Method for obtaining xenotransplants for ophthalmology

FIELD: medicine, ophthalmology.

SUBSTANCE: the present innovation deals with creating safe and efficient heteroplastic material for treating progressing myopia, stabilization of dry maculodystrophy and manufacturing antiglaucomatous drainages at maximal decrease of antigenic properties and guaranteed sterility of biomaterial that provides the absence of post-operational complications (chemosis, tenonitis, pridocyclitis) at operative therapy of progressing myopia and guarantees stabilization of myopia progressing, the stabilization of pathological processes at dry maculodystrophy, normalization of intra-ocular pressure at operative treatment of glaucoma at applying the drainage out of the material suggested. In the course of the method in question one should treat cattle pericardium after mechanical purification with 10%-ammonia solution for about 4-5 h, washed with distilled water followed by a 4-fold freezing at -10-12° C and defrosting at +40-45° C, for 7.5 h it should be treated with 6%-hydrogen peroxide solution at +15° C, after cutting the material it should be treated with ultrasound to repeat the whole procedure at mixing device, then material should be dehydrated in alcohols of initial concentration ranged 30-70 volumetric percentage to be applied into the vials with 70%-ethanol and sterilized with ionizing radiation at the dosage of 1.5 Mrad.

EFFECT: higher efficiency.

3 ex

 

The proposed method relates to medicine, more precisely to ophthalmology, and can be used for the manufacture of xenografts used in ophthalmology for surgical treatment of progressive myopia medium and high, for the treatment of dry macular degeneration and manufacturing antiglaucoma drains.

Previously, that was used cadaveric alloplastic material: sclera, Dura, Achilles tendon, aorta and other, and as heterotransplantation - the pericardium.

A method of processing scleroplastic material derived from the connective tissue of animals and humans, which includes cleaning tissues from mechanical impurities and blood, washing with cold water, cut into strips of the desired size with perforations, placement in a 6%solution of hydrogen peroxide is from 1 to 3 hours, then in 4 M urea solution is not less than 15 hour incubation in 2 m solution of sodium chloride, washing material, placing the material in a mixture of chloroform : ethanol in a ratio of 1:1, washing with water, lyophilization and sterilization radiation method at a dose of 1.7-2.5 Mrad (patent No. 2234289 dated 20.08.2004).

The disadvantages of this method is that it does not provide maximum removal of soluble proteins and glycoproteins that define the antigenic properties of biological material, it is not possible to exclude ricotta in the material of some dispute, bacteria and viruses, as well as Pranovich proteins that cause such rare new diseases like "mad cow disease", a disease of Kranzfelder-Jacobs and others

The technical result of the invention is to provide a safe and effective geterotsiklicheskikh material for the treatment of progressive myopia, stabilization of dry macular degeneration and manufacturing antiglaucoma drains, which will be removed soluble proteins and glycoproteins, there will be no disputes, bacteriae, viruses and prion proteins that cause "mad cow disease", a disease of Kranzfelder-Jacobs and other rare new disease, with a maximum reduction of antigenic properties and guaranteed sterility of the biomaterial, which reduce the rate of postoperative complications and ensure the stabilization of pathological processes. This in turn ensures that the complications of type Chemezov, tenonitis and iridotsiklity after operational use of materials, stabilization of the progression of myopia average and a high degree of stabilization of the pathological process in the fundus when dry macular degeneration, the normalization of intraocular pressure during the operational use of the drainage from the proposed material.

This technical result is possible because the proposed method on especial the most complete liberation of the pericardium from cellular elements, blood, soluble proteins and glycoproteins due to mechanical degradation of proteins with 4 freeze - thawing of the material with obligatory observance of a temperature mode and processing of material by ultrasound. The removal of proteins and glycoproteins are also the result of the hydrolysis of proteins by treatment with ammonia before and after cutting of the material, and in the latter case, using a mixing device. It should be noted that the ammonia provides the maximum hydrolysis of proteins in comparison with urea. The result of this treatment, the material at the microscopic level was found the blood pigment and any cellular elements, and the content of soluble protein by Lowry Folio in the hood of the material was decreased compared with the initial value of 45 times and accounted for 2.7±1.2 g/L. Qualitative reaction on proteoglycans in the resulting material was negative.

Moreover, the limited delivery of material after the slaughter of animals allowed to avoid post-mortem degradation in the tissue of the pericardium, and the delivery of the pericardium in clean containers reduced the degree of their contamination microbial flora.

The method provides guaranteed not only the lack of microbial flora, but a dispute, Pranovich proteins that cause diseases like "mad cow of berenst the", as a result of processing of the material 6%hydrogen peroxide solution before and after cutting the material, and in the latter case, even in a mixing device. Re-treatment with hydrogen peroxide ensures the destruction of all pathogenic organisms, because after the first treatment with hydrogen peroxide further processing of the material, as a rule, goes under non-sterile conditions.

In addition, this reduces the effective dose of radiation for sterilization of the material up to 1.5 Mrad and thus to prevent the possible additional destruction of proteins, including increased protein content in the extract from the material and thereby prevent postoperative complications when using the material.

Failure on our part chloroform, which when working with the pericardium there is no need, provides a more environmentally friendly production.

The absence of perforations in the material and cutting it into small fragments than in the early treatment provides sufficient strength of the material that is essential for the surgeon during the operation and may affect results of operations.

This technical result is achieved by the fact that in the known method of obtaining grafts for ophthalmic surgery, including removing the pericardium cattle, mechanical separation, washing with cold water is, fragmentation of the material with subsequent perforation, processing 6%solution of hydrogen peroxide, alcohol treatment, and sterilization by ionizing radiation, optionally, the pericardium bovine directly after mechanical cleaning is treated with ammonia solution, mainly 10%within 4-5 hours, then washed with distilled water, after which the material is repeatedly frozen at a temperature of minus 10÷12°C and thawed at a temperature of + 40÷45°Since, further, the material for 7-8 hours is treated with 6% the hydrogen peroxide solution at a temperature of up to plus 15°, fragmenting, fragmented material is treated with ultrasound, re-mixing device is treated with a 10%solution of Amica again repeatedly freeze - thawed, re-treated with 6%hydrogen peroxide and washed with distilled water, after which the material dehydration in ascending alcohols concentration of from 30 to 70 volume percent, placing them in vials with 70%ethanol and sterilized by ionizing radiation dose of 1.5 Mrad.

The method is as follows.

1. Extract 1 kg of pericardium cattle within 1 hour after slaughter, shipping containers.

2. Carry out mechanical cleaning material from Shust the s blood, the stuck particles and vessels with a scalpel and cut it on the plate 15×10 see

3. Treated with ammonia solution, mainly 10%within 4-5 hours with a 3-fold change of the solution.

4. Wash the material with distilled water for 30 min with 3-fold change of water 10 litres.

5. Frozen material at a temperature of minus 10-12°in the refrigerator and thawed at a temperature of 40-45°C - warm water - 4 cycle.

6. Handle material 6%hydrogen peroxide solution at a temperature of up to +15°With 3 cycles for 2.5 hours.

7. Washed material in distilled water 3 times for 10 min in each cycle to use 10 litres of water.

8. Cut the material into strips of size (10×20)mm and (10×70) mm or disks with a diameter of 11 mm,

9. Handle material with ultrasound for 1 hour on the device Retona USU - 0707 when the oscillation frequency emitters 125±6 kHz, a power of 9 watts.

10. Re-process the raw materials 10%ammonia solution in the mixing device PE-M 3 hours with three changes of solution.

11. Washed material in distilled water for 30 min with three changes of water.

12. Re-freeze the material at a temperature of 10-12°in the refrigerator and thawed at a temperature of 40-45°in warm water - a total of 4 cycles.

13. Re-process the raw materials 6%dissolve the om hydrogen peroxide in a mixing device PE-M within 30 minutes

14. Repeatedly washed with distilled water for 30 min with three changes of 10 l of water.

15. Dehydration in ascending alcohols concentration of from 30 to 70 volume percent, in each alcoholic solution stand for 24 hours in the mixing device. In the material there is no pigment.

16. Display material in vials with 70%ethanol, to produce the bottles, sterilization with ionizing radiation dose of 1.5 Mrad γ-installation of Co60. Bacteriological examination of the material sterile.

Examples of the method

Example 1. Getting xenograft for the treatment of progressive myopia, dry macular degeneration and antiglaucoma drainage.

1. 1 kg of the pericardium bovine extracted within 45 minutes after slaughter and transported in a container.

2. Conducted mechanical removal of material from blood clots adhering particles and vessels with a scalpel and the material is cut into plates 15×10 see

3 the Material is treated with ammonia solution 10%within 4.5 hours with a 3-fold change of the solution.

4. The material is washed with distilled water for 30 min with 3-fold change in water for 10 litres.

5. The material is frozen at a temperature of -10°in the refrigerator and thawed at a temperature of +40°in warm water - a total of 4 cycles.

6. The material is treated with 6%of rastvoronasosy hydrogen at temperatures up to +15° With only 3 cycles for 2.5 hours.

7. The material is washed in distilled water 3 times for 10 min in each cycle used 10 litres of water.

8. The material is cut into strips of size (10×20) mm discs with a diameter of 13 mm

9. The material is treated with ultrasound for 1 hour on the device Retona USU - 0707, when the oscillation frequency emitters 125±6 kHz, a power of 9 watts.

10. The material is again treated with a 10%ammonia solution in the mixing device PE-M for 3 hours with three changes of solution.

11 the Material was washed in distilled water for 30 min with three changes of water. Monitoring for the presence of ions NH4+conducted using Nessler reagent. Reaction negative ions NH4is missing.

12. The material is frozen at a temperature of -12°in the refrigerator and thawed at a temperature of +40°With warm water - just 4 cycles of freeze - thawing.

13. The material is treated with 6%hydrogen peroxide solution at a mixing device PE-M within 30 minutes

14. The material is washed with distilled water for 30 min with three changes of 10 l of water.

15. Material dehydration in ascending alcohols concentrations of 30, 50 and 70 volume percent of each alcoholic solution, is maintained for 24 hours in the mixing device.

16. The material decomposed in vials with 70%ethanol JV the mouth, sealed, sterilized with ionizing radiation dose of 1.5 Mrad γ-installation of Co60.

Obtained by the present method the material examined for the presence of soluble proteins, glycoproteins, blood elements. Monitoring for the presence of soluble proteins, glycoproteins, cellular elements was performed using UV-spectroscopy by studies of aqueous extracts of the material (the method of Lowry Polina). It turned out that the optical density of the aqueous extracts of the material at wavelengths of 205 and 210 nm was not more than 0.1 units. Qualitative reaction on glycoproteins was negative. This result suggests that there was a full release of the pericardium from the cellular elements of blood, soluble proteins and glycoproteins.

Thus obtained material was used:

a) in progressive myopia of an average degree.

Patient B. 25 years OU myopia - 4,0 D. Gradient progression 1,0 D per year. Produced scleroplastic operation Pivovarov on OD, but after 3 months on the OS using pericardium treated by our proposed technique.

The postoperative period was uneventful, and after 1 year after surgery myopia on OU still 4 OD. VIS-corrected sph - 4 OD=1.0 in.

b) if you have dry macular degeneration

Patient M. aged 45, OD dry macular degeneration. Vision gradually whosales is and the time of treatment VIS 0.3 nm/K. In the fundus in the macular area petechial lesions and dissemination of pigment. Produced by implantation of the disc from the pericardium in subtenons space in the posterior pole.

The postoperative period was uneventful. After 1 year, blurred vision and picture of the fundus is not marked.

C) with primary open-angle subcompensated glaucoma

Patient S. 62, OD glaucoma primary open-angle subcompensated. IOP 28 mm Hq. Processed non-penetrating deep sclerectomy with the use of drainage of the pericardium.

The postoperative period was uneventful. IOP is normalized. IOP at 1 year after surgery, 18 mm Hq.

The method of obtaining xenografts for ophthalmic surgery, including removing the pericardium cattle, mechanical separation, washing with cold water, the fragmentation of the material with subsequent perforation, processing 6%solution of hydrogen peroxide, alcohol treatment, and sterilization with ionizing radiation, wherein the pericardium bovine directly after mechanical cleaning is treated with a solution of ammonia primarily 10%within 4-5 hours, then washed with distilled water, after which the material is repeatedly frozen at a temperature of 10-12°and razmerjih the t at a temperature of 40-45° Since, further, the material for 7-8 h treated with 6%hydrogen peroxide solution at a temperature of up to 15°, fragmenting, fragmented material is treated with ultrasound, re-mixing device is treated with a 10%solution of ammonia, again repeatedly freeze-thawed, re-treated with 6%hydrogen peroxide and washed with distilled water, after which the material dehydration in ascending alcohols concentration of from 30 to 70 vol.%, put in vials with 70%ethanol and sterilized by ionizing radiation dose of 1.5 Mrad.



 

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