Method for making plate from modified xenogeneic enteral submucosa

FIELD: medicine.

SUBSTANCE: method for making a plate from modified xenogeneic enteric submucosa involves mechanical evacuation of small bowel, processing in hypertonic saline in a combination with ultrasonic exposure, enzymatic treatment, subsequent washing in acetic acid and sodium hydrocarbonate, sodium chloride processing, additional ultrasonic exposure and enzymatic treatment, storage in acetic acid and sodium hydrocarbonate, storage in an antimicrobial agent and multiply changed ascending glutaric dialdehyde. The biotissue is stored in multiply changed ascending glutaric dialdehyde in the melted form between the two plates of porous non-woven material.

EFFECT: preparing the non-immunogenic biomaterial uniformly cross linked throughout with particularly damaged collagen structure that provides the fast recipient's cell penetration into the transplant and smooth invasion of vessels and tissues in the postoperative period.

4 ex

 

The present invention relates to medicine, in particular for prosthetics affected areas of organs and tissues, dentistry, combustology, as well as to the field of tissue engineering, in particular to the manufacture of substrates, scaffolds to check their donor cells.

There are many ways of processing submucosal membrane of the small intestine with the purpose of its further application in medicine.

So, one of the patents describes a tissue graft, comprising a shell submucosal basis of a segment of small intestine of warm-blooded vertebrate. Shell submucosal basics layer is separated from the muscle sheath and at least from the abdominal part of the mucous membrane (U.S. Pat. RF 2037317 C1 IPC6, A61B 17/00). Further, the biomaterial is placed in a solution of neomycin sulfate. The disadvantage of this method of preparing a tissue graft is that the fabric is not subjected to decellularization and chemical processing, which would reduce the degree of immunogenicity of the biomaterial, as well as to monitor its biodegradation. Also the material is processed with a stabilizing agent, then its strength is increased.

Closest to the claimed method of manufacturing submucosal membrane of the small intestine is the method described in the patent of the Russian Federation 2438713 navigated machining thin the intestine, her sterilization, degreasing, the fixation cross stitching, minimizing antigenic activity and adherence of the active layer. However, this method of processing biological tissue does not provide for the complete removal of cellular elements of the collagen matrix, which may lead to a possible immune response in the patient's body.

The proposed method provides mechanical cleaning of the small intestine from the mucous and muscular layers, pre-treatment in hypertonic salt solutions of sodium chloride with simultaneous exposure to ultrasound, the enzymatic treatment of submucosal membrane of the small intestine, washing in a solution of acetic acid and sodium bicarbonate, treatment with sodium chloride, the additional impact of ultrasound and re enzymatic processing with subsequent curing of the biomaterial solution of acetic acid and sodium hydrogen carbonate solution, exposure to antimicrobial solution, and then cross-linking agent.

The purpose of this method is to reduce the risk of immune response to the transplant, partial destruction of collagen structures submucosal membrane of the small intestine to provide a more rapid and unimpeded sprouting of blood vessels and body tissues of the patient at the plate on the basis of the modified xenogeneic submucosal membrane thin the intestine.

The essence of the method lies in the fact that biological tissue prior to enzymatic treatment is cleaned mechanically and placed in hypertonic solutions of sodium chloride and exposed to ultrasound at least once during 20-300 minutes, then treated with a proteolytic enzyme at a concentration of 0.1-10 PE 1 gram of wet tissue, washed in a solution of acetic acid and sodium bicarbonate, and incubated in hypertonic solutions of sodium chloride, then further exposed to ultrasound and enzymatic processing in acetate buffer solution with a concentration of the enzyme is not less than 10.1 PU per gram of wet tissue with subsequent curing of the biomaterial solution of acetic acid and sodium hydrogen carbonate solution, whereupon the submucosal membrane of the small intestine is treated with an antimicrobial agent and repeatedly smenauscheesa solutions of glutaraldehyde increasing concentrations straightened as between the two plates of a porous non-woven material (for example, a porous film material made from a mixture of cellulose acetates) and sterilized.

Mechanical cleaning of the small intestine, muscular and mucous layer is carried out by exposure to the gut set shaft, compresses and extrudes the above layers. It is also possible application is the development of other methods of mechanical treatment of the small intestine to get out of it submucosal shell: rubbing, the scraping and so on.

In the pre-treatment in hypertonic solutions of sodium chloride purified submucosa membrane of the small intestine is 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, using 0.1-10 PE of proteolytic enzyme per gram of wet tissue.

The processing time of the biomaterial with ultrasound, smaller than 20 minutes is 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.

The enzymatic processing guarantees the destruction left after ultrasonic treatment of cellular elements submucosal membrane of the small intestine and proteins as the main carriers of antigenicity.

The concentration of the proteolytic enzyme is less than 0.1 PE may not be effective for the destruction of the remaining cells, tissues and proteins. The concentration of more than 10 PE is not appropriate, because the concentration of enzyme in 10 NE enough to process the complete destruction of the cell.

Order inactivation effects of proteolytic enzymes on submucosal membrane of the small intestine change the parameters on which depends the activity f is rment, namely, the temperature of the acetate buffer solution, pH. For this biomaterial is kept in a cooled acidic solution and repeatedly washed in running distilled water.

To neutralize remaining after washing with acetic acid biomaterial submucosal membrane of the small intestine is placed in a solution of sodium bicarbonate, then incubated in hypertonic salt solutions to extract the products of proteolysis of cells and proteins from tissues and washed with distilled water.

Repeated exposure of ultrasound on submucosal membrane of the small intestine and re-processing enzyme solution at a concentration of more 10,1 PE per gram wet tissue with subsequent curing of the biomaterial solution of acetic acid and sodium hydrogen carbonate solution leads to the partial destruction of collagen structures submucosal membrane of the small intestine without significant changes in its physical and mechanical properties that will provide a more rapid penetration of the tissue cells of the recipient to the graft and smooth the sprouting of blood vessels and body tissues of the patient in the postoperative period. The concentration of the proteolytic enzyme is less than 10.1 PE per gram wet tissue is not sufficient to start the destruction of collagen fibers.

Keeping podkisst the second membrane of the small intestine in antimicrobial agent will eliminate microbial activity on the surface of the submucosal membrane of the small intestine. As the antimicrobial agent can be an antibiotic of broad or narrow spectrum of activity, or the agents based on silver.

Treatment of submucosal membrane of the small intestine solutions of glutaraldehyde increasing concentrations necessary to stabilize the fabric, reducing its immunogenicity. Keeping submucosal membrane of the small intestine in solutions of glutaraldehyde is straightened as between the two plates of a porous non-woven material. This allows to get a smooth plate, without folds, uniformly impregnated with a crosslinking agent. The use of porous material provides a sufficient degree of contact with the submucosal membrane of the small intestine with a solution glutarovogo aldehyde.

A method of manufacturing a plate on the basis of the modified xenogeneic submucosal membrane of the small intestine submucosal membrane of the small intestine is performed in the following way.

The small intestine thoroughly washed several times with internal and external parties running water and cut in the longitudinal direction. Then produce a division of the small intestine into sections with a length of 10-40 cm by cutting.

Next, perform a mechanical cleaning of the small intestine, muscular and mucosal layers by extrusion between the two shafts. You can also use other methods IU onicescu cleaning of the small intestine to get out of it submucosal shell: rubbing, the scraping and so on.

Received submucosal membrane of the small intestine 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 of sodium chloride in the container. During this processing submucosal membrane is exposed to ultrasound for 50 minutes. After processing, the material is washed in distilled water. Next, perform a processing of a proteolytic enzyme. To do this, calculate the amount of proteolytic enzyme, 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 treatment with the enzyme submucosal membrane 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 a 0.1 M solution of sodium bicarbonate, then washed with distilled water. Then hold postfermentation processing hypertonic solutions of sodium chloride 2% and 7% concentration within 24-76 hours, then exposed to ultrasound for 40 minutes, washed in running distilled water and re-produce enzymatic processing solution with a concentration of proteolytic the ski enzyme 10,1 PE over one gram of tissue with subsequent curing of the biomaterial solution of acetic acid and sodium hydrogen carbonate solution, washed in running distilled water. Then make processing of submucosal membrane of the small intestine by triclosan and solutions of glutaraldehyde increasing concentrations straightened as between two porous nonwoven plates.

Example 1.

The small intestine clear of muscular and mucosal layers by scraping them into submucosal membrane of the small intestine, which is placed in a container with a hypertonic solution of sodium chloride at a concentration of 3% and leave for 68 h, producing daily replacement of a hypertonic solution of sodium chloride, then hold shift for a 7% solution of sodium chloride and leave submucosal membrane of the small intestine for 5 hours in this solution. During the processing of biological tissue once exposed to ultrasound for 110 minutes. After a time pre-treatment samples repeatedly washed in distilled water and carry out enzymatic processing. To do this, calculate the amount of proteolytic enzyme 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 of acetic acid (6-60 g of acetic acid, 100 g of sodium chloride, 840-894 g of distilled water) for at least 20min, washed repeatedly in distilled water and placed in 0.1 M sodium hydrogen carbonate solution and again washed with distilled water. The material is then placed in a hypertonic salt solutions at concentrations of 3% and 7% for 15 and 6 h, respectively, and washed in running distilled water. Then hold the additional impact of ultrasound on submucosal membrane of the small intestine within 60 minutes and re-enzymatic treatment with a concentration of proteolytic enzyme 12,5 PE per gram wet tissue for 5 minutes, followed by curing of the biomaterial solution of acetic acid and sodium hydrogen carbonate solution, as described above. Then submucosal membrane of the small intestine incubated in 0.3% solution of triclosan within 24 hours and placed in a solution of glutaraldehyde concentration was low in the unfolded between the two plates of a porous non-woven material for 72 h, then make shift solution of glutaraldehyde low concentration in a solution of glutaraldehyde higher concentration.

Example 2.

Differs from example 1 in that submucosal membrane of the small intestine is placed in a container with a hypertonic solution of sodium chloride at a concentration of 3% at 48 h, and then at 7% hypertonic sodium chloride solution for 12 h, is subjected to the action of ultra is sound for 50 minutes, processing proteolytic enzyme is carried out at 5 PE 1 gram of wet tissue and a second enzymatic treatment is carried out at 10,2 PE per gram wet tissue for 10 minutes.

Example 3.

Differs from example 1 in that submucosal membrane of the small intestine is treated with an antimicrobial agent, which is gentamicin.

Example 4.

Differs from example 1 in that the small intestine clear of muscular and mucosal layers by punching through the shafts.

Application of the proposed method of manufacture plate on the basis of the modified xenogeneic submucosal membrane of the small intestine will get a non-immunogenic, evenly stitched throughout the volume of the biomaterial with partially destroyed collagen structure that will provide a more rapid penetration of recipient cells in the graft and smooth the sprouting of blood vessels and body tissues of the patient in the postoperative period.

Sources of information

- RF patent №2037317,

- RF patent №2438713.

A method of manufacturing a plate on the basis of the modified xenogeneic submucosal membrane of the small intestine, intended for use in tissue engineering and artificial organs and tissues, including mechanical cleaning of the small intestine, the treatment in hypertonic solutions of chloride of soda is I with the simultaneous influence of ultrasound, enzymatic processing, sequential washing in a solution of acetic acid and sodium bicarbonate, treatment with sodium chloride, additional processing ultrasound high frequency and re-enzymatic processing carried out in acetate buffer solution with a concentration of the enzyme is not less than 10.1 PE over one gram of tissue, the shutter speed in the solution of acetic acid and sodium hydrogen carbonate solution, the exposure to the antimicrobial agent and repeatedly samenaide solutions of glutaraldehyde increasing concentrations, with the extract of tissues in repeatedly samenaide solution of glutaric aldehyde is carried out in expanded form between the two plates of a porous non-woven material.



 

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