DE2557947C2 - - Google Patents

Description

The invention relates to a prosthesis with one Content of negatively charged groups antithrombogenic intimate surface.

In a summary of known products of the specified type (V.L. Gott and A. Furuse, Federation Proceedings, Vol. 30, No. 5 (1971) is out leads to the fact that it is there according to the knowledge at that time exhaustive list of all basic possibilities to create antithrombogenic prostheses. The following options are mentioned:

• 1. Heparinized surfaces
  • 1.1. Graphite-benzalkonium-heparin coating on polymer plastic;
  • 1.2. Heparin coating on silicone rubber and polymer plastic;
  • 1.3. Epoxy resin with embedded heparin.
• 2. Surfaces with anionic residues or negative electrical charges
  • 2.1. Cationic polymer with excess anionic polymer;
  • 2.2. Acrylic monomers with anionic surfactants;
  • 2.3. Cellulose with carboxyl groups;
  • 2.4. Electrets with negative surface charge
• 3. Surfaces made of essentially inert materials
  • 3.1. Pyrolytic carbon;
  • 3.2. Albuminated polystyrene;
  • 3.3. Fluorinated silicone rubber;
  • 3.4. Acrylic hydrogel;
  • 3.5. Epoxy resin with surface active properties;
  • 3.6. Polyether glycol / urethane / urea polyurethane;
  • 3.7. Polyurethane-polydimethylsiloxane copolymer;
  • 3.8. Graphite-polyurethane-polyvinyl coating;
  • 3.9. Metallographic sebum polish on Stellite 21 metal
• 4. Plastic fabric on which one quickly becomes in use Precipitates fibrin layer.

As is also indicated in the above illustration, the Use of surfaces with heparin and / or negative elec charges in line with Sawyer's work, according to which it was to be assumed that the normal lining of blood vessels was a Has negative charge, and with the fact that that is strongly antithrom bow heparin is the most negatively charged organic substance is in the human organism.

The practical application of the numerous concepts described but made trouble; in particular was with heparin coated surfaces the heparin after a relatively short Use time replaced and thus the antithrombogenic effect ver loren. Furthermore, the compatibility of the carrier substances was a difficult problem. While it was known that graft carriers materials pose fewer problems in this regard, yes the natural, strongly structured surface of such trans plantat prostheses for the described possibilities of generation antithrombogenic surface layers as obvious appear unsuitable, although in the case of a heparin coating also the same rapid wear and tear as with other carriers materials was to be expected.

The invention is based on the task of being easy to manufacture and the prosthesis remains functional over long periods of use with an antithrombogenic intimal surface.

According to the invention, this task with the prosthesis after Claim 1 solved.

According to the invention is obvious despite the described Obstacles provided a graft prosthesis, so that their basic advantages, especially the good tolerance and the long service life can be used; who feared Obstacles are avoided in that the antithrombogenic quality the intimal surface not by coating or on storage of foreign material, but through a certain chemical Conversion has been brought about, so that the surface structure doesn't matter and a chemically bound and therefore there is a particularly durable antithrombogenic layer.

It was from another source (K.H. Gustafson, Aktiv. For Kemi 17: 541 (1961)) that solubilized collagen About 95% succinylate the epsilon amino groups into free car can convert boxyl groups, but this provided no suggestion for this represents this procedure on solid collagen in a prosthesis to create an antithrombogenic surface. This applies all the more than in the combination mentioned at the beginning  comprehensive representation of God and Furuse only from Prostheses with plastic carriers was discussed.

Preferably, the starting material for the trans plantat uses a collagen that consists of a Fig protease (ficin) treated or degraded Bovine carotid artery material was made and also is known as collagen tanned with dialdehyde starch.

A starting material is preferably in the form of a Tube used, one end closed and in a liquid medium is used, e.g. B. in ethanol, with a liquid chemical reagent in the lumen of the Tube is introduced to the negative surface charge to increase the intimal area. Acting on the reagent it is preferred (as explained below) around succinic anhydride in a basic solution.

In particular, a NaHCO₃ buffer solution in the lumen used in portions in approximately 10 ml aliquots, for what about 0.1 g of crystals of succinic anhydride each be added.

The above object and features of the invention will be explained by the detailed description that follows.  

However, before going into a detailed description of the invention is given, there will be another short brief first given the purpose for which the Investigation was carried out to the present Invention led.

Vascular collagen prostheses were used, which are by enzymatic treatment of bovine head arteries with figs protease were manufactured. Sections of this vascular Dentures have been chemically modified to be more intimate change electrochemical surface configuration, and to achieve a positive total surface charge, a negative total surface charge and a neutral one Total surface charge. The modified vascular Grafts were made along with sections of unmodi vascular collagen prostheses in head arteries, external jugular veins, femoral arteries and femoral veins of Dog bastards for periods of 1 min, 15 min, 30 min and used for 2 hours. The grafts were then removed distant and macroscopic on the existence and degree thrombotic occlusions examined. The same prostheses were further using a scanning electron microscope examined closely to the nature of the occluding thrombi to determine. It was found that by increasing the total negative surface charge density on the  intimal surface of this vascular prosthesis thrombo table occlusions are essentially excluded could while training a more positive Prosthesis blood interface potential the occurrence of Thrombosis was greatly accelerated.

Sections of these vascular prostheses were also shown in the vascular system of dog bastards is implanted, and it were in vivo arterial and vein flow potential measurements carried out. The polarity of the flow potential reflects the interface potential between the vascular Prosthesis and blood flow through the graft again, being a positive flow potential at the downstream Electrode placed on the negative surface total charge density on the intimal surface of the Indicating graft versus blood components. The arterial and vein flow potentials measured in vivo, the vascular collagen prostheses modified for "negative" obtained showed an evenly positive polarity, while the flow potentials that are at "positive" modified grafts were obtained evenly were negative. It was also found that the Flow potentials obtained with grafts which chemically neutralize the intimal surfaces  charge has been modified at the physiological pH positive polarity of smaller size than in had negatively modified transplants. The unmodifi graced bovine head arteries broken down with fig porotease had flow potentials with slightly negative polarity. These flow potential measurements served the to check and guarantee chemical modification processes afford the electrical modification of the surfaces charge density proceeded as predicted.

The invention is described below with reference to the drawing explained.

Fig. 1 shows schematically an apparatus for performing the invention;

Fig. 2 illustrates schematically a technique for measuring streaming potentials in accordance with the invention; and

Fig. 3 shows schematically a section of Fig. 2 on an enlarged scale.

Studies have been done to determine the antithrom bowel activity of chemically modified collagen to be elucidated by treating with fig protease in vivo or dismantled vascular cattle artery heterograft  prostheses evaluated in the vascular system of Dog bastards were used.

A device according to FIG. 1 was provided for the chemical modification of bovine carotid arteries treated with fig protease. It has a container 10 with an opening 12 through which the shaft 14 of an electric stirrer 16 extends. A glass stopper 18 is provided at the lower end of the bovine carotid artery 20 treated with fig protease, which is immersed, for example, in 40% ethanol.

To the positive total surface charge density of the intimal surface of the vascular collagen prosthesis to increase, was an amide image promoted by cyanamide tion reaction carried out to be the main source of negative Total charges (free carboxyl groups of aspartic acid and glutamic acid) in amide groups.

There were 100 ml of a reaction solution at room temperature containing 0.5 m EDC 1-ethyl-3,3'-dimethylaminopropyl cyanamide HCl (7.75 g / 100 ml), 7.5 m urea (45 g / 100 ml ) and 5.0 m NH₄Cl (26.75 g / 100 ml) in triple distilled water (adjusted to pH 5) and added to the lumen of the vascular prosthesis in 10 ml aliquots, the graft in 40% ethanol was suspended, as shown in Fig. 1.

The reaction solution was changed every hour, with the last aliquot remained in the lumen overnight. After a 24 hour reaction time, the vascular Prosthesis removed from the modifier and in given a sterile salt pan in which the long tubes are closed Segments 3.5 cm long for implantation sub searches or a length of 10.0 cm for current pots tial measurements were cut.

The total negative surface charge density of the intimal Vascular collagen prosthesis surface area has been increased an acylation of the free amino groups (especially the Lysine and hydroxylysine) of the protein with amber acid anhydride performed in a solution that is more basic than when it corresponds to a pH of 7.0. This reaction not only blocks the main source of free positive Charges, but also converts them into anionic residues (Means, G.E., and Feeney, R.E .: Chemical Modification of Protein. Holden-Day, Inc., San Francisco, Calif., 1971, P. 74, 75). The chemical reaction proceeds as follows:

A basic solution was prepared from 75 ml of 100% ethanol and 25 ml of a 1 m NaHCO₃ buffer solution and added to the lumen of the vascular prosthesis in 10 ml aliquots in the course of 5 hours, the graft being in 40% Ethanol was suspended, as shown in Fig. 1. Crystals of succinic anhydride were added to each of these 10 ml aliquots of the basic solution. 1.0 g of succinic anhydride was distributed evenly over the 10 aliquots. At the end of the reaction period, the vascular prosthesis was removed from the modifier and placed in a sterile salt bowl where the long tubes were cut into segments 3.5 cm long for implantation studies and 10.0 cm long for flow potential measurements.

To the electrical surface charges on the intimal Neutralize the area of the vascular collagen prostheses, became an internal amide formation promoted by cyanamide reaction carried out to the free carboxyl groups and Connect amino groups of adjacent peptide chains. A such reaction removes the main source of the electronega activity (carboxyl groups) and electropositivity (free amino groups). The chemical reaction is as follows measure:

100 ml of a solution of 0.5 m EDC (7.75 g / 100 ml) in triple distilled water was prepared and added to the lumen of the vascular prosthesis in 10 ml aliquots, with the graft in 40% ethanol was suspended, as shown in Fig. 1. The reaction solution was changed every hour with the last aliquot remaining in the lumen overnight. After a 24 hour reaction period, the vascular prosthesis was removed from the modifier and placed in a sterile salt bowl where the long tubes were cut into segments 3.5 cm long for implantation studies and 10.0 cm for flow potential measurements.

As a starting material in the above case, for example wise vascular dialdehyde collagen prostheses with dialdehyde Starch-denatured collagen including ent speaking heterografts from bovine collagen used which are commercially available (Johnson + Johnson, New Brunswick, New Jersey).

Surgical implantation of the modified vascular Collagen prostheses included a carotid artery implantation and a jugular vein implantation in dog bastards weighing 10 to 28 kg (average 18.6).

The vascular prostheses (with a length of 3.5 cm for macroscopic studies and an SEM assessment and  10.0 cm for flow potential measurements were made on her Place left for the specified amount of time, where the wound was covered with salt-soaked gauze bandages.

The inside diameter of the blood vessels and the prostheses were checked calculated in such a way that the outer diameter of the Vessels and the grafts were measured with a micrometer, being Blood flowed through the system, and two wall thicknesses of subtracted from these diameters. The difference between that Inner diameter of the receiving blood vessels and the prosthetic blood vessels can be found in Table 1.  

Table 1

Difference between the inside diameter of the receiving and the prosthetic blood vessel

For flow potential measurements, a pair of silver / silver chloride electrodes 22 and 24 ( Figs. 2 and 3) were inserted into the lumen 26 of the vascular prosthesis 27 through the middle of two "pockets" 28 and 30 made of Dacron sewn into the top surface and were separated by a distance that corresponded to at least 10 radii. The electrodes were connected to a Kiethley electrometer 32 (Sawyer, PN, Himmelfarb, E., Lustrin, I., and Ziskind, H .: Measurement of streaming potential of mammalian blood vessels, aorta, and vena cava , in vivo. Biophys. J. 6: 641 (1966).

For removal, fixation and macroscopic observation at the end of each period, the blood vessels upstream and downstream of both the artery graft tats as well as the venous graft double crossed usual Kelly brackets and bracketed between the two Brackets separated. Both prostheses (arterial prosthesis and Prosthetic vein) filled with blood and on both ends were closed with the Kelly brackets immediately removed and placed in a 10% formalin solution. Under the liquid level of the formalin, the Kelly clamps together with the stainless steel cannulas and the silk ligatures removed, and the vascular trans plantates were placed in appropriately labeled tubes,  which contained 10% formalin. The degree of thrombotic occlusion was based on a scale of 0 to 4 + by three independent observers in one Single blind test rated. The average of all three values determined for each vessel and each time period given in Table 2.

Table 2

Macroscopic assessment of vascular prosthesis transplants from modified collagen *)

The degree of thrombotic occlusion in different Modified vascular, treated with fig protease Bovine heterografts can be found in Table 2 above be removed. Unmodified artery and vein hetero transplants did not immediately lead to thrombosis, if they have been exposed to the blood components. With increasing However, implantation duration can be determined that both the vascular arterial prostheses and larger ones  Extent of vascular prosthesis thrombotic were closed. It was found that a corresponding sequence of operations at neutral modifi adorned collagen prostheses and those with dialdehyde starch tanned vessels. In the latter case they ran thrombotic processes more slowly.

The chemical treatment that was carried out to make a Increase in the total positive surface charge density the intimal surface of vascular heterografts to cause accelerated thrombotic Operations. From Table 2 it can be seen that already the contact of the blood components with the vascular Prosthesis surface for a noticeable occlusion of the Vascular lumen led (more clearly in the vein graft with slower flow). A total thrombotic occlusion entered the vein graft immediately and became after Observed for 15 minutes in the artery grafts.

The negative modification reaction that occurs on the collagen tubes led to transplants with the cheapest properties. It can be seen from Table 2 that the thrombotic processes involved in the intimal Surface of the vascular prostheses occur considerably were delayed. It was a macroscopically clean one intimal surface in both artery grafts as well  with venous grafts when blood flows through them achieved up to 15 min. After two hours of contact with the Components of the blood flow were merely contiguous Thrombi at the interface between the cannula made of rust free steel and the prosthesis.

The results of arterial and veins performed in vivo flow potential measurements during the implantation of vascular prostheses from modified collagen are in Table 3 indicated.  

Table 3

Flow potential measurements (in vivo) of vascular prostheses from modified collagen

The polarity of the flow potential reflects the limit area potential between the vascular prosthesis and the blood flow through the graft again, with a positive flow potential at the downstream located electrode is recorded, a negative Total surface charge density at the intimal Surface of the graft against the blood components displays.

The polarity of the arterial and Vein flow potential measurements taken during implantation the vascular, unmodified, with fig protease treated bovine heterograft was obtained in the order of magnitude of -0.2 or -0.1 mV. These values show that a slightly positive interface potential between the intimal surface of the vascular trans plantats and the flowing blood components. A even greater negative flow potential (positive Interface potential) was at arterial and vein hetero grafts found that have been chemically modified were to increase the positive surface charge density (-0.7 mV - arterial; -0.4 mV - venous).

Vascular collagen heterografts that are chemically used for Neutralization of the surface electric charges on the intimal wall of the prosthesis had been modified,  provided arterial and vein flow potentials in the Magnitude of +0.23 or +0.13 mV. The vascular Collagen grafts that chemically increase negative surface charge density has been modified were positive flow potential measurements larger amounts (+0.6 mV - arterial; +0.4 MV - venous). The latter values show that there is indeed a meaning Lich negative interface potential at the intimal Surface of the vascular grafts through the chemical modification was trained.

The springback should be an ideal vascular prosthesis properties of an intact elastic material and have the strength of a collagen structure. The Firmness of this collagen structure, again for many months to withstand sweeping pulsating arterial pressure and as prosthetic vascular heterograft is known (Rosenberg, N., Henderson, J., Lord, G.H. and Bothwell, J. W .: Collagen arterial prosthesis of heterologous vascular origin; physical properities and hehavior as an arterial graft. In: Biophysical Mechanisms in Vascular Homeostasis and Intravascular Thrombosis, P.N. Sawyer, Ed. Appleton Century Crofts, New York 1965, pp. 314-321). Furthermore, many of the desirable features of a vascular prosthesis (Wesolowski, S.A., Fries, C.C., and Sawyer, P.N .: Some desirable physical characteristics of prosthetic vascular grafts.  

In: Biophysical Mechanisms in Vascular Homeostasis and Intravascular Thrombosis, P.N. Sawyer, Ed. Appleton Century Crofts, New York, 1965, pp. 322-336) with with fig protease treated bovine carotid artery preparations. Through the Enzyme treatment becomes the parenchyma components that one Subject to necrosis, removed; this prevents Ent ignition response that affects the integrity of the graft wall impaired and favored intimal thrombosis. they also provides some degree of vascular porosity Prosthesis wall, the correct penetration of connective tissue favored by the host; due to the relative absence an inflammatory reaction also favors an early one Endothelization.

In addition, a new type of prosthesis according to the invention provided that it was treated with a fig protease Artery that acts with anionic dialdehyde starch is modified. This prosthesis is tanned; you will negative charges incorporated in one step. That's how it is resulting product by modifying (tanning) with Dialdehyde starch increases and has excess negative Charges so that a better, less thrombogenic surface is provided. A suitable dialdehyde starch derivative is the 6-carboxymethyl derivative I:  

This material is easy to manufacture and is in Commercially available ("Water Soluble Anionic Polymeric Dialdehyde", DASOL A, Miles Laboratories, Inc.); it is as a tanning agent and suitable charge carriers of interest.

One treated with fig protease, with dialdehyde starch modified prosthesis can continue with succinic anhydride modified to introduce negative charges. The Reak dialdehyde starch may mainly be used with the epsilon Amino groups of the lysine under tanning or modifying conditions run off (pH 8.8). A certain reaction occurs with the Guanidino groups of arginine, since it is known that these Groups react with neighboring dicarboxyl groups. However that may be, there may be positions that are still for an attack by succinic anhydride is available  stand. This leads to a tanned, negatively charged one Prosthesis.

It also takes place through the reaction with succinic anhydride exhaustive decationation takes place. Succinic acid anhydride mainly reacts with the epsilon amino groups of lysine, creating a negatively charged carboxy propeonoyl group is introduced. If you assume that all lysine groups are blocked positive charge on the arginine residues and on the histidine residues before (with some imidazole groups of histidine the positive charge must be removed at pH 7.4). A modification the arginine residues can be achieved with phenylglyoxal that under mild conditions with the positively charged guanidinium groups reacts, whereby a neutral derivative is obtained. Histidine can be converted into a neutral derivative by using Diethyl pyrocarbonate can be converted to an ethoxyformyl derivative is obtained on the imidazole ring.

The techniques of negative charging described above Modifying and optionally tanning collagen to Protection against intravascular thrombosis can be on prostheses for other areas of the body and especially in heterotrans implant materials used by different Animal species are obtained and used in humans. The The most notable prosthetic devices are homotrans plantation valves that are obtained from a human and at  another human being used, and heterotrans implant flaps, usually obtained from pigs and are used in the aortic valve area. It is known that these flaps offer certain advantages because they allow a central flow. The basic problem with various aspects of their application is always that their Usability in the mitral valve area was questionable while they work satisfactorily in the aortic area. That negative supercharging modifications of such flaps increase their Usability.

According to a further embodiment of the invention, a Heterograft valve under semi-sterile conditions a pig heart removed. It is described in the Modified negatively charged way. The modification will performed by using succinic anhydride or given if dasol to achieve a highly negatively charged collagen surface used on the flap, giving the prosthesis a receives more anticoagulant character than usual Conditions would be the case. This also increases life duration of the heterograft valve used due to its Resistance to fibrin separation and degradation. The same also applies to homograft valves.

Claims (14)

1. Prosthesis with a content of negatively charged groups antithrombogenic intimal surface, characterized in that the prosthesis is a collagen graft prosthesis, in the intimal surface free amino groups of the collagen are midified by succinylation. 2. Prosthesis according to claim 1, characterized in that that the collagen is modified with dialdehyde starch. 3. Prosthesis according to claim 2, characterized in that modified the collagen with anionic dialdehyde starch is.   4. Prosthesis according to one of the preceding claims, characterized characterized as being a vascular prosthesis. 5. Prosthesis according to one of claims 1 to 3, characterized characterized as having a vascular valve prosthesis is. 6. Method of making a prosthesis after a of the preceding claims, characterized in that the prosthesis from a collagen graft Manufactures starting material in which at least in one for the formation of the intimal surface of the prosthesis certain surface of the prosthesis certain surfaces area free amino groups of the collagen by succinylation be converted into carboxyl groups. 7. The method according to claim 6, characterized in that that one uses a starting material that at least in modified the surface area with dialdehyde starch and then subjected to the succinylation reaction is.   8. The method according to claim 7, characterized in that that the surface area with anionic dialdehyde Strength has been modified.   9. The method according to claim 7, characterized in that the surface area also with phenylglyoxal has been modified. 10. The method according to claim 7, characterized in that the surface area also with diethyl pyrocarbonate has been modified. 11. The method according to any one of claims 6 to 9, characterized characterized in that the succinylation reaction the surface area with a basic solution treated by succinic anhydride. 12. The method according to claim 11, characterized in that one a NaHCO₃ buffer solution with a concentration of 10 g / l succinic anhydride used. 13. The method according to claim 12, characterized in that that one is a collagen graft starting material used in the form of a tube and that one end of the pipe closes, the pipe with the closed Put the end in a liquid medium and aliquot 10 ml portions of the treatment solution to which 0.1 g of succinic anhydride crystals each have been filled into the tube one after the other.   14. The method according to any one of claims 6 to 13, characterized characterized in that as a collagen graft Starting material used bovine head arteries have been treated with fig protease.