RU2384309C2 - Method for preimplantation preparation of bioprostheses - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves chemical stabilisation of biotissue with 0.625 % aqueous solution of glutaraldehyde, pH 7.4, followed with preparation with a surface-active substance and quadruple change of a working solution. Immediately ahead of implantation, bioprostheses are thoroughly washed with sterile physiologic saline sixfold changed, at 500 ml of the solution for 100 g of biotissue. Then it is processed with 0.05-0.5% aqueous solution of chitosan N-sulphosuccinate of molecular weight 50-150 kDa in intensive stirring during 0.5-2 h with pH within 5 to 8, and to temperature 22±2°C. Further, it is fixed in sterile absolute ethanol and put in sterile physiologic saline, and stored at temperature 6-8°C before implantation.

EFFECT: improved durability of bioprostheses.

5 ex, 4 tbl

Description

The invention relates to medicine, namely to preimplantation processing of bioprostheses for cardiovascular surgery. Bioprostheses for cardiovascular surgery are artificial heart valves made in whole or in part from biological tissues in accordance with GOST 26997-2003 “Artificial heart valves. General specifications "and the international standard ISO 5840: 2005" Cardiovascular implants - Cardiac valve prostheses ", NEQ.

Chemical stabilization of biological tissue is necessary for the prevention of calcification of the biological tissue of the prosthesis after implantation. A known method of preimplantation treatment of bioprostheses (RF Patent No. 2120212 from 10.20.1998, IPC ⁶ A01N 1/02), in which the biological tissue is chemically stabilized with a 0.625% glutaraldehyde solution, followed by treatment with a surfactant with a 4-fold change of working solution.

The disadvantage of this method is the fragility of bioprostheses, namely, calcification of the bioprosthesis by 6-8 years after implantation. In addition, stabilization of the biological tissue with glutaraldehyde leads to the formation of a significant amount of free aldehyde groups in the biological tissue, which provokes the cytotoxicity of the biological implant.

The technical result of the proposed method is to increase the durability of bioprostheses by reducing calcification and imparting antimicrobial properties to tissue of bioprostheses, which is achieved due to their modification with chitosan N-sulfosuccinate (N-CCX). A chitosan derivative soluble in water at neutral pH values can be obtained using the original method proposed by one of the authors of the present invention (RF Patent No. 2048475, 1995, IPC ⁶ C08B 37/08). Immediately prior to implantation, bioprostheses are thoroughly washed with sterile physiological saline with a 6-time change based on 500 ml of solution per 100 g of biological tissue, then treated with a 0.05-0.5% aqueous solution of chitosan N-sulfosuccinate with a molecular weight of 50-150 kD with intensive stirring for 0.5-2 hours at a pH in the range from 5 to 8 and a temperature of 22 ± 2 ° C, then fixed in sterile absolute ethanol, then placed in a sterile physiological solution and stored at a temperature of 6-8 ° C until implantation .

The invention is as follows.

Biological tissue, namely: calf or pig xenopericardium, calf liver glisson capsule, pork aortic or pulmonary complex, is chemically stabilized with 0.625% aqueous solution of glutaraldehyde, pH 7.4, followed by surfactant treatment with 1% sodium dodecyl sulfate solution with 4 -fold change of working solution. Bioprostheses are made from the obtained chemically stabilized biological tissue.

Immediately prior to implantation, bioprostheses are modified, for which the bioprostheses are thoroughly washed with sterile physiological saline with a 6-fold change based on 500 ml of solution per 100 g of biological tissue. Washed bioprostheses are treated with a 0.05-0.5% solution of chitosan N-sulfosuccinate with a molecular weight of 50-150 kD with vigorous stirring for 0.5-2 hours, at a pH in the range from 5 to 8 and a temperature of 22 ± 2 ° C . The amount of bound N-CCX is determined spectrophotometrically by the difference in concentration of the initial and current N-CCX solutions. Then the bioprostheses are fixed in sterile absolute ethanol, after which they are placed in sterile physiological saline and stored at a temperature of 6-8 ° C until they are implanted.

Example 1

The calf xenopericardium is subjected to chemical stabilization, as described above, and frame bioprostheses of aortic, tricuspid or mitral heart valves are made from it.

The finished sterile skeleton bioprosthesis is thoroughly washed with sterile physiological saline with a 6-fold change of sterile saline at the rate of 500 ml of solution per prosthesis, then placed in a 0.25% aqueous solution of N-CCX with a molecular weight of 100 kD (10 g xenopericardium / 50 ratio ml of solution), the pH of the solution is adjusted to 7.50 ± 0.50 by adding 4M NaOH or 6N HCl, then intensively stirred on a shaker for 1 hour at a temperature of 22 ± 2 ° C. At the end of the treatment, the bioprosthesis is completely immersed in absolute ethanol, the exposure time is 10 minutes, after which it is placed in sterile physiological saline and stored at a temperature of 6-8 ° C until implantation. The content of bound N-CCX, determined by spectrophotometric method, is 80 ± 9 μg / cm ² xenopericardium.

Example 2

Pig xenopericardium is subjected to chemical stabilization, as described above, and frame bioprostheses of aortic, tricuspid or mitral heart valves are made from it.

Ready sterile framework bioprosthesis is thoroughly washed with sterile physiological saline with 6-fold change of sterile physiological saline at the rate of 500 ml of solution per prosthesis, then placed in a 0.05% aqueous solution of N-CCX with a molecular weight of 25 kD (10 g xenopericardium / 50 ratio ml of solution), the pH of the solution is adjusted to 6.00 ± 0.50 by adding 4M NaOH or 6N HCl, then intensively stirred on a shaker for 2 hours at a temperature of 22 ± 2 ° C. At the end of the treatment, the bioprosthesis is completely immersed in absolute ethanol, the exposure time is 10 minutes, after which it is placed in sterile physiological saline and stored at a temperature of 6-8 ° C until implantation. The content of bound N-CCX, determined by spectrophotometric method, is 45 ± 14 μg / cm ² xenopericardium.

Example 3

The calf liver glisson capsule is subjected to chemical stabilization, as described above, and frame bioprostheses of the tricuspid valve of the heart are made from it.

The finished sterile skeleton bioprosthesis is thoroughly washed with sterile physiological saline with a 6-fold change of sterile physiological saline at the rate of 500 ml of solution per prosthesis, then placed in a 0.5% aqueous solution of N-CCX with a molecular weight of 50 kD (ratio of 10 g of a glisson capsule / 50 ml of the solution), the pH of the solution is adjusted to 8.00 ± 0.50 by adding 4M NaOH or 6N HCl, then intensively stirred on a shaker for 0.5 hour at a temperature of 22 ± 2 ° C. At the end of the treatment, the bioprosthesis is completely immersed in absolute ethanol, the exposure time is 10 minutes, after which it is placed in sterile physiological saline and stored at a temperature of 6-8 ° C until implantation. The content of bound N-CCX, determined by spectrophotometric method, is 100 ± 19 μg / cm ² glisson capsule.

Example 4

The calf's xenopericardium is subjected to chemical stabilization, as described above, and frameless bioprostheses of the aortic or pulmonary heart valves are made from it.

Ready sterile frameless bioprosthesis is thoroughly washed with sterile physiological saline with 6-fold change of sterile physiological saline at the rate of 500 ml of solution for one prosthesis, then placed in a 0.25% aqueous solution of N-CCX with a molecular weight of 100 kD (10 g xenopericardium / 50 ratio ml of solution), the pH of the solution was adjusted to 7.00 ± 0.50 by adding 4M NaOH or 6N HCl, then intensively stirred on a shaker for 1 hour at a temperature of 22 ± 2 ° C. At the end of the treatment, the bioprosthesis is completely immersed in absolute ethanol, the exposure time is 10 minutes, after which it is placed in sterile physiological saline and stored at a temperature of 6-8 ° C until implantation. The content of bound N-CCX, determined by spectrophotometric method, is 90 ± 9 μg / cm ² xenopericardium.

Example 5

The pig’s aortic or pulmonary xenocomplexes are chemically stabilized as described above, and frameless bioprostheses of the aortic or pulmonary heart valves are made from it, respectively.

Ready sterile frameless bioprosthesis is thoroughly washed with sterile physiological saline with 6-fold change of sterile physiological saline at the rate of 500 ml of solution for one prosthesis, then placed in a 0.25% aqueous solution of N-CCX with a molecular weight of 90 kD (10 g xenocomplex / 50 ratio ml of solution), the pH of the solution was adjusted to 6.50 ± 0.50 by adding 4M NaOH or 6N HCl, then intensively stirred on a shaker for 1.5 hours at a temperature of 22 ± 2 ° C. At the end of the treatment, the bioprosthesis is completely immersed in absolute ethanol, the exposure time is 10 minutes, after which it is placed in sterile physiological saline and stored at a temperature of 6-8 ° C until implantation. The content of bound N-CCX, determined by spectrophotometric method, is 70 ± 9 μg / cm ² xenocomplex.

The tables show the results of tests of bioprostheses that underwent pre-implantation treatment with the proposed method.

Table 1. Toxicological studies. Cytotoxicity in short-term suspension culture of motile cells. Permissible value of the number of surviving cells,% Prototype Example 1 Example 2 Example 3 Example 4 Example 5 70-120 50 ± 5% 100 ± 5% 100 ± 5% 100 ± 5% 100 ± 5% 100 ± 5%

The data presented indicate that the modification of bioprostheses with N-sulfosuccinate chitosan significantly reduces the cytotoxicity of bioprostheses.

Table 2. In vivo rat calcification assay The method of chemical stabilization of biological tissues Number of samples Ca content, mg / g dry tissue Prototype 53 8.22 ± 0.42 The proposed method Example 1 41 0.9 ± 0.06 Example 2 38 1.2 ± 0.11 Example 3 44 0.7 ± 0.15 Example 4 40 1.3 ± 0.19 Example 5 42 0.8 ± 0.10

The data presented indicate that the modification of bioprostheses with N-sulfosuccinate chitosan reduces the calcification of bioprostheses.

Table 3. Microbiological studies. Biofilm Formation S. epidermidis The method of chemical stabilization of biological tissues Test results Prototype one hundred% The proposed method Example 1 ≤1% Example 2 ≤1% Example 3 ≤1% Example 4 ≤1% Example 5 ≤1%

The data presented indicate that the modification of bioprostheses with chitosan N-sulfosuccinate gives antimicrobial properties to bioprostheses.

Table 4. Elastic strength properties of chemically stabilized biological tissues The method of chemical stabilization of biological tissues Strength (in two directions), σ _pc , MPa Modulus of elasticity (in two directions), E, MPa Maximum deformation before discontinuity (in two directions), ε _max ,% Prototype 15.08 ± 0.71 71.40 ± 1.34 41.43 ± 1.62 7.32 ± 0.43 37.21 ± 1.10 41.30 ± 1.43 The proposed method Example 1 13.60 ± 0.58 71.51 ± 1.45 37.18 ± 1.49 7.02 ± 0.39 36.78 ± 1.39 38.89 ± 1.31 Example 2 14.21 ± 0.38 70.28 ± 1.23 40.18 ± 1.56 6.96 ± 0.37 37.34 ± 1.15 39.89 ± 1.19 Example 3 11.33 ± 0.41 15.51 ± 1.37 36.18 ± 1.36 12.87 ± 0.56 14.78 ± 1.21 35.89 ± 1.22 Example 4 13.60 ± 0.58 71.51 ± 1.45 37.18 ± 1.49 7.02 ± 0.39 36.78 ± 1.39 38.89 ± 1.31 Example 5 22.64 ± 1.83 7.68 ± 0.82 - 9.98 ± 1.74 11.61 ± 0.21

The data presented indicate that the modification of bioprostheses with N-sulfosuccinate chitosan does not reduce the mechanical strength of the prosthesis biotissue.

Thus, the invention in the presented set of features obviously ensures the achievement of a technical result, namely, it reduces the degree of calcification and cytotoxicity of biological prostheses, while at the same time giving them antimicrobial properties, thereby increasing their durability and reducing the risk of repeated expensive cardiosurgical interventions.

New biological prostheses being developed, characterized by high biological compatibility and resistance to the formation of microbial biofilms, will be able to find the widest application in cardiac surgical clinics of the Russian Federation and abroad.

Claims (1)

A method for preimplantation processing of bioprostheses, including chemical stabilization of biological tissue with a 0.625% aqueous solution of glutaraldehyde, pH 7.4, followed by treatment with a surfactant with a 4-fold change of working solution, characterized in that immediately before implantation, the bioprostheses are thoroughly washed with sterile physiological saline with 6-fold change, based on 500 ml of solution per 100 g of biological tissue, then treated with 0.05-0.5% aqueous solution of chitosan N-sulfosuccinate with a molecular weight of 50-150 kD at vigorous stirring for 0.5-2 hours at a pH in the range from 5 to 8 and a temperature of 22 ± 2 ° C, then fixed in sterile absolute ethanol, then placed in sterile saline and stored at a temperature of 6-8 ° C to implantation.