Method for preimplantation preparation of bioprostheses

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

 

The invention relates to medicine, namely to preimplantation processing bioprocesos for cardiovascular surgery. Under bioprocesses for cardiovascular surgery refers to artificial heart valves made entirely or partially of biological tissue in accordance with GOST 26997-2003 "heart Valves artificial. General technical conditions" and the international standard ISO 5840:2005 "Cardiovascular implants - Cardiac valve prostheses", NEQ.

Chemical stabilization of the tissue is necessary for the prevention of calcification of the tissue prosthesis after implantation. The known method preimplantation processing bioprocesos (RF Patent No. 2120212 from 20.10.1998, IPC6A01N 1/02), in which chemical stabilization tissues conduct of 0.625% solution of glutaraldehyde followed by treatment of the surface-active substance with a 4-fold change of the working solution.

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

The technical result of the proposed method is to increase the durability of bioprocesos by reducing calories is tificatio and impart antimicrobial properties of tissue bioprocesos, what is achieved by modification of the N-sulfosuccinate chitosan (N-CCX). Derived chitosan soluble in water at neutral pH values, can be obtained using the original method proposed by one of the authors of the present invention (Patent RF №2048475, 1995, IPC6SW 37/08). Immediately prior to implantation of Bioprocess thoroughly washed with sterile saline solution with 6 times its change at the rate of 500 ml per 100 g of tissue, then treated with 0.05 to 0.5% aqueous solution of N-sulfosuccinate chitosan with molecular weight of 50 to 150 KD under 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, and then placed in sterile saline and stored at a temperature of 6-8°C prior to implantation.

The invention is carried out as follows.

Biological tissue, namely: kenopanishad calf or pig, glassonby capsule veal liver, porcine aortic or pulmonary complex is subjected to chemical stabilization of 0.625% aqueous solution of glutaraldehyde, pH 7.4, followed by treatment of the surfactant is 1% solution of dodecyl-sulfate sodium 4-fold change of the working solution. From the obtained chemically stabilized tissues are made bioprothesis.

Immediately before implantation PR is lead modification of bioprocesos, what bioproces thoroughly washed with sterile saline solution with 6 times its change at the rate of 500 ml per 100 g of tissue. The washed bioproces process of 0.05-0.5% solution of N-sulfosuccinate chitosan with molecular weight of 50 to 150 KD under vigorous stirring for 0.5-2 hours, at pH in the range from 5 to 8 and a temperature of 22±2°C. the Amount of bound N-CCX determined spectrophotometrically according to the difference of concentration of the source and the current solutions of N-CCX. Then bioproces fix in sterile absolute ethanol, and then placed in sterile saline and stored at a temperature of 6-8°C up to their implantation.

Example 1.

Kenopanishad calf is subjected to chemical stabilization as specified above, and make of him framed bioproces aortic, tricuspid or mitral heart valves.

Ready sterile framed bioproces thoroughly washed with sterile saline solution with a 6-fold change in sterile saline at the rate of 500 ml for one denture, and then placed in a 0.25% aqueous solution of N-CCX with a molecular mass of 100 KD (ratio of 10 g xenopericardial/50 ml), the pH of the solution was adjusted to 7.50±0,50 by addition of 4M NaOH or 6N HCl, then intensively stirred on a shaker for 1 hour at a temperature of 22±2°C. On okonomiyaki bioproces fully immersed in absolute ethanol, the exposure time 10 min, then placed in sterile saline and stored at a temperature of 6-8°C until implantation. The content of the associated N-CCX determined by the spectrophotometric method, is 80±9 µg/cm2xenopericardial.

Example 2.

Kenopanishad pigs subjected to chemical stabilization as specified above, and make of him framed bioproces aortic, tricuspid or mitral heart valves.

Ready sterile framed bioproces thoroughly washed with sterile saline solution with a 6-fold change in sterile saline at the rate of 500 ml for one denture, and then placed in a 0.05% aqueous solution of N-CCX with molecular weight of 25 KD (ratio of 10 g xenopericardial/50 ml), the pH of the solution was adjusted to 6.00±0,50 by addition of 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 processing Bioprocess fully immersed in absolute ethanol exposure time of 10 min, after which placed in sterile saline and stored at a temperature of 6-8°C until implantation. The content of the associated N-CCX determined by the spectrophotometric method, 45±14 µg/cm2xenopericardial.

Example 3.

Glassonby capsule of the liver of a calf is subjected hee is practical stabilization as described above, and is made from her frame bioproces tricuspid valve of the heart.

Ready sterile framed bioproces thoroughly washed with sterile saline solution with a 6-fold change in sterile saline at the rate of 500 ml for one denture, and then placed in a 0.5% aqueous solution of N-CCX with a molecular mass of 50 KD (ratio of 10 g of glassonby capsules/50 ml), the pH of the solution was adjusted to 8.00±0,50 by addition of 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 processing Bioprocess fully immersed in absolute ethanol exposure time of 10 min, after what is placed in sterile saline and stored at a temperature of 6-8°C until implantation. The content of the associated N-CCX determined by the spectrophotometric method is 100±19 µg/cm2glassonby capsules.

Example 4.

Kenopanishad calf is subjected to chemical stabilization as specified above, and make of him frameless bioproces aortic or pulmonary heart valve.

Ready sterile frameless bioproces thoroughly washed with sterile saline solution with a 6-fold change in sterile saline at the rate of 500 ml for one denture, and then placed in 0.25% wagnerstr N-CCX with a molecular mass of 100 KD (ratio of 10 g xenopericardial/50 ml), the pH of the solution was adjusted to 7.00±0,50 by addition of 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 processing Bioprocess fully immersed in absolute ethanol exposure time of 10 min, and then placed in sterile saline and stored at a temperature of 6-8°C until implantation. The content of the associated N-CCX determined by the spectrophotometric method is 90±9 µg/cm2xenopericardial.

Example 5.

Aortic or pulmonary kinokompleks pigs subjected to chemical stabilization as specified above, and make of him frameless bioproces aortic or pulmonary heart valve, respectively.

Ready sterile frameless bioproces thoroughly washed with sterile saline solution with a 6-fold change in sterile saline at the rate of 500 ml for one denture, and then placed in a 0.25% aqueous solution of N-CCX with a molecular mass of 90 KD (ratio of 10 g kinokompleks/50 ml), the pH of the solution was adjusted to 6.50±0,50 by addition of 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 processing Bioprocess fully immersed in absolute ethanol exposure time of 10 min, after which placed in sterile physiological rest the R and stored at a temperature of 6-8°C until implantation. The content of the associated N-CCX determined by the spectrophotometric method, 70±9 µg/cm2kinokompleks.

Tables give the results of tests of bioprocesos held preimplantation processing of the proposed method.

Table 1.
Toxicological studies. Cytotoxicity on short-term suspension culture of motile cells.
A valid value for the number of surviving cells, %The placeholderExample 1Example 2Example 3Example 4Example 5
70-12050±5%100±5%100±5%100±5%100±5%100±5%

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

Table 2.
The study of calcification in vivo in rats
The method of chemical stabilization of biological tissuesThe number of samplesThe content of CA, mg/g dry cloth
The placeholder53by 8.22±0,42
The proposed methodExample 1410,9±0,06
Example 2381,2±0,11
Example 3440,7±0,15
Example 4401,3±0,19
Example 5420,8±0,10

The data presented indicate that modification of bioprocesos N-sulfosuccinate chitosan reduces calcification of bioprocesos.

Table 3.
Microbiological studies. The formation of biofilms of S. epidermidis
The method of chemical stabilization of biological tissuesThe results of the tests
The placeholder100%
The proposed methodExample 1≤1%
Example 2≤1%
Example 3≤1%
Example 4≤1%
Example 5≤1%

The data presented indicate that modification of bioprocesos N-sulfosuccinate chitosan gives bioprothesis antimicrobial properties.

Table 4.
Uprugoopticheskii properties of chemically stabilized tissues
The method of chemical stabilization of biological tissuesTensile strength (in two directions), σFC, MPaThe modulus of elasticity (in two directions), E, MPaThe maximum strain to the discontinuity (in two directions), εmax/sub> , %
The placeholder15,08±0,7171,40±1,3441,43±1,62
7,32±0,4337,21±1,1041,30±1,43
The proposed methodExample 113,60±0,5871,51±1,4537,18±1,49
7,02±0,3936,78±1,3938,89±1,31
Example 214,21±0,3870,28±1,2340,18±1,56
of 6.96±0,3737,34±1,1539,89±1,19
Example 311,33±0,4115,51±1,3736,18±1,36
12,87±0,5614,78±1,2135,89±1,22
Example 413,60±0,5871,51±1,4537,18±1,49
7,02±0,3936,78±1,39 38,89±1,31
Example 522,64±1,83of 7.68±0,82-
9,98±1,74to 11.61±0,21

The data presented indicate that modification of bioprocesos N-sulfosuccinate chitosan does not reduce the mechanical strength of the tissue prostheses.

Thus, the invention presents a combination of traits obviously ensures the achievement of the technical result, namely, reduces the degree of calcification and cytotoxicity biological prostheses, at the same time giving them antimicrobial properties, thereby increasing durability and reducing the risk of costly duplication of cardiovascular interventions.

Developing new biological prostheses with high biological compatibility and resistance to the formation of microbial biofilms will be able to find a wide application in cardiac clinics of the Russian Federation and abroad.

The way preimplantation processing bioprocesos, including chemical stabilization of the tissues of 0.625%aqueous solution of glutaraldehyde pH 7.4 followed by treatment of the surface-active substance with a 4-fold change of the working solution, great for the present, however, that just prior to implantation of Bioprocess thoroughly washed with sterile saline solution with 6 times its change rate of 500 ml per 100 g of tissue, then treated with 0.05 to 0.5%aqueous solution of N-sulfosuccinate chitosan with molecular weight of 50 to 150 KD under vigorous stirring for 0.5-2 h at pH in the range from 5 to 8 and a temperature of 22±2°C, then fixed in sterile absolute ethanol, and then placed in sterile saline and stored at a temperature of 6-8°C prior to implantation.



 

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