RU2558089C1 - Method for pre-implantation preparation of biological prostheses for cardiovascular surgery - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves sampling and washing native tissues and preserving them for 2-21 days in mixed ethylene glycol diglycyl ester and oligoepoxy compound with the branched structure of a hydrocarbon chain prepared in a 0.05-0.2 M phosphate buffer; that is followed by heparinisation of the biological tissue surface at 20-40°C for 2-14 hours.

EFFECT: method enables improving viscoelastic and thromboresistant characteristics of the biological prosthesis, increasing the sterilising effect and biomaterial resistance to calcification.

3 cl, 3 dwg, 4 tbl, 3 ex

Description

The invention relates to medicine, namely to cardiovascular surgery, and can be used for preservation, sterilization and modification of biological material in the manufacture of bioprostheses of blood vessels and heart valves.

In the development of cardiovascular prostheses, particular importance is attached to the physicomechanical characteristics of the biomaterial, such as strength and elasticity, which are evaluated by the values of breaking stress and elongation under tension. In addition to the mechanical characteristics, bioprostheses of the valves of the heart and arteries should have thromboresistant properties, since they are in constant contact with the blood. Monomeric epoxy compounds, in particular ethylene glycol diglycyl ether (DEE), have been successfully used to preserve cardiovascular biological prostheses. However, the search for more promising preservatives does not lose its relevance.

A known method of preserving biological tissue for cardiovascular surgery, based on the use of a preserving solution of diglycidyl ether of ethylene glycol (DEE) (Pat. 2008767, Russian Federation. IPC A01N 1/00. Method of preserving biological tissue for prosthetics of heart and blood valves [Text] / L . S. Barbarash, S.P. Novikova, I.Yu. Zhuravleva et al .; patent holder L. Barbarash (RU) - No. 5024513/14; filed January 23, 1992; published March 15, 94). The method allows to achieve a high density cross-linking and sufficient sterilizing effect. However, the concentration of DEE on a standard phosphate buffer solution decreases by 21 days from 5 to 0.7%. In this case, the pH of the DEE solution by the 9th day reaches 11.5-12 (compared with the initial 7.4-7.5), which may have an adverse effect on the structure of collagen. The osmotic pressure of the solution prepared in accordance with this method is 140-145 mOsm / l, which is 2 times lower than the osmolarity of blood plasma. Low osmotic pressure and alkaline environment (pH 11.5-12) of the preserving and sterilizing solution in phosphate buffer cause swelling of the leaf and muscle parts of the aortic complex.

A known method of preservation and sterilization of biological prostheses, comprising treating the biomaterial with a solution of ethylene glycol diglycidyl ether at a temperature of 5-25 ° C for 2-21 days, using 0.05-0.1 M phosphate-tetraborate buffer pH as the basis for the preservation solution 7.4, and the sterilizing effect is provided by adding 1-15% ethanol (Pat. 2350075, Russian Federation: IPC A01N 1/02, A61L 27/00. Method for the conservation and sterilization of biological prostheses for cardiovascular surgery [Text] / I.Yu. Zhuravleva, L.S. Barbarash, . .A Kudryavtsev, IL Gantimurova;. ZHuravleva IY patent (RU) - №2007125733 / 15; appl 06.07.07; published 27.03.09, Bulletin 9. - 5)..... This technology has a sterilizing effect due to exposure to biomaterial with small concentrations of ethyl alcohol. However, the conservation of monomeric DEE limits the possibility of additional modification of bioprostheses, and the strength properties of DEE-treated biomaterial are significantly inferior to the strength of native tissue.

A known method of sterilization and preservation of biological prostheses for cardiovascular surgery (application WO 9209309: IPC A01N 43/20, A61L 2/00, A61L 2/18 Method of liquid sterilization [Text] / TU Roger (US); Thyagarajan Kalathi (US ); Myers Harriet Chan (US); applicant and copyright holder Baxter int (US) .- PCT / US91 / 08900; claimed 25.11.91; publ. 11.06.92). The essence of the method lies in the fact that for the sterilization of biological prostheses use a 2-4% solution of polyepoxide compounds prepared on 0.1 M carbonate buffer at a pH of 9.5-10.0. To enhance the sterilizing effect, up to 20 parts of ethanol is added to the solution of polyepoxide compounds, while the greatest sterilizing effect is achieved at a solution temperature of 37 ° C to 45 ° C and adding 20% ethanol. The main disadvantages of this method include: the use of high temperatures of the solution; the use of a buffer solution with an alkaline pH value in excess of physiological; the addition of 20% ethanol, which ultimately has a tanning effect on biological tissue, giving it rigid properties. All this can adversely affect the structural stability of the biomaterial and lead to prosthesis dysfunction after implantation.

Closest to the claimed is a method of processing biological prostheses, in which the native tissue from freshly killed animals is treated with a mixture of epoxy compounds at a temperature of 4-45 ° C for 2-21 days, followed by treatment with a solution of heparin with a concentration of at least 75 u / ml at a temperature of 20- 45 ° for 2-16 hours and ethylation with a 70% aqueous solution of ethanol (Pat. 2122321 Russian Federation. IPC A01N 1/02, Method for processing biological prostheses for cardiovascular surgery [Text] / I.Yu. Zhuravleva, L.S. Barbarash, S.P. Novikova, I.L. Gantima moat; applicant Kemerovo Cardiology Center (RU); patentee I. Zhuravleva (RU). - No. 96104575/14; filed March 6, 1996; publ. 11/27/98. The disadvantages of this method include the use of distillation products of DEE, which, having a toxic effect, damage the cells of biological tissue. In addition, the treatment of xenobioprostheses pre-modified with heparin in a solution of 70% ethanol leads to leaching of the anticoagulant and rigidity of the biomaterial, which impairs its biomechanical characteristics.

The technical result of the invention is to improve the elastic and thromboresistant characteristics of a biological prosthesis, as well as to increase the sterilizing effect and resistance of the biomaterial to calcification.

The technical result is achieved due to the additional introduction of oligoepoxy compounds with a branched hydrocarbon chain structure and an increased number of reactive epoxy groups into the preservative solution of diglycyl ethylene glycol ether, which allows additional modification of biological tissue, giving it the desired properties. In addition, the technical result is achieved due to the use of a buffer solution in the preservative solution with the osmolarity of the corresponding blood plasma.

It is known that during the conservation of biological tissues with a solution of monomeric epoxy compounds, the majority of reactive epoxy groups react with amino acids of the collagen matrix, which significantly reduces the possibility of further immobilization of biologically active substances on the surface of the biomaterial. The inclusion of oligoepoxy compounds in the preservative will not only increase the strength and elasticity of the biomaterial by enhancing the chemical crosslinking of collagen fibers, but also improve the antithrombotic characteristics of the solution by involving additional reaction epoxy groups in the process of modifying biological tissues and increasing the amount of immobilized heparin on the surface of the bioprosthesis.

Based on the studies, including analysis of the effect of epoxy preservatives on the surface structure of xenogenic biomaterial, 1,2,3,4,6-penta-O- {1 - [(glycidyloxy) ethoxy] ethyl} -α- was selected from the synthesized original compositions D-glucopyranose (PE), with the optimal ratio of epoxide in the mixture from 5 to 30 parts.

As a solvent, preservatives used a solution of 0.05-0.2 M phosphate buffer prepared in a 0.9% solution of sodium chloride (NaCl). The pH of the resulting buffer solution was 7.3-7.4, and the osmolarity of 280-300 mOsm / l, which is similar to the osmolarity of blood plasma. These indicators make it possible to ensure the safety of collagen fibers, avoid swelling of biological tissue and, as a result, prevent dysfunction of bioprostheses after implantation in patients.

The phosphate buffer was prepared from a concentrate No. 1 (sodium phosphate disubstituted - 12 water grade “chemically pure”) and a concentrate No. 2 (sodium phosphate monosubstituted monohydrate brand - “chemically pure”) previously dissolved in 0.9% sodium chloride solution. At the same time, 120 ml of concentrate No. 1 and 880 ml of concentrate No. 2 were used to prepare 1 liter of buffer.

The preservation solution was prepared as follows: each of the preservatives was previously dissolved in 250 ml of a buffer solution, then it was stirred and the volume of the preservative was adjusted to 1 liter with a buffer solution, while the ratio of parts of DEE: PE in the preservative was 95-70: 5-30, respectively.

Preservation and sterilization of biological material with the proposed solution was carried out at a temperature of 5-25 ° C for 5-21 days. After that, bioprostheses were modified in a solution of unfractionated and low molecular weight heparins at a concentration of 75-150 Edml, such as Enoxaparin sodium (Clexan), Nadroparin (Fraxiparin), Dalteparin (Fragmin), Reviparin (Klivarin), Bemiparin sodium (Zibor. Heparinization was performed at a temperature of from 20 to 40 ° C for 2-14 hours. Carrying out the modification at a temperature below 20 ° C is not advisable, because significantly increases the time of heparinization, and at temperatures above 40 ° C there is the possibility of thermal damage to tissues.

To study the effect of the proposed method for preimplantation processing of bioprostheses on the structure of the biomaterial, a histological study of the valves of the aortic valve of pigs, pericardium and cattle arteries, preserved in experimental solutions, was performed. When visualizing histological tissue samples using electron microscopy, it was proved that the storage of biomaterial treated with the proposed preservative does not adversely affect the biomaterial — the collagen fibers are convoluted and are more compact compared to using a DEE solution. And the density of collagen fibers increases in the series DEE <DEE + 5% PE <DEE + 15% PE <DEE + 30% PE.

Assessment of the strength and elasticity of the biomaterial showed that the use of preservative solutions containing a mixture of DEE from 95-70% and oligoepoxy compounds 1,2,3,4,6-penta-O- {1 - [(glycidyloxy) ethoxy] ethyl} -α- D-glucopyranose from 5% to 30%, increases strength by 10-62% and does not adversely affect the elasticity of the biomaterial (table. 1).

The use of oligoepoxy compounds in a preservative solution allows you to bind biologically active substances having amino, caboxy or hydroxy groups, while maintaining a high anticalcinosis effect. The amount of calcium in experimental studies for three months remains at the metabolic level (table. 2).

In addition, the use of 1,2,3,4,6-penta-O- {1 - [(glycidyloxy) ethoxy] ethyl} -α-D-glucopyranose and performing heparinization of the samples after the conservation step allows increasing the amount of immobilized heparin on the surface of the biomaterial (tab. 3).

The study of thrombotic resistance of the biomaterial was performed after contact of the samples with native blood for 90 minutes. The results obtained indicate that adding to the composition of the preservation solution from 5% to 30% 1,2,3,4,6-penta-O- {1 - [(glycidyloxy) ethoxy] ethyl} -α-D-glucopyranose significantly reduces the amount of blood proteins adsorbed on the surface of the biomaterial, especially after modification of samples in a solution of low molecular weight heparin (Table 4).

The increase in the thromboresistant properties of the biomaterial is due not only to an increase in the adsorption of heparin, but also to a smoothing of the surface relief of the biological tissue due to more dense cross-linking of collagen fibers during the conservation of the biomaterial.

The proposed method has a good sterilizing effect - after a day of storage of biomaterial contaminated with St. aureus, E. coli Ent. faecalis and fungal flora in a solution prepared by the proposed method, the growth of all microorganisms is completely suppressed.

The invention is illustrated by drawings, which shows the results of scanning electron microscopy of the inner surface of the xenoarteries, with an increase of × 1000. Moreover, in FIG. 1 shows a sample treated with a 5% DEE solution; in FIG. 2 - a sample treated with a solution of epoxy compounds containing 70% DEE + 30% PE; in FIG. 3 - a sample treated with a solution of epoxy compounds containing 70% DEE + 30% PE with additional modification in a solution of Enoxaparin sodium.

In the study of biomaterial, a change in the surface relief of the biomaterial was observed, and its smoothing was observed. The addition of an oligoepoxy compound to the preservative solution enhances the chemical cross-linking of collagen fibers during the conservation of the biomaterial due to the branched structure of the hydrocarbon chain, which also affects the surface relief of the biological tissue — it becomes smoother than FIG. 2. Subsequent modification in a solution of low molecular weight heparin of FIG. 3 allows you to enhance the smoothing effect on the surface topography of the biological tissue, which in combination with the anticoagulant properties of heparin significantly increases the thromboresistance of bioprostheses.

The following are examples of the proposed method.

Example 1. Prepared by existing methods of selection of the native artery tissue is washed with 0.9% sodium chloride solution, then placed in a 2-5% solution containing 95% DEE and 5% 1,2,3,4,6- penta-O- {1 - [(glycidyloxy) ethoxy] ethyl} -α-D-glucopyranose prepared in 0.05-0.2 M phosphate buffer in 0.9% NaCl. After 5-21 days of preservation, bioprostheses are washed from the preservation solution three times for 20 minutes in excess of physical. solution and immersed in a solution of low molecular weight heparin Enoxaparin sodium with a concentration of 75 IU / ml and a temperature of 20-40 ° C for 2-14 hours. Using a concentration of heparin solution of less than 75 Units / ml will increase the modification time, and using a concentration of more than 150 Units / ml is also impractical, because the amount of the drug to be added does not increase, but the consumption of low molecular weight heparin only increases.

Example 2. Prepared by existing selection methods, native heart valve flaps are washed with 0.9% sodium chloride solution, then placed in a 2-5% solution containing 85% DEE and 15% 1,2,3,4,6 -penta-O- {1 - [(glycidyloxy) ethoxy] ethyl} -α-D-glucopyranose prepared in 0.05-0.2 M phosphate buffer in 0.9% NaCl. After 5-21 days of preservation, bioprostheses are washed from the preservation solution three times for 20 minutes in excess of physical. solution and immersed in a solution of low molecular weight heparin Bemiparin sodium with a concentration of 100 IU / ml and a temperature of 20-40 ° C for 2-14 hours.

Example 3. Prepared according to existing selection methods, native xenopericardium is washed with 0.9% sodium chloride solution, then placed in a 2-5% solution containing 70% DEE and 30% 1,2,3,4,6-penta -O- {1 - [(glycidyloxy) ethoxy] ethyl} -α-D-glucopyranose, prepared in 0.05-0.2 M phosphate buffer in 0.9% NaCl. After 5-21 days of preservation, bioprostheses are washed from the preservation solution three times for 20 minutes in excess of physical. solution and immersed in a solution of low molecular weight heparin Dalteparin with a concentration of 150 PIECES / ml and a temperature of 20-40 ° C for 2-14 hours.

Thus, the proposed method for preimplantation treatment of biological prostheses for cardiovascular surgery allows to increase the strength of the biomaterial, provides resistance to calcification of the biological tissue and the sterilizing effect, and the use of low molecular weight heparins makes it possible to smooth the surface relief and increases the thromboresistance of bioprostheses.

Claims (3)

1. A method for preimplantation treatment of biological prostheses for cardiovascular surgery, including sampling and washing of native tissues, followed by preservation for 2-21 days in a solution of a mixture of diglycyl ether of ethylene glycol and an oligoepoxy compound with a branched hydrocarbon chain structure prepared at 0.05-0, 2 M phosphate buffer and subsequent heparinization of the biological surface of the tissue at a temperature of 20-40 ° C for 2-14 hours, characterized in that from 5 to 30 parts of oligoepoks are added to the preservation solution isoedineniya by weight of a diepoxide, a phosphate buffer prepared in 0.9% sodium chloride solution with osmolarity of 280-300 mOsm / l. 2. The method of preimplantation treatment of biological prostheses according to claim 1, characterized in that 1,2,3,4,6-penta-O- {1 - [(glycidyloxy) ethoxy] ethyl} -α- are used as oligoepoxy compounds in the mixture D-glucopyranose. 3. The method of preimplantation treatment of biological prostheses according to claim 1, characterized in that the modification of biological tissue is carried out in a solution of unfractionated and low molecular weight heparins at a concentration of 75-150 U / ml.

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