Antibacterial surfaces on expanded polytetrafluoroethylene; penicillin attachment

Expanded polytetrafluoroethylene (ePTFE) was chemically modified to retard the growth of Staphylococcus aureus bacteria. This was accomplished by microwave plasma reactions in the presence of maleic anhydride (MA) to create acid functional groups on ePTFE surfaces, followed by esterification reactions with 200 and 600 molecular weight linear polyethylene glycol (PEG). Such surfaces were utilized for further reactions with penicillin (PEN) through etherification reactions to create anti-microbial surfaces. These reactions resulted in surface morphological changes, and spectroscopic analysis using attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) revealed the formation of ester linkages resulting from reactions between PEN and PEG functionalities. Antibacterial activities were evaluated by a series of experiments where PEN-modified ePTFE specimens were immersed in a liquid aureus culture, and the bacteria growth was quantified by measuring % absorbance of the suspension at 600 nm wavelength. The lowest absorbance was observed for the solution containing PEN-PEG-MA-ePTFE specimens, thus showing highly effective anti-bacterial activity toward gram-positive Staphylococcus aureus bacteria. To our best knowledge, this is the first study that shows PEN-ePTFE surface modifications that are effective against gram-positive aureus bacteria.     

Attachment of ampicillin to expanded poly(tetrafluoroethylene): surface reactions leading to inhibition of microbial growth

The broad spectrum antibiotic, ampicillin (AM), was reacted to expanded poly (tetrafluoroethylene) (ePTFE) surfaces and resulted in the formation of antimicrobial surfaces effective against gram-positive, Staphylococcus aureus, Bacillus thuringiensis, and Enterococcus faecalis, and gram-negative, Escherichia coli, Pseudomonas putida, and Salmonella enterica bacteria. These ePTFE surface modifications were accomplished by utilization of microwave maleic anhydride (MA) plasma reactions leading to the formation of acid groups, followed by amidation reactions of heterofunctional NH 2/COOH-terminated polyethylene glycol (PEG). The final step, the attachment of AM to the PEG spacer, was achieved by amidation reactions between COOH-terminated PEG and NH 2 groups of AM. This approach protects the COOH-AM functionality and diminishes the possibility of hydrolysis of the antimicrobial active portion of AM. These studies also show that approximately 90% of AM molecules are still covalently attached to PEG-MA-ePTFE surfaces after exposure to the bacteria solutions. Even after a 24 h period, the AM volume concentration changes only from 2.25 to 2.04 microg/m3, and depending upon the bacteria type, the bacteria suspensions containing AM-PEG-MA-ePTFE specimens retain 85-99% of their initial optical density.     

Optimization of human endothelial cell attachment to vascular graft polymers

Endothelial cells (EC) covering the blood-contacting surface of a prosthetic material could potentially enhance the subsequent nonthrombogenicity of the surface. In order to create such a surface, the EC must become attached to the surface, spread and ultimately form a monolayer. In this study we examined several factors that influence these processes. On ePTFE surfaces, surface pretreatment with human serum for 30 minutes at a concentration of 1.4 gm percent protein resulted in significantly more attached EC when compared to other concentrations or when compared to fetal calf serum or human serum albumin. The rate of EC spreading was strongly influenced by temperature, with a maximum occurring at 37 degrees C. During real-time video microscopy, it was noted that the rate of EC attachment and spreading was primarily dependent on arrival of the EC to the surface rather than attachment and spreading. Thus as a method of increasing EC delivery, the concept of filtering EC onto the graft lumenal surface was tested by pressurizing the graft lumen to speed EC delivery to the surface. This technique produced a 2 to 5-fold increase in EC attachment when compared to gravity forced cell deposition. We conclude that an ePTFE graft can be rapidly endothelialized using these simple measures.     

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

Poly(styrenesulfonate)/poly(allylamine) multilayers: a route to favor endothelial cell growth on expanded poly(tetrafluoroethylene) vascular grafts

Small-diameter synthetic vascular grafts of expanded poly(tetrafluoroethylene) (ePTFE) polymer concern one of the most common alternatives for the replacement of diseased vessels. However, high failure rates arise especially due to the lack of endothelial cells (ECs). EC seeding was developed to build a monolayer on the luminal surface. Because ECs show little or no adhesion on synthetic prostheses, it is necessary to promote their retention. On ePTFE surfaces we successfully deposited polyelectrolyte multilayer films (PMFs) consisting of poly(ethylenimine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) to obtain PEI-(PSS-PAH)3 films. EC adhesion and spreading on modified ePTFE were assessed by scanning electron and confocal microscopies. Cell viability was evaluated by Alamar Blue assay. After 7 days of culture, the ePTFE modified with PMF exhibited improvements of EC viability as compared to that of the controls (nonmodified ePTFE) or even ePTFE coated by a PAH monolayer (p < 0.05). Moreover, the spreading of ECs was largely enhanced compared to that of the same controls, resulting in a healthy confluent cell monolayer formation. Positive staining for the von Willebrand factor confirmed the EC phenotype. Promoting EC attachment and function on ePTFE modified with PMFs could become in the future a promising treatment for synthetic small-diameter vascular grafts.     

Engineered Si(1 0 0) surfaces for the gas-phase anchoring of metal β-diketonate complexes

A synthetic strategy for the covalent anchoring of nickel β-diketonate complexes on Si(1 0 0) has been examined. Engineered Si(1 0 0) surfaces were prepared by the Si-grafting of 10-undecylenic acid methyl ester followed by hydrolysis of the ester to free the carboxylic functions suited for the anchoring of the Ni complex. Bis(pentane-2,4-dionate)Ni(II) was bonded to the functionalized surface from the gas phase by the exchange of the acetylacetonate ligand with the grafted acid. The surface density of the anchored Ni complex was controlled by tuning the surface concentration of carboxylic groups adopting a mixed monolayer of undecylenic acid and 1-decene used as a spectator spacer. The nickel decorated silicon surfaces were characterized by attenuate total reflectance infrared absorption spectroscopy (ATR-IRAS) and angle resolved X-ray photoelectron spectroscopy (AR-XPS).     

On-line monitoring of cell growth and cytotoxicity using electric cell-substrate impedance sensing (ECIS)

An on-line and continuous technique based on electric cell-substrate impedance sensing (ECIS) was developed for measuring the concentration and time response function of fibroblastic V79 cells exposed to mercury chloride and 1,3,5-trinitrobenzene (TNB). Attachment, spreading and proliferation of V79 fibroblastic cells cultured on a microarray of small gold electrodes precoated with fibronectin were detected as resistance changes. The response function was derived to reflect the resistance change as a result of cell attachment, spreading, mitosis and cytotoxicity effect. Exposure of V79 cells to mercury chloride or TNB led to alterations in cell behavior, and therefore, chemical cytotoxicity was easily screened by measuring the response function of the attached and spread cells in the presence of inhibitor. The half inhibition concentration, the required concentration to achieve 50% inhibition, was obtained from the response function to provide information about cytotoxicity during the course of the assay. A simple mathematical model was developed to describe the responses of ECIS that were related to the attachment, spreading, and proliferation of V79 fibroblastic cells. The novel results of this paper are mainly characterized by the systematic study of several parameters including the cell number, detection limit, sensor sensitivity, and cytotoxicity, and they may motivate further research and study of ECIS sensors.     

FRET studies with factor X mutants provide insight into the topography of the membrane-bound factor X/Xa

FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of Fl-FPR (fluorescein-D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56+/-1 A (1 A=0.1 nm) and 63+/-1 A for fXa-desEGF1 compared with 72+/-2 A and 75+/-1 A for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming kappa2=2/3. In comparison, an L value of 95+/-6 A was obtained for a S195C mutant of fXa in the absence of fVa in which fluorescein was attached directly to Cys(195) of fXa. These results suggest that (i) EGF1 plays a spacer function in holding the active site of fXa above the membrane surface, (ii) the average distance between fluorescein attached to Fl-FPR in the active site of fXa and OR at the vesicle surface may not reflect the actual distance of the active-site residue relative to the membrane surface, and (iii) fVa alters the orientation and/or the height of residue 195 above the membrane surface.     

Seeding of expanded polytetrafluoroethylene (ePTFE) vascular grafts. A morphological study of porcine endothelial and fibroblast cells.

The need to improve clinical results with small and medium calibre grafts has led to extensive research on cell seeding of prosthetic materials. Numerous problems remain regarding identification, seeding, adhesion and survival of the cells attached. We have studied the behaviour of seedings of endothelial and fibroblast cells on ePTFE grafts. Scanning electron microscopy allows us to observe the morphological characteristics and their interaction with the biopolymers. It has been possible to differentiate both cellular types by their characteristics and interactions with the ePTFE. At the same time, from this "in vitro" study it can be concluded that the time needed to obtain a stable and confluent monolayer on ePTFE pretreated with fibronectin is between 18 hours to 4 days for endothelial cells, and 24 hours for fibroblasts. These would be the optimal time periods for "in vivo" grafting of the seeded prostheses.     

Synthesis of novel cationic poly(ethylene glycol) containing lipids.

In this paper, the synthesis of novel divalent cationic lipids with poly(ethylene glycol) segments is described. The lipids consist of an unsaturated double-chain hydrophobic moiety based on 3, 4-dihydroxy benzoic acid, attached to a hydrophilic poly(ethylene glycol) spacer which contains a divalent cationic end group. As poly(ethylene glycol) spacers monodisperse triethylene glycol and telechelic poly(ethylene glycol)s with an average degree of polymerization of 9, 23, and 45 were used. The divalent cationic end group was attached by coupling a protected dibasic amino acid to the PEG spacer and following cleavage of the protecting groups. These novel class of cationic lipids is of particular interest for nonviral gene delivery applications.     

Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV)

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.     

Conjugation of Arg-Gly-Asp (RGD) sequence in copolymer bearing sugar moiety for insulinoma cell line (MIN6) culture

Copolymers composed of an Arg-Gly-Asp (RGD) sequence for the adhesion molecule and sugar moieties were synthesized for an insulinoma cell (MIN6) culture. MIN6 cells attached on the poly(N-p-vinylbenzyl-D-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VMA-co-VBGRGDS))-coated dishes were in a more aggregated form than other polymer-coated surfaces. P(VMA-co-VBGRGDS) also shows faster proliferation of MIN6 cells (about 18% higher) than with p(VLA-co-VBGRGDS). By interaction between cell and matrix, about 80% greater insulin secretion from MIN6 cells was produced with the p(VMA-co-VBGRGDS), and about 50% greater insulin secretion was produced with the poly(N-p-vinylbenzyl-D-lactonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VLA-co-VBGRGDS) as compared with unstimulated cells. Moreover, attachment of MIN6 cells treated with RGD monomer was suppressed approximately 50% for the p(VMA-co-VBGRGDS) surface. This result supported the idea that conjugation of adhesion molecules of RGD peptide in p(VMA-co-VBGRGDS) copolymer specifically interact with integrin families on MIN6 cell membrane.     

Conjugation of Arg-Gly-Asp (RGD) Sequence in Copolymer Bearing Sugar Moiety for Insulinoma Cell Line (MIN6) Culture

Copolymers composed of an Arg-Gly-Asp (RGD) sequence for the adhesion molecule and sugar moieties were synthesized for an insulinoma cell (MIN6) culture. MIN6 cells attached on the poly(N-p-vinylbenzyl-D-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VMA-co-VBGRGDS))-coated dishes were in a more aggregated form than other polymer-coated surfaces. P(VMA-co-VBGRGDS) also shows faster proliferation of MIN6 cells (about 18% higher) than with p(VLA-co-VBGRGDS). By interaction between cell and matrix, about 80% greater insulin secretion from MIN6 cells was produced with the p(VMA-co-VBGRGDS), and about 50% greater insulin secretion was produced with the poly(N-p-vinylbenzyl-D-lactonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VLA-co-VBGRGDS) as compared with unstimulated cells. Moreover, attachment of MIN6 cells treated with RGD monomer was suppressed approximately 50% for the p(VMA-co-VBGRGDS) surface. This result supported the idea that conjugation of adhesion molecules of RGD peptide in p(VMA-co-VBGRGDS) copolymer specifically interact with integrin families on MIN6 cell membrane.     

Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture

A novel surface modification method of biodegradable polymers was investigated for inducing the attachment of specific cells onto the polymer surface via ligand-receptor interactions. Galactose, a targeting ligand specific to asialoglycoprotein receptors present on cell membrane of hepatocytes, was introduced on the surface of poly(D,L-lactic-co-glycolic acid) (PLGA) films. A terminal end group of carboxylic acid in PLGA was activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide for the direct conjugation of lactose by reductive amination reaction. Di-block copolymers of PLGA-b-poly(ethylene glycol) (PEG) having a free terminal amine group were also synthesized and used for the conjugation of galactose for the introduction of a PEG spacer between PLGA and galactose. The presence of galactose moieties on the blend film surface was characterized by measuring water contact angle and X-ray photon spectroscopy, and the amount of galactose was indirectly determined by a specific lectin-binding assay. With increasing the galactose concentration on the blend film surface, the initial attachment as well as the cell viability of hepatocyates concomitantly increased. The introduction of PEG spacer reduced the cell attachment and viability. Albumin secretion rate from hepatocytes was enhanced for galactose modified surfaces, whereas it was reduced for the surfaces not having galactose moieties.     

Stable immobilization of rat hepatocytes as hemispheroids onto collagen-conjugated poly-dimethylsiloxane (PDMS) surfaces: importance of direct oxygenation through PDMS for both formation and function

The highly oxygen-permeable material, poly-dimethylsiloxane (PDMS), has the potential to be applied to cell culture microdevices, but cell detachment from PDMS has been a major problem. In this study, we demonstrate that a combination of collagen covalently immobilized PDMS and an adequate oxygen supply enables the establishment of a stable, attached spheroid (hemispheroid) culture of rat hepatocytes. The bottom PDMS surfaces were first treated with oxygen plasma, then coupled with aminosilane followed by a photoreactive crosslinker, and they were finally reacted with a collagen solution. X-ray photoelectron spectroscopy (XPS) and contact angle measurements showed that the covalent immobilization of collagen on the surface occurred only where the crosslinker had been introduced. On the collagen-conjugated PDMS surface, rat hepatocytes organized themselves into hemispheroids and maintained the viability and a remarkably high albumin production at least for 2 weeks of culture. In contrast, hepatocytes on the other types of PDMS surfaces formed suspended spheroids that had low albumin production. In addition, we showed that blocking the oxygen supply through the bottom PDMS surface inhibited the formation of hemispheroids and the augmentation of hepatocellular function. These results show that appropriate surface modification of PDMS is a promising approach towards the development of liver tissue microdevices.     

Cytotoxicity study of high gold content Degutan surfaces of various degrees of roughness with fibroblasts (BALB 3T3) and osteoblasts (hFOB 1.19)]

The cytotoxicity of Degutan surfaces with different degrees of roughness, and the effect of surface structures on osteoblast proliferation and differentiation, was investigated with standardised cell culture systems. Fibroblast cell lines (BALB/3T3) and osteoblast cell lines (hFOB 1.19) were used. The number and variability of the cells were determined for assessment of proliferation and alkaline phosphatase activity, collagen I and osteocalcin production were used as parameters for differentiation. In the early phase, the largest numbers of cells and greatest proliferation were measured on polished Degutan surfaces. In the late phase, however, larger numbers of cells and a greater degree of proliferation were to be seen on sandblasted and sandblasted/heat-treated Degutan surfaces. No differences were found for collagen I, osteocalcin production or alkaline phosphatase activity. Neither the osteoblasts nor the fibroblasts revealed a toxic effect of Degutan. The results for osteoblast differentiation correlate with recent studies on identical structured titanium surfaces. In view of the immeasurable amount of ion release, Degutan may be considered an ideal model for an inert material surface.     

Investigation of spacer length effect on immobilized Escherichia coli pili-antibody molecular recognition by AFM

The immobilization of antibodies to sensor surfaces is critical in biochemical sensor development. In this study, Poly(ethylene glycol) (PEG) and Jeffamine spacers were employed to tether Escherichia coli K99 pilus antibody to silicon wafer surfaces for the purpose of improving the orientation of antibody as well as reducing the steric hindrance. To illustrate the effect of spacer length, a variety of linear polymers were used to covalently attach the antibodies to silicon surfaces. Atomic Force Microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface morphology and chemical composition at each reaction step. The effect of spacer length in improving the specificity of immobilized antibody was investigated by attaching E. coli on the end of an AFM tip. The distribution of unbinding force and rupture distance from the force-distance curves obtained by AFM showed that the introduction of PEG spacer facilitates bacterial recognition which can improve the incidence of interactions by up to 90%. J600 proved to be the most effective spacer overcoming the steric hindrance seen with direct immobilization of antibody. In addition, the force spectroscopy reveals the elementary force quantum of E. coli-antibody to be 0.3 nN.     

Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays.

We have investigated the use of spacer molecules to reduce steric interference of the support on the hybridisation behaviour of immobilised oligonucleotides. These spacers are built up from a variety of monomeric units, using phosphoramidite chemistry, by condensation onto an amine-functionalised polypropylene support. The optimal spacer length was determined to be at least 40 atoms in length, giving up to 150-fold increase in the yield of hybridisation. The effects of different charged groups in the spacer were also examined, and it was shown that both positively and negatively charged groups in the spacer diminish the yield of hybridisation. Steric hindrance in hybridisation can also be a problem if the oligonucleotides attached to the support are too close to each other. Surface coverage was varied using a combination of cleavable and stable linkers, giving the highest hybridisation yields for surfaces containing approximately 50% of the maximum concentration of oligonucleotides.     

The fine structure of muscle attachments in an acarid mite, Caloglyphus mycophagus (Megnin) (Acarina)

The fine structure of the myo-cuticular junction in an acarid mite, Caloglyphus mycophagus, is described. The muscle fibres are attached to the cuticle via flattened, much invaginated, epidermal cells. Unlike the situation described for other arthropods, the stress across these epidermal cells does not appear to be transmitted by microtubules but rather by desmosome-like structures which form intraepidermal cell bridges where invaginations from the outer and inner surfaces of the epidermal cells lie close together. The muscles are attached to the inner surface of this complex desmosome and the outer surface is linked to the cuticle by extracellular fibrils.     

Heparin-coupled poly(poly(ethylene glycol) monomethacrylate)-Si(111) hybrids and their blood compatible surfaces

Well-defined (nearly monodispersed) poly(poly(ethylene glycol)monomethacrylate)-Si hybrids were prepared via surface-initiated atom transfer radical polymerization (ATRP) of the poly(ethylene glycol)monomethacrylate (PEGMA) macromonomer on the hydrogen-terminated Si(111) surface (Si-H surface). Both the active chloride groups at the chain ends (from the ATRP process) and the chloride groups converted from some ( approximately 32%) of the -OH groups of the Si-C bonded PEGMA polymer, or P(PEGMA), brushes were used as leaving groups for the covalent coupling of heparin. For the heparinized P(PEGMA)-Si hybrid surfaces, protein adsorption and platelet adhesion were significantly suppressed. The well-defined and dense P(PEGMA) brushes, prepared from surface-initiated ATRP, had allowed the immobilization of a relatively high concentration of heparin (about 14 mug/cm(2)). The resulting silicon surface exhibited significantly improved antithrombogenecity with a plasma recalcification time (PRT) of about 150 min. The persistence of high bioactivity for the immobilized heparin on the hybrid surfaces can be attributed to the biocompatibility of the PEGMA units, as well as their role as spacers in providing the immobilized heparin with a higher degree of conformational freedom in a more hydrophilic environment. Thus, the heparin-coupled P(PEGMA)-Si hybrids with anti-fouling and antithrombogenic surfaces are potentially useful in silicon-based implantable devices and tissue engineering.