RU2008767C1 - Method for conservation of biological tissues for prosthetics of heart and vessel valves - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: traditional preservative in form of glutar aldehyde is replaced with new suturing and sterilizing agent from class of epoxy compounds - 2.5% solution of diglycidilether ethylene glycol which enables full suppression of biological tissue calcification, increased resistivity to thrombosis of prostheses. This effect is intensified by covalent addition of heparin by means of treatment with heparin solution having concentration not less then 100 E/ml. Method allows preimplantation treatment of biological prostheses in no more than two stages. EFFECT: higher efficiency and promising results of method were confirmed by model of chronic hypodermic implantation of biological prostheses cusps to rats and by experiments on ex vivo vessels.

Description

 The invention relates to surgery of the heart and blood vessels, mainly to the biological prosthetics of heart valves and blood vessels and can be used in the manufacture of xenobioprostheses of heart valves and blood vessels.

 A known method of preserving biocall valves in glutaraldehyde. However, this treatment leads to the loss of tissue elasticity, hydrophilicity and provokes calcification and preserves the thrombogenicity of bioprostheses in the recipient's body.

 A known method for the prevention of tissue calcification according to Dewanjee, which consists in the fact that in order to prevent calcification, the fabric is sequentially treated with a solution of sodium dodecyl sulfate, glutaraldehyde, aminodiphosphonate, sodium borohydride and glutaraldehyde. However, this method does not allow complete suppression of calcification and does not increase the atrombogenicity of the prosthesis, being, at the same time, technologically complex, multi-stage.

 A known method for the step-by-step modification of heart valve bioprostheses with papain, aminodiphosphonates and heparin immobilized onto the tissue with an ion-covalent method, followed by storage in glutaraldehyde. Its disadvantages are: incomplete suppression of calcification, the complexity and multi-stage technology, a small amount of immobilized heparin and the possible reversibility of this method of heparinization.

 A known method of heparinization of polymers by direct binding of heparin to the epoxy group of the previously grafted glycidyl methacrylate. This method is also characterized by multi-stage and stringent conditions of grafted copolymerization of glycidyl methacrylate on a polymer base, unsuitable for biological tissue. In addition, glycidyl methacrylate is a monoepoxy compound, the epoxy group of which can be used only for one type of interaction, for example, for heparin binding.

 A known method of impregnating vascular bioprostheses with protamine, followed by treatment with a 5% solution of the polyepoxy compound, ionic binding of heparin and storage in 70% ethyl alcohol. However, this processing method leads to the rapid leaching of heparin into the bloodstream (since ionic binding does not provide a strong fixation of heparin on the biomaterial), and, therefore, to the rapid loss of the atrombogenic properties of the prosthesis. In addition, pre-impregnation with protamine complicates the treatment and leads to the expenditure of the epoxy groups of the polyepoxide for interaction with protamine, which may impair the quality of cross-linking.

 Thus, existing technologies for processing bioprostheses of heart and vascular valves either solved separately the problems of inhibition of calcification and thrombosis, or were extremely difficult and multi-stage. The prototype method is proposed to reduce immunogenicity, improve physical and mechanical properties and increase the atrombogenicity of biological tissue. The authors did not set the task of preventing calcification and did not study the effect of this method of conservation on the calcium-binding activity of biological tissue. In addition, this method is also technologically sophisticated.

 We believe that ethylene glycol diglycidyl ether is an optimal preservative for biological prostheses of heart valves and blood vessels, as it has high collagen crosslinking activity, sufficient sterilizing activity, and the possibility of using the unreacted second epoxy group (for some molecules that bind to the collagen amino groups with one epoxy group) covalent immobilization of heparin. In addition, the epoxy groups of diglycidyl ether of ethylene glycol, interacting with the amino groups of collagen, form bonds of a fundamentally different nature than glutaraldehyde. Theoretically, the presence of an oxygen atom in this bond should prevent the crystallization of calcium salts on the collagen matrix.

 The aim of the invention is the simultaneous suppression of calcification and thrombus formation on bioprostheses of heart valves and blood vessels by preimplantation processing, as well as the simplification of the technology of conservation of bioprostheses.

 The goal is achieved in that, as a preservative for the pre-implantation treatment of valve and vessel bioprostheses, a diepoxide compound is used - ethylene glycol diglycidyl ether, which is both a cross-linking and sterilizing agent for prosthetic biotissue and provides a covalent bond with heparin. In this case, the process of preimplantation processing is carried out in only two stages.

 The method is as follows.

Taken from freshly slaughtered animals, cleaned and washed by blood biomaterial is immersed in a 5% solution of ethylene glycol diglycidyl ether at pH = 7.4 and temperature of 20 ^{° C} where it is preserved for 21 days, and then incubated for 1 day with heparin in solution concentration of 100 IU / ml and stored in a 2% solution of diglycidyl ether of ethylene glycol until use.

 An example of the practical implementation of the method.

Variant 1. 70 g taken from freshly slaughtered pigs, washed and purified from the blood of the aortic valve leaflets were placed in a vessel containing 350 ml of a 5% solution of ethylene glycol diglycidyl ether at pH = 7.4 and temperature ^of 20 ° C, where a biological tissue for canned 21 days The canned material was immersed in a heparin solution with a concentration of 100 IU / ml for 1 day at ²⁰ ° C, after which heparin was removed, the container was filled with a 2% solution of ethylene glycol diglycidyl ether, wherein the biological tissue, and stored prior to implantation.

2. Option 4 segments bovine internal thoracic artery 20 weight each, washed and purified from blood, was immersed in a 2% ethylene glycol diglycidyl ether solution at pH = 7.4 and temperature of 20 ^{° C} for 21 days. followed by heparinization were performed by incubating in a solution of heparin (100 IU / ml) for 1 day at ²⁵ ° C and again immersed in a 2% solution of ethylene glycol diglycidyl ether. where they were stored until the study.

 The anti-calcifying effect of this treatment method was studied on the classical model of accelerated calcification during subcutaneous implantation of rats xenvial valve cusps. The adequacy of such a model has been proven previously (Shoen F. J. et al., 1986).

 In this experiment, the model was reproduced on 80 young non-linear male rats with a body weight of 70-80 g, which were divided into 4 groups of 20 animals each.

 Under aseptic conditions, 6–8 hypodermic pockets were formed for each animal from separate sections along the spine, into which samples were implanted in the form of disks from the valves of xenographic valves, preserved by 2 analogue methods (glutaraldehyde and Dewanjee), the prototype method and the method we proposed.

 The calcium content in the implants was determined by atomic absorption spectroscopy. The results are shown in table. 1.

 The thromboresistant properties of the biomaterial were determined by the number of thrombotic masses deposited on the walls of the test vascular segments upon contact with native blood in ex vivo experiments.

 For this, a special 12-channel device was used, connected to the animal by the method of arteriovenous shunt. Exposure with blood was carried out in three time modes: 8, 16 and 24 minutes. A comparative evaluation of the test samples was carried out. As a control, samples treated with glutaraldehyde were used. The number of thrombotic masses deposited on their surface at each time regime was taken as 100%. The results are presented in table. 2.

 The results obtained allow us to conclude that the proposed method of conservation allows us to make xenomaterial almost completely resistant to calcification, since for all periods of subcutaneous implantation, the calcium content in the samples does not exceed the metabolic level and, in essence, does not increase over time. At the same time, the analogue method and the prototype method protect the tissue to a much lesser extent (p <0.01).

 The results of chemical analysis correlated with data from a morphological study. So, the tissue treated by the proposed method, at all stages of implantation, was characterized by a high degree of preservation of the bulk of the collagen and the absence of signs of calcification.

 Biological tissue treated by the proposed method has a higher thrombotic resistance under the conditions of a model experiment.

 (56) Nairi C. et. al. -J. Thorac. Cardiovasc. Surg, 1987-v. 93, N 6, p. 867-877.

Claims (1)

 METHOD FOR CONSERVING BIOTABLES FOR PROSTHESIS OF HEART VALVES AND VESSELS, including treating them with ethylene glycol diglycidyl ether, characterized in that, in order to suppress calcification and thrombosis processes, the treatment is carried out with a 2-5% solution of diglyc solution of heparine ethylene glycol with ethylene glycol dilated with ethylene glycol solution not less than 100 IU / ml.

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