In vivo evaluation of ammonia plasma modified ePTFE grafts for small diameter blood vessels replacement. A preliminary report.

BACKGROUND: Today, saphenous veins are most frequently used to reconstruct occluded or diseased small diameter vessels (< or =6 mm in diameter). However, these veins are unavailable in 30-40% of patients. In such a situation, prosthetic grafts provide the only alternative. Since an endothelial cell (EC) lining, important for maintaining a haemostatic-thrombotic balance, does not develop onto the intima of implanted grafts in humans, these grafts occlude within a short period of time. The failure of vascular grafts is attributed to their characteristics which are nonconducive towards endothelial cell adhesion, spreading and growth. In order to examine whether the patency of vascular grafts can be improved by the surface modification of grafts' intima, small diameter grafts were modified by a novel ammonia plasma treatment to enhance their interactions with EC. METHODS: Through laparotomy, ammonia plasma treated ePTFE (4 mm in diameter) grafts (n=3) and control untreated grafts (n=6) were implanted into the distal infrarenal aorta of rabbits. At appropriate time, grafts and adherent tissue were removed, fixed, stained and embedded in Poly/Bed 812. Light microscopic examination of thin sections cut from the proximal and distal anastomatic and midportion segments of explanted grafts was carried out. RESULTS: In control group studies, all animals developed lower limb paraplegia within seven days of implantation. Light microscopic examination of explanted control grafts showed that control grafts were obstructed by thrombosis/intimal hyperplasia. However, ammonia plasma modified explanted grafts, after one month of transplantation, revealed an endothelial cell-like lining that covered the grafts' inner surfaces. Accordingly, these grafts remained patent in animals. CONCLUSIONS: The grafts' surfaces that are made conducive to EC adhesion and proliferation and host response may influence endothelial regeneration. It is hoped that the combination of angiogenic molecules (i.e. endothelial cell specific growth factors such as VEGF) with ammonia plasma modified grafts may provide further insight into the development of ideal small diameter prosthesis.

Antithrombotic and fibrinolytic system of human endothelial cells seeded on PTFE: the effects of surface modification of PTFE by ammonia plasma treatment and ECM protein coatings.

The aim of this study was to determine the effects of ECM protein coatings and surface modification of PTFE on the ability of seeded human endothelial cells (EC) to secrete prostacyclin (PGI2), plasminogen inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA). PTFE surfaces were modified by a novel surface modification technique based on ammonia plasma. Fibronectin, collagen type-1 and gelatin-coated ammonia plasma modified PTFE and unmodified PTFE surfaces were employed and compared in this study. All ammonia plasma modified surfaces showed similar secretions of PGI2 compared to non-modified PTFE surfaces. With the exception of gelatin-coated modified PTFE, seeded EC seeded on all modified PTFE showed lower levels of PAI-1 secretion compared to those seeded on unmodified PTFE. The specific activity of t-PA secreted by EC seeded on ammonia plasma modified and fibronectin coated modified PTFE showed increases of 100 and 30%, respectively, when compared to their unmodified counterparts. Our studies show that EC seeded on modified PTFE have ability to secrete PGI2 that modulates the early phase of thrombus formation. Furthermore, superior t-PA profile, along with lower levels of PAl-1 suggest that ammonia plasma modification and use of appropriate ECM proteins can modulate antithrombotic and fibrinolytic properties of in vitro endothelialized vascular prostheses. Accordingly, these surfaces may be suitable to further develop protocols and other strategies for arterial and venous reconstruction.

Enhanced growth of animal and human endothelial cells on biodegradable polymers.

Biodegradable polymers such as poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA) and PGA coated with PLLA are being employed for cell transplantation and for in vivo regeneration of vascular tissue. Ingrowth and organization of fibrovascular tissue inside polymer scaffolds lead to the occlusion of the regenerated blood vessel. In order to provide regulatory mechanisms to control the development of an inner capsule, endothelialization of these materials is necessary. To achieve this, we employed a novel ammonia plasma technique to surface modified PLLA substrates. Human endothelial cell (HUVEC) and rabbit microvascular endothelial cell (RbMVEC) growth was studied on modified PLLA and control PLLA. Our studies show that modified PLLA and fibronectin (Fn)-coated modified PLLA exhibited statistically significant improvement in HUVEC and RbMVEC growth (P<0.001) when compared to PLLA and Fn-coated PLLA. Therefore, ammonia plasma treatment gives us the unique capability of modifying prosthetic biomaterials of various constructs with the eventual transplantation of mammalian cells to be used in tissue engineering or as biological implants.

Biocompatibility aspects of new stent technology.

Stent implantation represents a major step forward since the introduction of coronary angioplasty. As indications continue to expand, better understanding of the early and late biocompatibility issues appears critical. Persisting challenges to the use of intracoronary stents include the prevention of early thrombus formation and late neointima development. Different metals and designs have been evaluated in animal models and subsequently in patients. Polymer coatings have been proposed to improve the biocompatibility of metallic stents or to serve as matrix for drug delivery and they are currently undergoing clinical studies. The promises of a biodegradable stent have not yet been fulfilled although encouraging results have recently been reported. Continuous low dose-rate brachytherapy combining the scaffolding effect of the stent with localized radiation therapy has witnessed the development and early clinical testing of radioactive stents. The combined efforts of basic scientists and clinicians will undoubtedly contribute to the improvement of stent biocompatibility in the future.

Transplantation of human endothelial cell monolayer on artificial vascular prosthesis: the effect of growth-support surface chemistry, cell seeding density, ECM protein coating, and growth factors.

The failure rates of synthetic vascular grafts, when placed in low blood flow environments in humans, are not acceptable. Thus, endothelial cell (EC) seeding technology of vascular grafts was developed to prepare prostheses lined with a human monolayer expressing optimal thromboresistant properties. In a clinical setting, endothelialization of a graft can be achieved using higher cell seeding densities, or by creating a surface on which EC can adhere and grow to confluence. But, human endothelial cells show little or no proliferation on the currently available graft materials. In this study, surface modification of PTFE and ePTFE by ammonia plasma treatment was carried out to enhance its interactions with ECM protein, EC growth factors, and with EC harvested from human umbilical vein (HUVEC), and from human saphenous veins (HSVEC). Our data shows that various vascular graft materials generated from ammonia plasma treated PTFE and ePTFE exhibited statistically significant improvements in HUVEC and HSVEC growth when compared to their respective controls (p values < 0.001). Growth of HSVEC on ammonia plasma treated ePTFE without ECM protein coating was also found to be statistically significant in comparison to that on fibronectin coated ePTFE (p < 0.001). The final HSVEC cell densities found on various ePTFE surfaces prepared from ammonia plasma treated ePTFE, suggests that transplantation of HSVEC monolayers on vascular prostheses can be established within clinically relevant times. Ammonia plasma treatment process provides an unique opportunity to surface modify prosthetic materials of various construct to transplant mammalian cells including those that have undergone ex vivo gene transfer, and to deliver angiogenic molecules to a target area for tissue development.

High-efficiency transformation of human endothelial cells by Apo E-mediated transfection with plasmid DNA.

Endothelial cells, because of their proximity to the blood stream, provide an attractive system for gene transfer and delivery of gene products that control foci of vascular disease processes. We describe a simple, new methodology to achieve highly efficient transformation of cultured human endothelial cells derived from umbilical veins (HUVEC). A plasmid pCH110 containing coding region for beta-galactosidase driven by SV 40 early promoter region was employed to transfect HUVEC. The developed protocol exploits the role of apolipoprotein E (Apo E) in the metabolism of Apo E-containing lipoproteins and its high affinity binding to LDL receptors. DNA transfection of cultured HUVEC was carried out using standard transfection methods including calcium phosphate precipitation, polybrene mediated transfection, and lipofection. The new methodology of transfecting HUVEC employed Apo E adsorbed lipofection reagent-DNA complex, and was found to be the most efficient procedure to transform HUVEC in comparison to the standard methods used in this study.

Enhanced attachment and growth of human endothelial cells derived from umbilical veins on ammonia plasma modified surfaces of PTFE and ePTFE synthetic vascular graft biomaterials.

Ammonia plasma generated by electrical discharge at low pressure was employed for the surface modification of PTFE and ePTFE. A new chemistry at the plasma treated surfaces is reported. X-ray photoelectron spectroscopy studies showed the incorporation of C-N, C-O, C = O etc functional groups on the plasma treated surfaces. Human endothelial cells derived from umbilical veins (HUEC) were used to seed the plasma treated PTFE and ePTFE surfaces to assess the attachment and growth. Enhanced attachment and growth of HUEC was observed on the plasma treated surfaces. In addition, the performance of these surfaces in this respect was found to be considerably superior to human collagen or human fibronectin or collagen-fibronectin coated PTFE. HUEC attachment and growth on these plasma treated surfaces was further enhanced by immobilizing collagen or fibronectin or collagen-fibronectin. Ammonia plasma treated and untreated ePTFE vascular graft samples were seeded with 3.6 X 10(4) cells/sample. At 24 hrs after seeding, HUEC cell attachment was studied. Although, HUEC attachment on collagen or fibronectin coated ePTFE was improved, but there was no significant difference between the number of cells attached to these surfaces when compared with those adhered to plasma treated ePTFE without collagen or fibronectin coating. Collagen or fibronectin coated plasma treated surfaces showed better performance over their respective controls.

X-ray photoelectron spectroscopy studies, surface tension measurements, immobilization of human serum albumin, human fibrinogen and human fibronectin onto ammonia plasma treated surfaces of biomaterials useful for cardiovascular implants and artificial cornea implants.

XPS studies of untreated and ammonia plasma treated surfaces of PTFE, ePTFE, Dacron, P(HEMA), PMMA, Silastic and PS were carried out. Ammonia plasma treatment caused significant changes in the surface composition. The curve-fitting results confirmed the incorporation of nitrogen and oxygen in the form of functional groups such as C-N, C=O, C-O, Si-N, Si-OH etc. Increases in the values of surface tension occurred. The surface tension of plasma treated surfaces varied between 44-48 erg/cm2 with the exception of Dacron which became wettable. Enhanced immobilization of human albumin on plasma treated surfaces was achieved. When washed with 0.2% Tween in buffer, these albuminated surfaces were found to be stable compared to control samples. Increased immobilization of human fibrinogen was also observed. The ammonia plasma treated surfaces showed high binding properties and retention for human fibronectin. Ionic interaction between proteins solution and plasma treated surfaces may be cause of the increase attachment of these biological molecules.

The enhanced attachment and growth of endothelial cells on anhydrous ammonia gaseous plasma modified surfaces of polystyrene and poly(tetrafluoroethylene).

Anhydrous ammonia gaseous plasma technique was used for the surface modification of polystyrene petri dishes and poly(tetrafluoroethylene) (PTFE) membranes. Amino groups were added onto surfaces by exposing them to ammonia plasma. Plasma modified polymeric surfaces and control polymeric surfaces were seeded with bovine pulmonary artery endothelial cells (EC). It was found that attachment of EC to control polystyrene surface was negligible. On the plasma modified polystyrene surface, there was improved attachment and growth of EC. At 96 hours, plasma modified surfaces yielded an order of 3 magnitudes more cells compared to those on control. Twenty four hours after seeding the cells, the percentage of EC attachment to control PTFE surfaces and modified surfaces were found to be about 36% and 92% respectively.

Towards an artificial cornea: surface modifications of optically clear, oxygen permeable soft contact lens materials by ammonia plasma modification technique for the enhanced attachment and growth of corneal epithelial cells.

The advent of high water content, oxygen permeable contact lens materials has made the intracorneal implants more feasible. A major obstacle encountered is the regrowth of a stable epithelium over the implant. Therefore, ammonia gaseous plasma modification technique was used to modify the surface chemical properties of soft contact lens material such as poly(2-hydroxyethyl methacrylate and methacrylic acid), PHEMA-MAA copolymer, in an attempt to enhance the cell attachment and growth of rabbit corneal epithelial cells.

Enhanced oxidation of bilirubin by an immobilized tri-enzyme system of glucose oxidase, bilirubin oxidase and horseradish peroxidase.

Bilirubin oxidase was immobilized to nylon fibres. A tri-enzyme system composed of glucose oxidase, bilirubin oxidase and horseradish peroxidase was also immobilized to the fibres. Both immobilized systems were tested and it was found that the latter gave enhanced oxidation rates for bilirubin.

Immobilization and kinetics of lactate dehydrogenase at a rotating nylon disk.

Lactate dehydrogenase (LDH) was covalently attached to an impervious nylon surface by an improved technique. The procedure allowed the kinetics of the rotating enzyme disk reactor to be successfully explored. This enzyme-disk configuration has potential applications in assays for lactic acid or pyruvic acid in fluids of biological importance (e.g., urine). In order to evaluate and understand the physics and chemistry underlying the kinetics of the heterogeneous biocatalyst, a mathematical model based on the von Karman-Levich theories of rotating electrodes, was developed. It applied well to LDH attached to a disk, under variable NADH concentrations and fixed pyruvic acid. The new theory, leads to the conclusion that the apparent Michaelis constant K(m)(app), varies linearly with f(-1/2), where f is the speed of rotation of the disk. Extrapolation of f(-1/2) to zero gives the Michaelis-Menten constant, K(m), corresponding to the diffusion-free behavior. With immobilized LDH, the diffusion-free K(m) for NADH obtained at 25 degrees C, in phosphate buffer (pH 7.5) using the extrapolation method was 84 muM. This value was in good agreement with the previously published value of 87 muM, obtained with LDH attached to the inner surface of a nylon tubing. However, when compared to the K(m) for a free enzyme system, the 84 muM was about nine times larger, indicating an inherent reduction in the activity of the bound LDH. Since, at extrapolated infinite rotation speeds, diffusion effects were assumed eliminated, the drop in the activity was thought to be due to sterric hinderances imposed on the substrate NADH as a result of having LDH bound to another polymer.

Immobilization of enzymes on polypropylene bead surfaces by anhydrous ammonia gaseous plasma technique.

Anhydrous ammonia gaseous plasma technique was used for the surface modification of polypropylene beads. Amino groups were added onto the surfaces of beads by exposing them to ammonia plasma. Through these amino groups covalent immobilization of glucose oxidase and peroxidase were carried out. The total amounts of immobilized glucose oxidase and immobilized peroxidase were found to be 52 and 43 micrograms/cm2, respectively. To assess the stability of enzyme-polypropylene linkage, beads with covalently immobilized glucose oxidase and peroxidase were washed with phosphate buffer. It was found that after the removal of the adsorbed enzymes, the concentration of covalently immobilized enzymes tended to reach a steady state. After additional washing with buffer for 5 to 6 h, 40-55% of the immobilized enzymes were found to be in the active form.

FTIR-ATR spectra of protein A immobilized on to functionalized polypropylene membranes by gaseous plasma of oxygen and of anhydrous ammonia.

Polypropylene membranes were treated in a gaseous plasma of oxygen or anhydrous ammonia, in order to add hydroxyl groups (oxygen plasma) or amino groups (ammonia plasma) on their surfaces. The presence of these functional groups was detected by FTIR-ATR spectrometry. The FTIR-ATR spectrum of free Protein A was recorded and absorption bands for amide I and amide II at 1650 cm-1 and 1552 cm-1 were identified. The surfaces of immobilized Protein A on to hydroxylated polypropylene membranes and on to polypropylene membranes having amino groups, were characterized by FTIR-ATR spectrometry. The presence of amide I and amide II absorption bands confirmed the immobilization of Protein A on to functionalized polypropylene membranes by gaseous plasma modification.

Enhanced albumin binding to polypropylene beads via anhydrous ammonia gaseous plasma.

Plasma surface modification technique was used to add amino groups onto the surfaces of polypropylene beads by exposing them to anhydrous ammonia plasma. Through these amino groups, albumin was attached to the polypropylene beads. Attached albumin was further stabilized by crosslinking with glutaraldehyde. The effect of washing albuminated polypropylene beads with saline and human plasma was investigated. It was found that after initial rapid removal of albumin, the concentration of attached albumin tended to reach a steady-state. After 52 h of washing, the amount of albumin retained on the beads varied between 125 and 171 micrograms/cm2.

Characterization of plasma polymerized polypropylene coatings.

Propylene was polymerized at low pressure in a radio frequency plasma reactor. The plasma polymerized propylene (PPP) films were insoluble in toluene. Thermal analysis of PPP showed no phase transition took place up to 255 degrees C. From these results, it was concluded that PPP is a highly crosslinked polymer. To further characterize the deposited PPP, Fourier Transform infrared (FTIR) spectroscopy in the attenuated total reflection (ATR) mode was used. The deposited material was shown to be polymerized propylene as a spectrum of the material showed absorption bands characteristic of polypropylene.

Characterization of plasma polymerized silicone coatings useful as biomaterials.

Plasma polymerization techniques were used to deposit a layer of filler-free silicone rubber on a variety of substrate materials. The thickness of the deposited film was 0.5-0.8 micron. As it is the surface of the biomaterial that comes in direct contact with the body fluids, the surface of the biomaterial is of paramount importance. In this study, the plasma polymerized biomaterials were characterized. Thus, the scanning electron microscope (SEM) showed the surfaces to be smooth. To study the surface layer of the deposited polymer, Fourier-transform infrared spectrometry in the attenuated total reflection (ATR) mode was used. The deposited material was indeed silicone polymer with adsorption bands at 1262, 1020, and 802 cm-1 for the Si-CH3 bending, Si-0-Si stretching, and Si-CH3 bending, respectively. To find the bonding nature of the polymer, electron spectrometry for chemical analysis (ESCA) was used. The silicone polymer was shown to be highly cross-linked. To find the molecular weight between cross-links, swelling studies were done. Thus the results of the study show that the plasma polymerization could produce a filler-free silicone layer on a variety of substrate materials.

Albuminated polymer surfaces for biomedical application.

Amino groups were added on to the surfaces of Celgard-2400 membranes by exposing them to an ammonia plasma. The presence of amino groups on the surfaces was detected by an attenuated total reflection Fourier Transform infrared spectrometer and by the Auger electron spectrometer. Through these amino groups, albumin was attached to the membranes. In some experiments, the attached albumin was further stabilized by cross-linking with glutaraldehyde. The effect of washing the albuminated membranes with saline and with plasma was investigated. It was observed that after the initial wash-out of albumin, the concentration of attached albumin tends to level off. The amount of albumin retained on the membranes varied between 275 to 357 micrograms/cm2.