obtained thanks to the association of cold plasma and wet chemistries of the inner surface of the titration well. The PP surface was cold plasma-activated, then coated with different amphiphilic molecules bearing either ionic charges and/or long hydrocarbon chains. The support treated and coated with hexatrimethylammonium bromide improves the signal detection of proteins while reducing the background due to non-specific associations of biomolecules such as hyperphosphorylated Tau protein. However, coating with 3-butenylamine hydrochloride could also be suitable.

Study of the adhesion of neurodegenerative proteins on plasma-modified and coated polypropylene surfaces.

The inner polymeric surface of an ELISA titration well is plasma-modified and coated with different surfactant molecules. The titration of neurodegenerative proteins markers (prion, Tau and ß-synuclein), previously demonstrated as more efficient with such modified tubes, is related to the adhesion behaviour of these proteins and their corresponding capture antibodies. The adhesion process is studied in terms of anchoring and specific mechanisms. The proteins and antibodies binding onto such modified surfaces is related to the substrate hydrophilic character calculated from the angle contact measure, to the polymer surface charge measured through the streaming potential determination at different pH and the inner surface roughness determined from AFM images. Furthermore, the influence of the blocking agent used during the ELISA titration is also studied.

Kinetics of insulin secretion from MIN6 pseudoislets after encapsulation in a prototype device of a bioartificial pancreas.

Xenotransplantation of insulin-secreting cells from nonhuman sources is an alternative therapeutic approach to bypass the shortage of human pancreatic islet tissue for transplantation in order to treat insulin deficiency in type 1 diabetes mellitus. Therefore, we studied the suitability of pseudoislets generated from insulin-secreting MIN6 tissue culture cells to serve as a surrogate for replacement of pancreatic islets after encapsulation in a minicell, representing a prototype of a new bioartificial pancreas device. MIN6 pseudoislets showed an excellent insulin secretory responsiveness with a typical biphasic secretory pattern to glucose stimulation. When encapsulated in the minicell, insulin release from the pseudoislets in response to glucose stimulation was reduced. The initial first phase insulin secretory response was greatly attenuated. In contrast, the first phase insulin secretory response of the encapsulated pseudoislets was restored on stimulation with the sulfonylurea drug tolbutamide. Our results indicate that the reason for the attenuated first phase of release is the restricted permeability of the pores in the separating membrane in the minicell for the hydrophilic glucose molecule rather than a limited permeability for the secretion product insulin. The reduced release of insulin from the encapsulated pseudoislets could be compensated by overexpression of glucokinase in MIN6 cells, which resulted in an increased glucose responsiveness of the pseudoislets for stimulation with glucose. Thus, this minicell is a well-suited miniature test system for the evaluation of the feasibility of encapsulation of insulin-secreting cells and allows the testing of permeability properties of separating membranes in bioartificial pancreas devices.

Radio frequency plasma treatments on titanium for enhancement of bioactivity.

Titanium and its alloys, when treated in alkali solutions, are able to form calcium phosphate coatings on their surface after immersion in supersaturated solutions. In this study, the surfaces of titanium alloy discs were modified by an alkali treatment and a radio frequency (RF) plasma procedure (150 W and 13.56 MHz) in N(2), CO(2) or N(2)/O(2) (80/20%) atmospheres. After the alkali treatment, atomic force microscopy showed differences in the surface roughness of the samples. X-ray photoelectron microscopy indicated that the chemical composition of the surfaces changed after the different alkali and RF plasma treatments. The contact angles were also modified by approximately 5 degrees , making the original titanium surface more hydrophilic. Immersion in a supersaturated calcium phosphate solution was used to evaluate the bioactivity of the RF plasma-treated samples in vitro. Alkali-treated samples gave more homogeneous and thick coatings that those without alkali treatment. The use of RF plasma treatments enhanced the bioactivity of the samples, in particular for treatments performed in N(2) or N(2)/O(2) atmospheres. Energy-dispersive X-ray analysis indicated that coatings had Ca/P ratios between the values of octacalcium phosphate and hydroxyapatite. X-ray diffraction confirmed the presence of these two phases in most of the coatings. This study shows that an RF plasma treatment enhanced the bioactivity of titanium surfaces.

New surfaces with hydrophilic/hydrophobic characteristics in relation to (no)bioadhesion.

The possibility of biosurfaces with high or low adhesiveness for protein, bacteria or eukaryotic cells is discussed. At the interface surface object/biological milieu, biocompatibility, (no) bioadhesion and (no) biocontamination are shown to be correlated with physico-chemical surface characteristics. First consequence is the (no) possibility of biofilm formation. Substrates with low surface energy could interact only with hydrophobic biomolecules. On substrates with high surface energy, a water monolayer spontaneously formed. Modification of a surface by plasma techniques is a way for engineering biomaterials. Plasma techniques are dry processes and more suitable for biomedical applications. In the field of biomaterial medical devices, in hygienic prevention of nosocomial diseases, in food packaging, the use of substrates with a very hydrophilic character may help to prevent the proliferation of cells and bacteria. Such a technique is so efficient that antibiotic molecules are not necessary. Therefore, surface engineering is a tool for modifying and adapting materials to specific biological applications.

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Effects of the length of crosslink chain on poly(2-hydroxyethyl methacrylate) (pHEMA) swelling and biomechanical properties.

Polymers are widely used in medicine for vascular prostheses, bone substitutes, and devices for controlled release. Among these polymers, poly(2-hydroxyethyl methacrylate) (pHEMA) is the most employed. To confer particular properties, pHEMA can be copolymerized with other monomers or in the presence of plasticizers or crosslinking agents. The influence of the length of crosslink chains on swelling, surface rugosity, hardness, and stiffness of crosslinked pHEMA were studied by several techniques, including fractal analysis and AFM. Four crosslinking agents (divinyl benzene, DVB; ethylene glycol dimethacrylate, EGDMA; tetraethylene glycol diacrylate, TEGDA; and polyethylene glycol diacrylate, PEGDA) were added to the bulk polymerization mixture. Only linear and PEGDA-pHEMA presented a significant decrease in surface roughness confirmed by fractal analysis. Differences in hardness and biomechanical properties were evidenced on dried polymers but the highest differences were exhibited for hydrated pHEMA. Correlations between the length of the crosslink chain and hardness or stiffness of hydrated crosslinked pHEMA were evidenced. TEGDA and PEGDA appeared to be the two most suitable crosslinking agents for controlled release of bioactive molecules in bone.

Culture of melanoma cells as aggregates on cellulose substratum.

Cells aggregate on an original cellulose substratum (CEL). This influences the signaling programs of adhering cells. CEL thus appears to be a suitable tool for studying the regulation of cell-substratum and cell-cell interactions.

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation.

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.

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.

Influence of VEGF on the viability of encapsulated pancreatic rat islets after transplantation in diabetic mice.

After pancreatic islet transplantation, insufficient blood supply is responsible for the loss of islet viability. The aim of our study was: 1) to determine the influence of vascular endothelial growth factor (VEGF) on the survival of encapsulated rat islets transplanted into healthy and diabetic mice and 2) to evaluate the metabolic efficiency of the VEGF-supplemented grafts. Twenty-four hours after culture, 50 rat islets immobilized into collagen in the presence of VEGF (100 ng/ml) and encapsulated (AN69 membrane, HOSPAL) were grafted in the peritoneal cavity of healthy or streptozotocin-induced diabetic mice (n = 6). Seven, 14, and 28 days after implantation, the encapsulation device and tissue surrounding the device were removed and the following parameters were analyzed: the number and the diameter of buds, the distance between devices and buds, the amount of cellular adhesion on the capsule surface, and the level of insulin secreted by encapsulated islet. For reversal of diabetes, 1000 rat islets encapsulated in the presence of VEGF were implanted in the peritoneal cavity of diabetic mice and fasting glycemia was analyzed. After 7 days of islet implantation in the absence of VEGF, the bud diameter was 16.1 +/- 6.9 microm in diabetic mice and 34.4 +/- 3.9 microm in healthy mice. However, the number of buds increased by a factor 2.5 in the presence of VEGF in both types of mice. Furthermore, when islets were transplanted in the presence of VEGF, the distance between the device and the buds was significantly decreased in both types of mice (p < 0.001) after 7, 14, and 28 days of islet implantation. Capsule analysis showed a decrease in cellular adhesion when the islets were encapsulated in the presence of VEGF. Insulin secretion of the islets was higher in the presence of VEGF compared with islets alone at all steps of the study. When 1000 rat islets were transplanted in the presence of VEGF, the glycemia level decreased to 6.2 +/- 0.8 mmol/L after 3 days and remained stable until at least 28 days. In contrast, in the absence of VEGF, the initial decrease in the glucose level was rapidly followed by a relapse in hyperglycemia. In summary, VEGF increased the viability of engrafted encapsulated islets, increasing the duration of a normalized glycemia in diabetic mice following transplantation. Local adjunction of VEGF may therefore improve the clinical outcome of islet transplantation.

Induction of angiogenesis in omentum with vascular endothelial growth factor: influence on the viability of encapsulated rat pancreatic islets during transplantation.

Transplantation of pancreatic islets is proposed as a treatment for type 1 diabetes, but insufficient blood supply can cause the loss of viable grafted islets. In the present study, we investigated the influence of vascular endothelial growth factor (VEGF) on the angiogenesis of omentum during encapsulated islet allotransplantation and consequently on islet survival. Fifty rat islets, cultured for 24 h, were encapsulated in the presence or absence of human VEGF and implanted in the peritoneal cavity of rats (n = 6). After 7, 14 and 28 days of implantation, encapsulation devices with surrounding omentum were removed. Histological analysis of this tissue was performed. Cellular adhesion at the membrane surface was characterized by a phagocytosis test. The morphological aspect of the islets was analyzed and their functionality was evaluated by measuring insulin secretion. At each step of the study, there was a two-fold increase in the number of vessels in the presence of VEGF. In addition, VEGF increased the vessel diameter and the surface area of the angiogenic pedicle. Moreover, the presence of VEGF significantly decreased the distance between the devices and vessels (16.2 +/- 5.6 vs. 51.6 +/- 10.1 microm, p < 0.001). Membrane surface analysis showed a decrease in macrophage adhesion in the presence of VEGF. Furthermore, islet structure and functionality was preserved in the presence of VEGF. Stimulation of angiogenesis of omentum induced by VEGF is associated with preservation of islet viability. Local delivery of VEGF proved to be a relevant approach to ameliorate the outcome of islet transplantation.

Interaction between hydroxypropyl methylcellulose and biphasic calcium phosphate after steam sterilisation: capillary gas chromatography studies.

The purpose of this study was to check the chemical stability of an injectable bone substitute (IBS) composed of a 50/50 w/w mixture of 2.92% hydroxypropyl methylcellulose (HPMC) solution in deionized water containing biphasic calcium phosphate (BCP) granules (60% hydroxyapatite/40% beta-tricalcium phosphate w/w). After separation of the organic and mineral phases, capillary gas chromatography (GC) was used to study the possible modification of HPMC due to the contact with BCP granules following steam sterilisation and 32 days storage at room temperature. HPMC was extracted from IBS in aqueous medium, and a dialytic method was then used to extract calcium phosphate salts from the HPMC. The percentage of HPMC extracted from BCP was 98.5%+/-0.5%, as measured by UV. GC showed no chemical modifications after steam sterilisation and storage.

In vivo study of a fluorocarbon polymer-coated intraocular lens in a rabbit model.

PURPOSE: To evaluate the biocompatibility in rabbit eyes of poly(methyl methacrylate) (PMMA) intraocular lenses (IOLs) that were surface modified using Teflon AF. SETTING: Hôtel-Dieu Hospital, Paris Cedex, France. METHODS: The IOLs were coated with Teflon AF, an amorphous, transparent, and highly hydrophobic fluorocarbon polymer, by immersing them in Teflon AF 5% and evaporating the solvent (C8F18). The surface quality of the Teflon-coated IOLs was evaluated by scanning electron microscopy (SEM). Teflon-coated (n = 20) and control PMMA (n = 10) IOLs were implanted in rabbit eyes. The presence of iris-IOL synechias and the number of deposits on the IOL surfaces were clinically evaluated in both groups to assess the antiadhesive effect of Teflon AF. The Teflon-coated IOLs were removed, their surfaces were evaluated by SEM, and their elemental composition was checked by EDXA and Raman spectrometry. RESULTS: The PMMA IOLs were completely coated with Teflon AF. The Teflon group had no iris-IOL synechias and the control group, two extensive synechias. There were significantly fewer deposits on the surfaces of Teflon-coated IOLs than on the control IOLs 30 and 60 days postoperatively (P < .0001). Scanning electron microscopy showed lens epithelium proliferation and spindle-shaped cells on the surfaces of the PMMA IOLs and cell deposits on the irregular regions of the Teflon-coated IOLs. White-yellow spots were present on the surfaces of both IOL types. The elemental composition of Teflon-coated IOLs was stable. CONCLUSION: Teflon AF had an antiadhesive effect that increased the biocompatibility of PMMA IOLs in vivo.

Endothelial damage caused by uncoated and fluorocarbon-coated poly(methyl methacrylate) intraocular lenses.

PURPOSE: To assess endothelial damage induced by poly(methyl methacrylate) (PMMA) intraocular lenses (IOLs) coated with a fluorocarbon polymer, Teflon AF, to make them highly hydrophobic. SETTING: Department of Ophthalmology. Hôtel-Dieu Hospital, Paris, France. METHODS: Ten Teflon-coated and 10 uncoated PMMA IOLs were used in an in vitro static touch model. The corneal endothelium was placed in direct contact with the IOL for 15 seconds and then stained with trypan blue and alizarin red. The endothelial damage produced by each IOL in the area of contact was assessed semiquantitatively and quantitatively. RESULTS: Teflon-coated IOLs produced significantly less endothelial damage than uncoated PMMA IOLs (P < .0001). Endothelial cells in contact with Teflon-coated IOLs did not usually adhere to the IOL surface. In contrast, the uncoated IOLs produced large areas of endothelial cell loss. CONCLUSION: Teflon-coated PMMA IOLs have an antiadhesive effect that reduced endothelial damage after IOL insertion in an in vitro model.

Physicochemical and biological studies of corona-treated artificial membranes used for pancreatic islets encapsulation: mechanism of diffusion and interface modification.

The artificial AN69 membrane (Hospal), a synthetic copolymer composed of acrylonitrile and sodium methallyl sulphonate suitable for pancreatic islet encapsulation, was submitted to physicochemical treatment (Corona discharge) to improve its insulin permeability. X-ray photoelectron spectroscopy (XPS) analysis of the AN69 membrane indicated the presence of up to two molecular layers of glycerol at its surface while the surface energies revealed the presence of hydrophilic sites (-SO3Na/glycerol) located at the membrane surface and acrylonitrile hydrophobic groups inside the material. The Corona discharges decreased the number of glycerol molecules at the membrane surface and from a biological point of view, produced a threefold increase in insulin diffusion. Furthermore, the biocompatibility of the treated membrane was preserved after 1 year of intraperitoneal implantation. The increase in insulin permeability should result from a decrease of the membrane polarity and of a steric hindrance in pores. Thus, Corona discharge treatment may serve to optimize the properties of artificial membranes used for pancreatic islets encapsulation.

Influence of corona surface treatment on the properties of an artificial membrane used for Langerhans islets encapsulation: permeability and biocompatibility studies.

An artificial membrane (AN69 Hospal) suitable for pancreatic islets encapsulation was submitted to a physicochemical treatment (corona discharge) to improve its insulin permeability. This effect depends on the duration of the electrical discharge (expressed as the speed of a conveyor belt) and the distance between the electrodes and the membrane. Among the various treatments tested, the most efficient (distance of 5 cm and a speed of 2 cm s-1) produced a three-fold increase in insulin diffusion. This improvement persisted after a protein-coating test which mimics in vivo conditions. At 1 y after the peritoneal implantation, the corona-treated membrane remained biocompatible. Thus, corona discharge treatment may serve to optimize the properties of artificial membranes used for pancreatic islets encapsulation.

In vitro evaluation of inflammatory cell response after CF4 plasma surface modification of poly(methyl methacrylate) intraocular lenses.

The inflammatory cell response of tetrafluorocarbon (CF4) plasma surface modification of poly(methyl methacrylate) intraocular lenses (IOLs) was investigated in vitro. After two hours of lens contact with human granulocytes, scanning electron microscopy showed significantly less cell activation and granulocyte adhesion on the surface-modified IOL than on the untreated IOL (P < .01). The x-ray photo-electron spectroscopy analysis of the CF4 plasma surface modification demonstrated that new compounds containing fluorine were homogeneously grafted onto the PMMA lens surface, resulting in a marked increase of contact angle. These in vitro results must be confirmed by in vivo studies of CF4 plasma surface modification of IOLs.