Method for obtaining scleroplant biomaterial usable in ophthalmology

FIELD: medicine.

SUBSTANCE: method involves mechanically cleaning biological connective tissue and treating animal or human connective tissue with solutions. After mechanical cleaning, the animal or human connective tissue is hold in 3-6% hydrogen peroxide solution 2-4 h long, frozen at temperature below 0°C in wet state, thawed and treated with alkaline detergent solution of pH 7.0-14.0. Then, it is treated with 3-6% hydrogen peroxide solution 2-4 h long , heat treated at 40-90°C, shaped as required and sterilized.

EFFECT: improved quality of allergen-free biomaterial.

14 cl

 

The invention relates to medicine, and more specifically to ophthalmology, and can be used in the manufacture of materials for surgical treatment of myopia, for plastics defects of the connective tissue; in the manufacture of sutures for surgical treatment of glaucoma; to obtain solutions of collagen manufacture collagen eye coverings, protector of the cornea, composite materials for intraocular and contact lenses, to strengthen the sclera.

A method of obtaining a biomaterial for use in ophthalmology by treatment of connective tissue in animals or humans, which is first mechanically cleaned and then treated with solutions of hydrogen peroxide, urea, sodium chloride, a mixture of chloroform and ethanol, washed with purified water, dried, lyophilizers and sterilized dose of from 1.7 to 2.5 Mrad (U.S. Pat. Of the Russian Federation No. 2234289 dated 20.08.2004,).

The disadvantages of this method is that the urea used for treatment of connective tissue by the described method, is a good solvent of collagen and partially destroys the structure of the tissue, which affects its mechanical properties. In addition, the described method is used the treatment with chloroform, which is a strong allergen.

The objective of the invention is to provide a simple, effective, and b the safe method of obtaining a biomaterial for use in ophthalmology, having high biocompatibility.

The technical result that is achievable with the use of the invention is to obtain biocompatible material, maximum freed from allergens, which can be used for the manufacture of medical devices. And what is also important, and the simplification of manufacturing technology biomaterial.

The technical result is achieved by the fact that after mechanical treatment of connective tissue in animals or humans stand in 3-6% hydrogen peroxide solution, frozen at a temperature below 0°C, treated with an alkaline solution of detergent with a pH 7,0-14,0, and then 3-6% solution of hydrogen peroxide or boric acid, washed with water purified, subjected to heat treatment at 40-90°To give the necessary form and sterilized.

As a source of raw materials used connective tissue in animals or humans, for example, pericardium, Dura mater, the peritoneal membrane, the skin, interstitial membrane of the small intestine, the basal membrane (capsule kidney, placenta), scleral layer of the eye, the cornea and other

The processing of 3-6% hydrogen peroxide solution is performed with the aim of destruction of tissue pigments, lipid structures and disinfection. Freezing wet leads to the destruction of cellular elements, Raza is Tate which they become available for removal in subsequent stages of processing.

As the alkaline solution of detergent can be used from 0.01 to 5.0% solution of ethylenediaminetetraacetate, sodium and/or sodium dodecyl sulfate in 0.1 to 10.0% solution of sodium bicarbonate and/or sodium tetraborate, and/or ammonia and/or sodium hydroxide, and/or ethyl alcohol. The alkaline solution of detergent has a double action. An alkaline environment promotes the loosening of the tissue, the saponification of the lipid components and detergent, as surface-active substance that promotes the transition into the solution and removal restrukturisasi components of the connective tissue protein and lipid nature. Fabric processing at a temperature of 25-40°speeds up the process of washing.

Subsequent processing of 3-6% solution of hydrogen peroxide or boric acid is carried out for the neutralization of the residual alkali, as well as for disinfection.

All the processing stages of connective tissue solutions can be accelerated by constant stirring, as well as by simultaneous exposure to ultrasound in the cavitation regime, which is especially important when processing thicker connective tissue such as Dura.

Subsequent heat treatment of the connective tissue is carried out at 40-90°C. thus, the collagen fibers are reduced and the fabric is compacted, which improves its physical and mechanical properties. This is luceat frame properties of the implants of this fabric, and also makes it easier for the surgeon to implant insertion into the formed cavity during surgery, for example, when the operation scleroplasty. Temperature below 40°With no effect reduction and seal tissue. At temperatures above 90°connective tissue elementary welded, turning into a shapeless conglomerate.

The biomaterial can be given the form of a disk, or ellipse, or a polygon with rounded corners, for example rectangular, or elongated line, resembling a petal. The biomaterial can be formed through holes with a diameter of 0.3 to 3.0 mm, and the perimeter can be formed notches. Through holes and notches on the perimeter of the implant to facilitate penetration of tissue fibroblasts recipient in the implanted biomaterial and thereby accelerate the process of fusion of the implant with its own tissue recipient.

The biomaterial before sterilization can be additionally blended with glycosaminoglycans and/or biologically active substances and/or drugs. Glycosaminoglycans additionally structure of the collagen framework stroma of connective tissue, improving its physical and mechanical properties. To the class of glycosaminoglycans include hyaluronic acid and its salts, chondroitin and keratinolytic, dermatooncology, heparin and C is resultat. As biologically active substances can be used antioxidants, steroids, hormones, vitamins, peptides and polypeptides that affect the normalization of metabolic and regenerative processes in the intervention area. As medicines can be used with a wide range of drugs, for example antibiotics, anesthetics and other

Sterilization can be carried out using ionizing radiation.

Another option sterilization is dehydration in ethanol. Thus the final concentration of ethanol may not be less than 70°as the solution of lesser concentration does not have a sterilizing effect.

The next option sterilization is the dehydration of the biomaterial with subsequent irradiation with ionizing radiation.

The biomaterial was obtained as follows.

Raw materials are carefully mechanically cleaned of residues surrounding tissues, blood and washed under running water.

Then the fabric is placed in a 3-6% hydrogen peroxide solution for 2-4 hours to destruction of tissue pigments and disinfection, and then washed under running water and freeze in a moist condition at a temperature below 0°C. When this cellular elements collapse and become available for removal in subsequent stages of processing.

The tissue was thawed, washing etc is accurate water at a temperature not exceeding 40° C. and Then placed in an alkaline solution of detergent with a pH of 7.0-14.0 and vyderjivaut within 8-24 hours, shaking occasionally. When this occurs, the loosening of the tissue, lipid and non-structural protein components interact with the alkali and removed with detergent.

Then the fabric is washed with water from the alkaline solution by controlling the pH value of wash water, and pour a 3-6% solution of hydrogen peroxide or boric acid for 2-4 hours to fully neutralize residual alkali, as well as for disinfection. After that, the fabric is rinsed with purified water and subjected to heat treatment at 40-90°C.

From the treated tissue form the implant of desired shape, packaged and sterilized by one of the following ways, for example by using ionizing radiation.

Obtained in this way, the biomaterial has been studied in vitro and in vivo. Extract from the biomaterial did not cause changes in the morphology and number of cells in the culture of fibroblasts of mice. Toxic effects have been identified.

Sensitizing action on 5 white rats were observed. When subcutaneous injection of the biomaterial obtained by the proposed method, and provocative skin sample extract from a biomaterial reaction was negative, degranulation of mast cells was not observed.

Thus, the proposed method can receive the e biomaterial, having high biocompatibility and maximum freed from allergens.

The invention is illustrated by examples.

Example 1. Patient A. Diagnosis: high myopia. Surgery meridional scleroplasty using biomaterial of the pericardium cattle, processed by the proposed method using an alkaline solution of detergent with a pH of 14 of the following composition, wt.%: caustic sodium 0.1%, ethyl alcohol, 5%sodium dodecyl sulphate - 3%. Then the pericardium was washed with water flowing, treated with 3% solution of boric acid until complete neutralization of residual alkali, washed with water purified, subjected to heat treatment at a temperature of 45°and formed implants in the form of rectangles with rounded corners length of 2 cm and a width of 1 cm After the implants were obezvozhivani in ethanol solution of ascending concentration to 50°. Sterilization was performed using ionizing radiation.

During the operation in the channels formed between the sclera and tenon's membrane was implanted on 4 implant in each eye. The early postoperative period without complications. Allergic reactions were not observed, which indicates the high biocompatibility of the biomaterial. The observation period of 6 months. Increased anterior-posterior segment of the eye is not you who go.

Example 2. Patient K. the Diagnosis of myopia of an average degree, complicated Central chorioretinal dystrophy. An operation of a scleroplasty using implants of the pericardium bovine received on the proposed method, in the form of discs with a diameter of 14 mm as the alkaline solution of the detergent solution was used with a pH of 7.0 the following composition, in wt.%: sodium bicarbonate - 10%, sodium ethylenediaminetetraacetate - 5%. Before sterilization, the implant is saturated with a 1% solution of carnosine. Sterilization method using ionizing radiation. During the operation with a spatula-raspatory at the posterior pole of the eye shaped pocket between tenon's capsule and the sclera, in which were placed the implant, covering them to the posterior pole of the eyeball in the projection of macular and paramacular area. In the early polarisation period complications and allergic reactions were observed. Visual acuity with best correction is reduced compared to the preoperative level. The observation period of 8 months.

According to the proposed method was obtained 60 implants that have been used in clinical practice when performing operations meridional scleroplasty for the treatment of progressive myopia in children and adults, as well as operations regarding the treatment of chorioretinal maculopathy the different Genesis. In all cases of postoperative complications associated with allergic reactions were noted. Thus, proposed an efficient method of obtaining a biomaterial for ophthalmology, which has high biocompatibility and low allergenicity.

Example 3. Tracheal cartilage of cattle mechanically cleaned, cut into pieces no longer than 5 cm, kept in 3% hydrogen peroxide solution, 3 hours, washed with water, froze wet, thawed under running water and treated with an alkaline solution of detergent at a temperature of 40°With the following composition, wt.%: ethylenediaminetetraacetate sodium - 0,01%, caustic sodium - 1,0%, sodium bicarbonate - 3%, sodium tetraborate - 5%. From a solution of detergent cartilage was washed with water and then treated with 6% hydrogen peroxide solution for 4 hours with constant stirring. Next, the connective tissue was rinsed with water and subjected to heat treatment at a temperature of 60°S. Of fragments of cartilage formed strip with a length of 5-6 mm, a width of 2-3 mm, thickness 0.5-1.0 mm and were placed in a 2% solution of glycosaminoglycans before saturation. Then strips of cartilage freeze-dried and irradiated with ionizing radiation. The implants of the biomaterial obtained in this way are used as drains when running antiglaucoma OPE the Nations.

Example 4. Dura person was mechanically cleaned, cut into pieces with an area of about 30-60 cm2, stood at 6% solution of hydrogen peroxide for 4 hours, washed with water and froze wet at a temperature of - 18°C. For melting the fragments of connective tissue was rinsed with warm running water and treated with an alkaline solution of a detergent of the following composition, wt.%: sodium dodecyl sulphate - 5%, caustic sodium (6%), ethyl - 4%. Then the fabric was washed with water followed by treatment with 6% solution of boric acid for 4 hours. Thermal treatment of tissue was carried out at a temperature of 40°S. Of fragments processed tissue formed implants were placed for saturation consistently in the solution anesthetic lidocaine therapeutic concentrations in the solution of anabolic steroid. Then the implants of the biomaterial was placed in a sealed package and sterilized using ionizing radiation. Received the implants used for scleroplastic operations and episcleral filling.

Example 5. Amniotically membrane person was kept in 3% hydrogen peroxide solution for 2 hours, washed with water and treated with an alkaline solution of a detergent of the following composition, wt.%: sodium dodecyl sulphate - 0,01%, ammonia - 0,05%, Speer is ethyl - 0,05%. From a solution of detergent connective tissue was rinsed with water and pour 3% hydrogen peroxide solution for 2 hours. Thermal treatment of tissue was carried out at 40°C. the Treated fabric was saturated in a solution of glycosaminoglycans and powder of the following composition, wt.%: glycosaminoglycans - 2%, gentamicin - 0,1%. After that was cut out fragments in the form of an ellipse and sterilized using ionizing radiation. The resulting biomaterial used for plastic surgery of the conjunctiva of the eyelids.

Example 6. Scleral shell of the eyes of cattle after mechanical cleaning was kept in 3% hydrogen peroxide solution for 2 hours, washed with water and froze it wet, then thawed, rinsing with running water and treated at room temperature with an alkaline solution of a detergent of the following composition: sodium ethylenediaminetetraacetate - 0,005%, sodium dodecyl sulphate - 0,005%, sodium bicarbonate - 3%, sodium tetraborate - 5%, ammonia - 0,5%, caustic sodium 0.5%, ethyl alcohol and 1%. Thereafter, the fabric was rinsed with water and treated with 6% solution of boric acid for 4 hours with constant stirring. The treated fabric was subjected to heating at 55°and was placed in a 2% solution of glycosaminoglycans containing 0.1% biologically active substances - antioxidant carnosine. Then formed implants, freeze-dried and irradiated ionize the respective radiation. The implants of the biomaterial obtained in this way is used for the operation of a scleroplasty in chorioretinal macular degeneration.

Example 7. The pericardium cattle after mechanical treatment were placed in a 6% solution of hydrogen peroxide for 2 hours, washed with water and froze it wet, then thawed, rinsing with warm running water and treated at room temperature with an alkaline solution of a detergent of the following composition: sodium ethylenediaminetetraacetate 0.5 percent sodium dodecyl sulphate and 0.5%, sodium bicarbonate - 3%, caustic sodium 0.5%, ethyl alcohol and 5%. Thereafter, the fabric was rinsed with water and treated with 6% hydrogen peroxide solution for 4 hours with constant stirring, washed with water purified, subjected to heat treatment at 45°and shape of squares with sides of 1.5 to 2.5 cm with rounded corners. Then the fragments of the treated fabric was obezvozhivani in solutions of ethanol of the rising concentration of 30° to 70°, were placed in sterile vials with 70° ethyl alcohol and corked. The biomaterial obtained in this way is used for the plastic scleral defects.

1. A method of obtaining a biomaterial for use in ophthalmology, including mechanical treatment of connective tissue in animals or humans, treatment with a solution of peroxide is hydrogen, water purified, and sterilization, wherein after machining connective tissue in animals or humans kept in a 3-6%solution of hydrogen peroxide 2-4 hours, washed with water and freeze in a moist condition at a temperature below 0°C, then thawed, washing with running water is not higher than 40°C, treated with an alkaline solution of detergent with a pH 7,0-14,0, and then 3-6%solution of hydrogen peroxide or boric acid for 2-4 h, washed with water purified, subjected to heat treatment at 40-90°To give the necessary form and sterilized.

2. The method according to claim 1, characterized in that the alkaline solution of the detergent used is 0.01 to 5.0%solution of ethylenediaminetetraacetate, sodium and/or sodium dodecyl sulfate in 0.1 to 10.0%solution of sodium bicarbonate and/or sodium tetraborate, and/or ammonia and/or sodium hydroxide, and/or ethyl alcohol.

3. The method according to claim 1 or 2, characterized in that the treatment with an alkaline solution of detergent is carried out at a temperature of 25-40°C.

4. The method according to claim 1, characterized in that the processing solution carried out with constant stirring.

5. The method according to claim 1, characterized in that the processing solution is carried out at the influence of ultrasonic cavitation mode.

6. The method according to claim 1, wherein the biomaterial is formed into the shape of a disk, or ellipse, or mogogo which of these lamps with rounded corners.

7. The method according to claim 1 or 6, characterized in that the biomaterial to form through holes with a diameter of 0.3 to 3.0 mm

8. The method according to any of claim 1 or 6, or 7, characterized in that the perimeter of the biomaterial to form a notch.

9. The method according to claim 1, characterized in that before sterilization biomaterial additionally saturate glycosaminoglycans, and/or biologically active substances and/or drugs.

10. The method according to claim 1, characterized in that the sterilization is performed with the use of ionizing radiation.

11. The method according to claim 1 or 10, wherein the biomaterial is sterilized in a solution of salts of sodium ionizing radiation.

12. The method according to claim 1, wherein the biomaterial is sterilized by freeze drying and subsequent exposure to ionizing radiation.

13. The method according to claim 1, wherein the biomaterial is sterilized by dehydration in solutions of ethanol ascending concentration, while the final concentration of ethanol should be at least 70°.

14. The method according to claim 1, wherein the biomaterial is sterilized by dehydration in solutions of ethanol, followed by irradiation with ionizing radiation.



 

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