WO2014112956A1

WO2014112956A1 - Method for treatment of a vascular graft

Info

Publication number: WO2014112956A1
Application number: PCT/SI2013/000003
Authority: WO
Inventors: Ita Junkar; Martina MODIC; Alenka Vesel; Karin Stana KLEINSCHEK; Zdenka PERŠIN; Miran Mozetič; Aleš DOLIŠKA
Original assignee: Center Odličnosti Polimerni Marteriali In Tehnologije
Priority date: 2013-01-17
Filing date: 2013-01-17
Publication date: 2014-07-24

Abstract

Method of treatment of a vascular graft addresses the need for improved methods of increasing hemocompatibility of vascular grafts made from polyethylene terephthalate polymers against the state of the art. The present invention does not involve deposition or grafting of any anti-thrombogenic coating on the inner walls of vascular grafts. Hence, it provides a method for improved hemocompatibility of vascular grafts by treatment of inner walls by excited oxygen molecules. The efficiency of the method which is the subject of this invention is confirmed by behavior of blood platelets on modified vascular grafts. Against the state of the art the methods of the invention prevents transformation of platelets from normal state in healthy blood to highly activated states, which would further lead to undesired thrombus reactions. Thus the present invention improves hemocompatibility of artificial polyethylene terephthalate surfaces, as transformation of platelet shape from its normal state- round or discoid and dendritic to spread dendritic, spread and fully spread shape is highly reduced.

Description

Center odlicnosti polimerni material^'! in tehnolog

Tehnoloski park 24

1000 Ljubljana

Slovenia

Method for treatment of a vascular graft

FIELD OF TECHNOLOGY

The present invention relates to methods for manufacturing of grafts. In particular the present invention relates to optimization of vascular grafts made from polyethylene terephthalate polymers by treatment of inner walls.

TECHNICAL PROBLEM

Technical problem addressed by this invention is a need for improved methods of increasing hemocompatibility of vascular grafts made from polyethylene terephthalate polymers, current methods in some cases showing hemocompatibility problems.

STATE OF THE ART

Atherosclerotic cardiovascular disease is still the largest cause of mortality in Western society. Arterial damage produces localized reductions in the calibre of arteries (stenosis) which ultimately stops the flow of blood through the affected vessels. The disease is treated surgically by bypassing the segment of affected vessels to restore blood flow (Chandy, T.; Das, G. S.; Wilson, R. F.; Rao, G. H. R.; Biomaterials 21, 699 (2000)). Wherever possible, an autograft is the best choice for a replacement vessel. However many patients, especially those with pre-existing vascular diseases and patients who have already undertaken autograft procedures, do not have blood vessels healthy enough to adequately serve as replacements. In such cases, the most common form of treatment is by using synthetic polymeric materials, such as Dacron (PET - polyethylene terephthalate) or ePTFE (extended polytetra fluoroethylene), because they exhibit a unique through-pore microporous wall structure and because they also have highly flexible mechanical properties. These synthetic vascular grafts have been used successfully to replace large-diameter blood vessel, however the long-term patency for small-diameter vascular grafts is still unsatisfactory; this is primarily due to thrombus formations (Wissink, M.J.B.; Beernink, R.; Poot, A.A.; Engbers, G.H.M.; Beugeling, T.; van Aken, W.G.; Feijen, J.; Improved endothelialization of vascular grafts by local release of growth factor from heparinized collagen matrices. Journal of Controlled Release 64, 103 (2000)). Significant efforts have been made in recent years to develop novel vascular grafts, and some novel grafts have indeed been produced, however the long term patency is still disappointing. At every 10 % of patients post-surgical complications are observed, mostly due to inflammatory reactions, infections and aneurysm. In such cases the artificial blood vessel has to be removed and the new autologous material for vascular graft should be implemented, which increases the expenses of treatment for at least two times.

When polymer materials come into contact with blood they can cause various undesirable host responses such as thrombosis, inflammatory reactions, infections and various other responses. Such responses are minimised if the materials are hemocompatible. In fact surface-induced thrombosis is one of the main problems associated with blood-contacting biomaterials. Polyethylene terephthalate (PET- Dacron grafts) and polytetrafluoroethylene (PTFE) are the two most common prosthetic materials available today for vascular grafts. Though PTFE and Dacron vascular grafts possess many of the properties of ideal vascular prostheses, successful results for small-calibre vascular grafts have not yet been reported.

Normal blood is a metastable state stabilized between two opposing driving forces coagulation and anticoagulation. Almost any external perturbation in the form of an artificial surface tends to trigger the coagulation system, i.e. blood clotting may occur (Kasemo, B.; Biological surface science. Surface Science 500, 656; 2002). Once biomaterials are exposed to blood the adsorption of blood proteins immediately follows. The type, the amount and the conformational state of the adsorbed proteins determine whether platelets will adhere and become activated or not. The adsorption of fibrinogen (FNG), which is present in blood plasma, has been shown to be closely related to surface-induced thrombosis. Fibrinogen is the most abundant protein in blood plasma and has been closely linked to adhesion and activation of platelets. As thrombus formation begins with protein adsorption, the main efforts to improve hemocompatibility of materials have been directed towards controlling (mainly preventing) protein adsorption. Therefore, a modification of the material surface with protein-repulsive molecules has become a widely used approach for improving hemocompatibility of materials (Tzoneva-Velinova et al.; Journal of Material Science 19, 3203, 2008). The modification of these grafts materials with a suitable protein coating may improve cell adhesion and proliferation. Commercially available collagen-, gelatin-, heparin- and albumin- coated vascular grafts have been developed to prevent the procedure of clotting of the vascular graft before implantation, some of them are disclosed in patents: WO 9608149, EP 1501565, FR 2793693, DE 19724869, WO2005053765, US 4326532, US 20060228391, US 4987181 in US 20040234575. Heparin impregnation is also usually performed to prevent coagulation as disclosed in patents US 4613517 and US 4521564.

Another method for surface modification of a vascular grafts is by gaseous plasma in which various gases have been employed in order to obtain desirable surface features and enhance and optimize adhesion of coatings and/or biological materials. Some of these methods are disclosed in patents: US60033582 and US7803393. Gasous-plasma treatment is used together with various protein coatings to to tailor surface properties and improve biocompatibility/hemocompatibility.

DESCRIPTION OF NEW INVENTION

Method of treatment of a vascular graft addresses the need for improved methods of increasing hemocompatibility of vascular grafts made from polyethylene terephthalate polymers against the state of the art. The present invention does not involve deposition or grafting of any anti- thrombogenic coating on the inner walls of vascular grafts. Hence, it provides a method for improved hemocompatibility of vascular grafts by treatment of inner walls by excited oxygen molecules. The efficiency of the method which is the subject of this invention is confirmed by behavior of blood platelets on modified vascular grafts. Against the state of the art the methods of the invention prevents transformation of platelets from normal state in healthy blood to highly activated states, which would further lead to undesired thrombus reactions. Thus the present invention improves hemocompatibility of artificial polyethylene terephthalate surfaces, as transformation of platelet shape from its normal state- round or discoid and dendritic to spread dendritic, spread and fully spread shape is highly reduced.

Vascular grafts are cardiovascular implants which replace injured or diseased blood vessels. The methods of invention deals with vascular grafts made from polyethylene terephtha late in any forms; tubes made from foils, or prosthetic grafts made by weaving or knitting. The term "polyethylene terephthalate" will be used identically as its abbreviation "PET". Expression PET polymers relates to at least one PET polymer. Vascular grafts are of any length but usually of length between 0.5 and 50 cm. They are of any diameter but typically of diameter between 1 and 20 mm. The wall thickness is typically between 0.05 and 1 mm. These figures are given for information purposes in order to better present embodiment of this invention, and do not, in themselves, limit scope of this invention.

The disease or condition to be treated with vascular graft according to this invention is selected from group comprised of atherosclerotic cardiovascular disease, inflammatory reactions, infections, aneu rysm.

Electronically excited oxygen molecules are molecules of oxygen gas containing two atoms bonded by covalent forces for which one of the electrons is found in an energetically less favorable orbital. Against normal state of oxygen molecules which have an electron configuration in energetically most favorable state the electronically excited molecules have one electron in an energetically higher orbital. Electronically excited molecules are often described with symbols. A normal oxygen molecule is often known as an oxygen molecule in the ground state and has been assigned the state (X ³∑g^"). Electronically excited oxygen molecules are found either in the first excited state which has been assigned the state (a ³A_g ^"), or in the second excited state which has been assigned the state (b ¹∑_g ⁺). Electronically excited molecules do not de-excite by electrical di pole radiation and are often referred as metastable oxygen molecule excited states. The radiative life time of electronically excited molecules in the first excited state is about 45 minutes, and the radiative life time of the electronically excited molecules in the second excited state is about 10 seconds. Radiative life time means an average time an electronically excited molecule remains in the excited state until it is transformed to a molecule in the ground state by simultaneous emission of a light quantum (a photon).

Broadly, this new invention provides a method for improved hemocompatibility of vascular grafts by treatment of inner walls by excited molecules, preferably oxygen. The efficiency of the method which is the subject of this invention is confirmed by behavior of blood platelets on modified vascular grafts. Against the state of the art the methods of the invention prevents transformation of platelets from normal state in healthy blood to highly activated states, which would further lead to undesired thrombus reactions. Thus the present invention improves hemocompatibility of artificial polyethylene terephthalate surfaces, as transformation of platelet shape from its normal state- round or discoid and dendritic to spread dendritic, spread and fully spread shape is highly reduced.

In preferred embodiments a vascular graft made from PET is subjected to the electronically excited molecules, preferably oxygen, in a suitable processing chamber. The electronically excited molecules will siowly react chemically with PET materials leaving excessive hydroxyl, ether, carbonyl, ester and carboxyl groups on the surface. These groups will form hydrophilic surface with a negative surface charge which will in turn reduce adsorption of fibrinogen and lower adhesion of platelets and their activation. The enzyme thrombin acts on the release of small peptides from fibrinogen, which causes the polymerization of fibrinogen monomers into fibrin polymer. Thus the fibrin structures, if appearing on a surface of vascular grafts, may cause accumulation of blood proteins, and further activation of blood platelets which will with high probability result in aggregation of platelets and surface induced thrombosis.

In further preferred embodiments the electronically excited molecules are dosed to vascular grafts in such a way that the flux of the electronically excited molecules will be applied at a temperature range between 0 and 50°C or even at room temperature said temperature ranging from 15 and 25 ^CC. This temperature range is favorable because it prevents condensation of water molecules on the surface and allows for good stability of hydroxyl, ether, carbonyl, ester and carboxyl functional groups on the surface. Higher temperatures, at more than 50°C, are avoided in preferred embodiments since the excessive hydroxyl, ether, carbonyl, ester and carboxyl groups on the surface of PET polymers are thermally not stable and decay spontaneously at elevated temperatures, i.e. above about 50°C.

Preferably the treatment of vascular grafts made from PET polymers is performed under reduced pressures, i.e. pressures below atmospheric pressure. Reduced pressure is favorable since it allows for rapid desorption of water molecules that might be present on PET polymers and since it prevents a loss of electronically excited molecules by gas phase thermallization. The term thermallization stands for transformation of electronically excited molecules to molecules in the ground state through supereliastic collisions with other molecules at three body collisions. A three body collision is referred as a collision of at least one electronically excited molecule with two other molecules that may or may not be electronically excited.

Brief description of the drawings

Other advantages of the invention will become apparent upon reading the following descriptions and accompanying drawings, said drawing forming part of this application, presenting as follows:

Figure 1. shows schematic of the formation of excessive hydroxyl, ether, carbonyl, ester and carboxyl functional groups on the surface of PET polymers upon exposure to electronically excited oxygen molecules

Figure 2. shows measured values of hydroxyl, ether, carbonyl, ester and carboxyl functional groups by ESCA (Electron Spectroscopy for Chemical Analyses) on the surface of untreated materia! and materials treated by electronically excited oxygen molecules

Figure 3. an image from SEM (Scanning Electron Microscopy) of a standard vascular graft after incubation with whole blood

Figure 4. an image from SEM (Scanning Electron Microscopy) of a standard vascular graft after treatment by the method of invention and incubation with whole blood Figure 5. shows the surface density of selected forms of blood platelets: ( )- round or discoid; (D) dendritic or early pseudopodia; (SD) spread-dendritic or intermediate pseudopodial and (FS) fully spread on untreated vascular grafts and vascular grafts treated by the method of the invention

Figure 6. shows the adsorbed mass of fibrinogen on untreated PET surface and on PET surface treated by the method of the invention

Detailed description of the invention

The hemocompatibility of standard vascular grafts is not optimal. The blood platelets often adhere to PET material, and transform from inactive to active types. The active types release an enzyme called thrombokinase which favors transformation of blood proteins, in particular fibrinogen to fibrin.

Platelets are self sufficient as they contain all the necessary ingredients needed for adhesion, aggregation and formation of thrombi. In fresh blood platelets have spheroidal form, but have a tendency to extrude hair-like filaments from their membranes and can adhere to each other (Shi et al.; Biomaterials and Tissue Engineering, Springer-Verlag, Berlin; 2004). Their function is to arrest bleeding through the formation of platelet plugs and to stabilise platelet plugs by catalyzing coagulation reactions which lead to the formation of fibrin, as described above.

The basis for understanding platelet function is their structure. In an un-stimulated state platelets have a discoid shape, which is maintained by a cytoskeleton of microtubules. The external surface coat of platelets contains membrane-bound receptors (glycoproteins lb and llb/llla) that mediate contact reactions of adhesion (platelet - surface) and aggregation (platelet - platelet). The membrane also provides a phospholipid surface, which accelerates the coagulation cascade and forms a spongy, canal-like open network that represents an expanded reactive surface to which plasma factors are selectively adsorbed (Ratner et al.; Academic Press, San Diego; 2008).

A possible way to asses the activation degree of adherent platelets is to study their shape and the number of adherent platelets. According to Goodman (Goodman et al.; Platelet responses to silicon-alloyed pyrolytic carbons. Journal of Biomedical Materials Research Part A 83A, 64; 2006), their shape can be categorised on a scale from lower to higher level of activation as: round or discoidic (R); dendritic or early pseudopodial (D); spread-dendritic or intermediate pseudopodial (SD); spreading or late pseudopodial (S) and fully spread (FS).

According to the methods of invention, the adsorption of blood proteins and transformation to active types is prevented by formation of a stable negative charge on PET polymers. The negative surface charge is formed because of presence of excessive hydroxyl, ether, carbonyl, ester and carboxyl functional groups on the surface. In the preferred embodiment the functional groups are formed on the surface of PET polymers by exposure to a flux of electronically excited molecules. Unlike oxygen molecules in the ground electronic state which are not capable of chemical interaction with PET polymers at room temperature, the electronically excited molecules interact with PET polymers predominantly by breaking bonds between hydrogen and carbon in polymer materials and thus enrich the surface of PET polymers with hydroxyl, ether, carbonyl, ester and carboxyl functional groups. The schematic of the interaction between an electronically excited molecule and PET polymers is presented in Figure 1. The enrichment of surface with functional groups is determined by the ESCA method. ESCA is the abbreviation for "Electron Spectroscopy for Chemical Analyses". The atomic concentration of these groups is presented in Figure 2. Increase in hydroxyl, ether, carbonyl, ester and carboxyl groups is observed after treatment by the method of the present invention.

Presence of excessive hydroxyl, ether, carbonyl, ester and carboxyl functional groups on the surface of vascular grafts improves hemocompatibility of vascular grafts made from PET polymers. Namely, the amount and orientation of blood proteins, which always adsorb on a polymer surface to form a thin film, influence on the biological response. A change in the surface charge will result in the amount and reorientation of blood proteins adsorbed on the surface, which further influence on adhesion and activation of platelets. The long term equilibrium is always established in such a manner that the blood proteins orient on the surface in energetically most favor state. This state is often called the equilibrium state. When an equilibrium state is reached the surface becomes passivized by blood proteins so further accumulation is unlikely to occur. The major task is to obtain an appropriate amount and orientation of blood proteins, which adsorb on the inner walls of vascular grafts made from PET polymers, to prevent activation of blood platelets on the surface of vascular grafts. According to the methods of invention, an optimal amount and orientation of blood proteins is obtained by functionalization of the inner walls of vascular grafts with excessive hydroxyl, ether, carbonyl, ester and carboxyl functional groups. The presence of these functional groups in a preferred concentration will cause a change in the surface charge and thus favorable amount and orientation of blood proteins adsorbing on the vascular graft surface from blood plasma. Such a film of blood proteins is stable since it is found in energetically most favorable state.

Electronically excited oxygen molecules could be produced in large quantities by two different procedures: i) heating of gas containing molecules in the ground state to very high temperatures (typically several 1000 K), and ii) formation from neutral oxygen atoms in the ground state and association of said oxygen atoms in the ground state collisions in the gas phase. Since PET materials will not stand heating to high temperatures the first method should be applied in such a way that gas is heated at high temperature and then cooled down to room temperature by adiabatic expansion to a chamber pumped continuously in order to achieve pressure much lower than in the hot chamber. The second method could be applied at lower temperatures. In a preferred embodiment oxygen gas is leaked to a dissociation chamber. The pressure in the dissociation chamber should be in the range of rough vacuum, typically between 100 and 10000 Pa. In the dissociation chamber, the molecules found essentially in the ground state are dissociated to neutral oxygen atoms also essentially in the ground state by irradiation with free electrons. The kinetic energy of electrons used for dissociation of oxygen molecules in the ground state should be in a narrow bracket between 1 and 6 eV. If the electron energy is lower the molecules would not be likely to dissociate. If the electron kinetic energy is higher than about 6 eV, the dissociation process might lead to heating of gas which is an effect that should be minimized, otherwise the gas would be heated and a need for cooling down would become an important consideration. Once the atoms are dissociated the gas is lead from the dissociation chamber to an association chamber. In the association chamber the atoms associate to molecules in electronically excited states. The gas in the association chamber is essentially kept at almost the same pressure as the gas in the dissociation chamber. Since it is essential to have a flow of gas from the dissociation to the association chamber a small pressure gradient is needed anyway, but this .pressure gradient is essentially so small that the ratio of pressures in the dissociation and association chambers rarely exceeds a factor of 1.05. Association of neutral oxygen atoms in the ground state to electronically excited oxygen molecules is slightly exothermic process so in a preferred embodiment the association chamber is cooled by forced air. Despite cooling the gas in the association chamber is still found at elevated temperature (about 400 K) so it should be cooled down by expansion to the treatment chamber. The pressure difference between the association and treatment chambers should allow for cooling the gas of electronically excited molecules down to the room temperature. In practice, this means that the ratio of pressures in the association and treatment chambers is a factor of about 1.5. This value depends on the temperature of gas in the association chamber - a higher temperature in the association chamber will dictate a higher pressure difference between the association and the treatment chambers.

Once the electronically excited molecules are prepared they are let to interact with vascular grafts made from PET polymers. In cases when the ratio between the vascular graft diameter and its length is rather large (say above a factor of 0.3 cm) the electronically excited molecules are left to diffuse freely throughout the treatment chambers. Many electronically excited molecules will enter the vascular graft and will interact chemically with the inner walls of the vascular graft. The chemical reactions are presented schematically in Figure 1. Since the vascular grafts during treatment with electronically excited molecules are kept essentially at room temperature, where the interaction probability and thus a loss of electronically excited molecules due to surface chemical reactions is low, many electronically excited molecules will reach the entire surface inside the vascular graft so the surface functionalization will be uniform throughout the entire length of the vascular graft.

If the ratio between the vascular graft diameter and its length is rather small (say below a factor of 0.3 cm) the electronically excited molecules will not reach all surfaces inside the vascular graft so free diffusion is not enough to assure for uniform surface modification. In the case of small ratio between the vascular graft diameter and its length the electronically excited molecules should be forced to flow through a vascular graft. In a preferred embodiment forcing is realized by establishing a pressure gradient across the length of a vascular graft. Such a pressure gradient is commonly established by pumping gas containing electronically excited molecules through the vascular graft. Presence of water molecules adsorbed on surfaces of vascular grafts may prevent chemical interaction between electronically excited molecules and PET polymers and thus prevent successful functionalization of the inner walls of vascular grafts with excessive hydroxyl, ether, carbonyl, ester and carboxyl functional groups. I n the preferred embodiment the vascular grafts are dried before treatment using any of available drying techniques. Still, some water molecules may be present either on vascular grafts or in the treatment chamber. In order to minimize the influence of adsorbed water molecules on functionalization of vascular grafts with excessive hydroxyl, ether, carbonyl, ester and carboxyl functional groups by electronically excited molecules the following precautions are adopted: gas leaked into the dissociation chamber is of high purity and/or is dried by any available technique prior to leakage into the dissociation chamber; the whole system is made from materials that contain water molecules only in minimum quantities; rubber materials are particularly avoided; the system is pumped by powerful vacuum pumps; the entire system is heated to elevated temperature and pumped during continuous leakage of dry gas prior to treatments and then cooled down to room temperature; after the treatment the entire system is filled with dry gas rather than air from laboratory room.

In the preferred embodiment the treatment of vascular grafts with electronically excited molecules is essentially performed at room temperature. This temperature assures for favorable reactivity between electronically excited molecules and vascular grafts made from PET materials, allows for desorption of water molecules that might be formed on the PET polymer surface during chemical reactions and prevents structural changes of PET polymers that might otherwise occur due to thermal effects.

Subject of this invention is also vascular graft product made of PET polymer or plurality thereof which has been treated by method as decribed in this application.

Said vascular graft products are used when the blood flow through the natural vessel is restricted. In such cases the part of the natural blood vessel has to be replaced by an artificial one. In therapeutic treatment said vascular graft products may be used for replacement or re-inforcing of already existing blood vessel.. Example 1

In the example disclosed here a vascular graft made from PET polymer has been treated in a treatment chamber by electronically excited molecules. The electronically excited molecules were produced by dissociation and subsequent association of oxygen molecules following the procedure described in details in the text above. The vascular grafts have been dried under vacuum conditions for prolonged time at essentially room temperature. A dried graft has been mounted into the treatment chamber containing electronically excited molecules at the density of about 2xl0²² m^"3. The vascular graft made from PET polymer was left in the treatment chamber for 100 seconds and the electronically excited molecules were left to diffuse freely. The electronically excited molecules reacted with vascular graft made from PET polymer following reactions presented schematically in Figure 1. Excessive hydroxy!, ether, carbonyl, ester and carboxyl functional groups were formed on the surface of the vascular graft made from PET polymer and their concentration has been determined immediately after treatment by ESCA method. The results of the characterization are presented in Figure 2. The vascular grafts made from PET polymers have been incubated with whole blood taken by vein puncture from a healthy donor. The activation of blood platelets has been analyzed by scanning electron microscopy (SEM). Figure 3 represents an SEM image of untreated vascular grafts after incubation with whole blood. And on Figure 4 SEM image of vascular grafts treated by the methods of the invention after incubation with whole blood is presented. A huge difference is observed: high concentration of highly activated blood platelets is observed on the surface of an untreated vascular graft, while much lower number of platelets and mostly in normal- round state are seen on the surface of vascular graft treated by methods of the invention. An important effect governing the hemocompatibility of vascular grafts is transformation of blood platelets from inactive form (round shape) present in real blood of a healthy donor not in contact with any foreign materials to activated forms. Several forms have been indicated and the surface concentration has been determined using a common counting technique. The results are summarized in Figure 5. The results show that the vascular graft treated by methods of the invention prevent transformation of platelets to the most active- fully spread (FS) form, while the transformation to other forms such as dendritic (D), spread dendritic (SD) and spread (S) forms are strongly reduced as compared to the untreated vascular graft. Huge difference in the amount of adsorbed blood plasma protein fibrinogen is observed between untreated and treated PET vascular grafts by the method of the invention. The amount of fibrinogen was measured by quartz crystal microbalance (QCM) and much lower amount of fibrinogen was shown to adhere on treated PET surface, as can be seen from Figure 6. As lower adhesion of fibrinogen to artificial surfaces has been linked with lower activation of platelets and surface induced thrombosis, these results further support improved hemocompatibility of PET vascular grafts after treatment by the method of the present invention.

Claims

1. Method for treatment of a vascular graft made from PET polymer or plurality thereof, said method comprised of the following steps:

Drying said vascular graft;

Mounting of the said vascular graft into a vacuum chamber with essentially negligible amounts of water molecules;

Essentially evacuating said treatment chamber to achieve pressure below atmospheric pressure, preferably in a range between about 1 Pa to about 1000 Pa;

Applying electronically excited molecules into said treatment chamber, preferably electronically exited oxygen molecules;

Maintaining pressure in a range of 1 - 1000 Pa in said treatment chamber; Reacting said electronically excited oxygen molecules with said vascular grafts;

Venting said treatment chamber with essentially dry air.

2. Method according to claim 1, wherein the treatment procedure takes place at temperatures between 0 and 50 degrees Centigrade, preferably at temperatures between 15 and 25 degrees Centigrade.

3. Method according to claim 1, wherein water vapor partial pressure in said treatment chamber is below 10 Pa, preferably below 1 Pa.

4. Method according to claim 1, wherein the density of said electronically excited molecules, preferably electronically excited oxygen molecules is between about lxlO¹⁹ m^'3 and about lxlO²⁴ m^"3.

5. Method according to claim 1, wherein the treatment time is between 1 s and 10.000 s, preferably between 10 s and 1000 s.

6. Method according to claim 1, wherein the said electronically excited molecules, preferably electronically excited oxygen molecules are produced by any known technique including thermal excitation and formation by association of free oxygen atoms.

7. Method according to claim 1, wherein the said electronically excited molecules, preferably electronically excited oxygen molecules are at their temperature between -20 degrees Centigrade and 100 degrees Centigrade, preferably at temperatures between 15 and 25 degrees Centigrade.

8. Method according to claim 1, wherein the said electronically excited molecules, preferably electronically excited oxygen molecules are cooled down to the temperature according to claim 7 by essentially adiabatic expansion.

9. Vascular graft made from PET polymer or plurality thereof treated by a method of any of preceding claims.

10. Use of vascular graft of claim 9 in therapeutic treatment of disease or condition.

11. Use of vascular graft according to claim 10 sherein said therapeutic treatment is replacement or reinforcement of blood vessel.

12. Use of vascular graft according to any of claims 10 or 11 whereas the disease or condition is selected from the group comprised of atherosclerotic cardiovascular disease, inflammatory reactions, infections, aneurysm.

13. A product comprising vascular graft of claim 9.