Method for modifying biotissue for prosthetic repair

FIELD: medicine.

SUBSTANCE: before enzymatic treatment, a biotissue is placed into a hypertonic saline and exposed to ultrasound for 20-300 minutes. That is followed by terrilytin treatment in the concentration of 0.1-10 Production Units per 1g of the tissue, washing in an acetic acid solution and sodium hydrocarbonate, keeping in multiply changed glutaric dialdehyde solutions of the increasing concentrations, and sterilisation.

EFFECT: invention ensures maintaining a collagen-elastic structure of the biotissue, reducing a degree of its mineralisation, using fewer reactants for treatment, preparing a non-immunogenic biomaterial crossed throughout.

2 cl, 3 ex

 

The invention relates to medicine, in particular for prosthetics affected areas of organs and tissues in traumatology, surgery, dentistry, ophthalmology, bariatric surgery.

There are several ways of processing the tissue to reduce its immunogenicity and mineralization.

One way involves the manipulation of tissues 2-5% solution diglycidylether ether of ethylene glycol with further treatment with heparin in a concentration of not less than 100 IU/ml (U.S. Pat. RF 2008767 C1, MKI 6 A01N 1/100, 1994). The disadvantage of this method is that diglycidyl ether of ethylene glycol forms an additional crosslinking between collagen fibers only on the surface of the biological tissue, and any damage will result in violation of this protective layer, the result will be the development of calcification of the graft.

A method of chemical treatment of xenopericardial, including chemical stabilization xenopericardial of 0.625% solution of glutaraldehyde and subsequent treatment with 1% sodium dodecylsulfate, being chemically stable kenopanishad further treated 0,05÷0.25% aqueous solution of chitosan or metal-containing chitosan. The disadvantage of this method is the fact that tissues are not removed cellular elements that are guaranteed not to provide the rest of reducing immunogenicity (U.S. Pat. RF 2384348 C2, IPC, A61L 27/36, 2008).

Closest to the claimed method of modifying the biological tissue is a method of preparing tissues for xenoposeidon, in which the biological tissue prior to enzymatic treatment was incubated for between 15 and 72 h in hypertonic solution of sodium chloride 1-10%concentration, treated with enzyme in borate buffer solution at 32-37°C, washed first in the 0.6 to 6%solution of acetic acid, then in solutions of sodium bicarbonate and sodium chloride, can withstand multiple replaceable solution of glutaraldehyde in a buffer solution with increasing concentration and sterilized (U.S. Pat. RF 2197818 C1, MPK7, A01N 1/100, 2001).

The disadvantages of this method are the high concentration of enzyme used and prolonged enzymatic processing, and use as a solvent of terrilian borate buffer solution, which may lead to the destruction of collagen-elastic structure of the tissue during processing and to increase the degree of mineralization.

The proposed method will eliminate these shortcomings.

The purpose of this method is to reduce the risk of destruction of collagen-elastic structure of the tissue during enzymatic processing and, as a consequence, reduction of mineralization.

The essence of the method lies in the fact that biological tissue prior to enzymatic treatment room is up in hypertonic solutions of sodium chloride, during which the biological tissue is exposed to ultrasound at least once during 20-300 minutes, then treated with the enzyme terrintino in a concentration of 0.1-10 PE 1 gram of tissue, washed in a solution of acetic acid and sodium bicarbonate, can withstand multiple samenaide solutions of glutaraldehyde increasing concentration and sterilized.

The difference of the proposed method lies in the fact that in the pre-treatment in hypertonic solutions of sodium chloride biological tissue subjected to ultrasonic treatment at least once in the course of 20-300 minutes, and the enzymatic treatment is carried out in acetate buffer solution, the amount of enzyme used when this is reduced to 0.1-10 PU per gram of tissue.

Processing time less than 20 minutes, not enough for the effective destruction of cellular elements, and more than 300 minutes is not reasonable, because the process of destruction of cellular elements will be completed. When the effects of ultrasound on biological tissue within a specified time range of the destruction of collagen-elastically patterns does not occur.

Enzymatic processing guarantees the destruction left after ultrasonic treatment of cellular elements tissues and glycosaminoglycan as the main media antigen the STI. Replacement borate buffer solution of acetate will reduce the risk of destruction of collagen-elastic structure of the tissue.

The concentration of terrilian from 0.1 to 10 proteolytic units per gram of wet tissue due to the fact that the concentration of the enzyme is less than 0.1 PE not sufficient to destroy the remaining cells of the tissue, and the concentration of more than 10 PE will lead to the destruction of collagen and elastic fibers.

In order to stop the effects of terrilian on biological tissue, needed its inactivation. Do you want to change the parameters on which depends directly on the activity of the enzyme, namely the temperature of the acetate buffer solution, pH, concentration of enzyme in the catchment area. Therefore, the biological tissue is kept in a cooled acidic solution, and then washed in running distilled water.

To inactivate remaining after washing in tissues of acidic groups of the biological tissue is placed in a solution of sodium bicarbonate. Then incubated in hypertonic salt solutions to extract the products of proteolysis of cells from the tissue and washed with distilled water.

Treatment of biomaterial solutions of glutaraldehyde increasing concentrations necessary to stabilize the fabric and reduce its immunogenicity.

The modification of the tissue for procesorova the Oia affected areas of organs and tissues is carried out as follows.

Biological tissue is placed in a container with a hypertonic solution of sodium chloride at a concentration of 1-10% and leave on 24-76 hours, changing every day hypertonic solution in the container. During this processing, the biological tissue is exposed to ultrasound for 60 minutes. After the end of treatment in hypertonic solutions of sodium chloride biomaterial washed in distilled water. Then hold the enzymatic treatment. To do this, count the number of terrilian necessary for the process: 0,1-10 PE 1 gram of wet tissue and dissolve it in acetate buffer solution, heated to 37°C. After enzyme treatment, the biomaterial is kept in inactivating solution (6-60 g of acetic acid, 100 g of sodium chloride, 840-894 g of distilled water) not less than 20 minutes, washed in distilled water and placed in 0.1 M sodium hydrogen carbonate solution and again washed with distilled water. Then spend postfermentation processing hypertonic salt solutions 2%and 7%concentration for 17 and 6 h, respectively, and washed in running distilled water, placed in a solution of glutaraldehyde. Hold shift solution of glutaraldehyde in the solution with higher concentration and sterilized.

Example 1.

Biological tissue is placed in a container with a hypertonic solution chloridrate at a concentration of 2% and leave it for 72 h, changing every day hypertonic solution in the container, then hold shift for a 7%solution of sodium chloride and leave the biomaterial for 6 hours. During this processing biological tissue once exposed to ultrasound for 120 minutes. After a time pre-treatment samples washed in distilled water and carry out enzymatic processing. To do this, count the number of terrilian needed to process: 1 PE 1 gram of wet tissue and dissolve it in acetate buffer solution, heated to 37°C. After enzyme treatment, the biomaterial is kept in inactivating solution (6-60 g of acetic acid, 100 g of sodium chloride, 840-894 g of distilled water) not less than 20 min, washed in distilled water and placed in 0.1 M sodium hydrogen carbonate solution and again washed with distilled water. Then spend postfermentation processing hypertonic salt solutions at concentrations of 2% and 7% for 17 and 6 h, respectively, and washed in running distilled water, placed in a solution of glutaraldehyde. Hold shift solution of glutaraldehyde in the solution with higher concentration and sterilized.

Example 2.

Differs from example 1 in that the biological tissue is placed in a container with a hypertonic solution of sodium chloride at a concentration of 2% at 48 h,and then at 7% hypertonic solution for 12 h, subjected to the action of ultrasound for 60 minutes, the enzymatic treatment is carried out with the concentration of terrilian 5 PE 1 gram of wet tissue.

Example 3.

Differs from example 1 in that the biological tissue is subjected to the action of ultrasound for 100 minutes, the enzymatic treatment is carried out with the concentration of terrilian 5 PE 1 gram of wet tissue.

Application of the proposed method modifications of the tissue to the prosthesis will allow guaranteed to keep collagen-elastic structure of the biological tissue, to reduce the degree of mineralization, to reduce the consumption used for processing reagents, to obtain non-immunogenic, stitched throughout the volume, the biomaterial.

Sources of information

1. RF patent №2008767.

2. RF patent №2384348.

3. RF patent №2197818.

1. The modification of the tissue to replace the damaged areas of the organs and tissues, including pre-treatment in hypertonic salts of sodium chloride, processing the tissue by the enzyme in a buffer solution, sequential washing in a solution of acetic acid and sodium bicarbonate, extract solutions of glutaraldehyde and sterilization, characterized in that during the pre-treatment in hypertonic solutions of sodium chloride biological tissue is exposed to ultrasound at least once, during the s 20-300 minutes; the enzymatic treatment is carried out in acetate buffer solution with a concentration of enzyme of 0.1-10 PE over one gram of tissue.

2. A method of modifying tissue according to claim 1, characterized in that on stage postfermentation processing biological tissue incubated in hypertonic salt solutions.



 

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