CN107227330B - Method for extracting bovine achilles tendon type I collagen - Google Patents
Method for extracting bovine achilles tendon type I collagen Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 10
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- 238000002791 soaking Methods 0.000 claims abstract description 17
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 claims abstract description 16
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- VSZSIEBALNXIFG-UHFFFAOYSA-N 2-hydroxyethyl 2,2-bis(sulfanyl)acetate Chemical compound OCCOC(=O)C(S)S VSZSIEBALNXIFG-UHFFFAOYSA-N 0.000 claims abstract description 15
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- 239000007788 liquid Substances 0.000 claims abstract description 13
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 11
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 11
- 229940111202 pepsin Drugs 0.000 claims abstract description 11
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 claims abstract description 10
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 claims abstract description 10
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 10
- 239000007974 sodium acetate buffer Substances 0.000 claims abstract description 10
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims abstract description 10
- 229940031439 squalene Drugs 0.000 claims abstract description 10
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000007605 air drying Methods 0.000 claims abstract description 9
- 238000005520 cutting process Methods 0.000 claims abstract description 9
- 210000003205 muscle Anatomy 0.000 claims abstract description 9
- 238000005185 salting out Methods 0.000 claims abstract description 9
- 238000000227 grinding Methods 0.000 claims abstract description 8
- 230000010355 oscillation Effects 0.000 claims abstract description 8
- XHFLOLLMZOTPSM-UHFFFAOYSA-M sodium;hydrogen carbonate;hydrate Chemical compound [OH-].[Na+].OC(O)=O XHFLOLLMZOTPSM-UHFFFAOYSA-M 0.000 claims abstract description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 24
- 239000008367 deionised water Substances 0.000 claims description 14
- 229910021641 deionized water Inorganic materials 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 12
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 9
- 239000000463 material Substances 0.000 abstract description 6
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- 235000015278 beef Nutrition 0.000 description 7
- 210000003625 skull Anatomy 0.000 description 6
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 206010008164 Cerebrospinal fluid leakage Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
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- 230000023597 hemostasis Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
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- 101100008047 Caenorhabditis elegans cut-3 gene Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
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- 210000005013 brain tissue Anatomy 0.000 description 1
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- 238000005238 degreasing Methods 0.000 description 1
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- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000001037 epileptic effect Effects 0.000 description 1
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- 239000000835 fiber Substances 0.000 description 1
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- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
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- 230000008676 import Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
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- 230000003340 mental effect Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000003455 parietal bone Anatomy 0.000 description 1
- 210000003446 pia mater Anatomy 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
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- 230000036573 scar formation Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Abstract
The invention belongs to the technical field of biomedical materials, and particularly relates to a method for extracting bovine achilles tendon type I collagen; the method is realized by the following steps: cleaning fresh ox calcaneus tendon, removing fat, aponeurosis and muscle, cutting tendon, soaking in sodium bicarbonate water solution, cleaning, and air drying to obtain pure ox calcaneus; adding pure bovine achilles tendon into a myristic acid solution containing triethanolamine, soaking, cleaning and airing; adding the dried bovine achilles tendon into liquid nitrogen, and grinding into powder to obtain bovine achilles tendon powder; taking bovine achilles tendon powder, adding acetic acid-sodium acetate buffer solution, adding pepsin and squalene, stirring uniformly, performing enzymolysis, adding ethylene glycol dimercaptoacetate, and performing oscillation reaction to obtain an enzymolysis solution; salting out and dialyzing the enzymolysis liquid to obtain the bovine achilles tendon I type collagen. The preparation method provided by the invention is simple and has low requirements on equipment; the obtained product is high-purity I type collagen with a triple helix structure, can not generate mechanical damage to nerve tissues, and has good biocompatibility.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a method for extracting bovine achilles tendon type I collagen.
Background
Collagen is the most abundant class of proteins in vertebrates and is also one of the four major components of the extracellular matrix. It contains one or several regions of triple-helical structure composed of alpha chains, the unique triple-helical structure and covalent cross-linking between its molecules, which endow collagen fibers with extremely high tensile strength and thermal stability, and also the structural basis for collagen interaction with cells.
In the existing extraction method, the purity of the extracted collagen is low, and how to extract the high-purity I type collagen with a triple helix structure becomes a technical problem to be solved urgently. The dura mater/dura mater may cause different degrees of damage and defect of the dura mater/dura mater in case of trauma and timely invasion of tumor, which in turn may lead to leakage of cerebrospinal fluid and secondary infection. The current extracted materials may cause rejection reaction, and the reaction of local tissues can cause stimulation to brain tissues, form scar or package reaction, meningitis symptom or bleeding and other serious complications, and most of the current repair materials depend on import and are expensive.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the method for extracting bovine achilles tendon type I collagen, which is simple and has low requirements on equipment, and the prepared product has high purity and yield.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a method for extracting bovine achilles tendon type I collagen, which comprises the following steps:
(1) cleaning fresh bovine achilles tendon, removing fat, aponeurosis and muscle, cutting into pieces, adding sodium bicarbonate water solution, soaking for 16h, taking out, cleaning with deionized water, and air drying to obtain pure bovine achilles tendon;
(2) adding pure bovine achilles tendon into a myristic acid solution containing triethanolamine, soaking for 2-3h, taking out, washing with deionized water, and air drying;
(3) adding the dried bovine achilles tendon into liquid nitrogen, and grinding into powder after the liquid nitrogen does not boil to obtain bovine achilles tendon powder;
(4) taking 20g of bovine achilles tendon powder, adding 1000mL of acetic acid-sodium acetate buffer solution, adding pepsin and squalene, stirring uniformly, performing enzymolysis for 40-50h at 35-37 ℃, adding ethylene glycol dimercaptoacetate, and performing oscillation reaction for 2-3h at 28-30 ℃ to obtain an enzymolysis solution;
(5) salting out and dialyzing the enzymolysis liquid to obtain the bovine achilles tendon I type collagen.
Further, the mass fraction of the sodium bicarbonate aqueous solution is 10%.
Further, the mass fraction of the myristic acid solution is 5-8%.
The content of triethanolamine in 1L myristic acid solution is 0.1-0.15 g.
Further, the pH value of the acetic acid-sodium acetate buffer solution is 3.5.
Furthermore, the pepsin is added in an amount of 0.6-0.8% of the weight of the bovine achilles tendon powder.
Furthermore, the addition amount of squalene is 0.2-0.3% of the weight of bovine achilles tendon powder.
Further, the mass ratio of the ethylene glycol dimercaptoacetate to the bovine achilles tendon powder is 0.5: 1.
the beef tendon is creatively soaked in the myristic acid solution, and a small amount of triethanolamine is added, so that the beef tendon can be more thoroughly degreased, most of soluble esters and other substances are removed, the later extraction efficiency is improved, and the purity of the product is improved. The inventor finds that the extraction method provided by the invention can improve the enzymolysis efficiency, reduce the enzymolysis time and improve the purity of the obtained product through a large number of experiments. The salting-out dialysis method adopted by the invention can be realized by adopting the existing method.
The invention has the beneficial effects that:
(1) the preparation method provided by the invention is simple and has low requirements on equipment;
(2) the product obtained by the invention is high-purity I type collagen with a triple helix structure, can not generate mechanical damage to nerve tissues, and has good biocompatibility.
Detailed Description
In order to better understand the essence of the present invention, the technical scheme of the present invention is further illustrated by specific examples and clinical trial data.
Example 1
1.1, cleaning fresh bovine achilles tendon, removing fat, aponeurosis and muscle, cutting into blocks (the size of each block can be 0.5cm multiplied by 0.5cm), adding a sodium bicarbonate aqueous solution with the mass fraction of 10 percent, soaking for 16h (the sodium bicarbonate aqueous solution can be used for soaking beef), taking out, cleaning with deionized water, and airing to obtain pure bovine achilles tendon;
1.2, adding the pure achilles tendon into a 5% myristic acid solution containing triethanolamine, soaking for 2-3h, taking out, washing with deionized water, and air drying;
the content of triethanolamine in each 1L of myristic acid solution is 0.15 g;
1.3 adding the liquid nitrogen into the dried bovine achilles tendon, and grinding the mixture into powder after the mixture is not boiled to obtain bovine achilles tendon powder;
1.4 taking 20g of bovine achilles tendon powder, adding 1000mL of acetic acid-sodium acetate buffer solution, adding pepsin accounting for 0.6% of the weight of the bovine achilles tendon powder and squalene accounting for 0.2% of the weight of the bovine achilles tendon powder, stirring uniformly, stirring for enzymolysis for 40-50h at 35-37 ℃, then adding ethylene glycol dimercaptoacetate, and carrying out oscillation reaction for 2-3h at 28-30 ℃ to obtain an enzymolysis solution;
the mass ratio of the ethylene glycol dimercaptoacetate to the bovine achilles tendon powder is 0.5: 1;
1.5 salting out and dialyzing the enzymatic hydrolysate to obtain the bovine achilles tendon type I collagen.
Example 2
2.1 cleaning fresh bovine achilles tendon, removing fat, aponeurosis and muscle, cutting into blocks (the size of the blocks can be 0.5cm multiplied by 0.5cm), adding 10% sodium bicarbonate aqueous solution to soak for 16h (the sodium bicarbonate aqueous solution can submerge the beef), taking out, washing with deionized water, and air drying to obtain pure bovine achilles tendon;
2.2 adding the pure achilles tendon into a myristic acid solution containing triethanolamine and with the mass fraction of 6.5%, soaking for 2-3h, taking out, washing with deionized water, and airing;
the content of triethanolamine in each 1L of myristic acid solution is 0.12 g;
2.3 adding the liquid nitrogen into the dried bovine achilles tendon, and grinding the mixture into powder after the mixture is not boiled to obtain bovine achilles tendon powder;
2.4 taking 20g of bovine achilles tendon powder, adding 1000mL of acetic acid-sodium acetate buffer solution, adding pepsin accounting for 0.8% of the weight of the bovine achilles tendon powder and squalene accounting for 0.3% of the weight of the bovine achilles tendon powder, stirring uniformly, stirring for enzymolysis for 40-50h at 35-37 ℃, then adding ethylene glycol dimercaptoacetate, and carrying out oscillation reaction for 2-3h at 28-30 ℃ to obtain an enzymolysis solution;
the mass ratio of the ethylene glycol dimercaptoacetate to the bovine achilles tendon powder is 0.5: 1;
2.5 salting out and dialyzing the enzymatic hydrolysate to obtain the bovine achilles tendon type I collagen.
Example 3
3.1 cleaning fresh bovine achilles tendon, removing fat, aponeurosis and muscle, cutting into blocks (the size of the blocks can be 0.5cm multiplied by 0.5cm), adding 10% sodium bicarbonate aqueous solution to soak for 16h (the sodium bicarbonate aqueous solution can submerge the beef), taking out, washing with deionized water, and airing to obtain pure bovine achilles tendon;
3.2 adding the pure achilles tendon into a myristic acid solution containing triethanolamine and with the mass fraction of 8%, soaking for 2-3h, taking out, washing with deionized water, and airing;
the content of triethanolamine in each 1L of myristic acid solution is 0.1 g;
3.3 adding the liquid nitrogen into the dried bovine achilles tendon, grinding the mixture into powder after the mixture is not boiled to obtain bovine achilles tendon powder;
3.4 taking 20g of bovine achilles tendon powder, adding 1000mL of acetic acid-sodium acetate buffer solution, adding pepsin accounting for 0.7% of the weight of the bovine achilles tendon powder and squalene accounting for 0.3% of the weight of the bovine achilles tendon powder, stirring uniformly, stirring for enzymolysis for 40-50h at 35-37 ℃, then adding ethylene glycol dimercaptoacetate, and carrying out oscillation reaction for 2-3h at 28-30 ℃ to obtain an enzymolysis solution;
the mass ratio of the ethylene glycol dimercaptoacetate to the bovine achilles tendon powder is 0.5: 1;
3.5 salting out and dialyzing the enzymatic hydrolysate to obtain the bovine achilles tendon type I collagen.
Comparative example 1 (insufficient purity, affecting extraction efficiency) Triethanolamine was not added
1.1, cleaning fresh bovine achilles tendon, removing fat, aponeurosis and muscle, cutting into blocks (the size of each block can be 0.5cm multiplied by 0.5cm), adding a sodium bicarbonate aqueous solution with the mass fraction of 10 percent, soaking for 16h (the sodium bicarbonate aqueous solution can be used for soaking beef), taking out, cleaning with deionized water, and airing to obtain pure bovine achilles tendon;
1.2, adding pure bovine achilles tendon into a myristic acid solution with the mass fraction of 8%, soaking for 2-3h, taking out, washing with deionized water, and airing;
1.3 adding the liquid nitrogen into the dried bovine achilles tendon, and grinding the mixture into powder after the mixture is not boiled to obtain bovine achilles tendon powder;
1.4 taking 20g of bovine achilles tendon powder, adding 1000mL of acetic acid-sodium acetate buffer solution, adding pepsin accounting for 0.7% of the weight of the bovine achilles tendon powder and squalene accounting for 0.3% of the weight of the bovine achilles tendon powder, stirring uniformly, stirring for enzymolysis for 40-50h at 35-37 ℃, then adding ethylene glycol dimercaptoacetate, and carrying out oscillation reaction for 2-3h at 28-30 ℃ to obtain an enzymolysis solution;
the mass ratio of the ethylene glycol dimercaptoacetate to the bovine achilles tendon powder is 0.5: 1;
1.5 salting out and dialyzing the enzymatic hydrolysate to obtain the bovine achilles tendon type I collagen.
Comparative example 2 (Long extraction time, Low purity) No addition of Squalene
2.1 cleaning fresh bovine achilles tendon, removing fat, aponeurosis and muscle, cutting into blocks (the size of the blocks can be 0.5cm multiplied by 0.5cm), adding 10% sodium bicarbonate aqueous solution to soak for 16h (the sodium bicarbonate aqueous solution can submerge the beef), taking out, washing with deionized water, and air drying to obtain pure bovine achilles tendon;
2.2 adding the pure bovine achilles tendon into a myristic acid solution with the mass fraction of 8%, soaking for 2-3h, taking out, washing with deionized water, and airing;
2.3 adding the liquid nitrogen into the dried bovine achilles tendon, and grinding the mixture into powder after the mixture is not boiled to obtain bovine achilles tendon powder;
2.4 taking 20g of bovine achilles tendon powder, adding 1000mL of acetic acid-sodium acetate buffer solution, adding pepsin accounting for 0.7 percent of the weight of the bovine achilles tendon powder, stirring uniformly, stirring for enzymolysis for 40-50h at 35-37 ℃, then adding ethylene glycol dimercaptoacetate, and carrying out oscillation reaction for 2-3h at 28-30 ℃ to obtain an enzymolysis solution;
the mass ratio of the ethylene glycol dimercaptoacetate to the bovine achilles tendon powder is 0.5: 1;
2.5 salting out and dialyzing the enzymatic hydrolysate to obtain the bovine achilles tendon type I collagen.
Comparative example 3
3.1 cleaning fresh achilles tendon, removing fat, aponeurosis and muscle, cutting into pieces (the size of the pieces can be 0.5cm multiplied by 0.5cm), soaking in sodium chloride solution, removing impurities, and air drying for later use;
3.2 soaking the dried bovine achilles tendon in acetone for degreasing; then, after treatment with a dialysis bag, extraction was performed with pepsin.
Effects of the embodiment
The extraction rate and purity of the collagen extracted in examples 1-3 and comparative examples 1-3 were determined, and the specific results are shown in table 1.
TABLE 1
And secondly, stirring 18 g of high-purity I-type medical collagen material with a triple helix structure in hydrochloric acid environment at 28000 rpm for 15 minutes to prepare 2.5% collagen composite suspension. Then sodium hydroxide is used for adjusting the pH value and the 100 percent alcohol of the alcohol to make the viscosity of the alcohol meet the requirement. Stirring was continued for 15 minutes. And (3) pouring 500 ml of the dried meninges/spinal meninges biomembrane into a freezing tray of 50X50 cm, freezing for 3-6 hours at the low temperature of-75 ℃, and drying for 25 hours to prepare the meninges/spinal meninges biomembrane with pores of less than 20 microns and thickness of 2 mm.
Animal experiments: 3 male retrievers were subjected to right anterolateral craniectomy. A trephine with a diameter of 3 cm was used to perform a right anterolateral roof osteotomy. Does not damage any infracranial tissue. After careful hemostasis, the dura mater beneath it was lifted and carefully cut 3 cm diameter dura mater. Protecting the underlying arachnoid and pia mater from any damage.
The dural defect site was carefully implanted with the biofilms of examples 1-3, 3.5 cm in diameter, and the dural defect site and the surface of the brain parenchyma were covered with sutures. The excised skull flap is then placed back at the site of the skull defect after careful hemostasis.
The animals after the operation are all healthy and alive without any nerve, abnormal mental symptoms, leakage of cerebrospinal fluid and infection. After 6 weeks the animals were sacrificed and the skull was removed with an enlarged range using a trephine of 6 cm diameter with the center of the original skull after the first surgical flap reduction as the new center, exposing the dura mater and brain parenchyma below.
And (4) visual observation: the right antero-lateral parietal bones of 3 experimental animals healed well without any infection. After the bone flap is lifted, no obvious adhesion exists between the skull and the dura mater, the neogenetic dura mater tissue is smooth, and no obvious adhesion or scar exists between the dura mater, the arachnoid membrane and the brain parenchyma. The surface of the brain parenchyma is normal without any degenerative changes.
Histological examination: the skull/dura mater/brain parenchyma was entirely removed. Decalcifying, embedding in wax, preparing into 5-6 micron thick section, HE staining, and observing histological morphology.
The abnormal tissue cells are not found in routine section examination under a microscope. Fibroblasts and fibrodesmium tissue shown in their entirety as orders. There was no difference in structure, morphology and normal dura mater compared to normal dura mater except for active neocellular differentiation at the base of neodural mater. No adhesion and no scar formation. Displaying the newly-grown fibrous tissue under a polarizing microscope
The special structure-stage spectrum structure of the type I collagen is shown, and the implanted collagen membrane is degraded and absorbed without any residual collagen material.
However, in the case of the film produced in comparative example 3, the occurrence of the epileptic mark was observed during the repeated tests.
Claims (3)
1. The method for extracting bovine achilles tendon type I collagen is characterized by comprising the following steps:
(1) cleaning fresh bovine achilles tendon, removing fat, aponeurosis and muscle, cutting into pieces, adding sodium bicarbonate water solution, soaking for 16h, taking out, cleaning with deionized water, and air drying to obtain pure bovine achilles tendon;
(2) adding pure bovine achilles tendon into a myristic acid solution containing triethanolamine, soaking for 2-3h, taking out, washing with deionized water, and air drying;
the mass fraction of the myristic acid solution is 5-8%;
the content of triethanolamine in each 1L of myristic acid solution is 0.1-0.15 g;
(3) adding the dried bovine achilles tendon into liquid nitrogen, and grinding into powder after the liquid nitrogen does not boil to obtain bovine achilles tendon powder;
(4) taking 20g of bovine achilles tendon powder, adding 1000mL of acetic acid-sodium acetate buffer solution, adding pepsin and squalene, stirring uniformly, performing enzymolysis for 40-50h at 35-37 ℃, adding ethylene glycol dimercaptoacetate, and performing oscillation reaction for 2-3h at 28-30 ℃ to obtain an enzymolysis solution;
the adding amount of the pepsin accounts for 0.6 to 0.8 percent of the weight of the bovine achilles tendon powder; the addition amount of squalene accounts for 0.2-0.3% of the weight of the bovine achilles tendon powder;
the mass ratio of the ethylene glycol dimercaptoacetate to the bovine achilles tendon powder is 0.5: 1;
(5) salting out and dialyzing the enzymolysis liquid to obtain the bovine achilles tendon I type collagen.
2. The extraction method according to claim 1, wherein the aqueous sodium bicarbonate solution is present in an amount of 10% by mass.
3. The extraction process according to claim 1, wherein the acetic acid-sodium acetate buffer solution has a pH of 3.5.
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