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.

Non-saponifiable fraction of cocoa shell butter: effect on rat and human skin fibroblasts.

Synopsis Non-saponifiable lipid fraction (ICSB) extracted from cocoa shell butter was solubilized in dimethylformamide (DMF) and analysed for its biological activity on growth of rat and human fibroblasts. Non-saponifiables (10 mug ml(-1)) partially protected cells from toxicity of DMF (1%) and allowed the growth of fibroblasts cultivated in optimal conditions (10% fetal calf serum-FCS, 37 degrees C) or improved the survival of cells maintained in altered conditions (2.5% FCS, 35 degrees C). At higher concentration (ICSB 50 mug ml(-1), DMF 1%), the protective effect was suppressed. ICSB was fractionated by chromatography into four compounds: sterols, terpenic alcohols, tocopherols and hydrocarbons +/- carotenoids. We found that biological activity of ICSB was mostly due to the major fraction containing sterols.

Anticoagulant activity of a bacterial glycopeptide.

The anticoagulant properties of myxalin, a glycopeptide secreted by a Gram negative bacterium strain (Myxococcus xanthus) are studied and compared to those of heparin. This soluble material exhibits an anticoagulant activity which implies the inhibition of some serine proteases, thrombin and factor Xa. In the presence of normal and antithrombin III-depleted plasma, myxalin inhibits the amidolytic activity of thrombin on synthetic chromogenic substrate as a function of its concentration, but fails to increase thrombin inactivation significantly in the presence of purified AT III. However, crossed immunoelectrophoresis data suggests that its antithrombi effect is mainly mediated by binding to the enzyme, rather than to AT III and probably differs from the catalytic activity of heparin which requires the presence of AT III. The anticoagulant process occurs without degradation of fibrinogen and can be neutralized by protamine.

Chemically processed bovine heterografts of the second generation as arterial substitutes: a comparative evaluation of three commercial prostheses.

The use of chemically processed bovine heterografts is primarily confined to the construction of arterio-venous blood accesses in those patients requiring hemodialysis, plasmapheresis or chemotherapy. The grafts of the first generation i.e. Artegraft and Solcograft are now being supplanted by those of the second generation i.e. Reinforced Artegraft, Solco P and NCGT. We have investigated these three types of arterial prostheses as a biomaterial in terms of sterility, inflammatory response and cytocompatibility and as a blood conduit in dogs in terms of patency and healing. For each type of graft, two implantations were carried out for durations of 4 hours, 24 hours, 48 hours, one week, two weeks, one month, three months, and six months. Therefore a total of 48 grafts were implanted. All grafts but five were patent at sacrifice: thromboses were observed in two Reinforced Artegraft (after two weeks and after one month) and in three NCGT (after 24 hours, after 48 hours, and after one month). Therefore the following patencies were observed: Reinforced Artegraft 14/16, Solco P 16/16 and NCGT 13/16. In all the patent grafts, the healing was reduced to the formation of a pannus along both anastomoses; thrombotic accumulations were observed on the surface defects of the grafts, particularly the NCGT graft. The Reinforced Artegraft presents only minor advantages over the previous Artegraft; the Solco P, somewhat more acceptable is no longer commercially available since the manufacturer withdrew it after early clinical failures. The improvements noted in the bovine heterografts of the second generation appear to be marginal as compared to those of the first generation.

The cytocompatibility of compound polyester-protein surfaces using an in vitro technique.

To avoid the need to preclot porous polyester (Dacron) vascular prostheses, we have proposed the use of a protein coating that will promote the growth and adhesion of endothelial cells. This study assessed the relative advantages of coating woven, knitted, and velour polyester fabrics with albumin, collagen, and a albumin-collagen mixture after preservation in saline or drying by a commercial dehydration process. Preclotted fabrics were used as controls. The cytocompatibility of these biopolymers was measured by an organotypic culture technique which relies on the migration of chick embryonic endothelial cells. After 7 d of culture the cytocompatibility was quantified by counting the cells in the area of migration and the morphology of the endothelial cells was observed by scanning electron microscopy. In general, the knitted and velour fabrics showed superior compatibility than the woven one. The results confirmed that collagen, either alone or combined with albumin, provides in most cases a more cytocompatible surface than albumin alone. A cell morphology most closely resembling that of natural arterial endothelial cells was observed on the albumin-collagen substrate.

Polyester prostheses as substitutes in the thoracic aorta of dogs. II. Evaluation of albuminated polyester grafts stored in ethanol.

In an attempt to find an alternative procedure to the preclotting of porous textile vascular prostheses, the feasibility of an albumin coating and ethanol preservation technique has been evaluated by implanting albuminated polyester prostheses as substitutes in the thoracic aorta of dogs. Nine different grafts representing woven, knitted, and velour constructions were implanted for periods ranging from 4 h to 6 months. At the sacrifice, the grafts were excized for measurement of the thrombogenicity of the flow surface, for pathological examination by light microscopy and SEM, and for quantifying the changes in the textile structure during implantation. Also the kidneys were removed and examined for infarcts caused by trapped circulating emboli. The healing characteristics of the nine different grafts proved similar and followed the same sequence of events as preclotted control grafts. The albumin coating and ethanol preservation resulted in a somewhat slower rate of healing. Yet, given sufficient time, a more completely healed graft was achieved as evidenced by the presence of endothelial-like cells throughout the length of the graft. In addition, the albumin is believed to reinforce the textile structure by reducing the tendency to stretch and dilate in vivo.

Cytocompatibility of albuminated polyester fabrics.

An alternative to the usual technique of preclotting porous textile vascular prostheses prior to surgical implantation is to render them impermeable to blood by impregnation with a cross-linked albumin filler matrix. This material subsequently becomes the foundation for cellular development. The compatibility of such impregnated fabrics with newly formed endothelial cells has been evaluated by an in vitro organotypic culture method. This technique enables the characterization and numeration of cells that develop on blood contact surfaces and enables determination of their rate of development. Woven, knitted, and velour fabrics were evaluated following coating with albumin and either storage in Tyrode solution or 40% ethanol or desiccation by critical point drying. Preclotted cardiovascular repair fabrics prepared according to conventional surgical protocol served as controls. The identification of the newly formed cells was confirmed histologically. The most extensive and rapid cellular development was observed on the woven fabric and is believed may have resulted from the smoother surface topography of this substrate. Good cellular development was noted particularly on fabrics which had been stored in Tyrode solution. Ethanol had a deleterious effect on the apparent compatibility.

[Liquid storage media for vascular prosthesis of biological origins : their impact on biocompatibility (author's transl)]. / Influence des milieux de conservation sur la biocompatibilité des prothèses vasculaires biologiques.

Unlike synthetic arterial substitutes which are supplied as presterilized, dry-packaged products with potentially indefinite shelf lives, arterial prosthesis of modified biological origin must stored in less convenient liquid preservative media. These preservatives can affect the post-implantation performance and the biocompatibility of the devices. In order to identify the most suitable media, a modified "in vitro" cell culture test method was developed. It makes use of endothelial cells prepared from the aorta of chick embryo. In general, the prosthesis stored in liquid media had a disappointing performance from the point of view of neo-endothelium development and adhesion. Residual cytotoxicity was also noted for most of these currently used prosthesis storage and pre-surgical preparation protocols. Although devices stored in physiologic media showed adequate biocompatibility according to this cell culture test, it appears that neo endothelialization does not proceed concurrently with healing. Cellular development where present, adheres poorly to the substrate and cannot resist the shear forces of normal blood flow. Pre-implantation treatment of such prosthesis with cellular growth-promoting substances may enhance cell-prosthesis adhesion.

[In vitro study of cellular affinity to various protein supports suited for vascular prosthesis]. / Etude in vitro de l'affinité cellulaire de différents supports protéiques susceptibles d'être utilisés comme prothèses vasculaires.

We compare the affinity of different cellular samples with regard to variosu artificial materials by organotypic culture method. We use a technique which allows at once, to obtain the cells and to measure their migration on the artificial surface. The results show a high variation of the cellular affinity from one sample to another.