FR2722974A1 - METHOD FOR MODIFYING THE INTERNAL SURFACE OF SYNTHETIC PROSTHESES USED IN VASCULAR SURGERY - Google Patents

Abstract

The method comprises the formation, on the internal surface of synthetic prosthesis, of a physiological endothelial matrix obtained in vitro from arterial cells, said internal surface being preferably precoated with human fibronectine. The main steps of the method are as follows: coating the internal surface of the prosthesis by incubation with a human recombinant fibronectine solution, washing with a saline solution to remove the fibronectine in excess, seeding the internal face of the prosthesis with a suspension of arterial endothelial cells and cell culture by incubation of the prosthesis in a sterile container at 37 DEG C, elimination of the arterial cells by means of 2 M urea solution and storage of the prosthesis thus matrixed at 4 DEG C in a sterile environment.

Description

 The present invention relates to a method of modifying the internal surface of synthetic prostheses used in vascular surgery by producing in vitro a physiological endothelial matrix.

 In vascular surgery, bypass surgery is one of the most used techniques to treat arterial lesions. The ideal is to have an autologous arterial graft or, failing that, an autologous vessel of the venous type: saphenous vein most of the time.

 However, this preferential use of autologous vessel comes up against a major obstacle: the absence of a graft, either depending on the site of implantation, or because of the patient's history (saphenous vein stripped, previous use ...) or still because major alterations render the graft unusable.

 We therefore tried to find a valid alternative to autologous material. This is how the development and use, over the past 30 years, of synthetic materials, in particular Dacron (polyethylene terephthalate), woven or knitted, and PTFE (polytetrafluoroethylene) have allowed vascular surgery to '' advance considerably.

 These synthetic prostheses give results comparable, in terms of permeability, to those of an autologous vessel for large and medium-sized arteries. But their non-adaptation to small arteries (less than or equal to 4 mm) quickly became evident. This is explained, among other things, by the high thrombogenicity of the synthetic internal surface.

 Indeed, after implantation, and during exposure to blood flow, adhesion and platelet aggregation very quickly occur, followed by the development of an inflammatory fibrous tissue on the internal surface (fibrin, fibroblasts, macrophages, leukocytes ). The increase in platelet activity also induces the release of mitogens from smooth muscle cells, leading to hyperplasia in the areas of anastomosis with adjacent vessels.

In animals, spontaneous endothelialization of the surface can sometimes appear over time, endothelial cells originating from external capillaries, the blood circulation as well as adjacent vessels
In humans, on the other hand, there is no spontaneous colonization of the endoluminal surface with the exception of the juxta-anastomotic zones.

 In order to improve synthetic prostheses and to make them usable even in small calibers, it was thought that the "ideal" prosthesis should, among other things, reproduce on its internal face the natural vascular wall with a functional and stable endothelial coating.

 In 1978, Herring et al. (Surgery 78: 498 - 504) introduced the concept of seeding synthetic prostheses with autologous endothelial cells, in order to obtain an anti-thrombogenic surface. Subsequently, the development of harvesting and multiplication techniques in culture of endothelial cells, such as those described by Jarrel et al (1984 J. Vasc.

Surg. 1: 757-764) as well as the use of adhesive factors: fibronectin, laminagen collagen ... (Carabasi et al. 1991-Ann. Chir. Vasc. 5: 477-184 and Vohra et al. 1991
Br.J. Surg. 78: 417-420) have enabled the development of endothelialization trials of vascular prostheses.

 Numerous tests have thus been able to be carried out both in animals and in humans, on Dacron or PTFE.

These techniques allow:
- creation of a uniform functional cell monolayer before implantation; this results in a reduction in platelet activity and an improvement in permeability in the short and medium term
- inhibition of the proliferation of smooth muscle cells
- better resistance of prostheses to infection.

However, these techniques have limits:
- resistance of the monolayer to blood flow
- differences in morphology, fixation and growth potential of endothelial cells depending on their origin (venous or arterial cells) requiring their readjustment to the physiological conditions of implantation
- difficulty in organizing a procedure that is reliable clinically and over time: sampling, cell culture, need for absolute sterility throughout these stages.

 - Difficulties in clinical evaluation and time back to judge the stability of the monolayer.

 The inventors sought to circumvent the crippling limitations brought about by intraoperative endothelialization, and they chose to create a synthetic prosthesis whose internal surface is covered with a substrate allowing true endothelialization, in vivo, by endothelial cells. autologous and functional.

 We know that the adhesion, proliferation and migration of endothelial cells depend, above all, on the support on which they rest; it is this support that determines the polarity, orientation, morphology and response of the cell to stimulating factors.

 In the natural vascular environment, this support corresponds to the subendothelial basement membrane. This extracellular matrix (ECM) secreted by the endothelial cell at its basal pole has a defined organization and composition in matrix proteins and growth factors.

 The inventors have therefore endeavored to recreate this natural basement membrane on the internal surface of synthetic prostheses.

 Various studies have demonstrated the adhesive properties of the MEC of endothelial cells of the cornea or aorta (Gospodarowicz D. 1984 A.R. Liss.

Inc. N.Y. 275293). The MEC, secreted by these cells in culture and deposited at their basal pole, remains firmly attached to the culture support. Washing with a mild detergent of the cell monolayer releases the intact matrix free of cellular elements or debris.

 Using this principle, the inventors have developed an in vitro matrixing of synthetic prostheses with MEC of arterial cells; this preparation allows rapid and complete colonization of the endoluminal surface by the endothelial cells of the host during the in vivo implantation.

 The process for modifying the internal surface of synthetic prostheses used in vascular surgery according to the invention is characterized in that it consists in producing on said internal surface a physiological endothelial matrix obtained in vitro from arterial cells. This ensures true endothelialization, in vivo, by autologous and functional endothelial cells.

 According to a preferred embodiment of the invention, said internal surface is previously coated with human fibronectin.

 The present invention will be better understood, and its advantages will emerge clearly from the following description of the in vitro validation protocol.

 Preparation of cell lines Arterial lines, intended for the matrixing and endothelialization stage, were prepared from aortas or femoral arteries of mammals.

 Each arterial sample obtained, whatever its origin or nature, was treated in the same way, under rigorously controlled biosecurity conditions (sterility, harmlessness).

The arterial sample, placed on a tray, is incised longitudinally and then gently rinsed with a saline solution to remove residual blood elements. The endothelial cells are harvested by scraping the entire surface, then sown in a culture well (24-well plate). The culture plate is incubated at least 24 h at 37 "C before the first change of
culture centre.

 The culture medium for this work is preferably the EGM medium marketed by Clonetics: MCDB 131 medium supplemented with fetal calf serum, hydrocortisone, human recombinant epidermal growth factor (hEGF), bovine brain extract (Bovine Brain Extract BBE).

 Endothelial cells can be detached from their support for harvesting or reseeding by the action of a Trypsin-EDTA solution.

Validation of the matrix on PTFE
Conformation: In vitro endothelialization tests were carried out on PTFE + human fibronectin + MEC arterial cell disks compared to unmodified PTFE and PTFE disks coated with human fibronectin alone.

 A modification of Ependorf tubes made it possible to produce culture chambers with a surface area of 0.5 cm2, allowing immobilization of the prosthetic material (PTFE-Dacron); each disc thus formed is autoclaved before use.

 Coating with human fibronectin - The discs are incubated for 1 hour at 37 "C or overnight at 4" C with 5 μg / cm 2 of fibronectin from human plasma. The excess gel is sucked off, leaving a thin film on the surface.

Coating of an arterial matrix layer
The arterial cells are seeded at a density of 105 cells / cm2, on the prosthetic surface previously coated with fibronectin. They are then kept in culture for approximately 12 days in the culture medium.
EGM + 4% Dextran T 40. A regular change of the medium is carried out every 3 days. At the end of this culture time, the cells are detached by 2 successive 30 min incubations. in a 2 M urea solution at 37 "C, leaving the MEC firmly attached to the PTFE disc and devoid of cellular elements.

In vitro endothelialization
Pig aorta arterial cells are seeded, in parallel, on the various supports (naked PTFE, PTFE + human fibronectin, PTFE + human fibronectin + arterial ECM) at a density of 105 cells / disc; they are then cultivated for a period of 5 to 7 days in the EGM medium.

The endothelialization capacity of each support is determined by the following tests:
- cell count on days D + 1 and D + 5 of the culture of the detached cells by the action of a solution of Trypsin -EDTA (cell counter:
Coulter ZM). The results, expressed by the average of triplicate tests carried out for each support. are illustrated by the appended schematic drawing in which:
Figures 1, 2, and 3 are bar graphs of cell counts.

 In these figures, the number of cells is plotted on the abscissa and the number of days on the ordinate.

We see in Figure 1 the almost complete lack of adhesion of endothelial cells on the bare PTFE support (only 8% of cells have adhered to D + 1). This parameter is restored by coating the prosthetic surface with human fibronectin (FN H): 40% adhesion, and is significantly improved on the support PTFE + human fibronectin + arterial MEC (MEC
ART). The count performed on D + 5 highlights the proliferation of endothelial cells on this support: the number of cells is multiplied by 4 between D + 1 and
D + 5. The properties of rn'servoir growth factors of arterial MEC seem to be preserved thus allowing a stimulation of cell growth on this support.

 - balavaae microscopy examination: each endothelialized disc was fixed with 2.5% glutaraldehyde in a phosphate buffer (PBS: Phosphate Buffer Saline solution) for 1 h at room temperature. After 3 washes of 20 min in PBS, a post-fixation is carried out in 1% osmium tetroxide in 0.3 M cacodylate buffer for 1 h at 4 "C. The samples are dehydrated by passage through ethanol baths of increasing concentration: 2 min in ethanol at 30, 50, 70, 95, then 2 times 3 min in ethanol at 100. After drying by rapid stirring in air, the samples are coated with gold by sputtering and examined using a Philips XL20 microscope.

 It is found that the CEM secreted on the PTFE coated with human fibronectin allows a homogeneous and complete endothelial coverage. Seeding of arterial cells on the other two supports studied leaves the surface of the material visible in multiple places, even after coating with fibronectin.

 A possible modification of the matrixing was then studied.

In contrast to the endothelium, the basal lamina under endothelial represents a highly thrombogenic surface and I. Vlodavsky et al (1982
Thromb.Res. 28 179-191) conclude that there is a specific interaction between platelet matrix and it offers two palliative treatments:
- slight fixation of the matrix by glutaraldehyde: this treatment is known for its inhibitory action on cell proliferation but also for its high toxicity, which prohibits its use during implantation in humans.

 - denaturation of matrix proteins by heating; Platelet adhesion is inhibited by exposure of the matrix to a temperature of 90 ° C for 10 min. This protein denaturation also results in a reduction in the adhesion of the other cells.

 It is this technique, which is not toxic to humans, that the inventors have proposed to use, while checking, however, that the reduction in adhesion capacity does not significantly influence the endothelialization capacity. .

Treatment of CEM
PTFE discs, stamped by the MEC of arterial cells and heated to 70 "C and 90" C were seeded by pig arterial cells.

Cell adhesion and proliferation were evaluated by cell count on days D + 1 and D + 5 of the culture.

 The results, obtained for tests carried out in triplicate, are shown in Figure 2. They do not demonstrate a significant difference for the two parameters considered between the MEC heated to 90 "C and the control MEC; on the other hand, there is a slight decrease for the MEC heated to 70 "C.

Validation of the matrixing on Dacron
The same study methods were applied to the Dacron material and
We see, in Figure 3, the marked improvement in the endothelialization capacity of the
Dacron matrixed (MEC-ART) compared to other presentations of this naked material or coated with human fibronectin (FNH).

The results set out above have led the inventors to propose a protocol for the method of modifying the internal surface of synthetic prostheses which comprises the following steps:
- Coating of the internal surface of the prosthesis by incubation with a solution of human recombinant fibronectin
- washing with saline solution to remove excess fibronectin
- inoculation of the internal surface of the prosthesis with a suspension of arterial endothelial cells, then cell culture by incubation of this prosthesis in a sterile container at 37 "C
- elimination of arterial cells by the action of a 2 M urea solution
- storage of the prosthesis thus stamped at 4 "C in an environment
sterile.

Advantageously, the step of eliminating arterial cells by the action of a urea solution is followed by a step of heating the prosthesis at 90 "C for 10 minutes.
A specific protocol for modifying the internal surface of the synthetic prostheses according to the invention is described below:
coating of the internal surface of the prosthesis by incubation with a solution of human recombinant fibronectin: 2 to 10 g / cm 2 for 1 hour at 37 ° C.

- washing with saline solution (PBS) to remove excess fibronectin
- inoculation of the internal surface of the prosthesis with a suspension of arterial endothelial cells, at a density between 0.5.105 and 2.105 cells per cm2 and incubation of this prosthesis in a sterile container at 37 "C with regular rotations, at the rate of 4 per hour for 2 to 3 hours.

- culture in a bath of EGM medium + 4% of Dextran T40 for 10 to 15 days with change of the medium 2 times per week
- at the end of this period, elimination of arterial cells by the action of a 2 M urea solution for 30 minutes at 37 "C
- heating of the prosthesis at 90 "C for 10 minutes
- storage of the prosthesis thus stamped at 4 "C in an environment presenting guarantees of perfect sterility for a period which can range from 3 to 6 months.

 In vivo validation tests were then carried out and will now be described.

Operating procedure
During two consecutive months, the inventors implanted 4 molded prostheses in 4 pigs.

 This experiment was preceded by two trials in two pigs to assess the feasibility and develop a reproducible protocol.

 The pigs were one month old, weighed 20 kg, the sex ratio was 2 males and 2 females.

 The prostheses were all stamped according to the technique of the invention. These were 4 straight tubes, 3 in PTFE and 1 in Dacron with a diameter of 6 mm, except for 1 PTFE tube with a diameter of 5 mm and 6 cm in length.

 The pigs were operated on under general propofol anesthesia after premedication with ketamine, atropine and midazolam. They were intubated and ventilated with a peripheral venous route.

 The intervention consisted in the installation, after median laparotomy of a right aorto-aortic tube under renal. The proximal and distal terminal anastomoses were made with an overlock of non-absorbable polypropylene threads.

 The pigs were operated on heparin therapy 500 IU by intravenous injection before clamping and then 500 IU at closure, combined with 250 mg of lysine acetyl salicylate intravenously. The administration of lysine acetyl salicylate per os (continued directly into the pigs' throat) was continued at a dose of 500 mg every other day for the 8 postoperative days.

The explantations were made for the PTFE prostheses 8 days, 15 days and 1 month after the first intervention and at 1 month for the prosthesis in
Dacron.

 On the day of explantation, all the pigs were in good condition, with no functional signs, the lower limbs were warm and there were peripheral pulses perceived on the finger.

 The implantation, carried out under general anesthesia, consisted in the ablation of the prosthesis with location and removal of the proximal and distal anastomoses; the animals were not sacrificed until after the intervention.

Results
Macroscopic observation
All the prostheses were permeable. They were sectioned in two in the longitudinal direction and no significant wall thrombosis or fibrino-platelet deposits were macroscopically observed.

Scanning electron microscopy
As soon as they were removed, the prostheses were rinsed thoroughly in order to remove the residual blood elements and then they were fixed for one hour in 2.5% glutaraldehyde in PBS at room temperature. They were then studied by scanning electron microscopy according to the preparation protocol described during the in vitro validation. The implantation chronology of 8 days, 15 days and 1 month made it possible to follow the progressive (8 and 15 days) and complete (1 month) endothelialization of the entire internal surface of the prostheses, whether PTFE or Dacron:
- 8 days: start of endothelial coverage in a monolayer of about 1 cm from the anastomoses and appearance of endothelial cellular areas within the fibrin deposits observed throughout the central part of the prosthesis
- 15 days: the endothelial cover is practically completed; macrophages visible at the very center of the prosthesis ensure the cleaning of fibrin areas
- 1 month (PTFE and Dacron): total and exclusive endothelialization of the internal surface of the prosthesis.

Transmission electron microscope
The prosthetic samples fixed with glutaraldehyde are post-fixed with 1% osmium tetroxide in cacodylate buffer for 1 hour at 4 "C. They are then dehydrated using a progressive range of ethanol 5 minutes in the ethanol at 30, 50 ", 70, 95, 3 times 10 minutes in ethanol at 100" then 3 times 10 minutes in propylene oxide. After inclusion of the samples in an epoxy resin, ultrafine sections are made at 1 using an ultramicrotome then contrasted with uranyl acetate and lead citrate before their observation.

 Observation of the endoluminal cell layer reveals, in the light-wall direction, the successive presence of adjoining and quiescent endothelial cells, cells of macrophagic nature within a fibrin network and a layer of cells. myofibroblastic type which have formed a connective matrix in direct contact with the prosthetic matrixing (highlighting of collagen fibers). This organization anticipates the formation of normal endoluminal tissue.

Claims (5)

 1. Method for modifying the internal surface of synthetic prostheses used in vascular surgery. characterized in that it consists in producing on said internal surface a physiological endothelial matrix obtained in vitro from arterial cells.  2. Method according to claim 1, characterized in that said internal surface is previously coated with human fibronectin.  3. Method according to claim 2, characterized in that it comprises the following steps:  - Coating of the internal surface of the prosthesis by incubation with a solution of human recombinant fibronectin  - washing with saline solution to remove excess fibronectin  - inoculation of the internal surface of the prosthesis by a suspension of arterial endothelial cells, then cell culture by incubation of the prosthesis in a sterile container at 37 "C  - elimination of arterial cells by the action of a 2 M urea solution  - storage of the prosthesis thus stamped at 4 "C in a sterile environment.  4. Method according to claim 3 characterized in that the step of eliminating arterial cells by the action of a urea solution is followed by a step of heating the segment at 90 "C for 10 minutes  5. Method according to claim 3 and claim 4, characterized in that it comprises the following steps:  - Coating of the internal surface of the prosthesis by incubation with a solution of recombinant human fibronectin: 2 to 10 g / cm2 for 1 hour at 37 "C.  - inoculation of the internal surface of the prosthesis with a suspension of arterial endothelial cells, at a density between 0.5.105 and 2.105 cells per cm2 and incubation of the prosthesis in a sterile container at 37 "C with regular rotations, at the rate of 4 per hour for 2 to 3 hours.  - washing with saline solution (PBS) to remove excess fibronectin  - storage of the prosthesis thus stamped at 4 "C in an environment presenting guarantees of perfect sterility for a period which can range from 3 to 6 months.  - heating the prosthesis at 90 C for 10 minutes  - at the end of this period, elimination of arterial cells by the action of a 2 M urea solution for 30 minutes at 370C  - culture in a bath of EGM medium + 4% of Dextran T40 for 10 to 15 days with change of the medium 2 times per week