Endothelial cell behavior on vascular prosthetic grafts: effect of polymer chemistry, surface structure, and surface treatment.

When implanting any vascular prosthetic grafts, one important goal to ensure long-term patency is achieving complete endothelialization of the luminal surface, a process that has rarely been observed clinically in humans. Seeding vascular grafts with endothelial cells has been seen as an attractive approach but has not been clinically convincing. A determining factor may be the type of polymer and surface structure. Using organotypic culture assays, the present investigation studied the effect of different polymers, surface structures, and surface treatments on endothelial cell behavior. The materials tested were polyester (PET), polytetrafluoroethylene (PTFE), polyesterurethane (PESU), and polyetherurethane (PETU) grafts with different surface structures. The surface treatments on the PET grafts included impregnation with cross-linked albumin, collagen, and gelatin, and treatments with fluoropolymer and electrically conducting polypyrrole polymer. Low density polyethylene (LDPE) and polydimethylsiloxane (PDMS) sheets (smooth surface, plain wall) were used as controls. After incubation for 7 days at 37 degrees C, cell adhesion and migration on the different polymers and structures were as follows: woven and knitted PET (high porosity) > PTFE, PESU, PETU hydrophobic (low porosity) > PETU hydrophilic, LDPE, PDMS (no porosity). Cell density results showed no difference between polymers and porous structures and a higher cell density on smooth nonporous surfaces. Compared with the nonimpregnated PET structures, knitted PET treated with albumin, collagen, or gelatin showed slight decreases of cell adhesion. No differences in cell migration and density were reported between any of the PET grafts, except for one polyester graft with a different chemistry than Dacron, which exhibited greater cell migration and lower cell density. Polyester grafts with a fluoropolymer treatment showed lower cell adhesion and migration and higher cell density than the nontreated PET. Finally, the woven PET grafts treated with electrically conducting polypyrrole exhibited contrasting cell behavior depending on the conductivity involved.

In vitro cellular response to polypyrrole-coated woven polyester fabrics: potential benefits of electrical conductivity.

Electrically conducting polypyrrole-treated films have recently been shown to influence the morphology and function of mammalian cells in vitro. This type of polymer represents a possible alternative biomaterial for use in vascular implantation. The present study compared the in vitro biocompatibility of the five different polyester woven fabrics having increasing levels of electrical conductivity ranging from 4.5 x 10(4) to 123 omega/square with that of low density polyethylene and polydimethylsiloxane primary reference materials. Biocompatibility was measured in terms of four different types of in vitro cellular response, including (a) an indirect and (b) a direct control organotypic culture assay using endothelial cells, (c) a polymorphonuclear (PMN) cell activation study using flow-cytometric measurements of CD11/CD18 integrin molecule expression, and (d) a semiquantification of interleukin (IL)-6 mRNA expression on monocytes/macrophages using reverse-transcriptase polymerase chain reaction. The organotypic culture study revealed that the fabrics with high levels of conductivity exhibited lower cell migration, proliferation, and viability. The PMN activation study of blood from 10 healthy adult donors demonstrated that the two most conductive fabrics were able to identify the more reactive donors. The levels of IL-6 mRNA expression by monocytes/macrophages decreased as the conductivity level of the fabrics increased. The results of the present study therefore indicate that high levels of conductivity (< 200 omega/square) on polyester fabrics are detrimental to the growth, migration, and viability of endothelial cells; induce elevated PMN activation; and affect the intracellular metabolism of monocytes. They also point to a specific range of conductivity (10(3) < 10(4) omega/square) which is associated with an optimum in vitro cellular response.

Heparin immobilized on proteins usable for arterial prosthesis coating: growth inhibition of smooth-muscle cells.

Gelatin or a mixture of albumin and gelatin has been proposed for the coating of vascular grafts according to their surface thrombogenicity and biocompatibility, and the possibility of biodegradation. Heparin treatment of hemocompatible surfaces improved the patency of prostheses. In this study, different amounts of heparin were immobilized on these protein gels using a water-soluble carbodiimide [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]. The results showed a coupling of heparin with gelatin and/or albumin at the surface of the gels, stable for as long as 1 month. From 0.20 to 3.60 microg x cm(-2), heparin could be immobilized. The antiproliferative activity of immobilized heparin was controlled toward bovine smooth-muscle cells grown on these gels. Cell growth inhibition was dose dependent, but the percentages of inhibition were lower at day 8 than at day 4 at any heparin concentration used under experimental conditions. Referring to heparin in solution, immobilized heparin displayed an antiproliferative activity that improved the potential interest for coating.

Correlation between substratum roughness and wettability, cell adhesion, and cell migration.

Cell adhesion and spreading of chick embryo vascular and corneal explants grown on rough and smooth poly (methyl methacrylate) (PMMA) were analyzed to test the cell response specificity to substratum surface properties. Different degrees of roughness were obtained by sand-blasting PMMA with alumina grains. Hydrophilic and hydrophobic components of the surface free energy (SFE) were calculated according to Good-van Oss's model. Contact angles were determined using a computerized angle meter. The apolar component of the SFE gamma s(LW), increased with a slight roughness whereas the basic component, gamma s-, decreased. The acido-basic properties disappeared as roughness increased. Incubation of PMMA in culture medium, performed to test the influence if the biological environment, allowed surface adsorption of medium proteins which annihilated roughness effect and restored hydrophilic properties. An organotypic culture assay was carried out in an attempt to relate the biocompatibility to substratum surface state. Cell migration was calculated from the area of cell layer. Cellular adhesion was determined by measuring the kinetic of release of enzymatically dissociated cells. A slight roughness raised the migration are to an upper extent no matter which cell type. Enhancement of the cell adhesion potential was related to the degree of roughness and the hydrophobicity.

Selecting valid in vitro biocompatibility tests that predict the in vivo healing response of synthetic vascular prostheses.

We have investigated the usefulness of six in vitro biocompatibility tests in predicting the healing performance of polyester vascular prostheses as observed in previous canine in vivo trials. Vascular grafts were evaluated by using (i) a direct contact (DC) assay, (ii) an extract dilution (ED) assay on murine fibroblast cells, (iii) a DC assay on endothelial cells, (iv) a complement activation study, (v) a leucocyte activation study of CD18 integrin subunit expression on human polymorphonuclear cells (PMNs) and (vi) interleukin-2 receptor expression on lymphocytes. Uncleaned polyester grafts had previously been associated with poor healing and gelatin-impregnated polyester grafts with delayed but satisfactory healing, whereas commercially cleaned polyester grafts had demonstrated excellent healing. Lightweight and heavyweight knitted and woven polyester grafts supplied specifically for this project were studied, each with a different surface condition, i.e. commercially available (CP), uncleaned (UP) and impregnated with gelatin (GP). The UP grafts induced fibroblast cytotoxicity according to the ED assay, poor migration and viability of endothelial cells, and an elevated expression of CD18 and interleukin-2 receptor on PMNs and lymphocytes, respectively. In contrast, the CP grafts promoted good endothelial cell growth, no evidence of cytotoxicity and a weaker cell activation, and the GP grafts were found to be non-cytotoxic, to exhibit a good cellular response and to moderate cell activation. The complement activation assay and the DC assay on fibroblasts were found to be less useful and less discriminating. From this, it is concluded that the two cell activation measurements, the DC assay on endothelial cells and ED assay on fibroblasts, are useful in predicting the in vivo healing response of arterial polyester substitutes.

Cytocompatibility of calf pericardium treated by glutaraldehyde and by the acyl azide methods in an organotypic culture model.

Glutaraldehyde (GTA) is used to cross-link collagen-based biomaterials, but these materials are often cytotoxic. In order to overcome this problem, we have proposed the use of the acyl azide methods with either hydrazine or diphenylphosphoryl azide (DPPA) as reagents. In this paper we determine the cytocompatibility of acyl azide- and GTA-treated pericardium in vitro, by an organotypic chick aorta culture technique developed for the evaluation of the propensity of vascular cells (both endothelial and smooth muscle cells) to migrate and grow on the surface of biomaterials. We first examined pericardium stabilization as a function of GTA concentration and time, so that we could minimize residual GTA molecules in the material. Treatment for 72 h with 0.05% GTA was optimal for thermal stabilization of the pericardium with a denaturation temperature (Td) of 86.8 degrees C, providing similar results to treatment with 0.6% GTA for 4 h (Td = 85.1 degrees C). Pericardium treated in this way was, however, poorly cytocompatible with little vascular cell migration and growth when compared with tissues treated by the acyl azide methods. The best results were obtained with 0.5% DPPA; treated tissues showed a high level of cross-linking (Td = 82.4 degrees C) and three-fold increases in cell growth and migration over those in a non-toxic control.

In vitro organotypic culture method to evaluate the biocompatibility of heparin-surface-modified intraocular lenses.

Using an organotypic culture method, we evaluated the biocompatibility of two kinds of intraocular lenses: conventional poly(methyl methacrylate) (PMMA) and heparin-surface-modified (HSM) PMMA. Chicken corneal endothelium from embryos incubated for 14 days was placed on an agar medium and covered with one of three types of materials: PMMA, HSM PMMA, and a control, Thermanox. Experiments at five different times measured cell migration, cell multiplication, and cell adhesion. Scanning electron microscopy showed a low level of corneal endothelial adhesion on the HSM IOL surface. There was a significant difference between the HSM and untreated lenses in migration surface and cell density, with the HSM lenses having better biocompatibility.

Prostacyclin secretion by human endothelial cells grown on carbodiimide cross-linked proteins: an assessment of the cytocompatibility of a substratum.

Prostacyclin production by human umbilical vein endothelial cells cultured on carbodiimide cross-linked albumin and/or gelatin was quantified during the exponential growth phase and in confluent cultures as a response to arachidonic acid stimulus. In confluent cultures, basal production of prostacyclin measured by radioimmunoassay of the stable metabolite 6-keto-PGF1 alpha was comparable for both substrates to a control culture. Maximal release of prostacyclin occurred during the first 24 h following cell seeding and these values were significantly higher in media from cultures performed on membranes. In both cases, PGI2 production decreased as cell density increased. After stimulation with 20 microM arachidonic acid for 20 min, media from confluent cells grown on membranes contained slightly greater amounts of PGI2 than control culture medium. These results indicate involvement of substratum in PGI2 Release. Early enhancement of PGI2 secretion could improve biocompatibility of membranes by preventing platelet aggregation.

A novel microporous polyurethane blood conduit: biocompatibility assessment of the UTA arterial prosthesis by an organo-typic culture technique.

An organotypic culture assay has been used to assess the biocompatibility and cytotoxicity of an arterial prosthesis developed at the University of Texas-Arlington (the UTA graft) from a structurally modified polyurethane (PU) elastomer (Tecoflex). The cell culture test was applied to the UTA graft after sterilization by ethylene oxide and by gamma radiation in two separate series. First, small specimens of the prosthesis were incubated for 7 days on a semisolid nutrient medium with their luminal surface in direct contact with endothelium explanted from the aorta of chick embryos. Second, the possibility of cytotoxic contaminants being leached from the polyurethane was assessed by immersing the biomaterial in the liquid culture medium for 5 days at 37 degrees C prior to conducting the organo-typic culture assay on a standard control surface. The structure of the UTA polyurethane prosthesis is porous, but the graft wall is impervious because it contains closed (i.e., noncommunicating) pores. In addition, four other vascular prostheses were included in the study for comparison. They were the Hydrophilic Mitrathane PU graft with a similar impervious, closed pore structure, an experimental Hydrophobic Mitrathane PU graft with a fibrous, open pore structure, and the commercial Impra and Reinforced Goretex expanded PTFE grafts. Following 7 days of cell culture, the biocompatibility and cytotoxicity of the various biomaterials were measured in terms of the area of migrating cells, the density of cells surrounding the explants, and the level of cell adhesion. Comparison of the results against control cultures demonstrated that the UTA graft, along with the other four prostheses, does not release cytotoxic extractables. Microscopic observations of its cultured surface indicated that the UTA graft promotes a high density of cell growth over a limited area, similar to the Hydrophilic Mitrathane graft. This level of biocompatibility is considered inferior to that of the two PTFE and the Hydrophobic Mitrathane prostheses, which promote more extensive cell migration, greater cell adhesion, and cell growth in a continuous single layer.

A new bioactive molecule for improving vascular graft patency: exploratory trials in dogs.

Myxalin is a new bioactive molecule that we have isolated from the culture medium of Myxococcus xanthus, a non-pathogenic Gram negative bacterium. This glycopeptide possesses an antithrombotic effect in vivo and has been shown to promote human endothelial cell growth in vitro. With the object of exploring its ability to improve vascular graft healing and patency, myxalin was immobilized on 6 mm diameter knitted polyester prostheses using gelatin as a carrier, and the prosthesis was then implanted as an infrarenal abdominal arterial substitute in dogs for a period of 2 weeks. Two additional series of implantations were conducted for control purposes: one with gelatin-coated prostheses without myxalin, the other following normal preclotting of the polyester grafts. In order to select adequate sterilization conditions which can preserve the biological activity of myxalin, the prostheses were sterilized according to 3 different sterilization processes (gamma radiation and ethylene oxide either at 63 degrees C or 37 degrees C). At the sacrifice, all grafts were patent. The myxalin treated prostheses exhibited improved blood compatibility in terms of fewer thrombotic deposits and significant inhibition of platelet and fibrinogen uptake on their luminal surfaces. In addition, the development of a thin collagenous internal capsule with endothelial cells secreting high levels of prostacyclin was observed at both anastomoses of the myxalin-treated grafts sterilized by gamma radiation.

Use of myxalin for improving vascular graft healing: evaluation of biocompatibility in rats.

Myxalin is a glycopeptide extracted recently from a gram-negative bacterium. It has blood anticoagulant properties and can enhance endothelial cell growth. With the ultimate objective of using this bioactive molecule to promote vascular graft healing, this study assessed its biocompatibility in vivo by comparing the cellular and immunological responses of gelatin-coated knitted polyester grafts with and without myxalin following implantation in the peritoneal cavity of rats for prescheduled periods of 3 days and 1, 2, and 4 weeks. A nongelatin-coated virgin polyester graft was included as the reference material. The biological response to gelatin alone was characterized by a slower rate of cellular infiltration into the implant, reduced collagen synthesis, and higher levels of acid phosphatase and esterase activity in the surrounding tissue. The addition of myxalin to this coating resulted in a significant reduction of hydrolase secretion in the tissue surrounding the implant and an enhancement of cellular ingrowth.

Biocompatibility of the Vascugraft: evaluation of a novel polyester urethane vascular substitute by an organotypic culture technique.

To evaluate the biocompatibility of chemically and structurally modified polyurethane elastomers for use as blood vessel replacements, small squares of vascular prostheses were cultured in direct contact with endothelium from chick embryo aorta using an organotypic culture assay. The polyurethane materials tested were: Vascugraft (fibrous, open pore structure); commercial Hydrophilic Mitrathane prosthesis (high porosity, smooth surface, non-permeable, closed pore structure); experimental hydrophobic Mitrathane (less porosity but a fibrous, open pore structure, similar to Vascugraft). The commercial expanded polytetrafluoroethylene prostheses Impra and reinforced GORETEX were included as controls on account of their extensive clinical application in the femoropopliteal position. After 5 d incubation at 37 degrees C biocompatibility was assessed in terms of average area of migrating cells on the biomaterial, total number of cells surrounding the explant and level of adhesion between the cells and the biomaterial. The Vascugraft prosthesis promoted the growth of a continuous monolayer of cells on its surface. This behaviour was equivalent to Impra and reinforced GORETEX materials in terms of cell density and area of cell migration but appeared to be superior for cell adhesion. From a second series of cell culture tests, in which the extractables leached from the biomaterials were added to the nutrient medium, it was concluded that none of the biomaterials tested released cytotoxic contaminants.

IL-1-induced procoagulant and fibrinolytic activities of human endothelium grown on carbodiimide crosslinked proteins.

We examined the regulation of procoagulant activity and the production of fibrinolytic components by human vascular endothelium grown on coating membranes of gelatin, pure or mixed with albumin, crosslinked by carbodiimide ((G)C, (AG)C) in comparison with plastic culture dishes. Confluent monolayers were stimulated by human recombinant interleukin (IL-1 beta) and responses in terms of tissue factors like procoagulant activity, tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were followed for up to 72 h. Procoagulant activity of cell extracts displayed similar patterns whatever the substratum tested. Quantitative immunological assays revealed a 2-fold increase in tPA antigen released from monolayers grown on (G)C and on (AG)C compared to cells grown on plastic. Exposure of monolayers to IL-1 beta reduced the secretion of tPA antigen which still reached higher values on coated than on uncoated substratum. We found that the quasi-totality of tPA formed stable complexes with PAI-1, thereby suppressing measurable fibrinolytic activity. IL-1 beta stimulated the release of PAI-1 antigen quantified by immunoassay and the kinetics of secretion were comparable on both coated and uncoated substratum.

Comparative behaviour of L-929 fibroblastic and human endothelial cells onto crosslinked protein substrates.

Studies were carried out to compare the behaviour of human umbilical vein endothelial cells (HUVEC) and L-929 fibroblastic cells towards proteins crosslinked by glutaraldehyde (GTA) or carbodiimide (CDI) proposed for coating of vascular prostheses. CDI crosslinking of bovine serum albumin used alone, or mixed with gelatin, allowed higher rates of cell growth and DNA synthesis than GTA crosslinking independent of cells. Assessment of the plating efficiency revealed a similar behaviour of both cells towards membranes and reference plastic surface in terms of percentages of bound cells. HUVEC proliferation onto CDI crosslinked gelatin and/or albumin membranes did not differ significantly whereas the growth of L-929 was enhanced onto gelatin albumin membranes in comparison with both gelatin membranes and the reference surface. The analysis of DNA synthesis corroborated the results of the growth curves and elicited a delay of the growth phases in HUVEC cultured onto CDI crosslinked membranes, unlike the L-929 fibroblast.

Comparative assessment of cell/substratum static adhesion using an in vitro organ culture method and computerized analysis system.

The trypsin sensitivity of chick embryo cellular layers cultivated by an in vitro organ culture method toward different biomaterials has been analysed. Monolayer cells grown on to tested samples were enzymatically dissociated in 5 min - 1 h. Cumulative cell numbers, expressed as the percentage of the totally detached cell number, were plotted versus time, thus permitting the calculation of the function. The mathematical treatment by a computerized system of this function represents the trypsin sensitivity. This value modulated by the migration cell number is the static adhesion modulated index (SAMI). The trypsin sensitivity expressed according to this index allowed the establishment of an abacus wherein several zones, A, B, C and D, define cell adhesion behaviour on different biomaterials. Scanning electron microscopy analysis performed on dissociation steps showed the selective activity of trypsin on cells toward different substrata, revealing the role of an extracellular matrix and cytoskeleton in the adhesion behaviour.

Polyester arterial grafts impregnated with cross-linked albumin: the rate of degradation of the coating in vivo.

One of the techniques used to avoid preclotting a porous textile arterial prosthesis is to coat the graft with a layer of a bioerodible polymer. The efficacy of this treatment is dependent in part on the rate at which the polymer degrades after implantation. The focus of this study was therefore to investigate the rate of in vivo degradation of albumin-coated polyester (Dacron)-knitted vascular prostheses. Two types of cross-linked albumin were included: one using glutaraldehyde, the other using carbodiimide as the cross-linking agent. Radioactively labeled albumin-coated prostheses were implanted in the thoracic aorta and peritoneal cavity of dogs, and their rates of biodegradation in vivo were monitored over a 4-week period. The rate of biodegradation was found to depend upon the site of implantation. It occurred more rapidly in the peritoneal cavity where less than 20% of the albumin coating remained after 4 weeks in vivo in comparison to the approximately 30% in the thoracic aorta. The nature and intensity of the cellular response appeared to be related to the cytotoxic potential of the cross-linking agent. Glutaraldehyde induced an inflammatory response and a delay in healing, whereas carbodiimide caused only a mild tissue reaction.

Characteristics of polyester arterial grafts coated with albumin: the role and importance of the cross-linking chemicals.

Preclotting is mandatory prior to implanting a knitted polyester arterial graft, unless the structure is made impermeable to blood by coating with a bioerodible material. Before achieving wide-spread clinical acceptance, the technique of impregnating with cross-linked albumin must be optimized in order to develop a graft that is immediately implantable, easy to handle and suture and has improved healing characteristics. The choice of the chemical to cross-link the albumin is of paramount importance. In this study two alternative candidates have been evaluated by using a series of tests to measure the physical properties, the morphology and the cytocompatibility of albumin-coated grafts. A carbodiimide cross-linking agent appears more promising than glutaraldehyde, since it is equally effective in producing a blood impermeable prosthesis, yet presents improved biocompatibility and provokes a less intense inflammatory response from the host.

In vivo evaluation of polyester arterial grafts coated with albumin: the role and importance of cross-linking agents.

Previous in vitro studies have predicted that the type of chemical used to cross-link albumin-coated polyester arterial prostheses may influence the rate of bioerosion of the albumin layer in vivo. This study has confirmed that the healing process of this type of compound prosthesis does indeed depend on the nature and concentration of the cross-linking agent used. Four series of implantations in the thoracic aorta of dogs for scheduled periods for 4 h up to 6 months were conducted using 1.6% glutaraldehyde, 2.5% glutaraldehyde and 0.2 M carbodiimide as the alternative cross-linking agents plus a nonalbuminated preclotted polyester prosthesis which served as the control. The pathology of the explanted grafts revealed that in the short and medium term the rate of healing and the extent of tissue ingrowth was dependent initially on the presence of and later on the rate of bioerosion of the albumin layer. After 3 months in situ, the prostheses coated with albumin cross-linked with 1.6% glutaraldehyde and carbodiimide had healed more rapidly and were invaded by more extensive tissue ingrowth than the one cross-linked with 2.5% glutaraldehyde or the preclotted control. Moreover, the migration of cells over the carbodiimide-treated surface was the most fully developed and most regularly organized of all four series. Immunostaining revealed that the presence of glutaraldehyde induced an inflammatory response which failed to support the growth of normal luminal cells with the endothelial phenotype.

Radiosterilization of albuminated polyester prostheses.

The study reported here is concerned with the radio-sterilization of Dacron vascular prostheses coated with crosslinked albumin. gamma-Radiations have no effect on the mechanical properties of the polyester fibres or on their crystallinity, whether irradiated in a dry state or immersed in saline. Special attention has been paid to the release of the albumin, or protein fragments from the reticulum using 125I-labelled albumin as a radiotracer. The albumin leakage depends upon the type of Dacron fabrics considered but the values derived from radioactivity measurements are always greater than those directly measured, which indicates a radio-induced break of the bond between iodine and albumin; this has nothing to do with the break of the association between albumin and Dacron. Moreover no cytotoxicity of the irradiated immersion medium has been observed using a test based on organotypic culture in liquid medium. Thus radio-sterilization of an albuminated polyester vascular prosthesis immersed in saline appears to be a suitable procedure.

F.p.l.c. analysis of the leakage products of proteins crosslinked on Dacron vascular prostheses.

The haemocompatibility of vascular, Dacron prostheses was improved by coating with albumin and/or collagen crosslinked with glutaraldehyde (GTA) or carbodiimide (CDI). F.p.l.c. (fast protein liquid chromatography) analysis of the products desorbed from polymeric matrices incubated in physiological conditions for periods extended up to 10 d did not detect the monomers or polymers of collagen and albumin but small amounts of degradation products, the molecular weights of which were less than 45,000, thus minimizing an eventual immunogenic response after implantation. However GTA and CDI matrices required extensive washing to neutralize the cytotoxic effect of GTA and achieve the release of CDI from protein complexes.