RU2161976C1 - Method of preparing collagen from osseous tissue - Google Patents
Method of preparing collagen from osseous tissue Download PDFInfo
- Publication number
- RU2161976C1 RU2161976C1 RU2000109783A RU2000109783A RU2161976C1 RU 2161976 C1 RU2161976 C1 RU 2161976C1 RU 2000109783 A RU2000109783 A RU 2000109783A RU 2000109783 A RU2000109783 A RU 2000109783A RU 2161976 C1 RU2161976 C1 RU 2161976C1
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- Russia
- Prior art keywords
- mixture
- sodium hydroxide
- ratio
- ethanol
- hydrolysis
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- 210000000988 bone and bone Anatomy 0.000 title claims description 20
- 102000008186 Collagen Human genes 0.000 title claims description 9
- 108010035532 Collagen Proteins 0.000 title claims description 9
- 229920001436 collagen Polymers 0.000 title claims description 9
- 238000000034 method Methods 0.000 title claims description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000203 mixture Substances 0.000 claims abstract description 12
- 108090000526 Papain Proteins 0.000 claims abstract description 7
- 239000004365 Protease Substances 0.000 claims abstract description 7
- 229940055729 papain Drugs 0.000 claims abstract description 7
- 235000019834 papain Nutrition 0.000 claims abstract description 7
- 239000002244 precipitate Substances 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 15
- 230000007062 hydrolysis Effects 0.000 claims description 13
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000006386 neutralization reaction Methods 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000002682 general surgery Methods 0.000 abstract description 2
- 239000007795 chemical reaction product Substances 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 239000000835 fiber Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 2
- 210000003041 ligament Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Materials For Medical Uses (AREA)
Abstract
Description
Изобретение относится к медицине и может быть использовано при приготовлении лекарственных форм и изготовлении медицинских изделий для использования в общей хирургии, стоматологии и дерматологии. The invention relates to medicine and can be used in the preparation of dosage forms and the manufacture of medical devices for use in general surgery, dentistry and dermatology.
Известен способ получения коллагена из костной ткани, включающий очистку, измельчение костной ткани, гидролиз и нейтрализацию (US N 4172128, A 61 K 35/32). A known method of producing collagen from bone tissue, including cleaning, grinding bone tissue, hydrolysis and neutralization (US N 4172128, A 61 K 35/32).
Однако применение коллагена, полученного указанным способом, может привести к аллергическим или воспалительным реакциям, так как он содержит свободные концы коллагеновых волокон. However, the use of collagen obtained in this way can lead to allergic or inflammatory reactions, since it contains the free ends of collagen fibers.
Задачей изобретения является повышение качества конечного продукта. The objective of the invention is to improve the quality of the final product.
Технический результат достигается тем, что в способе получения коллагена из костной ткани, включающем очистку, измельчение костной ткани, гидролиз и нейтрализацию; костную ткань измельчают до получения костной крошки размером от 700 до 2000 мкм, проводят гидролиз в течение 24-48 часов в смеси раствора 0,4 Н гидроксида натрия и этанола в соотношении 1:1, затем нейтрализуют до pH 6,8-7,8, центрифугируют при 3000-5000 об/мин в течение 20-40 минут, надосадок сливают, а осадок обрабатывают 0,125%-ным раствором папаина при температуре 37oC, затем повторно проводят гидролиз смесью раствора 0,4 Н гидроксида натрия и этанола в соотношении 1:1, диализуют против воды, замораживают и лиофилизируют.The technical result is achieved by the fact that in a method for producing collagen from bone tissue, including cleaning, grinding bone tissue, hydrolysis and neutralization; bone tissue is crushed to obtain bone chips ranging in size from 700 to 2000 microns, hydrolysis is carried out for 24-48 hours in a mixture of a solution of 0.4 N sodium hydroxide and ethanol in a ratio of 1: 1, then neutralized to a pH of 6.8-7.8 centrifuged at 3000-5000 rpm for 20-40 minutes, the supernatant is drained, and the precipitate is treated with 0.125% papain solution at a temperature of 37 o C, then hydrolysis is repeated with a mixture of a solution of 0.4 N sodium hydroxide and ethanol in the ratio 1: 1, dialyzed against water, frozen and lyophilized.
Костную ткань измельчают до крошки размером 700-2000 мкм, так как при размере костной крошки менее 700 мкм значительно повреждаются коллагеновые волокна, а при ее размере более 2000 мкм процесс гидролиза удлиняется. Bone tissue is crushed to a crumb size of 700-2000 microns, since collagen fibers are significantly damaged when the size of bone crumbs is less than 700 microns, and when its size is more than 2000 microns, the hydrolysis process lengthens.
Гидролиз костной крошки проводят в течение 24-48 часов в смеси раствора в 0,4 Н раствора гидроксида натрия и этанола в соотношении 1:1, что позволяет полностью выделить и обезжирить весь костный коллаген. Hydrolysis of bone crumbs is carried out for 24-48 hours in a mixture of a solution in a 0.4 N solution of sodium hydroxide and ethanol in a ratio of 1: 1, which allows you to completely isolate and degrease all bone collagen.
Удаляют надосадок центрифугированием. При 3000-5000 об/мин в течение 20-40 минут, так как при центрифугировании менее 3000 об/мин, осаждается менее 50% гидролизата, а при более 5000 об/мин часть основного продукта уходит в осадок. Remove the supernatant by centrifugation. At 3000-5000 rpm for 20-40 minutes, since centrifugation is less than 3000 rpm, less than 50% of the hydrolyzate precipitates, and at more than 5000 rpm, part of the main product precipitates.
Обработку 0,125%-ным раствором папаина при 37oC позволяет провести полный гидролиз свободных неколлагеновых белков кости и удалить свободные концы коллагеновых волокон, так как папаин, являясь протеолитическим ферментом, гидролизует белки животного происхождения (Диксон М. и др. Ферменты, т. 1, М., 1982, с. 20-40).Treatment with a 0.125% papain solution at 37 ° C allows complete hydrolysis of free non-collagenous bone proteins and removal of the free ends of collagen fibers, since papain, being a proteolytic enzyme, hydrolyzes animal proteins (Dixon M. et al. Enzymes, vol. 1 , M., 1982, p. 20-40).
Повторное проведение гидролиза смесью раствора 0,4 Н гидроксида натрия и этанола в соотношении 1:1 позволяет удалить из ткани гидролизированные белки и их остатки. Repeated hydrolysis with a mixture of a solution of 0.4 N sodium hydroxide and ethanol in a ratio of 1: 1 allows you to remove hydrolyzed proteins and their residues from the tissue.
Способ осуществляется следующим образом. The method is as follows.
Кость очищают от мышц, связок и прочего, измельчают до получения костной крошки размером 700-2000 мкм, затем проводят гидролиз в течение 24-48 часов в смеси раствора 0,4 Н гидроксида натрия и этанола в соотношении 1:1, нейтрализуют до pH 6,8-7,8, центрифугируют при 3000-5000 об/мин в течение 20-40 минут, надосадок сливают, а осадок обрабатывают 0,125%-ным раствором папаина при температуре 37oC, затем повторно проводят гидролиз в смеси раствора 0,4 Н гидроксида натрия и этанола в соотношении 1:1, диализуют против воды, замораживают и лиофилизируют.The bone is cleaned of muscles, ligaments and other things, crushed to obtain bone chips with a size of 700-2000 microns, then hydrolysis is carried out for 24-48 hours in a mixture of a solution of 0.4 N sodium hydroxide and ethanol in a ratio of 1: 1, neutralized to pH 6 , 8-7.8, centrifuged at 3000-5000 rpm for 20-40 minutes, the supernatant is drained, and the precipitate is treated with 0.125% papain solution at a temperature of 37 o C, then hydrolysis is repeated in a mixture of a solution of 0.4 H, sodium hydroxide and ethanol in a ratio of 1: 1, dialyzed against water, frozen and lyophilized.
Пример. Example.
1 кг костей быка очищают от мышц, связок и прочего. Распиливают на кусочки, измельчают до получения костной крошки размером 800 мкм. Затем проводят гидролиз в течение 24 часов в смеси раствора 0,4 Н гидроксида натрия и этанола в соотношении 1:1, затем нейтрализуют до pH 6,8, центрифугируют при 3000 об/мин в течение 40 минут, надосадок сливают, а осадок обрабатывают 0,125%-ным раствором папаина при температуре не выше 37oC, затем повторно проводят гидролиз в смеси раствора 0,4 Н гидроксида натрия и этанола в соотношении 1:1, диализуют против воды, замораживают и лиофилизируют.1 kg of bones of a bull is cleaned of muscles, ligaments and other things. Sawed into pieces, crushed to obtain bone chips with a size of 800 microns. Then hydrolysis is carried out for 24 hours in a mixture of a solution of 0.4 N sodium hydroxide and ethanol in a ratio of 1: 1, then neutralized to a pH of 6.8, centrifuged at 3000 rpm for 40 minutes, the supernatant is drained, and the precipitate is treated with 0.125 % papain solution at a temperature not exceeding 37 o C, then re-hydrolysis is carried out in a mixture of a solution of 0.4 N sodium hydroxide and ethanol in a 1: 1 ratio, dialyzed against water, frozen and lyophilized.
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Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2000109783A RU2161976C1 (en) | 2000-04-21 | 2000-04-21 | Method of preparing collagen from osseous tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2000109783A RU2161976C1 (en) | 2000-04-21 | 2000-04-21 | Method of preparing collagen from osseous tissue |
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| Publication Number | Publication Date |
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| RU2161976C1 true RU2161976C1 (en) | 2001-01-20 |
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| RU2000109783A RU2161976C1 (en) | 2000-04-21 | 2000-04-21 | Method of preparing collagen from osseous tissue |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2240818C1 (en) * | 2003-12-24 | 2004-11-27 | Общество с ограниченной ответственностью "МАГНЭС" | Collagen-containing preparation "kololen" for medicinal designation and method for its preparing |
| RU2273489C1 (en) * | 2004-08-17 | 2006-04-10 | Андрей Федорович Панасюк | Method for obtaining collagen from bone tissue |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4172128A (en) * | 1975-03-26 | 1979-10-23 | Erhard Thiele | Process of degrading and regenerating bone and tooth material and products |
| EP0083868B1 (en) * | 1982-01-11 | 1986-04-30 | COLLAGEN CORPORATION (a California corporation) | Collagen implant material for augmenting soft tissue |
| RU2033165C1 (en) * | 1989-10-13 | 1995-04-20 | Межотраслевой научно-технический комплекс "Микрохирургия глаза" | Process for manufacture of plastic material from collagen |
| US5411887A (en) * | 1991-04-05 | 1995-05-02 | Collagen Casing Einar Sjolander Ab | Method for the production of collagen: collagen produced through the method and use of collagen |
| RU2136297C1 (en) * | 1998-10-22 | 1999-09-10 | Иванов Сергей Юрьевич | Method of preparing transplant "bio-matrix implant" for stomatology |
-
2000
- 2000-04-21 RU RU2000109783A patent/RU2161976C1/en not_active IP Right Cessation
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4172128A (en) * | 1975-03-26 | 1979-10-23 | Erhard Thiele | Process of degrading and regenerating bone and tooth material and products |
| EP0083868B1 (en) * | 1982-01-11 | 1986-04-30 | COLLAGEN CORPORATION (a California corporation) | Collagen implant material for augmenting soft tissue |
| RU2033165C1 (en) * | 1989-10-13 | 1995-04-20 | Межотраслевой научно-технический комплекс "Микрохирургия глаза" | Process for manufacture of plastic material from collagen |
| US5411887A (en) * | 1991-04-05 | 1995-05-02 | Collagen Casing Einar Sjolander Ab | Method for the production of collagen: collagen produced through the method and use of collagen |
| RU2136297C1 (en) * | 1998-10-22 | 1999-09-10 | Иванов Сергей Юрьевич | Method of preparing transplant "bio-matrix implant" for stomatology |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2240818C1 (en) * | 2003-12-24 | 2004-11-27 | Общество с ограниченной ответственностью "МАГНЭС" | Collagen-containing preparation "kololen" for medicinal designation and method for its preparing |
| RU2273489C1 (en) * | 2004-08-17 | 2006-04-10 | Андрей Федорович Панасюк | Method for obtaining collagen from bone tissue |
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Effective date: 20050422 |