RU2122321C1 - Method of treating biological prosthetic means for cardiovascular surgery - Google Patents

Abstract

FIELD: cardiovascular surgery. SUBSTANCE: native tissue from freshly slaughtered animals is treated with mixture of epoxy compounds at temperature from 4 to 45 C for 2 to 21 days and then with solution of heparin with concentration 75 unit/ml at 20-45 C for 2 to 16 hr, after which tissue is ethylated with 70% aqueous ethanol. EFFECT: increased tromb resistance of tissue and suppressed calcification. 4 cl, 1 tbl, 2 ex

Description

 The invention relates to medicine, namely to preimplantation treatment of biological prostheses for cardiovascular surgery.

 Currently, the development of thrombotic resistance of products in contact with blood is carried out in two main directions: the use of hydrogels and the immobilization of physiologically active substances.

 The use of hydrogels is a surface treatment of the material with polyalkylene glycol [1, 2], which consists in its binding to the surface.

 This treatment allows you to achieve a very short-term effect of increasing thromboresistance of the surfaces of materials by increasing hydrophilicity.

 When immobilizing physiologically active substances, mainly anticoagulants are used: gerudin [3], heparin-like substances (sulfonated chitosan) [4] and heparin.

 The most reliable, effective, stable in properties is heparinization of the surface of materials.

 Heparinization of materials and products is carried out using various types of heparin binding on the surface.

 When using ionic mechanisms [5, 6, 7, 8, 9] of binding, heparin does not attach sufficiently strongly to the surface of the tissue, which determines its rapid leaching from the surface by blood flow. In this regard, it becomes necessary to attach a sufficiently large amount of heparin (from 0.05 to 50% of the total mass of the material) to maintain its optimal concentration in the surface layer.

 Stronger covalent binding of heparin is preferred [10].

 A known method for the preparation of antithrombogenic material containing heparinized collagen as an antithrombogenic component [11], which is provided by the binding of collagen protamine through a polyepoxide medium and collagen heparinization by binding of heparin and protamine.

 A known method of preserving biological tissue for prosthetics of heart valves and blood vessels [12], in which the traditional preservative - glutaraldehyde is replaced by a new crosslinking and sterilizing agent from the class of epoxy compounds - 2-5% solution of diglycidyl ether of ethylene glycol, which allows to completely suppress the calcification of biological tissue , increase the thromboresistance of prostheses and enhance this effect by additional covalent addition of heparin by treatment with a heparin solution with a concentration of at least 100 IU / ml.

Taken from freshly killed animals, the biomaterial purified and washed from the blood is immersed in a 5% solution of ethylene glycol diglycidyl ether at a pH of 7.4 and a temperature of 20 ^o C, where it is preserved for 21 days, after which it is incubated for 1 day in a concentration of heparin 100 IU / ml and stored in 2% ethylene glycol diglycidyl ether solution until use.

 The most effective and larger heparin binding due to epoxy groups that did not react with the biomaterial can be carried out using not mono- and diepoxy compounds, but mixtures of diepoxy compounds with polyepoxy compounds.

 An object of the invention is to increase the thrombotic resistance of biological tissue while suppressing calcification.

The problem is achieved by using as a crosslinking and sterilizing agent a mixture of epoxy compounds of various compositions, followed by washing in physiological solution, treatment with heparin at a heparin concentration of at least 75 u / ml at a pH of 3.0 - 8.0, a temperature of 20-45 ^o C, and by ethylation.

 The method is as follows. Material processing is carried out in three stages.

1. Native tissue from freshly killed animals (pigs, calves), taken no more than 6 hours from the time of slaughter, is placed in a 2-5% solution of epoxy mixtures prepared on a buffer at pH 3.0-11.0 at a temperature of 4-45 ^o C, within 2-21 days. The concentration of the solution of epoxy mixtures below 2% does not ensure the sterility of the material during the preservation process, when the concentration of the solution is above 5%, processing is impractical, because complete crosslinking is achieved.

Epoxy mixtures are specific fractions of substances obtained after distillation of the monomeric basic substance (diglycidyl ether of ethylene glycol) in the temperature range of vacuum distillation 100-120 ^o C (VAI-1) and 120-140 ^o C (VAI-2) or after distillation of the main monomer diethylene glycol diglycidyl ether in the temperature range of vacuum distillation 120-140 ^o C (VAI-3).

 The use of a particular fraction of epoxy compounds allows you to adjust the biomechanical properties (elasticity, strength) and biological porosity of the material obtained as a result of processing. The higher the molecular weight of the fraction (VAI-1 <VAI-2 <VAI-3), the higher the biological porosity and elastic deformation properties of the tissue.

The processing of tissues at temperatures below 4 ^o C leads to mechanical destruction of the tissue due to crystallization of water, at a temperature above 45 ^o C temperature coagulation of the protein occurs.

 When preserving within 1-21 days, sufficient collagen crosslinking occurs. The crosslinking activity of the preservative depends on the composition of the fraction of epoxy compounds and on the preservation temperature.

 At the end of the preservation of the fractions of epoxy compounds in the tissue, a large number of free epoxy groups remain, due to which additional tissue modification can be performed, namely, the binding of biologically active substances that can increase thrombotic resistance (heparin, etc.) and antibacterial activity due to the addition of antimicrobial agents having in their structure, basic or acidic groups.

2. Washing the tissue with physiological saline for one hour with at least three-fold change of excess saline and treatment with heparin solution with a concentration of at least 75 u / ml at a pH of 3.08-8.0, a temperature of 20-45 ^o C for 2 -16 hours. With increasing temperature, the required heparin treatment time decreases.

Carrying out processing at a temperature below 20 ^o C is not advisable, because significantly increases the time of heparinization, and at temperatures above 45 ^o C there is the possibility of thermal damage to tissues.

 After that, washing is carried out until there is no heparin in the washings. The winding is judged by the absence of violet staining of the wash water when the dye is dissolved - toluidine blue.

3. Treatment with a 70% aqueous ethanol solution for 40 ^° C. for 60 minutes, followed by washing in physiological saline with at least a two-time change of solution.

 Ethylation allows to increase the binding strength, thereby increasing the amount of immobilized heparin.

 For storage, the treated prostheses are placed in a 2-5% solution of the epoxy compound used. Thus, at the first stage of processing, a higher crosslinking density and the possibility of its regulation, as well as the ability to control biomechanical properties, a greater heparin agent and the ability to bind the treated tissue with any biologically active substances having an amino, carboxy, or hydroxy group arise in including antimicrobial drugs.

Example 1. (prototype)
Samples of the valves of xenobioprostheses of heart valves are placed in a 5% solution of diglycidyl ether of ethylene glycol (DEE) in a buffer solution with a pH of approximately 7.4 at 20 ^o C and incubated for 21 days, then washed with saline and treated with an aqueous solution of heparin with a concentration of at least 75 IU / ml at pH 4.5 at a temperature of 20 ^o C for 8 hours, then washed 5 times with excess distilled water.

 Treated samples are stored for storage in a 5% solution of the epoxy compound used.

 The amount of heparin in the samples (in μg) is evaluated photocolorimetrically using a dye of toluidine blue and calculated per 1 g of dry biological tissue.

 Depending on the DEE treatment conditions, the amount of attached heparin can be obtained from 600 ± 50 to 1100 ± 100 μg / g.

 Example 2

Samples of the valves of the aortic valves from freshly slaughtered pigs are placed in a 2% solution of a mixture of (di- and poly-) epoxy compounds (VAI-1) in a buffer solution at pH 4.5 at a temperature of 20 ^o C incubated for 21 days. Then washed three times with excess saline and treated with an aqueous solution of heparin with a concentration of not less than 75 IU / ml at pH 3.0 at a temperature of 20 ^o C for 8 hours. After that, 5 times washed with excess distilled water and placed for storage in the 2nd solution of the used epoxy compounds, after evaluating the amount of heparin in the samples of biological tissue (µg / g dry biological tissue). Distilled water in the example is used only in connection with the need to assess the amount of heparin.

 The amount of heparin under these processing conditions is 1800 ± 100 μg / g.

 The processing results depending on the parameters of the processing conditions are shown in the table.

 In examples 4, 10, 13 and 14, segments of the bull’s internal thoracic artery are treated.

 In examples 13 and 14, after treatment with heparin and washing with physiological saline, the biological tissue is treated with 70% ethanol for 1 hour, after which the biological tissue is washed in physiological saline and placed in a solution of the corresponding epoxy compound for storage. The amount of added heparin in Example 13 is 2000 ± 100 μg / g, which exceeds the amount of heparin in Example 4 (1820 ± 100 μg / g), where treatment with 70% ethanol was not carried out.

 Similarly, in example 14, the amount of heparin is 1850 ± 100 μg / g compared with example 10, where the amount of heparin is 1600 ± 200 μg / g.

 A comparison of the results of processing biological tissue in a known manner and proposed using various modes shows that the amount of added heparin, and therefore the thromboresistant effect in the proposed method without ethylation, is 1.6-3.0 times, and with the use of ethanol treatment this difference increases to 1 , 8-3.3 times more than in the prototype. This is achieved both by the use of specially isolated (obtained) fractions of epoxy compounds containing an increased number of epoxy groups, and by the proposed technological methods.

 The increased content of epoxy groups allows you to bind any other biologically active substances with amino, caboxy or hydroxy groups, including antimicrobial agents. At the same time, a high anticalcinous effect remains. The amount of calcium in experimental studies for more than three months remains at a metabolic level.

Sources of information
1. Japanese application N 3-66904, MKI A 61 L 33/00.

 2. Japanese application N 3-64146, MKI A 61 L 33/00.

 3. Application EPO N 0357242, MKI A 61 L 33/00, 1990.

 4. Application of Japan N 1-49503, MKI A 61 L 33/00, 1989.

 5. Copyright certificate N 261635, MKI A 61 F 2/06, 1970.

 6. Japanese application N 61-20309, MKI A 61 L 33/00, 1987.

 7. PCT Application N 91/01767, MKI A 61 L 33/00, 1991.

 8. Japanese application N 61-20309, MKI A 61 L 33/00.

 9. Application EPO N 0350161, MKI A 61 L 33/00, 1990.

 10. Application EPO N 0351314, MKI A 61 L 33/00, 1990.

 11. US patent N 4806595, MKI A 61 F 2/04, 1989.

12. RF patent N 2008767, MKI ⁵ A 01 N 1/00, 1994.

Claims (4)

 1. A method for processing biological prostheses for cardiovascular surgery, including treating them with a solution of epoxy compounds and a heparin solution, characterized in that the treatment is carried out with a 2-5% solution of a mixture of epoxy compounds of various compositions, and after treatment with a solution of heparin, ethanol is additionally treated. 2. The method according to claim 1, characterized in that the treatment with a 2 - 5% solution of a mixture of epoxy compounds of various compositions is carried out at a pH of 3.0 - 11.0, a temperature of 4 - 45 ^o C for 1 to 21 days. 3. The method according to p. 1, characterized in that the heparin treatment is carried out with a solution of heparin with a concentration of 75 u / ml at a pH of 3.0 - 8.0, a temperature of 20 - 45 ^o C for 2 to 16 hours  4. The method according to claim 1, characterized in that the ethanol treatment is performed for 40-60 minutes.

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